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Arctigenic acid, the key substance responsible for the hypoglycemic activity of Fructus arctii.

We have reported the antidiabetic activity of the total lignans from Fructus arctii (TLFA) against alloxan-induced diabetes in mice and rats. In this study, arctigenic acid was found to be the main metabolite in rat plasma detected by UPLC/MS and HPLC/MS/MS after oral administration of TLFA. For the first time, its hypoglycemic activity and acute oral toxicity were evaluated in Goto-Kakizaki (GK) rats, a spontaneous type 2 diabetic animal model, and ICR mice respectively.

GK rats were orally given arctigenic acid (50 mg/kg) twice daily before each meal for 12 weeks. The treatment reduced the elevated plasma glucose, glycosylated hemoglobin and showed significant improvement in glucose tolerance in glucose fed hyperglycemic GK rats. We found that the hypoglycemic effect of arctigenic acid was partly due to the stimulation on insulin secretion, whereas the body weight was not affected by arctigenic acid administration in GK rats. Meanwhile, there was no observable acute toxicity of arctigenic acid treatment at the dosage of 280 mg/kg body weight daily in the acute 14-day toxicity study in mice.

This study demonstrates that arctigenic acid may be the main metabolite in the rat serum after oral administration of TLFA, which showed significant hypoglycemic effect in GK rats, and low acute toxicity in ICR mice. The result prompts us that arctigenic acid is the key substance responsible for Fructus Arctii antidiabetic activity and it has a great potential to be further developed as a novel therapeutic agent for diabetes in humans.

Keywords:

Arctigenic acid

Fructus Arctii

Hypoglycemic activity

GK rats

Diabetes

Acute oral toxicity

Introduction

Diabetes mellitus (DM) is a metabolic disorder characterized by defects in insulin secretion, insulin action, or both (Callahan and Mansfield 2000). The global burden of type 2 diabetes mellitus T2DMposes enormous societal costs and has major implications for all healthcare systems (American Diabetes Association 2011).

World Health Organization has suggested the evaluation of potential plants as effective therapeutic agents, especially in areas in which we lack safe modern drugs (Ivorra et al. 1989). Arctigenic acid (AA, CAS Registry Number: 42320-79-6, Fig. 1) was isolated and identified from germinating burdock (Arctium lappa) seeds at the first time in 2005 (Keiko et al. 2005). But its pharmacological activity has not been reported yet.

Fructus arctii, called "Niubangzi" in China (Great burdock achene in English), is a well-known Chinese Materia Medica. It is the dried ripe fruit of Arctium lappa L. (family Asteraceae) and was included in the Chinese pharmacopoeia for its traditional therapeutic actions. But it has been reported recently that the clinical use of Fructus arctii, in a relatively large quantity (15 g/day), resulted in a satisfactory hypoglycemic effect in patients T2DM (Chen 1999). Modern pharmacological studies revealed that the powder or extracts of Fructus arctii were effective for the treatment of high blood glucose and diabetic nephropathy (Yan and Li 1993; Wang and Chen 2004; Zhang and Piao 2007; Liu et al. 2011).

We have reported (Xu et al. 2008) the antidiabetic activity of the total lignans from the ethanol extract of Fructus arctii against alloxan-induced diabetes in mice and rats. In our prior study, TLFA exhibited significant antidiabetic activity, and a notable increase in the serum insulin level was observed at the same time.

In this study, we analyzed the constituents in the serum of the GK rats after TLFA was given orally. The serum samples were detected by UPLC/MS (Waters, US) and HPLC/MS/MS (Finnigan, US), AA was found to be the main ingredient in the serum.

Then we studied acute oral toxicity and hypoglycemic activity of AA, which was prepared with arctigenin by alkaline hydrolysis reaction. The pharmacodynamic study was performed in GK rats, a spontaneous type 2 diabetic animal model, and insulinotropic drug nateglinide was chosen as positive control drug.

Besides common evaluation indexes of hypoglycemic activity such as fasting blood glucose (FBG), postprandial glucose (PBG), oral glucose tolerance test (OGTT), as well as glycated hemoglobin, particular attention was paid to the effects of AA on insulin secretion and pancreas tissue sections, with a view to investigate its mechanism of hypoglycemic activity and providing a pharmacological rationale for the future development of it for diabetes.

Materials and methods

Plant material

The Fructus arctii purchased from Xuzhou Pharmaceutical Co., Ltd. was collected from Xuzhou, Jiangshu Province. The plant material was identified by Botanical Professor Zhong Liu at the School of Pharmacy, Shanghai Jiao Tong University. The voucher specimen is deposited in the Engineering Research Center of Modern Preparation Technology of Traditional Chinese Medicine, Ministry of Education, Shanghai University of TCM, under the number 20121211. The whole plant was then dried under air, insolation and mechanically powdered separately to obtain a coarse powder, which was then subjected to extraction.

