Rapid Isolation of Geraniin from Nephelium lappaceum rind waste and its anti hyperglycemic activity

See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/229409186 Rapid isolation of geraniin from Nephelium lappaceum rind waste and its antihyperglycemic activity Article in Food Chemistry · July 2011 DOI: 10.1016/j.foodchem.2010.12.070 CITATIONS READS 62 716 4 authors: Uma Devi Palanisamy Lai Teng Ling 60 PUBLICATIONS 626 CITATIONS 7 PUBLICATIONS 183 CITATIONS Monash University (Malaysia) SEE PROFILE University of Malaya SEE PROFILE Thamilvaani Manaharan David Ross Appleton 14 PUBLICATIONS 202 CITATIONS 55 PUBLICATIONS 587 CITATIONS University of Malaya SEE PROFILE Sime Darby SEE PROFILE Some of the authors of this publication are also working on these related projects: Gut Microbiota Dysbiosis in Diet-induced Obesity and Insulin Resistance and Prebiotic Potential of Polyphenols View project All content following this page was uploaded by Uma Devi Palanisamy on 02 January 2017. The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the original document and are linked to publications on ResearchGate, letting you access and read them immediately. Food Chemistry 127 (2011) 21–27 Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem Rapid isolation of geraniin from Nephelium lappaceum rind waste and its anti-hyperglycemic activity Uma D. Palanisamy a,⇑, Lai Teng Ling b, Thamilvaani Manaharan b, David Appleton c a Jeffrey Cheah School of Medicine and Health Sciences, Monash University Sunway Campus, Jalan Lagoon Selatan, 46150 Bandar Sunway, Malaysia Department of Physiology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia c Department of Pharmacology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia b a r t i c l e i n f o Article history: Received 7 September 2010 Received in revised form 6 November 2010 Accepted 13 December 2010 Available online 21 December 2010 Keywords: Geraniin Alpha glucosidase Alpha amylase Aldol reductase AGE Nephelium lappaceum L. a b s t r a c t Recently we confirmed the ability of ethanolic Nephelium lappaceum L. rind extract to act as anti-hyperglycemic agent. Geraniin, an ellagitannin, was identified as the major bioactive compound isolated from the ethanolic Nephelium lappaceum L. rind extract. In this study, we describe the rapid isolation of geraniin from the above plant. In addition to its extremely high anti-oxidant activity and low pro-oxidant capability, geraniin is seen to possess in vitro hypoglycemic activity (alpha-glucosidase inhibition: IC50 = 0.92 lg/ml and alpha-amylase inhibition: IC50 = 0.93 lg/ml), aldol reductase inhibition activity (IC50 = 7 lg/ml) and has the ability to prevent the formation of advanced glycation end-products (AGE). Geraniin was observed to exhibit these properties at more significant levels compared to the positive controls acarbose (carbohydrate hydrolysis inhibitor), quercetin (aldol reductase inhibitor) and green tea (AGE inhibitor). Geraniin therefore, has the potential to be developed into an anti-hyperglycemic agent. Our findings also strongly support the use of a geraniin-standardised N. lappaceum extract in the management of hyperglycemia. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction During onset and development of type 2 diabetes, cellular balance of carbohydrate and lipid metabolism is affected by improper glucose metabolism, which leads to elevated post-prandial blood glucose levels. One therapeutic approach for treating diabetes is to decrease post-prandial hyperglycemia by retarding absorption of glucose through inhibition of carbohydrate hydrolysing enzymes like a-glucosidase and a-amylase in the digestive tract (Cheplick, Kwon, Bhowmik, & Shetty, 2010). Prolonged hyperglycemia with diabetes mellitus leads to the formation of advanced glycosylated end products (AGEs) which are involved in the generation of reactive oxygen species (ROS) and causes oxidative damage (Mohamed et al., 2009). The complex, fluorescent AGE molecules formed during the Maillard reaction can lead to protein cross-linking and contribute to the development and progression of several diabetic complications such as peripheral neuropathy, cataracts, impaired wound healing, vascular damage, arterial wall stiffening and decreased myocardial compliance (Thomas, Baynes, ⇑ Corresponding author. E-mail addresses: umadevi.palanisamy@med.monash.edu.my (U.D. Palanisamy), lai_teng@siswa.um.edu.my (L.T. Ling), melisavaani@yahoo.com (T. Manaharan), drappleton@hotmail.com (D. Appleton). