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Pak. J. Agri. Sci., Vol. 45(1), 2008 EFFECT OF ONION (Allium cepa L.) EXTRACT ON MAILLARD REACTION
UNDER in vitro CONDITIONS
Samina Kousar, Munir A. Sheikh, M. Asghar and *Robina Rashid Department of Chemistry, University of Agriculture, Faisalabad *Department of Biochemistry, Independent Medical College, Faisalabad
Plant extracts have their own importance and now being studied extensively due to having little or no side effects.
Protein glycation takes place when elevated levels of reduced sugars react with amino groups in proteins,
reaction known as Maillard reaction. If this process continues, it will lead to the formation of complex, often
unstable, irreversible and reactive compounds "AGEs", a process that may take weeks or even months to
accomplish. In present study onion was selected and used to check the Maillard reaction inhibitory activity.
Different combinations of glucose, protein and onion extracts were made under in vitro conditions and their
activity was monitored with Trichloro acetic acid treatment method at 350 nm. Maillard reaction products/ AGEs
were more with high glucose and high protein concentration and these were decreased by highest concentration
of onion extract i.e. 25 mg/mL or 250 µL. Lower concentrations of plant extract produced either no or least
response against Maillard reaction.
Keywords:
Maillard reaction, AGEs, Onion extract, TCA treatment
INTRODUCTION
might provide useful sources for the development of drugs, in the treatment of diabetes mellitus. The In 1912, Louis Camille Maillard described the browning literature on medicinal plants with hypoglycemic activity of proteins in food and called it as Maillard reaction. is vast. As many of these plants were used for many This is also known as non-enzymatic glycation of centuries and some times as regular constituents of proteins, or a process which links chronic the diet, it is assumed that they do not have many side hyperglycemia to a series of physiopathological effects (Shnkar et al., 1980). alterations considered important in the development of Synthetic inhibitors and inhibitors from plant extracts chronic complications of different diseases like have their own importance and now are studied diabetes (Takeuchi et al., 2004). These glycated extensively. There are reports of some natural proteins further rearrange and give rise to a stable substances isolated from plants with AGE-inhibitory Amadori product that degrades into a variety of effects. One such compound is curcumin isolated from compounds which, more reactive than the sugars from Curcuma longa (Turmeric), commonly known as Haldi. which they are derived (Wautier and Schmidt, 2004). Ginger (Zingiber officinale Rosc.) is another spice These propagators again form yellow-brown, often useful for diabetic therapy (Broadhurst, 2000). When fluorescent (some are non fluorescent), irreversible type 2 diabetic rats are fed ginger, they show compounds, usually called Advanced Glycation End- hypoglycemic activity, thus improving their diabetic Products (AGEs) or Maillard products. Candidate condition (Kar et al., 2003). Now it is the need of time active AGE compounds include N-(carboxymethyl)-L- to develop or isolate new compounds either from lysine (CML) pyrraline, pentosidine and their cross- plants or synthetically to control diabetes and other age links (Kaysen, 2001). accelerating diseases. As plants have fewer side Plants have been the major source of drugs in the effects so these should be preferred to study. In this world and in sub-continent system of medicinal study onion (Allium cepa L.) extract was used to study therapy. Information on such plants in sub-continent its affect on glycation and Maillard products. The has been systematically organized (Satyavati et al., major object of this study was to investigate the effect 1987). It is known that medicinal plants have little or no of onion as inhibitor of AGE or Maillard reaction under side effects. Some of them are being used in traditional in vitro conditions and measure its activity against AGE systems of medicine from hundreds of years in many production or inhibition. countries of the world (Eshrat and Hussain, 2002).