Preparation of TLFA and AA

Simply comminuted dried Fructus arctii (10 kg) was extracted circumfluently with 95% aqueous ethanol (2 x 80 l) at 80[degrees]C for 6 h each time. The combined extracts were filtered and concentrated to yield liquid extract (EtOH-extract, 2.0 1, density 1.05 g/[cm.sup.3]) in a rotatory evaporator at 55[degrees]C under reduced pressure, and then defatted with petroleum ether (60-90[degrees]C) followed by extraction with ethyl acetate. The ethyl acetate extract was concentrated and dried in vacuum at 50[degrees]C, with a yield of 8.6% (w/w). Thus, the extracts containing total lignans (TLFA) were obtained.

Purity of TLFA was determined by ultraviolet spectrometry (Zhai et al. 2009). In detail, the precisely weighed 10.0 mg TLFA powder was dissolved with 25 ml MeOH, and 1.0 ml of dissolution was transferred into a 10 ml of volumetric flask to prepare sample solution. Arctigenin (purity > 99%, Lot number: A1854, Sigma-Aldrich Chemical Company) was dissolved in MeOH to 30 mg/ml as the control solution. Absorbance of the sample and the control were determined at 280 nm using ultraviolet spectrometry with MeOH as a blank. Finally, the purity of TLFA was calculated to be 86.0%.

The TLFA (200 g) was fractionated by column chromatography at atmospheric pressure over silica gel G using petroleum ether, chloroform, ethyl acetate, acetone and methanol successively, as a result, five fractions were obtained. The fraction eluted by chloroform was concentrated to one-tenth of the original volume in a rotatory evaporator at 40[degrees]C under reduced pressure, then the concentrate was crystallized at 4[degrees]C, followed by recrystallization with methanol to obtain purified arctigenin (yield 4.3%, w/w), which was identified by comparing the physical and chemical parameters with reference substance arctigenin.

The arctigenin was hydrolyzed in 12-fold (w/v) of 60% aqueous ethanol containing 4% NaOH (w/v) at 60[degrees]C for 3 h, then the reaction product was adjusted to neutral pH with 1 M of HCl. After standing overnight at room temperature, the reaction product was centrifuged, and the precipitate was washed by deionized water until washings become colorless, then it was dried in vacuum at 37[degrees]C to give a white amorphous powder with a yield of 96.6% (w/w). The chemical structure of this arctigenin hydrolysis product was further identified by spectral and elemental analysis.

Animals

The acute toxicity test was carried out on ICR mice of either sex weighing between 18 and 23 g. Male GK rats and Wistar rats of both sexes aged 9 weeks were used for serum pharmacochemistry study and hypoglycemic activity assessment.

All animals were obtained from Shanghai Laboratorial Animal Center, Chinese Academy of Science. All animals were housed in polycarbonate cages (five rats or mice per cage) with a wooden chip mat on the floor and tap water was available ad libitum. High fat diet (Suzhou Shuangshi Laboratory Animal Feed Science Co., Ltd, China) was provided to GK rats and standard chow was provided to mice and Wistar rats. The animal room was kept on a 12-h light/dark cycle, with a temperature range of 24 [+ or -] 1[degrees]C and a relative humidity of 55% [+ or -] 5% throughout the experimental period. All the procedures were in strict accordance with the PR China legislation on the use and care of laboratory animals and with the guidelines established by Institute for Experimental Animals of Shanghai University of TCM and were approved by the university ethical committee for animal experiments.

Analysis of the constituents in the rat serum after oral administration of TLFA

Sample collection

After 7 days acclimation, six experiment GK rats were fasted overnight before the experiments. Two milliliters of blood was taken from orbital sinus of every rat. Then vacuum dried powder of TLFA was dissolved with the solvent composed by ethanol:Tween 80:distilled water (1:1:8) as a stock solution (35 mg/ml) and was orally administrated to rats (1 ml/100 g body weight). 60 min after drug administration, the blood samples were collected from orbital sinus and immediately centrifuged at 13,000 rpm for 10 min at 4[degrees]C, the supernatant (serum) obtained were frozen immediately and stored at -40[degrees]C before analysis.