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.12.070 Thorpe, & Cooper, 2005; Wada & Yagihashi, 2005). Oxidative stress is believed to be a common pathway linking diverse mechanisms for the pathogenesis of complications of diabetes. There is strong evidence for increased levels of indicators of oxidative stress in diabetic individuals suffering from complications (Rahimi, Shekoufeh, Bagher, & Mohammad, 2005). In addition, during chronic hyperglycemia, excessive glucose uptake in tissues effect the key enzyme aldose reductase (AR) in the polyol pathway. This leads to reduction of various sugars to sugar alcohols, such as glucose to sorbitol, followed by nicotinamide adenine dinucleotide (NADH)-dependent sorbitol dehydrogenasecatalyzed fructose production. Increased fructose formation leads to reactive dicarbonyl species, key factors in AGE formation (Kawai et al., 2009). In addition, sorbitol and its metabolites accumulate in the nerves, retina, lens, and kidneys due to their poor penetration across membranes and inefficient metabolism, resulting in the development of diabetic complications, including retinopathy, neuropathy, nephropathy, and cataracts (Ramana & Srivastava, 2010). Traditionally, plant extracts have been used in the management of diabetes. Numerous studies have shown the effectiveness of the crude plant extract as well as its bioactive compounds in lowering blood glucose levels (Kaushik, Satya, Khandelwal, & Naik, 2010). Phenolic phytochemicals present in fruits and vegetable, apart from having anti-oxidant potential, have shown a-glucosidase 22 U.D. Palanisamy et al. / Food Chemistry 127 (2011) 21–27 and a-amylase inhibitory activity with minimal side effects (Cheplick et al., 2010; Da Silva Pinto et al., 2009; McDougall et al., 2005). The inhibition of aldol reductase (AR) and advanced glycation end-products (AGE) is yet another mode of diabetes treatment not dependent on the control of blood glucose level, and would be useful in the prevention or reduction of certain diabetic complications (Jung, Kim, & Choi, 2009; Tsuji-Naito, Saeki, and Hamano, 2009). Nephelium lappaceum L. belongs to the same family (Sapindaceae) as the sub-tropical fruits lychee and longan (Marisa, 2006) and it is native to Southeast Asia. This fruit is an important commercial crop in Asia, where it is taken freshly or processed. In Southeast Asia, the dried fruit rind has been employed in traditional medicine for centuries. Additionally, the rind is used in cooking and the manufacture of soap. The roots, bark, and leaves have various uses in medicine and in the production of dyes. Previous studies have shown N. lappaceum rind extract to exhibit high anti-oxidant activity (Palanisamy et al., 2008), antibacterial activity (Thitilertdecha, Teerawutgulrag, Kilburn, & Rakariyatham, 2010) and anti-Herpes Simplex virus type 1 (Nawawi, Hattori, Kurokawa, & Shiraki, 1999). Recently in our laboratory, N. lappaceum rind was also shown to have anti-hyperglycemic potential. The utilisation of N. lappaceum rind to manage hyperglycemia is seen as an important finding not only in traditional medicine but also in aspects of valorisation of food waste. Based on its beneficial health properties, N. lappaceum rind has potential to be developed into a nutraceutical (Palanisamy et al., 2008). It is therefore pertinent that we isolate and identify the active compounds in this extract that contributes to its biological activities. Structural characterisation of its purified compounds can thus lead to the formulation of the new therapeutic products. Thitilertdecha et al. (2010) recently identified the major phenolic compounds from N. lappaceum rind as ellagic acid, corilagin and geraniin. There is little evidence on the bioactivity of geraniin itself though it has been reported to have NO scavenging activities (Kumaran & Karunakaran, 2006), anti-infection activities against HSV-1 and HSV-2 (Yang, Cheng, Lin, Chiang, & Lin, 2007) and antihypertensive activity (Lin, Wang, Lu, Wu, & Hou, 2008). In this paper, we describe a rapid purification method to isolate and purify geraniin from N. lappaceum rind extract. The ability of geraniin to function as an ideal anti-oxidant, having low prooxidant capability, inhibit the carbohydrate hydrolysing enzymes, retard polyol and advance glycation end product formation is also described. (Buchs, Switzerland). Ethanol (absolute and denatured) was obtained from Scharlau Chemicals (Germany). 