aldose reductase ( ar ) is a key enzyme in the polyol pathway known to play important roles in the cataract formation and the pathogenesis of diabetic complications such as neuropathy , nephropathy , and retinopathy .
several naturally occurring and synthetic ar inhibitors with diverse structures have been studied in experimental animals and clinical trials to determine their effectiveness in preventing cataract formation and diabetic complications .
natural products are an excellent source of chemical structures with a wide variety of biological activities .
the development and progression of diabetic complications can be prevented by controlling blood glucose , but it is difficult to maintain normal blood glucose levels in a diabetic patient . ar inhibition is recognized as an important strategy for the prevention and attenuation of long - term diabetic complications .
ar inhibitors are being studied as potential therapeutic agents for use in treating diabetic complications [ 3 , 4 ] .
apart from diabetic microvascular disease , advanced glycation end products ( ages ) have also been implicated in a wide and seemingly disparate range of pathologies such as connective tissue diseases particularly in rheumatoid arthritis and neurological conditions such as alzheimer 's disease and end - stage renal disease . in vitro work
has mostly shown age to be part of complex interactions within oxidative stress and vascular damage , particularly in atherosclerosis and in the accelerated vascular damage that occurs in diabetes .
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 cataracts , atherosclerosis , nephropathy , and neuropathy . increased oxidative stress is a widely accepted contributor to the development and progression of diabetes and its complications .
diabetes is usually accompanied by an increased production of free radicals and impaired antioxidant defenses [ 7 , 8 ] .
therefore , inhibitors of ar and age formation provide an alternative mode of diabetes treatment , which is not dependent on the control of blood glucose level .
these inhibitors should be useful in the prevention or reduction of certain diabetic complications . 

prunella vulgaris , with almost 15 known individual species , is widely distributed in europe , asia , northwest africa , and north america , where it is generally known as self - heal herb .
p. vulgaris is used to cure high blood pressure , headaches , lymphatic system disorders , goiter , tuberculosis , and tumors [ 1113 ] .
more recently , a hot water infusion of this plant has been used to treat sores in the mouth and throat .
phytochemical studies indicate that p. vulgaris contains oleanolic , betulinic , ursolic , 2 , 3-dihydroxyurs-12-en-28-oic , and 2 , 3-ursolic acids , triterpenoids , flavonoids , tannins , and anionic polysaccharide called prunellin [ 11 , 14 ] .
the current study aimed to evaluate the ar and age inhibitory of p. vulgaris extract .
these studies are important in understanding the inhibitory effects of p. vulgaris on diabetic complications .

 dl - glyceraldehyde , the reduced form of nicotinamide adenine dinucleotide phosphate ( nadph ) , bovine serum albumin ( bsa ) , sodium phosphate , and quercetin used in this study were purchased from sigma ( st . louis , mo , usa ) .
human recombinant aldose reductase was purchased from wako pure chemical industries ( osaka , japan ) .
ric-1021 ) was deposited at regional innovation center , hallym university , republic of korea .
( 1  kg ) was extracted with water ( 5  l 2 times ) for 2  h at 100c .
the extract was suspended in distilled water and partitioned sequentially with n - hexane , methylene chloride ( ch2cl2 ) , ethyl acetate ( etoac ) , and n - butanol ( buoh ) , respectively .
ch2cl2 fraction was further purified by using a medium pressure liquid chromatography ( lichroprep rp-18 glass column ( 36 460  mm ; 2540  m particle size ) ; mobile phase meoh / h2o ( 40 to 50% ) ; flow rate 10 to 5  ml / min ) resulted in the isolation of compounds 5 ( 10.9  mg ) and 6 ( 13.5  mg ) . since the etoac fraction showed the ar inhibitory activity , this fraction ( 2.2  g ) was further purified by using a medium pressure liquid chromatography ( lichroprep rp-18 glass column ( 36 460  mm ; 2540  m particle size )
; mobile phase meoh / h2o ( 40 to 50% ) ; flow rate 10 to 5  ml / min ) resulted in the isolation of compounds 1 ( 102.8  mg ) , 2 ( 53.3  mg ) , 3 ( 10.6  mg ) , and 4 ( 27.9  mg ) .
crude rat lens aldose reductase ( rar ) was prepared as follows : lenses were removed from sprague - dawley rats weighing 250280  g and frozen at 70c until use . the rat lens homogenate was prepared according to the method of hayman and kinoshita with some modifications [ 1517 ] .
noncataractous transparent lenses were pooled and homogenate was prepared in 0.1  m phosphate buffer saline ( ph 6.2 ) .
after centrifugation at 10,000  rpm for 20  min in a refrigerated centrifuge , the supernatant containing the rar was collected .