HPLC grade ethanol (300 [micro]l) was added to 100 [micro]l of the above serum and vortexed for 1.5 min, centrifuged (13,000 rpm) at 4[degrees]C for 10 min, and the supernatant were dried under gentle nitrogen gas at 45[degrees]C. The residues were redissolved with HPLC grade methanol (100 [micro]l), ultrasonicated for 10 min and centrifuged (13,000 rpm) at 4[degrees]C for 10 min, the resultant supernatant was filtered through a 0.22 [micro]m-filter prior to UPLC/MS analysis.

A 10.5 mg of arctigenic acid was dissolved with 100 ml of HPLC grade methanol, filtered through a 0.22 [micro]m-filter, where the filtrate was used as the control for UPLC/MS analysis.

UPLC/MS analysis

Waters Acquity[TM] Ultra Performance LC system (Waters Corporation, Milford, USA) equipped with quaternary pump, vacuum degasser, autosampler, diode-array detector. The chromatographic condition was as follow: UPLC[TM] BEH [C.sub.18] column (1.7 [micro]m, 2.1 mm x 50 mm); mobile phase: a linear gradient system of A ([H.sub.2]O) and B (C[H.sub.3]OH), the gradient program is shown in Table 1; flow rate: 0.2 ml/min; column temperature: 40[degrees]C; detecting wavelength: 280 nm; injection volume: 2.0 [micro]l.

Mass analysis was performed on a Waters micromass ZQ2000 (Waters MS Technologies, Manchester, UK) mass spectrometer equipped with an ESI source operating in negative ion mode. The desolvation gas rate was set at 600 l/h at a temperature of 320[degrees]C, the cone gas rate was set at 50 l/h, and the source temperature at 120[degrees]C. The capillary and the cone voltages were set at 3000 and 45 V, respectively. Data were collected in SIR mode, which function type was SIR of nine channels, the channels mass is shown in Table 2.

HPLC/MS/MS analysis

HPLC/MS/MS analysis was performed using a Waters 2690 system (Waters, Milford, MA, USA) coupled with a TSQ triple quadrupole (Finnigan MAT, San Jose, CA, USA) equipped with an ESI interface. A CAPCELL PAK [C.sub.18] MG II column (100 mm x 2.0 mm, 3 [micro]m, Shiseido, Japan) was used to separate compounds at 40[degrees]C within 20 min using a mixture of [H.sub.2]O and C[H.sub.3]OH (6:4, v/v) as the mobile phase at a flow rate of 0.7 ml/min. A splitter was used to transfer only one-third of the flow into the mass spectrometer. The mass spectrometric conditions were optimized by manual optimization using infusion with a syringe pump to select the most suitable ion transitions for the target analytes. By automatically injecting standard solutions and comparing the ratio of signal/noise, other operation parameters, such as the heated capillary temperature (320[degrees]C), the electrospray voltage (5.0 kV), sheath gas flow rate (30 psig), and auxiliary gas flow (10), were also optimized. The MS/MS was operated in negative ion mode at collision energy of 40 eV, and the products of precursor (m/z = 389) were acquired with full scan range between m/z 105 and 400 and further processed using the Xcalibur 1.3 software.

Acute oral toxicity study of arctigenic acid

ICR mice were randomly divided into three groups, each group containing 10 mice (5 females and 5 males) and acclimatized for 1 week before starting the experiment. Mice were fasted overnight (12 h) with free access to water and then treated as follows: the first group was treated with AA (140 mg/kg b.w., dissolved in the solvent composed by ethanol, Tween 80 and distilled water (1:1:8)), the second group (negative control) was treated with blank solvent and the third group (normal control) was treated with distilled water in a single administration. Treatment was given by gavage of 4 ml/100 g mice BW. The general behavior of the mice was continuously monitored for 4 h after the first treatment, then they were dosed the second time at 4 h intervals if they all survived after 4 h of treatment and no clinical signs of toxicity were observed. All of the experimental animals were maintained under close observation intermittently during a 24-h period (Twaij et al. 1983), and thereafter daily up to 14 days.

All the mice were weighed once before the commencement of the dosing and then on Days 7 and 14. At the end of the study, all animals were sacrificed by clavicle dislocation, and vital organs were excised and weighed.

Pharmacodynamics experimental design

For precise evaluation of the influence of AA on postprandial glucose changes, all the rats (n = 40) were trained to consume the diet chow during a 1-h period, twice a day (9:00 AM to 10:00 AM and 3:00 PM to 4:00 PM) under the dark period (Yoshiro et al. 2002). The animal room was kept on a 12-h light/dark cycle (7 AM to 7 PM/dark, 7 PM to 7 AM/light). The animals were acclimatized to laboratory conditions for 2 weeks. After the dietary conditioning, the daily blood glucose profile showed a marked postprandial increase in GK rats, while there was no apparent change in that of Wistar rats.