2.2. Isolation of geraniin from N. lappaceum rind Nephelium lappaceum peels were harvested and collected in Malaysia from January to March 2009. The plants were authenticated by a botanist at the Herbarium of the Forest Research Institute of Malaysia (FRIM) in Kepong, Malaysia. Cleaned plants (1 kg) were dried at 40 °C in the oven before being powderised using the Fritsch dry miller. Ethanol extraction was carried out at room temperature for 24 h in an orbital shaker. The suspension thus obtained was filtered using a 114 Whatman filter paper and filtrate collected. Ethanol filtrate was concentrated using a rotary evaporator. Ethanolic extract (5 g) was dissolved in distilled water with a minimum amount of acetonitrile and eluted onto a glass column (100  30 cm i.d.) packed with LiChroprep RP-18 (40–63 um, 150 g) which was pre-equilibrated with water (200 mL). Elution was then carried out using 200 ml 0–5% acetonitrile to remove impurities. This was followed by eluting with 500 ml 80% acetonitrile until a yellowish fraction was obtained. The 80% acetonitrile eluent was rotary evaporated to dryness to yield a 3 g yellowish powder (F1). F1 (1.0 g) was further purified on a Gilson Preparative HPLC system, GX-281/322/156 Gilson Preparative HPLC using a Waters Xterra Prep RP18 OBD (19  50 mm) with a UV detector (210 and 275 nm). This step was repeated until all F1 was utilised. The mobile phase was 0.1% formic acid in acetonitrile and 0.1% formic acid in ultra pure water at a flow rate of 18 mL/min. The solvent gradient consisted of 0–10% acetonitrile for 3 min, 10–40% acetonitrile for 12 min, and finally 100% acetonitrile for 5 min to recondition the column. 2.3. HPLC–LCMS/MS analysis of geraniin Geraniin was analysed on a Shidmazu Prominence UFLC-LCMSIT-TOF. The analysis was performed in both the positive and negative modes. The capillary temperature was 250 °C and nitrogen gas was used as the sheath gas. The capillary voltage was 1.65 kV. A mass range of 200–2000 was scanned in both positive and negative full ion monitoring mode. Geraniin was monitored by measuring absorbance at 254 nm. The compound was separated using a Waters Xterra MS C18 (2.5  20 mm, 2.5 um) IS column eluted with 0.1% formic acid in water and 0.1% formic acid in acetonitrile. The flow rate was 0.5 mL/min with an injection volume of 10 uL at 40 °C. 2. Methods 2.4. NMR analysis 2.1. Chemicals and reagents L-Ascorbic acid, Galvinoxyl and potassium persulfate were obtained from Sigma–Aldrich (USA). DL-a-Tocopherol and potassium hexacyanoferrate (III) were obtained from Fluka biochemika (Germany). Ethanol (absolute) was obtained from Scharlau chemicals (Germany. ABTS Diammonium salt was obtained from Amresco (Ohio). Emblica™ was obtained from EMD (Darmstadt, Germany), Alpha glucosidase (Saccharomyces cerevisiae), alpha amylase (porcine pancreatic Type IV-B), DL-dithiothreitol (DTT), 4-nitrophenyl a-d-gluco-pyranoside (PNPG), and 3,5-dinitrosalicylic acid (DNS) were purchased from Sigma Aldrich (USA, CO). Human recombinant aldol reductase (Japan, ATCC) and trichloroacetic acid from Merck (Darmstadt, Germany). DL-Glyceraldehyde, b-NADPH, acarbose, soluble starch and quercetin (Standard Phenolic) were obtained from Sigma Chemicals Co. Ltd. (St. Louis, USA). Phosphate buffer saline (PBS), glucose anhydrous(+) and bovine serum albumin (BSA) were purchased from Fluka Biochemika NMR spectra were obtained using a Jeol ECA 400 (400 MHz) NMR spectrometer. Geraniin were characterised by 1H, 13C, 2D NMR. 2.5. Scavenging activity of geraniin onto Galvinoxyl and ABTS radicals Free radical scavenging ability of geraniin was evaluated using Galvinoxyl and ABTS assays as previously described by Palanisamy et al. (2008). 