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Rat Lens Aldose Reductase Inhibitory Activities of Cissus assamica var. pilosissima and Syzygium oblatum
Rat Lens Aldose Reductase Inhibitory Activities of Cissus assamica var. pilosissima and Syzygium oblatum
Natural Product Sciences. 2013. Dec, 19(4): 275-280
Copyright © 2013, The Korean Society of Pharmacognosy
  • Received : July 10, 2013
  • Accepted : August 28, 2013
  • Published : December 31, 2013
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About the Authors
Ki Ho Lee
Department of Integrative Plant Science, Chung-Ang University, Anseong 456-756, Korea
Dong Gu Lee
Department of Integrative Plant Science, Chung-Ang University, Anseong 456-756, Korea
Sangwoo Lee
International Biological Material Research Center, KRIBB, Daejeon 305-806, Korea
Wanyi Li
Yunnan Academy of Agricultural Sciences, Yunnan 650223, China
Sanghyun Lee
Department of Integrative Plant Science, Chung-Ang University, Anseong 456-756, Korea
slee@cau.ac.kr

Abstract
Aldose reductase (AR) has been shown to play an important role in the development of diabetic complications. To search for AR inhibitors from Chinese plants, the ethanol extracts of Chinese plants was tested against an inhibition of rat lens AR in vitro . Among Chinese plants tested, Cissus assamica var. pilosissima and Syzygium oblatum showed highest inhibition of AR (IC 50 values, 0.71 and 0.79 μg/ml, respectively). Cissus assamica var. pilosissima and Syzygium oblatum showed more potent inhibitory activity against AR than the positive control, TMG. Consequently, C. assamica var. pilosissima and S. oblatum have a possibility of new natural resources for the development of AR inhibitor for the prevention of diabetic complications.
Keywords
Introduction
Aldose reductase (AR) belonging to the aldo-keto reductase super family of enzymes in plants and animals is the first and rate limiting enzyme in polyol pathway (Ko et al ., 1997; Demaine ., 2000 ; Sree ., 2000 ). AR is monomeric and cytosolic protein that catalyzes reduction glucose to sorbitol (Wang ., 2009) . Sorbitol subsequently metabolized to fructose by sorbitol dehydrogenase which the second enzyme of polyol pathway (Ramana ., 2001 ; Wang ., 2009) . Accumulation of sorbitol leads to abnormalities of metabolism such as osmotic swelling and oxidative stress (Kao ., 1999) . And, stored sorbitol in the lens fiber is regarded as the main cause of blindness (Patel ., 2012) and cataract formation (Heyningen, 1959 ; Sugiyama ., 2000) . Chronic hyperglycemia is considered as the causative link on the onset and progression of diabetes chronic complications ( Demopoulos ., 2005 ; Chatzopoulou et al ., 2011). As a result, osmotic, oxidative, reductive, glycative and protein kinase C stress are induced with devastating manifestations for the cells (Alexiou ., 2009) . Under hyperglycemia environment, AR is highly activated by increasing glucose contents can cause increased accumulation of sorbitol rate by 2-4 times (Ramana ., 2001 ; Wang ., 2009) . The enzyme exists in the eye, nerves, retina, kidney, myelin sheath, and other tissues resulting in the development of diabetic complications (Enomoto ., 2004 ; Ha ., 2009) .
AR inhibitors (ARIs) have been proposed as possiblepharmacotherapeutics of diabetic complications (Miyamoto, 2002) . Numerous ARIs obtained from natural sources such as flavonoids, coumarins, stilbenes, monoterpenes, and related aromatic compounds have been reported in the past years, because of its high potency, promising efficacy, and insignificant adverse effect profile (Fuente and Manzanaro, 2003 ; Kawanishi ., 2003) . Therefore, the development of natural sources for ARIs will be able to better success of a potential treatment for diabetic complications, due to safer and more effective phytochemicals (Kawanishi ., 2003) .
The wide territory of China is a factor that can have complex natural environmental conditions. So, throughout China, about 32,000 higher plant species are present. Like this, China is the diversity of vegetation types and complex distribution. These factors provide the rich natural resources for human (Chang et al ., 2005). As described above, plants offer many required substances for humans. So the activity of the extract experiment to search for activity is very important.
In a series of investigations to evaluate potential ARIs from the natural products, we have shown that some Chinese plants exhibited a significant inhibition of AR in vitro . To search for ARIs from Chinese plants, the inhibition of rat lens AR in vitro using Chinese plants was investigated.
Experimental
Sample preparation − One hundred samples of 95% ethanol (EtOH) extract of Chinese plants (ECPs) were obtained from Plant Extract Bank in KRIBB, Daejeon, Korea. Table 1 shows scientific name of ECPs.
Sample list of the ethanol extracts of Chinese plants for aldose reductase inhibition
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Sample list of the ethanol extracts of Chinese plants for aldose reductase inhibition
General instruments and reagents − Fluorescence analysis was measured with a Hitachi U-3210 spectrophotometer. Solvents such as DL-glyceraldehyde, β-NADPH, sodium phosphate buffer, ammonium sulfate buffer, potassium phosphate buffer, and DMSO (Sigma-Aldrich Chemical Co.) were used for rat lens AR assay. 3,3-Tetramethylene glutaric acid (TMG), a typical AR inhibitor, was used as a positive control. A negative control was prepared using DMSO.