Then the rats were divided into four groups (n 10 each) and treated with either saline solution (0.9% NaCl, w/v, Wistar rats, normal control), vehicle alone (0.5% methylcellulose, GK rats, model control), nateglinide (50 mg/kg, GK rats, positive control) or AA (50 mg/kg, GK rats, sample) by oral gavage twice daily just before each meal for 12 weeks.

The dose of nateglinide (Product lot: NG-20130604, Jiangsu Yongda Pharmaceutical Co., Ltd., Jiangyin, China) was determined by the references (Yoshiro et al. 2002; Atsuko et al. 2006), while the dose of AA was determined according to the dose of nateglinide.

Measurement of blood glucose and body weight

Blood glucose and body weight was monitored once a week throughout the experimental period. Blood sampling from the tail vein was collected before the first meal (at 10 AM after overnight fasting) and 1 h after the first meal of the day to determine the FBG and the PBG. Blood glucose level was determined by the blood glucose meter and extremity whole blood glucose strips (OneTouch UltraEasy[TM] test strips and blood glucose meter, Johnson & Johnson Medical Ltd., Shanghai, China).

Oral glucose tolerance test (OGTT) and C-peptide

At the 10th week of the study, the experimental rats were subjected to an oral glucose tolerance test (OGTT). Blood samples of half of them were collected from the cut tails after fasting overnight and were measured and defined as zero time, then glucose (2 g/kg b.w.) was orally administered 30 min after an oral administration of the test samples or vehicle (for control). Blood glucose levels were measured at 30, 60 and 120 min after glucose administration.

Blood samples of the other experimental rats were collected from orbital sinus into chilled heparin microcentrifuge tubes immediately prior to the test samples or vehicle (for control) gavage and 1 h after the glucose gavage. Plasma was separated and stored at -80[degrees]C until analysis. C-peptide levels of these samples were measured by Iodine [125I] C-peptide Radio-immunoassay Kit (Beijing North Institute of Biological Technology, Product lot: 20110215, Beijing, China) according to the manufacturer's instructions.

Measurement of glycated hemoglobin (HbA1c), total cholesterol (TC), triglycerides (TC), low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) levels in rat serum

At the end of the study, all rats were fasted for 18 h, weighed and then anaesthetized with an intraperitoneal injection of Urethane (1.4 mg/kg). They were continually monitored until total loss of consciousness was reached, as indicated by a total lack of response after a foot pinch. Blood samples were collected from the abdominal aorta, allowed to clot on ice and subsequently subjected to centrifugation (3500 rpm at 4[degrees]C for 10 min), and after the resultant plasma aliquots were stored at -80[degrees]C for further analysis. Glycated hemoglobin was tested with ELISA kits according to the manufacturer's recommendations (Nanjing Jiancheng Bio-engineering Institute, Nanjing, China). The serum TC, TG, LDL-C and HDL-C levels were examined by an automatic biochemical analyzer (7080, HITACHI High-Tech Co., Ltd., Japan).

Histopathological examination of the pancreas

Histopathological analyses were performed by way of optical microscopy on paraffin material. After termination of experiment, the animals were euthanized and whole pancreas was excised from each rat group. Pancreas tissue sections were fixed in 10% buffered formalin and immediately histological preparations were made. 5 [micro]m thick sections were cut and stained with hematoxylene and eosin for histological examination (Luna 1990).

Statistical analysis

All the data were expressed as mean [+ or -] S.E.M. Significant difference between control and experimental groups were assessed by Student's f-test. A probability level of less than 5% (p < 0.05) was considered significant.

Results

Chemical structure identification o/AA

The hydrolysis product of arctigenin was obtained as a white amorphous powder, mp: 118-120[degrees]C; ESI-MS (m/z) [[M + Na].sup.+] = 413. The [sup.1]H and [sup.13]C NMR spectra were consistent with those published by Keiko et al. (2005), so it was identified as AA.

Analysis of the constituents in the rat serum after oral administration of TLFA

Typical TIC chromatograms of the control serum, the serum after oral administration of TLFA, AA and TLFA in methanol are shown in Fig. 2.

As shown in Fig. 2, the components of TLFA were almost not detected in rat serum after oral administration, while a metabolite whose retention time equaled with that of AA was detected in this sample using UPLC/MS.