ar activity was assayed spectrophotometrically by measuring the decrease in the absorption of nadph at 340  nm over a 4  min period according to the method of hayman and konoshita with some modifications , using dl - glyceraldehyde as the substrate .
each 1.0  ml cuvette contained equal units of the enzyme , 0.10  m sodium phosphate buffer ( ph 6.2 ) , 0.3  mm nadph , with or without 10  mm of the substrate , and an inhibitor [ 18 , 19 ] . the concentration of inhibitors giving 50% inhibition of enzyme activity ( ic50 ) was calculated from the least - squares regression line of the logarithmic concentrations plotted against the residual activity .
reaction mixtures consisted of 0.1  m potassium phosphate , 0.16  mm nadph , 2  mm of recombinant human aldose reductase ( rhar ) with varied concentrations of substrate dl - glyceraldehyde and ar inhibitor in a total volume of 200  l .
concentrations were ranged from 0.1 to 1  mm for dl - glyceraldehyde , and from 0.1 to 1  mm for active compound .
recombinant human aldose reductase activity was assayed spectrophotometrically by measuring the decrease in absorption of nadph at 340  nm after substrate addition using bio tek power wave xs spectrophotometer ( bio tek instruments , vt , usa ) .
lenses isolated from 10-week - old male rats were cultured for 6  d in tc-199 medium that contained 15% fetal bovine serum 100 units / ml penicillin and 0.1  mg / ml streptomycin under sterile conditions in an atmosphere of 5% co2 and 95% air at 37c .
the lenses were divided into 5 groups and cultured in medium containing 5  mm glucose , 30  mm galactose , and rosmarinic acid or caffeic acid ethylene ester .
galactitol was determined by hplc after its derivatization by reaction with benzoic acid to a fluorescent compound .
blood sample was collected in heparin - containing polypropylene tube from 10-week - old male rats . for sugar and sugar alcohol analysis , erythrocytes from heparinized blood
were separated from the plasma and buffy coat by centrifuging at 2000g for 10  min .
the cells were washed thrice with normal saline ( 0.9% nacl ) at 4c . in the final washing
, the cells were centrifuged at 2000  g for 10  min to obtain a consistently packed cell preparation .
the packed cells ( 1  ml ) were then incubated in a krebs - ringer bicarbonate buffer ( ph 7.4 ) containing 30  mm galactose in the presence or absence of samples at 37c in 5% co2 for 3  h. the erythrocytes were washed with cold saline by centrifuging at 2000  g for 10  min , precipitated by adding 6% of cold perchloric acid ( 3  ml ) , and centrifuged again at 2000  g for 10  min .
the supernatant was neutralized with 2.5  m k2co3 at 4c and used for galactitol determination .
hplc analysis for sugar and sugar alcohol in blood was performed with this supernatant of red blood cell homogenate after being benzoylated .
the modified procedure of lee et al .   was followed . bovine serum albumin ( 10  mg / ml ) was incubated with 5  mm methylglyoxal in sodium phosphate buffer ( 100  mm ; ph 7.4 ) .
all of the reagent and samples were sterilized by filtration through 0.2  m membrane filters , and the mixture was incubated at 37c for 7 days .
the fluorescence intensity was measured at an excitation wavelength of 330  nm and an emission wavelength of 410  nm with a ls50b fluorescence spectrometer ( perkin - elmer ltd .
the abts diammonium salt ( 2  mm ) and potassium persulfate ( 3.5  mm ) were mixed and diluted in distilled water kept in the dark at room temperature for 24  h before use .
after addition of abts solution to 10  l of antioxidant compounds were recorded at after 10  min reaction . the percentage inhibition of absorbance at 750  nm is calculated and potted as a function of concentration of antioxidants .
the constituents of the dried p. vulgaris were extracted with water . in order to identify the active fractions from p. vulgaris
the ch2cl2 soluble and etoac soluble fractions were isolated by using a medium pressure liquid chromatography .
their chemical structures were elucidated by chemical and spectral analysis as caffeic acid ( 1 ) , protocatechuic acid ( 2 ) , p - hydroxycinnamic acid ( 3 ) , rosmarinic acid ( 4 ) , caffeic acid ethylene ester ( 5 ) , and protocatechualdehyde ( 6 ) .
compounds 2 , 3 , and 5 were isolated for the first time from this plant .
the present study was conducted to identify new potential rar inhibitors from p. vulgaris , which might have potential uses in the treatment of diabetic complications .
we showed that water extract of p. vulgaris inhibited the activity of rar . in order to identify the active compounds from p. vulgaris the extract was divided into several fractions that were tested for their rar inhibitory activity .
the etoac fraction had high inhibitory activity against rar with ic50 value of 2.99  0.10  g / ml . these results are shown in table 1 .