2.6. Pro-oxidant assay Reducing power of iron ion was measured according to the method of (Tian & Hua, 2005) where 500 ll of geraniin or ethanolic N. lappaceum extract and 50 ll of 1% potassium ferricyanate [K3Fe(CN6)] were incubated at 50 °C for 20 min. An equal volume of 10% trichloroacetic acid was then added and mixture centrifuged 23 U.D. Palanisamy et al. / Food Chemistry 127 (2011) 21–27 at 3000g for 10 min. The upper layer of the solution (1.0 ml) was mixed with 1 ml of distilled water and 0.2 ml of 0.1% ferric chloride (FeCl3) and its absorbance was recorded at 700 nm. Ethanol or distilled water was used as negative control while Vitamin C and Emblica™ (a commercial anti-oxidant with very low pro-oxidant activity) was used as positive controls. Results are expressed in comparison with positive controls at concentrations ranging from 0.1 to 0.5 mg/ml. NADPH, 20 lL of geraniin or ethanolic N. lappaceum extract and 50 lL of 5 mM of DL-glyceraldehyde as the substrate. The reaction solution was incubated at 25 °C for 3 min. The AR activity was determined by measuring the decrease in NADPH absorption at 340 nm over 10 min period on a spectrophotometer. Quercetin was used as the positive control, while negative control was the assay performed without geraniin. The inhibition percentage% was calculated as: 2.7. Anti-hyperglycemic assays % Inhibition ¼ 2.7.1. Alpha glucosidase inhibitory activity The alpha-glucosidase inhibition assay was performed using the modified method of (Liu, Zhang, Wang, & Grinsgard, 2004). The assay was carried out using 96 well plates. Alpha-glucosidase from Saccharomyces cerevisiae (0.4 U/ml) was dissolved in phosphate buffer pH6.8 and supplemented with 0.2% BSA. 0.4 U/ml enzyme, 20 ll of DTT (1 mM), 20 ll of substrate PNPG (para-nitrophenly glucopyranoside) were added with 0.1 M sodium phosphate buffer pH 6.8 and incubated at 37 °C for 15 min with and without geraniin or ethanolic N. lappaceum extract The reaction was stopped with 80 ll of 0.2 M sodium carbonate and the yellow colour of para-nitrophenol was determined at absorbance 400 nm with a Cary 50 Bio UV–visible spectrophotometer (Varian, Inc., Palo Alto, CA). Acarbose, 3.5 mg/ml was used as the positive control while absence of geraniin was the negative control. Appropriate blanks were used to exclude background absorbance. The percentage inhibition was calculated as below: where, A sample/min represents the reduction of absorbance for 10 min with geraniin or extract and substrate, and A control/min represents the same, but with buffer instead of sample. The inhibitory activity of geraniin, ethanolic N. lappaceum extract and quercetin were assessed by plotting percentage inhibition against a range of concentrations (0.001–1 mg/ml) and determining the IC50. The IC50 values are expressed as means ± SEM of triplicate experiments. % inhibition ¼ 

 Aneg control  Ablank  Asample  Asample blank
 100 Aneg control  Ablank A = absorbance. Inhibitory activity was assessed by plotting percentage inhibition against a range of concentrations (0.01–1 mg/mL) and determining the IC50 (Concentration with 50% inhibition) value. Results are expressed as means ± SEM of triplicate measurements. 2.7.2. Alpha-amylase inhibitory activity The alpha-amylase inhibition assay was performed using the modified method of (Liu et al., 2004). The assay was carried out using 96 well plates. Porcine pancreatic a-amylase (Sigma Type IV-B) was dissolved in ice-cold distilled water to a concentration of 2U/ml. Potato soluble starch solution (1%) was prepared in 20 mM phosphate buffer pH6.9. DNS solution was prepared with 1 g DNS with 30 g sodium potassium tartrate dehydrate and 2 M NaOH in 100 ml solution. In the assay 80 ll of various dilutions of geraniin or ethanolic N. lappaceum extract and 40 ll of amylase enzyme were added and incubated at room temperature for 10 min. Then, 40 ll of starch added and incubated at 37 °C for 10 min. After which 80 ll of DNS solution was added and incubated in a water bath at 95 °C for 10 min, to detect the reducing sugar which will change the DNS from orange to brick red colour. The absorbance was measured at 540 nm with a Cary 50 Bio UV–visible spectrophotometer (Varian, Inc., Palo Alto, CA). Acarbose, 12 lg/ml was used as the positive control. Appropriate blanks were used to exclude background absorbance. The percentage inhibition and IC50 were determined as described for the alpha glucosidase assay. 2.8. Aldose reductase (AR) inhibitory activity The activities of recombinant human AR were measured according to the procedure of (Nishimura et al., 1991). AR with a specific activity of 0.2 U/mL, was used in the evaluations for enzyme inhibition. The reaction mixture consists of 870 lL of 100 mM sodium phosphate buffer (pH 6.2), 10 lL of AR enzyme, 50 lL of 3 mM  1  Asample=min  Ablank=min
Acontrol=min  Ablank=min
  100 2.9. Advanced glycation endproducts (AGE) formation inhibitory activity The measurement of fluorescent material based on AGEs in order to detect the inhibitory effect of test samples on the Maillard reaction was performed according to (Matsuura et al., 2002) using the BSA/glucose system. The reaction mixture consists of 4 mg of bovine serum albumin (BSA) in 400 lL of 50 mM sodium phosphate buffer (pH 7.4), 80 lL of 1 M glucose and 20 lL of geraniin (0.5 mg/ml) or ethanolic N. lappaceum extract (1 mg/ml). The reaction solution (500 lL) was incubated at 80 °C and the control was kept at 4 °C for 0, 1, 3, 5, and 7 days. Green tea (1 mg/ml) was used as the positive control in this assay. After the incubation period, the reaction was stopped with the addition of 100% TCA followed by centrifugation at 15,000 rpm for 10 min. The AGE–BSA precipitate thus formed was dissolved in 1 mL PBS (pH 10) and its fluorescence intensity determined with excitation and emission wavelengths at 385 nm and 415 nm, respectively on a spectrophotometer (Tecan microplate reader). The inhibition percentages were calculated as described for aldol reductase. The inhibitory activity of geraniin and ethanolic N. lappaceum extract was determined by plotting the% inhibition (fluorescence intensity) against a range of incubation time (days). 2.10. Statistical analysis All experiments were performed in triplicates. Analysis at every time point from each experiment was carried out in triplicates. Means, standard errors and standard deviations were calculated from replicates with in the experiments and analyses were done using Microsoft Excel 2003. 3. Results and discussion 3.1. Extraction, isolation and purification of geraniin Nephelium lappaceum rind was extracted with ethanol as previously described by our group (Palanisamy et al., 2008). We have also shown that the ethanolic extracts of N. lappaceum rind exhibited higher free radical scavenging and anti-hyperglycemic activity (Palanisamy, Manaharan, Ling, Radhakrishnan, & Masilamani, unpublished results) compared to other extraction methods. In this study, we focused on obtaining the major active compound, geraniin from the ethanolic extract. Ethanolic extract (5 g), extracted from 16.7 g plant material, was first separated on a RP-18 glass column to yield a 3 g yellowish fraction (F1). 1 g of F1 was used in 24 U.D. Palanisamy et al. / Food Chemistry 127 (2011) 21–27 subsequent purification on a preparative HPLC. Geraniin (211.2 mg) was obtained at the retention time of 13 min with 20% acetonitrile. (Fig. 1). Purification on a preparative HPLC was repeated (3) in the same manner until all F1 was used. Table 1 shows the method of extraction, yield and the quantification of geraniin throughout the extraction and purification steps. Thitilertdecha et al. (2010), in their work with N. lappaceum rind showed a 25.1% yield using methanol as the extraction solvent while we managed to obtain a 30.6% yield with ethanol. The same workers carried onto isolate geraniin from the methanolic extract on a Sephadex LH20 column with a yield of 56.8% and reported to obtain pure geraniin at 142 mg/g (14.2%) dry weight N. lappaceum rind. In our work, we showed a 60% yield using the LiChroprep RP-18 column, however further purification on a preparative HLPC was required to obtain pure geraniin at 37.9 mg/g (3.79% dry wt). We report a 27% lower geraniin content in N. lappaceum rind as compared to the earlier workers. It is acknowledged that additional purification steps (preparative HPLC) would compromise the final yield. There is also a possibility that geraniin, may have been hydrolysed to corilagin, ellargic acid and gallic acid (Luger et al., 1998). During this extra procedure, thus giving rise to lower geraniin yields. On the other hand, the possibility that the geraniin obtained from the 2 step procedure by Thitilertdecha et al. (2010) may not be of high purity, cannot be disputed. Table 1 Quantification of geraniin in the rapid purification method. Sample/fraction Extraction method Yield (%) Geraniin in sample (%)a N. lappaceum rind Ethanolic extract F1 Ethanol extraction LiChroprep RP-18 Preparative HPLC 30.58 60.00 21.15 3.79 12.68 21.13 a Calculation of the content of geraniin (%) is based on the assumption that 3 g of F1was in final purification step. the N. lappaceum ethanolic extract in both the assays studied. It can be concluded that the scavenging activity observed in the ethanolic extract is largely contributed by the 13% (Table 1) geraniin present in it. Geraniin has also been shown to possess superoxide radical and hydroxyl radical-scavenging activity in Mallotus japonicus leaves. It was also mentioned that geraniin exhibited a much stronger anti-oxidant activity than gallic acid, rutin, ellagic acid, quercetin, and chlorogenic acid, and was as active as epigallocatechin gallate (EGCG), a strong anti-oxidant found in green tea (Tabata et al., 2008). 3.4. Pro-oxidant assay The free radical scavenging ability of geraniin (IC50) using the radical DPPH has been shown by many workers to be in the range of 0.79–18.7 lM (Lin et al., 2008; Thitilertdecha et al., 2010;Yokozawa et al., 1998). We report here the free radicalscavenging activity of geraniin assessed using the radicals Galvinoxyl (IC50 = 1.9 lM) and ABTS (IC50 = 6.9 lM). Our results (Fig. 2) indicate that geraniin has similar anti-oxidant activity as Pro-oxidant anti-oxidant effect of compounds are due to the balance of two activities, free radical-scavenging activity and reducing power on iron ions, which may drive the Fenton reaction via reduction of iron ions. In a Fenton reaction, Fe2+ reacts with H2O2, resulting in the production of hydroxyl radical, which is considered to be the most harmful radical to biomolecules. In the Fenton reaction Fe2+ is oxidised to Fe3+. Many reductants, such as ascorbic acid can reduce the oxidised form of iron (Fe3+) to reduced form (Fe2+). This reaction could enhance the generation of hydroxyl radicals. The predomination of reducing power (on iron ions) over the free radical-scavenging activity results in the pro-oxidant effect. Evaluation of pro-oxidant activity of geraniin is crucial in order to ensure that at its effective concentrations geraniin does not act as a pro-oxidant in the presence of transition ions. In this Fenton reaction, Fe2+ reacts with H2O2, resulting in the production of hydroxyl radical which is a very harmful radical to biomolecules and Fe2+ is oxidised to Fe3+. Many reductants such as the oxidised form of vitamin C can be reduced to trigger the production of hydroxyl radicals (Tian & Hua, 2005). Every anti-oxidant is in fact Fig. 1. Purification on Prep-HPLC showing geraniin as the major compound in the ethanolic Nephelium lappaceum rind extract. The solvent gradient consisted of 0– 10% acetonitrile for 3 min, 10–40% for 12 min and finally 100% acetonitrile for 5 min to recondition the column. at a flow rate of 18 mL/min. Geraniin was obtained at the retention time of 13minutes. 1 was detected at 210 nm and 2 at 275 nm. Insert shows the chemical structure of geraniin. Fig. 2. Free Radical-scavenging activity of geraniin and N. lappaceum ethanolic extract. Data expressed in mean ± SD, n = 3. ⁄Palanisamy et al. (unpublished results). 3.2. Identification of geraniin with NMR and LCMS/MS The NMR profile obtained was the same as reported by previous studies (Gohar, Lahloub, & Niwa, 2003; Thitilertdecha et al., 2010). The negative mode of HRMS m/z: 951.0718 [MH] (calcd. for C41 H2 7O 27 , 951.0740) and the HRMS/MS m/z: 301, 445, 614, 933. The positive mode of HRMS m/z: 783.0592 [M–O-galloyl]+ and the HRMS/MS m/z: 259, 277, 303, 337, 463. 3.3. Free radical-scavenging activity 25 U.D. Palanisamy et al. / Food Chemistry 127 (2011) 21–27 a redox agent and might become a pro-oxidant to accelerate lipopolysaccarides and induce DNA damage under special conditions and concentrations. Studies have revealed the pro-oxidant effects of anti-oxidant vitamins and several classes of plant-derived polyphenols (Singh, Farhan Asad, Ahmad, Khan, & Hadi, 2001). Here, the pro-oxidant capacity of geraniin was compared with N. lappaceum ethanolic extract L-ascorbic acid and Emblica™, a commercial preparation of the plant Phyllantus emblica that possess very low reducing power (Fig. 3). Our results clearly show that geraniin had much lower reducing ability compared to N. lappaceum ethanolic extract, L-ascorbic acid and Emblica™ (Fig. 3), indicating that it is infact an ideal anti-oxidant. 