Further analysis was carried out to identify the metabolite by using HPLC/MS/MS (Finnigan MAT, San Jose, CA, USA). As shown in Fig. 3, product ion mass (MS/MS) spectra of the metabolite (Fig. 3(b)) were almost fully consistent with the AAs (Fig. 3(a)) in negative ionization mode. The metabolite were then provisionally identified from the product ion spectra, molecular weight, and structure information of AA, as benzenebutanoic acid, [alpha]-[(4-hydroxy-[beta]-methoxyphenyl)methyl]-/Hhydroxymethyl)-3,4- dimethoxy-, ([alpha]R, [beta]S)-. The proposed metabolic pathway is shown in Fig. 3(c).

Acute oral toxicity study of AA

In the acute 14-day toxicity study, AA, given at a dose of 280 mg/kg b.w. at the first day did not cause any visible signs of toxicity or mortality to the ICR mice (Table 3). The median lethal dose ([LD.sub.50]) of arctigenic acid was then not be detected.

The body weight of all the experimental mice increase gradually throughout the study period, and the AA group did not show any significant difference compared to the normal control group (Table 4). There was no significant difference between relative organ weights of the treated group and normal control group, except liver of the female mice in AA group (p < 0.05, Table 5).

In conclusion, AA has no observable acute effect on the experimental mice at 280 mg/kg BW a day.

Measurement of blood glucose

Fig. 4(a) shows the FBG levels of experimental rats each week, compared to the age-matched normal rats, GK rats displayed higher FBG levels than normal rats throughout the experiment. Among them, the FBG levels of model rats were elevated from 11.4 [+ or -] 1.98 mmol/l at the beginning to 17.32 [+ or -] 3.45 mmol/l at the end of the experiment representing 51.9% rise. However, from the third week of the treatment, FBG levels of both AA and nateglinide groups were decreased until the end of the experiment when compared to the model group. Interestingly, FBG level of AA group was further decreased during the last 6 weeks of the treatment, compared to nateglinide group. At the end of the experiment, AA decreased the FBG levels by 37.6% while nateglinide decreased that by 28.1% as compared to the model group.

Fig. 4(b) shows PBG levels of experimental rats in each week of the study, compared with age-matched normal rats, GK rats showed higher PBG levels throughout the treatment. Among them, the PBG levels of rats in model group increased from 16.7 [+ or -] 2.11 mmol/l at the beginning to 23.5 [+ or -] 2.42 mmol/l at the end of the experiment, representing 40.7% rise. However, from the second and the third week of the treatment respectively, PBG levels of AA and nateglinide group were significantly decreased until the end of the experiment when compared to the model group. At the end of the experiment, AA decreased the PBG levels by 43.7% while nateglinide decreased that by 19.6% when compared to the model control group.

Fig. 4(c) shows that, at the end of the experiment, the HbA1c value of rats in model group (p < 0.01) and nateglinide group (p < 0.05) were notably higher than the rats in normal group, while these values of rats in both nateglinide (p < 0.01) and AA (p < 0.01) groups were lower than rats in the model group significantly.

Fig. 4(d) shows body weight of experimental rats in each week, compared to age-matched normal rats. The body weights of GK rats were lighter throughout the experiment. The final body weight of nateglinide (p < 0.05) and AA (p < 0.05) group were significantly lighter than the normal group. However, there was no significant difference between the final body weight of the normal and the model group, as well as no significant difference between the final body weight of treatment (AA or nateglinide) group and model group.

Oral glucose tolerance test (OGTT) and C-peptide

Fig. 5 shows that, in the OGTT study, the blood glucose concentrations in the three diabetic groups all reached peak level at 30 min after glucose administration (2 g/kg) and then began to decrease. Compared with the model group, the plasma glucose levels of rats treated with nateglinide and AA showed a highly significant reduction of 21.9% and 33.6% respectively after 60 min. After 120 min, the plasma glucose levels of rats treated with nateglinide and AA were reduced by 22.0% and 33.7% (p < 0.01) respectively, compared to their diabetic control counterparts.

As shown in Fig. 6, 1 h after glucose gavage, the serum C-peptide levels of rats treated with nateglinide and AA increased 3.31-fold (** p < 0.01) and 2.88-fold (** p < 0.01), respectively compared to their fasting ones.

Histopathological examination of the pancreas

As shown in Fig. 7(a), the islets of normal rats were characterized with normal cellular pattern and diameter of pancreatic islets. While the islets of model rats displayed an irregular structure with reduced volume, and a reduction in the number of islet cells. The islets were shrunken with atypical cellular changes, such as mild hyperchromasia, coarse chromatin and pyknosis (Fig. 7(b)). The treatments of nateglinide (Fig. 7(c)) and AA (Fig. 7(d)) restored such irregularity to the structure similarly to that of normal control samples. Results showed that they enhanced the regeneration of islets in the pancreas and restoration of normal cellular size of the islet with hyperplasia.

Lipids characteristics of rats at the end of the experiment

Table 6 compares cholesterol, triglyceride, HDL-C and LDL-C of nateglinide and AA treated diabetic rats with control group. It found that all the four index of the model group were significantly different from the normal group. Among them, cholesterol, HDL-C and LDL-C were higher than their control counterparts while TG was lower. But all the four index of rats treated by AA were not significantly different from the model group. Table 6 also shows the significant decreased CHOI, while the remarkably increased TG in the nateglinide treated rats as compared to model group.

Discussion

In previous study, we have observed the hypoglycemic activity of TLFA in alloxan-induced diabetic mice and rats. We also noticed that TLFA decreased the blood glucose level only at hyperglycemic but not in basal conditions, which is similar to the glucose-dependent hypoglycemic effect of insulinotropic drug nateglinide (Keilson et al. 2000).

Nateglinide was approved in December 2000 for use in patients in the US with type 2 diabetes mellitus as an adjunct to diet and exercise to improve glycemic control. It is a derivative of D-phenylalanine ((-)-N-[trans-4-isopropylcyclohexane) carbonyl]-D-phenyl alanine) and has a relatively short onset of action (George 2011; Fig. 8). It works by closing the ATP-dependent potassium channels ([K.sub.ATP]) in the pancreatic islet [beta] cells by binding to the sulfonylurea SUR1 receptor, which causes depolarization of the [beta] cells followed by calcium influx and subsequent insulin secretion.

Hisashi (Hisashi et al. 1988) investigated the structure-activity relationships of nateglinide, found that not only the carboxyl group but also the R configuration at the asymmetric carbon was necessary for possessing hypoglycemic activity. Carboxyl group at the chiral carbon affects the activity, and the decarboxyl, esterification and acylation will result in different degrees of reduction of activity of the compounds.

We have isolated eight compounds from TLFA, they were all identified as lignans with dibenzyl butyrolactone structure (Xu et al. 2006). We found that if the lactone ring structure of arctigenin (2(3H)-furanone, 4-[(3,4-dimethoxyphenyl)methyl]dihydro-3-[(4-hydroxy3-methoxyphenyl)methyl]-, (3R, 4R)-), the main component of TLFA and the mother nucleus of chemical structure of the eight compounds, could be hydrolyzed in vivo, it will be converted to AA. The chemical structure of AA is similar to that of nateglinide, with the carboxyl group and the R configuration at the asymmetric carbon (Fig. 9).

So we speculated that AA probably was the main active metabolite of TLFA, which possessed hypoglycemic activity similar to that of nateglinide. It may be the key substance responsible for the hypoglycemic activity of Fructus Arctii. In this study, as we expected, AA was found to be the main ingredient in rats serum, which was cross-checked with the report of Gao, who found that AA was the major metabolite in vivo after oral administration of arctigenin in rats (Gao et al. 2013).

Given the complexity of diabetes, various experimental animal models have been developed for investigating the pathophysiology of T2DM. Among them, GK rat have been considered as one of the best spontaneous-onset, non-obese type 2 diabetic animal model (Akash et al. 2013), featuring mild fasting hyperglycemia, notable postprandial hyperglycemia, impaired insulin secretion, progressive reduction of [beta]-cell mass, and the development of long-term diabetic complications (Yoshiro et al. 2002).

Most of the characteristics that are common between GK rats and human diabetic patients are summarized in Table 7. As shown in the table, the characteristics of GK rats are comparably similar to human T2DM patients particularly relating to the underlying pathophysiological mechanisms. Thereby, GK rats may provide a vital tool to extrapolate the results in accordance with that of humans to study the new anti-diabetic agents. Accordingly, the GK rats were chosen for investigating the anti-diabetic activity of AA in this study.

Nateglinide can decrease fasting hyperglycemia as well as postprandial hyperglycemia, which will lead to exacerbations and complications of diabetes (Ceriello et al. 2006; David 2001), In 2007, International Diabetes Federation published the first edition of Guideline for management of postmeal glucose in diabetes (Ceriello et al. 2008). For this reason, nateglinide was selected as a positive control and we adopted the dietary protocol that enabled us to monitor both FBG and PBG levels in this study.

During the period of pharmacodynamics experiment, AA exhibited hypoglycemic activity against the fasting and postprandial hyperglycemia in GK rats, and showed significant improvement in glucose tolerance in glucose fed hyperglycemic GK rats. Its hypoglycemic activity is in agreement with the reported antidiabetic activity of TLFA in alloxan diabetic mice and rats. Furthermore, both AA and nateglinide well improved the HbA1c levels of the experimental animal. The FBG and PBG levels of rats treated with AA were even lower than those of nateglinide group. During the progression of diabetes, the excess of glucose present in blood reacts with hemoglobin to form glycated hemoglobin. Such biological process, is non-enzymatic, and reflects the average exposure of hemoglobin to glucose over an extended period of 120 days (red blood cells lifespan is 120 days) (Mohammadi and Naik 2008). Therefore, we think that the hypoglycemic activity of AA is potent and long-lasting. Furthermore, AA was given orally in this study, in view of the chemical structure of AA determines it is unstable under acidic conditions such as in the gastric juice, its enteric formulations should help improve its efficacy.

Owing to C-peptide and insulin released from pancreatic [beta]-cells in equimolar ratio to the blood, and C-peptide did not cross-react with insulin antibodies, so determine serum C-peptide levels helps to accurately evaluate the secretory function of pancreatic [beta]-cells. At the end of this study, postprandial serum C-peptide levels of all the experiment rats were higher than fasting. Among them, the C-peptide levels of both the nateglinide group and AA group were significantly higher than the normal group, of which nateglinide group showed higher levels than AA group. Given that the PBG levels of rats in AA group was lower than those in nateglinide group, we think that, despite the similarities of AA in some parts of the chemical structure with nateglinide, the stimulation of insulin secretion might just be one of the hypoglycemic mechanisms of AA, other mechanisms also deserve to be investigated including enhancement of glucose utilization of skeletal muscle and liver, inhibition of the absorption of glucose, and so on.

Histopathological examination showed that both AA and nateglinide enhanced the regeneration of islets in the pancreas and restoration of normal cellular size of the islet with hyperplasia, which may explain the efficiency of them in the management of hyperglycemia.

We found that AA could not affect the body weight of GK rats in this study. Meanwhile, biochemical parameters of blood in experimental rats also showed that AA had no significant effect on lipid metabolism in the GK rats. These results are somewhat different from our previous report (Xu et al. 2008), the reasons may be associated with the fact that GK rats are non-obese animal models. The influence of AA on plasma lipid profile needs to be further investigated.

The result of acute oral toxicity of arctigenic acid shows that it does not have obvious acute toxicity, which is conducive to the further development of its pharmacodynamic activity. However, there are limitations of the acute toxicity data since accumulative toxic effect may not be observed in a short period with a single dose application. A follow-up study with long-term exposure, the sub acute and chronic evaluation of the extracts will be carried out in evaluating the safety profile of AA.

To our knowledge it is the first report of the hypoglycemic effect of AA on spontaneous type 2 diabetic animal model, which confirmed our speculation that AA is the key substance responsible for the hypoglycemic activity of Fructus Arctii.

Conclusion

Arctigenic acid is the chief constituent in the rat serum after oral administration of TLFA and demonstrated significant hypoglycemic potential in GK rats, low acute toxicity in ICR mice. We identified that AA is the key substance responsible for the hypoglycemic activity of Fructus Arctii and has a great potential to be further developed as a novel therapeutic agent for diabetes in humans. The current finding indicates that stimulating insulin secretion may be one of the mechanisms of its hypoglycemic activity. However, further study is required for the verification of its hypoglycemic effect, the investigation of its mode of action, and the discovery of its more biological activity.

Abbreviations

AA,      arctigenic acid
CHOI,    cholesterol
DM,      diabetes mellitus
ELISA,   enzyme-linked immunosorbent assay
FBG,     fasting blood glucose
HbA1c,   glycosylated hemoglobin
HDL-C,   high density lipoprotein cholesterol
LDL-C,   low density lipoprotein cholesterol
OGTT,    oral glucose tolerance test
PBG,     postprandial blood glucose
TG,      triglycerides
T2DM,    type 2 diabetes mellitus


ARTICLE INFO

Article history:

Received 25 April 2014

Revised 21 September 2014

Accepted 15 November 2014

Acknowledgements

We are grateful for the grant by a Key Task of Innovation Project of Shanghai Municipal Education Commission (No: 09ZZ124), and a Project for Modernization of Traditional Chinese Medicine of Science and Technology Commission of Shanghai Municipality (No: 12401900300).

References

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Zhaohui Xua (a) *, Chenchen Gu (a), Kai Wang (b), Jiaxing Ju (a), Haiying Wang (c), Kefeng Ruan (a), Yi Feng (a)

(a) Engineering Research Center of Modern Preparation Technology of Traditional Chinese Medicine. Ministry of Education. Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China

(b) Center for Drug Safety Evaluation, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China

(c) School of Pharmacy, Shanghai University of Traditional Chinese Medicine. Shanghai 201203, China

* Corresponding author. Tel.: +86 021 51323094; fax: +86 021 51323094. E-mail address: lntcmx@163.com (Z. Xu).

http://dx.doi.org/10.1016/j.phymed.2014.11.006

Table 1
Solvent gradient program of UPLC analysis.