previous studies used quercetin as a positive control when comparing the inhibitory activity of active compounds isolated from natural products against lens or human recombinant aldose reductase [ 9 , 30 , 31 ] .
we compared the inhibition of rar and rhar by compounds 16 and quercetin , a natural aldose reductase inhibitor .
compounds 35 had ic50 values of 8.35  0.51 , 2.77  0.48 , and 3.2  0.55  m , respectively , for ar while compounds 4 and 5 had ic50 values of 18.62  0.40 and 12.58  0.32  m , respectively , for rhar .
of the compounds tested , rosmarinic acid had the most potent rar inhibitory activity , while caffeic acid ethylene ester showed almost the same activity at the same concentration .
compounds isolated from p. vulgaris had potent inhibitory effects on rhar , especially caffeic acid ethylene ester . to determine the type of inhibition activity produced by compounds 4 and 5 ,
a kinetic study was conducted using dl - glyceraldehyde as a substrate ( concentration : 0.11  mm ) at 3 different concentrations for each compound .
the lineweaver - burk plots of 1/velocity and 1/concentration for compounds 4 and 5 are shown in figure 2 .
changes in the concentration of the substrate dl - glyceraldehyde resulted in different slopes and different x axis intersects being obtained with the uninhibited enzyme and the 3 different concentrations for each compound .
the results indicated that the inhibition type of rhar by rosmarinic acid ( 4 ) and caffeic acid ethylene ester ( 5 ) was noncompetitive , showing that the inhibitor was unable to bind to either the substrate - binding region or the nadph - binding region of rhar .
we also studied the effects of compounds 4 and 5 on galactitol accumulation in rat erythrocytes and lenses ( table 3 ) .
rat lens incubation for 6  d in 30  mm galactose resulted in an increased intracellular accumulation of galactitol .
compounds 4 and 5 inhibited galactitol accumulation in rat erythrocyte by almost 35.5 and 31.7% at 5  g / ml , respectively .
quercetin ( the positive control ) inhibited galactitol accumulation in rat erythrocyte by 30.5% and in rat lenses by almost 36.9 and 22.5% at 5  g / ml , respectively .
the etoac fraction had high inhibitory activity against age with an ic50 value of 141.34  1.27  g / ml , showing almost the same activity with positive control ( table 4 ) .
inhibitory activity against age formation by the isolated compounds was tested using aminoguanidine as a positive control .
caffeic acid ethylene ester ( 5 ) exhibited potent inhibitory activity against age formation with an ic50 value of 33.16  0.54  m , compared with the positive control ic50 value of 1944.86  8.39  m ( table 5 ) .
oxidative stress might have a key role in the pathogenesis of vascular complications in diabetes , both microvascular and macrovascular , and it provides an early marker of such damage in the development of endothelial dysfunction [ 33 , 34 ] .
abts inhibitory activity of the isolated compounds was tested using trolox as the positive control .
p - hydroxycinnamic acid ( 3 ) had the strongest inhibitory activity with an ic50 value of 5.94  1.01  m , and caffeic acid ethylene ester ( 5 ) also had potent inhibitory activity with an ic50 value of 10.52  0.28  m , compared to a positive control ic50 value of 15.34  0.40  m ( see tables 6 and 7 ) .
the present study isolated six compounds from p. vulgaris by using a medium pressure liquid chromatography . among these compounds ,
caffeic acid ethylene ester ( 5 ) has potent ar , age inhibitory activity , and antioxidant .
specifically , this compound exhibited the most potent inhibition with age , with an ic50 of 33.16  0.54  m .
the inhibitory effect of this compound was 58.0-fold stronger than that of the positive control aminoguanidine ( ic50 = 1944.86  8.39  m ) .
these results suggest that this compound and p. vulgaris could potentially provide a new natural treatment for diabetic complications .
however , further studies on the mechanism of age action of caffeic acid ethylene ester ( 5 ) and more in vivo evidence from diabetic animals are required .
 to evaluate the aldose reductase ( ar ) enzyme inhibitory ability of prunella vulgaris l. extract , six compounds were isolated and tested for their effects . 
 the components were subjected to in vitro bioassays to investigate their inhibitory assays using rat lens aldose reductase ( rar ) and human recombinant ar ( rhar ) . among them , caffeic acid ethylene ester showed the potent inhibition , with the ic50 values of rar and rhar at 3.2  0.55  m and 12.58  0.32  m , respectively . in the kinetic analyses using lineweaver - burk plots of 1/velocity and 1/concentration of substrate , this compound showed noncompetitive inhibition against rhar . 
 furthermore , it inhibited galactitol formation in a rat lens incubated with a high concentration of galactose . also it has antioxidative as well as advanced glycation end products ( ages ) inhibitory effects . as a result 
 , this compound could be offered as a leading compound for further study as a new natural products drug for diabetic complications . 
1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusions