Inhibitory Effect of Chemical Constituents Isolated from Artemisia iwayomogi on Polyol Pathway and Simultaneous Quantification of Major Bioactive Compounds

Blocking the polyol pathway plays an important role preventing diabetic complications. Therefore, aldose reductase (AR) and advanced glycation endproducts (AGEs) formation has significant effect on diabetic complications. Artemisia iwayomogi has long been used as treatment of various diseases in Korea. However, no literatures have reported on AR and AGEs formation inhibitory activities of A. iwayomogi. For these reasons, we aimed to assess that A. iwayomogi had potential as anti-diabetic complications agents. We led to isolation of two coumarins (1 and 2), nine flavonoids (3–11), five caffeoylquinic acids (12–16), three diterpene glycosides (17–19), and one phenolic compound (20) from A. iwayomogi. Among them, hispidulin (4), 6-methoxytricin (6), arteanoflavone (7), quercetin-3-gentiobioside (10), 1,3-di-O-caffeoylquinic acid (13), and suavioside A (18) were first reported on the isolation from A. iwayomogi. Not only two coumarins (1 and 2), nine flavonoids (3–11), and five caffeoylquinic acids (12–16) but also extracts showed significant inhibitor on AR and AGEs formation activities. We analyzed contents of major bioactive compounds in Korea's various regions of A. iwayomogi. Overall, we selected Yangyang, Gangwon-do, from June, which contained the highest amounts of bioactive compounds, as suitable areas for cultivating A. iwayomogi as preventive or therapeutic agent in the treatment of diabetic complications.


Introduction
Aldose reductase (AR) belongs to the aldo-keto reductase family. It is the first and rate-controlling enzyme in the polyol pathway that reduces glucose to sorbitol using nicotinamide adenine dinucleotide 2 -phosphate (NADPH) as a cofactor [1,2]. Although blood sugar is high, the polyol pathway converts the excess glucose into sorbitol and then fructose. This accumulation of sorbitol and fructose has been demonstrated to be responsible for diabetic complications, including nephropathy, cataracts, neuropathy, and retinopathy [3,4]. In particular, sorbitol is implicated in the pathogenesis of sugar cataracts, while the accumulation of fructose induces the formation of advanced glycation endproducts (AGEs), which are strongly implicated in diabetic complications and Alzheimer's disease [5]. Therefore, research on AR and AGEs formation inhibition is on the rise. There is a wide range of literature demonstrating that the developments of AR and AGEs formation are blocked by natural sources, especially plants that have an enormous content of bioactive compounds [6,7].
Artemisia iwayomogi, locally called haninjin or dowijigi, is a member of the Compositae family and a perennial herb mainly found in Korea. A. iwayomogi has long been used in Korea in vegetables and foods such as tea, rice cake, and soup and also used for the treatment of various diseases including hepatitis, inflammation, and immune-related diseases as a protection for the liver, and a diuretic [8][9][10]. Previous studies have reported the isolation of scopoletin, esculetin 6methylether, scopolin, -sitosterol, chlorogenic acid, quebrachitol, essential oils, fatty acids, sesquiterpene lactones, and flavonoids in A. iwayomogi [11][12][13][14]. Among them, scopolin modulated the expression of obesity-associated genes and was shown to have pharmacological effects on obesity, fatty 2 BioMed Research International liver, and diabetes [15]. A. iwayomogi has also demonstrated various biological activities. For example, methanol extracts of A. iwayomogi were shown to act as a scavenger of peroxynitrile, a compound involved in inducing or maintaining many diseases, including inflammation and aging [16]. Methanol extract of A. iwayomogi also inhibits nitric oxide in the production of lipopolysaccharide activated macrophages [17]. In other studies, a water-soluble carbohydrate fraction from A. iwayomogi suppressed spontaneous or 2,3, 7,8-tetrachlorodibenzo-p-dioxin-induced apoptotic death of mouse thymocytes by downregulating Fas gene expression [18,19]. A water-soluble carbohydrate fraction from A. iwayomogi also repressed pulmonary eosinophilia and Th2-type cytokine production in ovalbumin-induced allergic asthma via downregulation of TNF-expression in the lungs [20], showed antitumor, immunomodulating activities [9], and modulated the functional differentiation of bone marrowderived dendritic cells [21].
However, A. iwayomogi's ability to inhibit AR and AGEs formation has never been previously studied. We investigated various components found in A. iwayomogi and their inhibitory activities on AR and AGEs formation. Our study also determined the bioactive compounds present in A. iwayomogi by region in Korea using an HPLC-PAD. Based on our study, we selected suitable areas for cultivating A. iwayomogi as medicinal foods.

Plant Materials.
The aerial parts of A. iwayomogi were purchased from Kyung-Dong market, Seoul, Korea. Moreover, the aerial parts of A. iwayomogi were collected from Seoul, Gyeongii-do, Chungcheongbuk-do, Jeollabukdo, Jeollanam-do, Gyeongsangbuk-do, Gyeongsangnam-do, Gangwon-do, and Jeju-do Korea for analysis. Professor Whang Wan Kyunn identified A. iwayomogi which was bought in Kyung-Dong market and collected in various specimens.

Measurement of AR Inhibitory Activity.
Rat lenses (one lens per 0.5 mL sodium buffer) were obtained from Sprague-Dawley rats (weighing 250∼280 g). The rat lenses were homogenized in 0.1 M sodium phosphate buffer (pH 6.2) and centrifuged at 10,000 rpm (4 ∘ C, 20 min). After centrifugation, the supernatant was used as an enzyme. AR activity was spectrophotometrically determined by measuring the decrease in the absorption of -NADPH at 340 nm for a 4 min period using dl-glyceraldehydes as substrates [22]. The assay mixture contained 1.6 mM -NADPH, 0.1 M potassium phosphate buffer (pH 7.0), AR homogenate, 4 M ammonium sulfate, 0.025 M dl-glyceraldehyde, and the sample in 100% DMSO. The total volume of the assay mixture was 0.3 mL and the reaction was performed in a 96-well plate. 3,3 -Tetramethylene glutaric acid (TMG), a typical AR inhibitor, was used as a positive control.

Measurement of AGEs Formation Inhibitory Activity.
Inhibition of AGEs formation was determined by an assay containing bovine serum albumin (10 mg/mL) in 50 mM phosphate buffer (pH 7.4) with 0.02% sodium azide, to which 0.4 M fructose and glucose was added. The assay mixture was incubated at 60 ∘ C for 2 days. After incubating, the reaction was measured on Fluorescence in a 96-black-well plate (excitation wavelength 350 nm, emission wavelength 450 nm) [23]. Aminoguaidine (AG) was used as a positive control.

For AR and AGEs Formation Inhibition Assays, the
Activity Was Calculated. Inhibition (%): (Ac − As/Ac) × 100. In AR inhibition activity, Ac is absorbance of control and As is absorbance of samples. In AGEs formation inhibition activity, Ac is fluorescence of control and As is fluorescence of samples. IC 50 is the concentration of inhibitor that gives a 50% inhibition in enzyme activity. IC 50 values were calculated from the least-squares regression line of the log of concentration plotted against residual activity. All assays were performed in triplicate. Data was presented as mean ± standard deviation (SD).

Sample Preparation for HPLC. Samples of A. iwayomogi
taken from the various regions in Korea and Yangyang, Gangwon-do, monthly between June and October were dissolved in 50% EtOH (20 mg/mL). The resulting solution was filtered with a 0.45 m syringe filter. The resulting solution was used for HPLC analysis.

HPLC Conditions.
A Waters Sunfire6 column C18 (4.6 × 250 mm, 5 m) was used for the determination of compounds , , , , , , , and . The mobile phase consisted of 0.1% formic acid (solvent A) and ACN (solvent B). The gradient solvent system was solvents A and B (85 : 15) and increased in linear gradients to 84 : 16 for 5 min, to 72 : 28 for 5 min, then to 69 : 31 for 5 min, and finally to 40 : 60 for 5 min. The injection volume was 10 L and the flow rate was 0.9 mL/min. The UV spectra were recorded at 330 nm for quantification of compounds. All injections were performed in triplicate.

Calibration
Curve. Stock solutions of compounds , , , , , , , and in 50% EtOH were prepared in several concentrations. Calibration curves of the eight standards were calculated using concentration ( , g/mL), peak area ( ), and mean value ( = 3) ± standard deviation. Contents of the analyte solutions were then determined from the calibration curves.

Determination of Limit of Detection (LOD) and Limit of Quantification (LOQ).
Quantification of the HPLC method for compounds , , , , , , , and as a standard compound was determined by LOD and LOQ. The LOD and LOQ were defined as detectable concentration of the compound with a signal to noise ( / ) ratio of ≥3.3 and ≥10, respectively. The percent recovery was evaluated by calculating the ratio of the amount detected versus the amount added. The quantity of analysis was subsequently obtained from the calibration curve.

AR Inhibitory Activities of the Extracts, Fractions, and Compounds 1-20 from A. iwayomogi. Our study investigated the inhibitory effects of A. iwayomogi on AR and
AGEs formation, which demonstrates the potential of these compounds to prevent diabetes complications. The extracts and fractions of the aerial parts of the A. iwayomogi were tested for AR inhibition. The results are summarized in Table 1. The extracts, 30%, 60%, and 100% MeOH fractions from A. iwayomogi showed significant inhibition of AR 1048.99 ± 0.11 * TMG b 6.58 ± 0.19 * -AG c -414.77 ± 5.96 * IC 50 calculated from the least-squares regression line of the logarithmic concentrations plotted against the residual activity. TMG was used as a positive control. AG was used as a positive control. ND was not detected. * Significant difference from control; * < 0.05, * * < 0.01.
(IC 50 value with 2.71 ± 0.72, 1.16 ± 0.10, 1.42 ± 0.17, and 0.79 ± 0.15 g/mL, resp.). In addition, compoundswere tested for their ability to inhibit AR. As can be seen in Table 2 Table 1. The extracts, CHCl 3 , 30% MeOH, and 100% MeOH fractions all showed greater inhibition than AG (IC 50 value with 45.85 ± 7.17 g/mL), the positive control. The IC 50 values  of extracts, CHCl 3 , 30% MeOH, and 100% MeOH fractions were 40.99 ± 0.50, 7.29 ± 0.05, 40.81 ± 0.05, and 38.40 ± 0.51 g/mL, respectively. Compounds -isolated from A. iwayomogi were also tested for inhibition of AGEs formation, as reported in Table 2. Similar to AR inhibitory activities, diterpene glycosides ( -) showed no inhibition of AGEs formation and compound showed only slight inhibition, whereas the two coumarins ( and ), nine flavonoids ( -), and five caffeoylquinic acids ( -) were all more inhibitory than AG (IC 50 value with 414.77 ± 5.96 M). Among them, compound , which exhibited minor inhibition on AR, exhibited the best inhibition of AGEs formation with an IC 50 value of 20.71 ± 1.38 M.  17.98, 15.41, 15.12, 11.59, 5.48, and 7.21 g/mL, respectively. Using optimized analytical methods, the amounts of bioactive compounds from A. iwayomogi in the 15 samples were determined ( Table 3). As a result, contents of compounds and were the highest in Seoul (1.38 ± 0.01, 4.80 ± 0.03 mg/g, resp.), compound was the highest in Gyeongju, Gyeongsangbuk-do (0.65 ± 0.06 mg/g), and compound was highest in Jeju-do (1.11 ± 0.07 mg/g). Furthermore, compounds , , , and were the most abundant in Yangyang, Gangwon  8.18 ± 0.10, and 68.87 ± 0.19 mg/g, resp.). Compound exhibited good inhibition activity of AGEs formation while compounds , , and were significant inhibitors of both AR and AGEs formation. Overall, the specimen with the highest total contents of the most compounds was acquired in Yangyang, Gangwon-do ( Figure 3). We therefore collected new specimens of A. iwayomogi in June, July, August, September, and October in Yangyang, Gangwon-do, and then analyzed the amounts of bioactive compounds in each specimen using HPLC ( Table 4). Amounts of compounds , , , and were taken in June (0.74 ± 0.02, 0.59 ± 0.12, 33.61 ± 0.48, and 8.18 ± 0.10 mg/g, resp.), that of compound was taken in July (73.99 ± 0.35 mg/g), those of compounds and were taken in August (3.67 ± 0.02 and 4.16 ± 0.04 mg/g, resp.), and that of compound was taken in September (0.86 ± 0.02 mg/g).