3.5. Alpha-glucosidase and alpha-amylase inhibitory activity The ability of geraniin to inhibit Saccharomyces cerevisiae a-glucosidase and porcine pancreatic Type IV-B a-amylase was investigated. It is evident in Fig. 4 that geraniin exhibited the highest inhibitory activity for both the enzymes (IC50 = 0.92 ± 0.10 lg/ml or 0.97 lM) compared to N. lappaceum ethanolic extract (a-glucosidase, IC50 = 2.7 ± 2.3 lg/ml; a-amylase, IC50 = 3.0 ± 1.4 lg/ml) and the positive control acarbose (a-glucosidase, IC50 = 25 ± 2 lg/ ml or 39 lM; a-amylase, IC50 = 36 ± 3 lg/ml or 56 lM). Acabose was chosen as the ideal positive control because of its use in the treatment of diabetes mellitus as a-glucosidase and a-amylase inhibitors. The inhibitory activities seen in N. lappaceum ethanolic extract could be due to the presence of geraniin in the extracts. Tadera and co-workers (Tadera, Minami, Takamatsu, & Matsuoka, 2006) using the same enzymes reported the inhibitory activities of a number of well-studied flavonoids. The decreasing order of Fig. 3. Reducing power of geraniin compared to N. lappaceum ethanolic extract, LAscorbic acid and EmblicaTM. Data expressed in mean ± SD, n = 3. ⁄Palanisamy et al. (2008).. a-glucosidase and a-amylase inhibition shown by them are as follows: epigallocatechin gallate (2 lM) > cyanidin > myricetin > quercetin> and >myricetin (0.38 mM) > quercetin > epigallocatechin gallate > cyanidin respectively. The ability of plant extracts and its active compounds to inhibit carbohydrate hydrolysing enzymes have been shown by other workers (Gao et al., 2008; Gunawan-Puteri & Kawabata, 2010; Ma, Sato, Li, Nakamura, & Hattori, 2010). Phuwapraisirisan, Puksasook, Aramruang, and Kokpol (2008), reported the ability of phenylethyl cinnamides isolated from Aegle marmelos leaves to inhibit a-glucosidase with an IC50 of 35.8 lM for its most active compound. This plant is widely used in Indian Ayurvedic medicine for the treatment of diabetes mellitus. In this study, we report that geraniin inhibits both a-glucosidase and a-amylase activity at significant levels far above any other compound studied thus far. It is also worth noting N. lappaceum extract’s ability to inhibit the carbohydrate hydrolysing enzyme far better than the drug acarbose. The use of the rind of N. lappaceum in the management of hyperglycemia is highly practical for it will be far more economical to produce than pharmaceuticals and has been shown to be safe for consumption (Palanisamy et al., 2008). 3.6. Aldose reductase inhibition activity Inhibition of the aldose reductase has been reported to reduce the development of diabetic complications, such as retinopathy, neuropathy, nephropathy, and cataracts (Ramana & Srivastava, 2010). Geraniin, N. lappaceum ethanolic extract and quercetin were subjected to aldol reductase inhibition studies. Geraniin displayed the highest IC50 value of 0.14 ± 0.04 lg/ml, followed by N. lappaceum ethanolic extract, 0.4 ± 0.2 lg/ml and finally quercetin, 1.74 ± 0.12 lg/ml (Table 2). Quercetin, a phenolic compound, has been used as a positive control in aldol reductase inhibition studies (Guvenc et al., 2010; Jung et al., 2009). In this study geraniin and N. lappaceum ethanolic extract were found to be about 40 and 15 times more effective than quercetin in inhibiting aldol reductase activity respectively. This was further substantiated when we compared the effectiveness of geraniin with published findings of quercetin (Table 1). It has been reported that (Guvenc et al., 2010) phenolic compounds from plants have the ability to promote aldose reductase inhibition. It is envisaged that the high inhibitory activity observed in the N. lappaceum ethanolic extract could be due to the presence of the other phenolics present in the crude extract. 3.7. Advanced glycation endproducts (AGE) inhibitory activity Endogenous AGE formation is known to contribute to the progression of pathogenesis in conditions associated with diabetic complications and aging (Wada & Yagihashi, 2005). A representa- Table 2 Aldose reductase inhibition activity of geraniin compared to N. lappaceum ethanolic extract and quercetin, as the positive control. Fig. 4. Alpha-glucosidase and alpha-amylase inhibitory activities of geraniin compared to N. lappaceum ethanolic extract and acarbose, as the positive control. Data expressed in mean ± SD, n = 3. ⁄Palanisamy et al. (unpublished results). Samples IC50 (lg/ml) IC50 (lM) Geraniin N. lappaceum ethanolic extract Quercetin Quercetin@ 0.14 ± 0.04 0.4 ± 0.2a 1.74 ± 0.12 0.15 ± 0.04 – 5.76 ± 0.40 2.48b 3.84c 5.54d Data expressed as mean ± SD, n = 3. @Published results. a Palanisamy et al. (unpublished results). b Wirasathien et al. (2007). c Guvenc et al. (2010). d Jung et al. (2009). 