Purification of rat lens AR − Normal eyes of Sprague-Dawley rats (weighing 250 - 280 g) were removed immediately after sacrificing through CO 2 and preserved by freezing it until use. After, these mixed with sodium buffer, the homogenate was and centrifuged at 10,000 rpm (4℃, 20 min) and the supernatant was used as an enzyme source.
Determination of AR activity − AR activity was spectrophotometrically determined by measuring the decrease in absorption of NADPH at 340 nm for a 4 min period at room temperature with DL-glyceraldehydes as a substrate (Bartels ., 1991) . For in vitro studies, mixed 0.1 M sodium phosphate buffer (pH 6.2), 0.1M potassium phosphate buffer (pH 7.0), 1.6 mM NADPH, and each sample of the extract in DMSO (1 mg/ml), 0.025 M DL-glyceraldehyde and 4M ammonium sulfate as substrate in quartz cell. IC 50 values, the concentration of inhibitors giving 50% inhibition of enzyme activity, were calculated from the least-squares regression line of the logarithmic concentrations plotted against the residual activity. TMG known as one of typical AR inhibitors was used as a positive control.
Results and Discussion
The EtOH extracts of Chinese plants were tested for their inhibitory effects on rat lens AR activity, and the results were summarized in Tables 2 and 3 . Table 2 shows the rat lens AR inhibition percentages, and appeared high activity in ECPs-001, -004, -006, -012, -015, -037, -047, -048, -063, -064, -078, and -087. However, these samples were repeated three times, and excluded ECPs-015, -037, -048, -064, and -078. As shown Table 3 , The EtOH extracts of ECPs-001, -004, -006, -012, -047, -063, and -087 were showed over 70% degree of inhibition on rat lens AR that are supposed to be far less deserving of further consideration. Among them, ECPs-001 and -087 were exhibited highest inhibitory percentages on rat lens AR (90.20% and 90.84%, respectively). And, ECPs-004, -006, -012, -047, and -063 were showed good inhibitory percentages of 86.03%, 72.98%, 87.96%, 69.78%, and 79.05%, respectively. ECPs-012 and -087 were measured higher inhibitory activity on AR than TMG and other samples. As results, ECPs-012 and -087 were exhibited higher inhibitory activity against AR than TMG, and showed promise of medication for blindness on the part of diabetes. There are many reports on inhibitory activities of Chinese herbal medicines against AR (Lee ., 2009 ; Lee ., 2010 ; Lee ., 2011 ; Lee ., 2013) .
Rat lens aldose reductase inhibitory activities of the ethanol extracts of Chinese plants
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Each sample concentration was 1 mg/ml DMSO. a Inhibition rate was calculated as percentage with respect to the control value.
IC50values of the ethanol extracts of Chinese plants on rat lens aldose reductase inhibition
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a Inhibition rate was calculated as percentage with respect to the control value. b IC50 value was calculated from the least-squares regression equations in the plot of the logarithm of at three graded concentrations vs % inhibition. c TMG was used as a positive control.
ECP-012 is one of Cissus species. Cissus species is a woody vines plants belonging to Vitaceae family, exist about 350 species. Among Cissus species, C. quadrangularis was reported anti-osteoporotic (Shirwaikar ., 2003) , analgesic, anti-inflammatory, and venotonic effects (Panthong ., 2007 ; Srisook ., 2011) , and bone tissue engineering (Soumya ., 2012) . And C. sicyoides was confirmed gastroprotective of microcirculation, endogenous sulfhydryls and nitric oxide and vasoconstrictor effect (Garcfa ., 1997 ; Ferreiraet ., 2008) , antiinflammatory and anti-bacterial activity (Garcia ., 1999 ; García ., 2000) .
ECP-087 is one of Syzygium species. Syzygium species is a tropical evergreen tree of Myrtaceae family and is native to the India and China. 1,300 species are known as medicinal plants in Indonesia called Jamu (Roosita ., 2007) . The fruit of S. samarangense was proved cytotoxic activity against the SW-480 human colon cancer cell line and known anti-oxidants were isolated including six quercetin glycosides (Simirgiotis et al ., 2008). S. cumini and S. travancoricum leaf were announced anti-bacterial activity (Shafi ., 2002) . Eugenol and eugenol acetate from the buds of S. aromaticum were involved melanin formation in B16 melanoma cells (Arung ., 2011) and these n -hexane extract was confirmed aphrodisiac effect by testosterone production in mice (Mishra and Singh, 2008) , anti-nociceptive activity of S. jambos (Ávila-Peña ., 2007) . In particular, AR inhibitory activity of S. cumini has been already reported (Rao ., 2013) .
Consequently, C. assamica var. pilosissima and S. oblatum has a possibility of new natural resources for the development of AR inhibitor for the prevention of diabetic complications. Further investigations on the bioactivity of constituents from C. assamica var. pilosissima and S. oblatum may prove the use of new medicinal plants for the prevention of diabetic complications.
Acknowledgements
The authors specifically thank Plant Extract Bank in KRIBB, Daejeon, Korea for the generous gift of the EtOH extracts of Chinese plants.
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