Metformin is the only ethical drug approved for the MATERIALS AND METHODS
treatment of non insulin dependent diabetes mellitus
Preparation of onion (Allium cepa L.) extract
(NIDDM) patients (Beisswenger et al., 1999), which is derived from a medicinal plant Galega officialis and Dried and ground onion plant (5 g) was extracted with historically used for treatment of diabetes (Oubre et al., 30 mL of 50% ethanol at 37 oC for 10 days and then 1970). There are many anti-diabetic plants, which filtered and stored at 4oC. Kousar, Sheikh, Asghar and Rashid Sample recovery
50% ethanol for extraction. Samples were recovered by Rotary Evaporator. Heat and liquid nitrogen method Sample was recovered by evaporating the ethanol were also tried but Rotary evaporator was cheaper, using Rotary Evaporator. Samples after drying were less time consuming and simple to perform than other dissolved in 25 mL of phosphate buffer saline and two. Samples were drawn for glycation inhibition stored at 4°C for further use. activity after 1st, 3rd and 5th week of incubation. In vitro glycation inhibition with onion (Allium cepa
Glucose concentration 250 mM and 5.5 mM was L.) extract
selected as it was cleared that 250 mM is a Conditions and concentrations selection for onion
hyperglycaemia condition (Rahbar and Figarola, 2003) (Allium cepa L.) extract
and 5.5 mM is a normal glucose level (human body). Bovine serum albumin (BSA) was used as protein for To measure glycation inhibition with onion extract, glycation and its concentration was i.e.10 mg/mL and 5 different concentrations of protein (BSA) and glucose mg/mL. Onion extracts (250 µL, 125 µL and 50 µL) in (two of each) and three concentrations or volumes of phosphate buffer were prepared. Each extract along onion extract were used. These are given in table 1. with glucose and BSA were incubated at 37oC and Table 1. Concentration of different components 50oC for five weeks to monitored glycation and Millard
used to study glycation inhibition under
reaction inhibitory activity. TCA treatment method in vitro conditions with onion (Allium
described by Matsuura et al. (2002) was adopted with cepa L.) extract.
modifications. The absorbance change based on Schiff base formation was measured by spectrophotometer at S.No. Components of reaction
350 nm. This gave the apparent inhibitory activity. Real inhibitory activity was estimated by subtracting the quenching effect from the apparent inhibitory activity. The results obtained by this method are explained Maillard reaction inhibitory activity by onion
(Allium cepa L.) extract
The results obtained from onion (Allium cepa L.) extract after TCA treatment at 37oC and 50 oC. At temperature 37oC (Figure 1), G1 P1 produced more In vitro glycation of BSA with onion (Allium cepa
Maillard products (0.099) after 5th week. While G2P2 L.) extract
produced minimum products 0.029 after 1st week at same temperature. G Glucose, BSA with or without inhibitor (plant extracts in 1P2 and G2P1 produce moderate level of products. Onion extract followed the pattern PBS pH 7.4) were prepared and their mixture was from F incubated at 37oC and 50oC for 5 weeks. During this, 1 to F3 as F1 (250 µL) generated maximum samples were drawn for glycation inhibition activity 3 (50 µL) generated least or no response at 37oC. after 1st, 3rd and 5th week of incubation. The samples BSA (10mg/mL, 5mg/mL), glucose (250mM, 5.5mM) kept at 4oC until analysis. with or without Onion (Allium cepa L.) extract (250 µL, Trichloracetic acid (TCA) method for Maillard 125 µL and 50µL) in phosphate buffer(0.075M), pH 7.4
reaction inhibitory activity of onion (Allium cepa L.)
and mixture was incubated at 37oC for five weeks. Samples were drawn for glycation inhibition activity after 1st, 3rd and 5th week of incubation. Absorbance This method is also known as TCA treatment method was recorded at 350nm. described by Matsuura et al. (2002) was followed with At 50oC (Figure 2) G1 P1 produced more Maillard some modification. products (0.109) after 5th week. While G2P2 produced minimum products 0.037 at same temperature after 1st RESULTS AND DISCUSSIONS
week of incubation. Here it was also seen that G2P2 generated maximum products at 3rd week (0.048) in Onion plant was selected for experiments as it is in spite of 5th week at 50oC. F1 (250 µL) generated routine use for daily food cooking and salads in maximum response while F3 (50 µL) generated least Pakistan. Onion plants were dried in hot air oven at response at 50oC and minimum inhibitory activity was 37oC. After drying, ground to powder form and kept in observed. At both temperatures F2 (150 µL) produced