26 U.D. Palanisamy et al. / Food Chemistry 127 (2011) 21–27 Fig. 5. AGEs inhibition activity of geraniin (20 lg/ml) compared to N. lappaceum ethanolic extract (40 lg/ml) and green tea (40 lg/ml). ⁄Palanisamy et al. (unpublished results). tive drug, aminoguanidine (AG) which is a hydrazine compound, prevents AGE formation by trapping intermediates at the initial glycation stages (Cheplick et al., 2010). Wu and coworkers (Wu, Hsieh, Wang, and Chen, 2009) reported that a commercial polyphenol extract of green tea, Polyphenon 60, was far more potent in inhibiting the glycation process than aminoguanidine, at the same concentration during a 21-day incubation period. In this study, BSA was chosen as the model protein for the formation of fluorescent AGEs and its inhibition was investigated using geraniin (20 lg/ml) and N. lappaceum extract (40 lg/ml) while green tea extract (40 lg/ml) was used as the positive control. It was observed that both geraniin and N. lappaceum ethanolic extract exhibited higher AGEs inhibition activity compared to the green tea throughout the 7-day incubation period (Fig. 5). The maximum inhibition activity was seen at the indicated incubation time of 7 days; 96% by geraniin, 43% by N. lappaceum ethanolic extract and 38% by green tea. It is worth mentioning that Psidium guajava leaf extracts (50 lg/ml) exhibited a 60% anti-glycation activity compared to Polyphenon 60 (40%) and aminoguanidine (8%) at the same concentration on a 21-day incubation (Wu et al., 2009). Rudnicki et al. (2007), reported that on a 3-day incubation period, Passiflora alata and Passiflora edulis extracts (10 lg/ml) showed an AGE inhibitory activity of 13% and 10%, respectively. Our results at 3-day incubation showed an inhibition of 21% in AGE formation. It was concluded in our previous studies that the N. lappaceum ethanolic extract had the highest AGE formation inhibition activity among the plant extracts studied and has comparable activity to green tea, an established inhibitor of AGE formation. Ho and coworkers, (Ho, Wu, Lin, & Tang, 2010) compared anti-glycation capacities of several herbal infusions with that of green tea and concluded that the amount of phenolics and flavonoid in the infusions highly correlated with their anti-glycation activity. The high phenolic content in N. lappaceum ethanolic extract (Palanisamy et al., 2008) may be the reason for its high anti-glycation activity. 4. Conclusion The rind of N. lappaceum, apart from being a highly efficient anti-oxidant, was shown to be effective in inhibiting carbohydrate hydrolysing enzymes, enzymes involved in the polyol pathway and able to prevent the formation of advanced glycation endproducts. Geraniin was found to be the major compound isolated from the rind of an ethanolic extract of N. lappaceum. In our interests to support further work on geraniin, a rapid and mid-scale purifica- tion of geraniin was achieved as described in this study. The compound with its extremely high free radical-scavenging activity and low pro-oxidant capability makes it an ideal anti-oxidant. Geraniin was also established to exhibit inhibition of the carbohydrate hydrolysing enzymes, alpha-glucosidase and alpha-amylase, at levels not reported by any other plant phenolic compound. The ability of phenolic compounds to prevent polyol (aldol reductase inhibition) and advance glycated endproduct formation has been documented (Guvenc et al., 2010; Ho et al., 2010). Our results not only supported their findings but also showed that geraniin was far more effective in preventing polyol and advance glycated endproduct formation compared to the positive controls quercetin and green tea respectively. Oxidative stress and its complications have been implicated in a number of diseased conditions, including diabetes mellitus. Geraniin with its ability to reduce free radicals, having a low pro-oxidant capacity, being an excellent inhibitor of carbohydrate hydrolysing enzymes and polyol and AGE formation makes it an ideal candidate for the management of hyperglycemia in diabetic individuals. In addition, our findings also add support for the use of a geraniin-standardised N. lappaceum extract, as a herbal formulation in the management of hyperglycemia. Acknowledgements This research work was supported in part by Research Grant from the Monash University Sunway Campus. Special thanks to Cheng Hwee Ming, Ammu K.C. Radhakrishnan, Theanmalar Masilamani and Thavamanithevi Subramaniam for their assistance in the project. References Cheplick, S., Kwon, Y.-I., Bhowmik, P., & Shetty, K. (2010). 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