Discussion
The worldwide prevalence of diabetes has become a massive health burden significantly decreasing quality of life and increasing morbidity, all at a huge economic cost. As diabetes is recognized as a serious disease, diabetic complications are also recognized as a serious disease. Accordingly, preventing treatment for diabetic complications is as important as diabetes and blocking the polyol pathway plays an important role preventing diabetic complications. Therefore, AR and AGEs formation inhibitors significantly affected diabetic complications. Artemisia iwayomogi, locally called haninjin or dowijigi, is a member of the Compositae family and a perennial herb mainly found in Korea. A. iwayomogi has long been used in Korea in foods such as tea, rice cake, and soup and for the treatment of various diseases. However, previous literatures did not have reported on inhibitory activities of A. iwayomogi on AR and AGEs formation. For these reasons, we aimed to assess Artemisia iwayomogi, which is native plant in Korea used as usual food, and has potential as anti-diabetic complications agent to inhibit AR and AGEs formation. Our research led to the isolation of two coumarins ( and ), nine flavonoids ( -), five caffeoylquinic acids ( -), three diterpene glycosides ( -), and one phenolic compound ( ). Among them, compounds , , , , , and were first reported on the isolation from A. iwayomogi. Compounds -have various bioactive properties such as antioxidant, anti-inflammation, antiangiogenesis, and cardiovascular activity [35][36][37].
After that, the extracts, fractions, and compoundsfrom A. iwayomogi were investigated for inhibition of AR. The extracts, 30%, 60%, and 100% MeOH fractions from A. iwayomogi showed significant inhibition of AR more than TMG, positive control. Among compounds isolated from A. iwayomogi, compounds , , , , , , , and were stronger inhibitors than TMG. Out of these, compound exhibited the strongest inhibition. Previous literature reported that caffeic acid had no inhibitory activity on AR but that caffeoyl derivatives are significant inhibitors of AR [24,38]. Existence of caffeoyl groups for substituents such as quinicacid is important parameter for not only AR inhibitory activity but also various biological activities [39]. Furthermore, other studies have demonstrated relationships between the inhibitory activity and structure of flavonoids [4]. They reported that monohydroxyflavones (such as compound ) are not inhibitors of AR, that flavonoid glucosides (compounds and ) are more active than flavonoid diglucosides (compounds and ), and that flavonoids with a substituted hydroxyl group (compound ) are more active than those with substituted methoxyl groups (compound ). Inhibition of AR by flavonoids isolated from A. iwayomogi in our study demonstrated similar results as previous studies. We demonstrated the inhibitory activities of not only AR but also AGEs formation. The CHCl 3 fraction from A. iwayomogi had no inhibitory activity on AR but showed the best inhibition on AGEs formation. The two coumarins ( and ), nine flavonoids ( -), and five caffeoylquinic acids ( -) exhibited also great inhibitory activities more than AG, positive control. Among them, compound showed minor inhibition on AR and exhibited the best inhibition of AGEs formation. Previous literature has reported that the presence of a methoxyl group at C-6 instead of a hydroxyl group and a hydroxyl group at C-7 in the coumarin structure are important parameters in the inhibition of AGEs formation [40]. Therefore, compound may be more active than coumarins with other substituents.

Conclusion
In summary, our study reported the isolation of two coumarins ( and ), nine flavonoids ( -), five caffeoylquinic acids ( -), three diterpene glycosides ( -), and one phenolic compound ( ). Among them, hispidulin ( ), 6-methoxytricin ( ), arteanoflavone ( ), quercetin-3gentiobioside ( ), 1,3-di-O-caffeoylquinic acid ( ), and suavioside A ( ) were isolated for the first time from A. iwayomogi. In addition, two coumarins, nine flavonoids, and five caffeoylquinic acids isolated from A. iwayomogi demonstrated biological activities against AR and AGEs formation. Since extracts of A. iwayomogi contained enough amounts of active compounds, A. iwayomogi extract can be used for the successful crude herbal drug against AR and AGEs formation. Because A. iwayomogi is native to Korea, we analyzed the bioactive compounds by region. We determined that it is best to harvest A. iwayomogi in Yangyang, Gangwondo, from June in order to get the highest content of active compounds. Based on these, our research demonstrates that A. iwayomogi containing active compounds is clearly a potential candidate for a new natural alternative medicine or health supplement and a preventive or therapeutic agent in the treatment of diabetic complications.