Effect of onion extraction on maillard reaction moderate response against Maillard products activity of onion extract i.e. their trend towards inhibition. It was also observed from both figures that inhibition is same. high temperature facilitate the production of Maillard Onion and garlic have significant blood sugar lowering products. Moreover, high temperature did not effect the action. Our results are supported by Demerdash et al. Kousar, Sheikh, Asghar and Rashid (2005).They carried out the study to investigate the Eshrat, H.M. and A. Hussain. 2002. Hypoglycemic, effects of onion (Allium cepa) and garlic (Allium hypolipidemic and antioxidant properties of sativum) juices on biochemical parameters, enzyme combination of curcumin from Curcuma longa, and activities and lipid peroxidation in alloxan-induced partially purified product from Abroma augusta, in diabetic rats. Their results showed that garlic and onion streptozotocin induced diabetes. Indian J. Clin. juices exerted antioxidant and antihyperglycemic Biochem. 17 (2): 33-43. effects. Our findings were contrary to Jelodar et al. Jelodar, G.A., M. Maleki, M.H. Motadayen and S. (2005) as they carried out the study to clarify the effect Sirus. 2005. Effect of fenugreek, onion and garlic of fenugreek, garlic and onion in the treatment of on blood glucose and histopathology of pancreas diabetes, on blood glucose and their possible effect on of alloxan-induced diabetic rats. Indian J. Med. Sci. pancreatic tissues. The results of their study indicated that only garlic was able to reduce blood glucose Kar, A., B.K. Choudhary and N.G. Bandyopadhyay. significantly compared with the control group. 2003. Comparative evaluation of hypoglycaemic However, most studies confirm our findings and activity of some Indian medicinal plants in alloxan showed onion decreased the hyperglycemic peak in diabetic rats. J. Ethno-pharmacol. 84 (1):105-108. rabbits (Romas et al., 1995). In addition, onion amino Kaysen, G. 2001. The microinflammatory state in acid s-methyl cysteine sulfoxide contributed to uremia: causes and potential consequences. J. antidiabetic effects in affected rats, controlling blood Am. Soc. Nephrol. 12:1549–1557. glucose in addition to other diabetic effects comparable Matsuura, N., T. Aradate, C. Sasaki, H. Kojima, M. Ohara, J. Hasegawa and M. Ubukata. 2002. to insulin (Shella, 1995). Screening system for the maillard reaction inhibitor from natural product extracts. J. Health Sci. 48 (6): CONCLUSIONS
Oubre, A.Y., T.J. Carlson, S.R. King and G.M. Reaven. Our studies concluded that onion has ability to inhibit 1970. From plant to patient, an Ethanomedical Maillard products that ultimately lead to AGEs approach to the identification of new drugs for the production. It was also cleared that 10mg/ mL treatment of NIDDM. Diabetologia, 40 (5): 614-617. concentration of BSA was more active towards Rahbar, S. and J.L. Figarola. 2003. Novel inhibitors of glycation of glucose. However, F3 (50 µL) advanced glycation end products. Arch. Biochem. concentration of onion extract was almost unable to Biophys. 419: 63-79. produce any effect at 37 oC. So it is suggested that Ramos, R.R., J.L. Flores-Saenz and F.J. Alarcon- more concentration of onion extract should be used to Aguilar. 1995. Anti-hyperglycemic effect of some stop or decrease glycation in human body level in case edible plants. J. Ethnopharmacol. 48:25-32. of diabetes mellitus and persistent hyperglycaemia. Satyavati, G.V., A. Gupta, T.N.B. Shankar, N.V. Abbreviations: AGEs, Advanced Glycation End-
Shanta, H.P. Ramesh, A.S. Murthy and I.V.S. Products; NIDDM, Non insulin dependent diabetes Murthy. 1987. Medicinal plants of India eds Indian mellitus; BSA, Bovine serum albumin; PBS, Phosphate Council, New Delhi. J. Exp. Biol. 18(2): 73- 74. buffer saline; TCA, Tri-chloro acetic acid. Sheela, C.G., K. Kumud and K.T. Augusti. 1995. Anti- diabetic effects of onion and garlic sulfoxide amino REFERENCES
acids in rats. Planta Med. 61:356-357. Shankar, T.N.B., N.V. Shanta, H.P. Ramesh, I.A.S. Beisswenger, P.J., S.K. Howell, A.D. Touchette, L. Murthy and V.S. Murthy. 1980. Toxicity Studies on Sundeep and B.S. Szwergold. 1999. Metformin Turmeric (Curcuma longa): Acute Toxicity studies Reduces Systemic Methylglyoxal Levels in Type 2 in rats, Guinea pigs and Monkeys. Ind. J. Exp. Bio. Diabetes. D i a b e t e s, 48:198–202. Broadhurst, C.L., M.M. Polansky and R.A. Anderson. Takeuchi, M., S. Yamagishi, M. Iwaki, K. Nakamura 2000. Insulin-like biological activity of culinary and and T. Imaizumi. 2004. Advanced glycation end medicinal plant aqueous extracts in vitro. J. Agric. product (AGE) inhibitors and their therapeutic Food Chem. 48(3):849-852. implications in diseases. Int. J. Clin. Pharm. Res. Demerdash, F.M., M.I. Yousef and N.I. El-Naga. 2005. Biochemical study on the hypoglycemic effects of Wautier, J.L. and A.M. Schmidt. 2004. Protein onion and garlic in alloxan-induced diabetic rats. Glycation: A Firm Link to Endothelial Cell Food Chem Toxicol. 43 (1):57-63. Dysfunction. Circulation Res. 95 (3): 233- 245.

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