Time     Flow
(min)   (ml/min)   A (%)   B (%)

  0       0.2       60      40
 1.3      0.2       60      40
 1.5      0.2       50      50
 7.0      0.2       50      50
 8.0      0.2       60      40
10.0      0.2       60      40

Table 2 Channels mass of UPLC analysis.

Channels    Dwell   Delay
mass        (s)     (s)     Constituents

357         0.1     0.02    Matairesinol
371         0.1     0.02    Arctigenin
389         0.1     0.02    Arctigenic acid
535         0.1     0.02    Lappaol A
553         0.1     0.02    Lappaol C
579         0.1     0.02    Arctiin
731         0.1     0.02    Arctignan E
749         0.1     0.02    Lappaol H
759         0.1     0.1     Lappaol F

Table 3
Acute oral toxicity of AA in mice.

Treatment      Mice         Latency   Symptoms of
dose (mg/kg)                          toxicity
               Sex    D/T

0 (normal      M      0/5   --        None
  control)     F      0/5   --        None
0 (negative    M      0/5   --        None
  control)     F      0/5   --        None
280            M      0/5   --        None
               F      0/5   --        None

Table 4
Body weight of control and rats treated with AA recorded
during acute toxicity study, measured weekly.

            Body weight

            Day 0

Group       Male                 Female

Normal
 control    21.4 [+ or -] 1.52   19.6 [+ or -] 1.34
Negative
 control    21.6 [+ or -] 0.89   19.4 [+ or -] 0.55
AA (a)      22.0 [+ or -] 1.0    19.6 [+ or -] 1.14

            Body weight

            Week 1

Group       Male                 Female

Normal
 control    28.2 [+ or -] 1.64   24.0 [+ or -] 1.41
Negative
 control    27.6 [+ or -] 0.89   23.0 [+ or -] 1.22
AA (a)      29.2 [+ or -] 1.79   24.2 [+ or -] 0.84

            Body weight

            Week 2

Group       Male                 Female

Normal
 control    31.8 [+ or -] 0.84   26.8 [+ or -] 1.64
Negative
 control    32.0 [+ or -] 1.22   25.0 [+ or -] 1.0
AA (a)      32.8 [+ or -] 1.10   25.6 [+ or -] 0.55

            Body weight

            Weight gained on Day 14

Group       Male                      Female

Normal
 control    10.4 [+ or -] 1.14        7.2 [+ or -] 0.45
Negative
 control    10.4 [+ or -] 0.89        5.6 [+ or -] 0.89 **
AA (a)      10.8 [+ or -] 1.10        6.0 [+ or -] 1.22 *

Values are expressed as mean [+ or -] standard deviation, n = 10.

(a) Group given 140 mg/kg BW of AA double dose orally 1 day
and observed for 14 days.

** p < 0.01, * p < 0.05 compared with the normal control.

Table 5
Relative organ weights of mice treated with double dose of AA.

Relative organ weights in gram per 100 g BW of control and
mice treated with double dose of arctigenic acid

Organs            Normal control         Negative control

Male

Heart          0.400 [+ or -] 0.033    0.375 [+ or -] 0.013
Liver          5.174 [+ or -] 0.22     5.264 [+ or -] 0.305
Spleen         0.433 [+ or -] 0.056    0.468 [+ or -] 0.035
Lungs          0.467 [+ or -] 0.011    0.486 [+ or -] 0.011 *
Kidney left    0.705 [+ or -] 0.027    0.741 [+ or -] 0.051
Kidney right   0.741 [+ or -] 0.026    0.731 [+ or -] 0.068

Female

Heart          0.357 [+ or -] 0.031    0.360 [+ or -] 0.020
Liver          4.271 [+ or -] 0.241    4.225 [+ or -] 0.224
Spleen         0.528 [+ or -] 0.095    0.447 [+ or -] 0.077
Lungs          0.477 [+ or -] 0.023    0.484 [+ or -] 0.043
Kidney left    0.572 [+ or -] 0.056    0.535 [+ or -] 0.028
Kidney right   0.561 [+ or -] 0.038    0.578 [+ or -] 0.022

Organs                AA (a)

Male

Heart          0.390 [+ or -] 0.028
Liver          4.968 [+ or -] 0.362
Spleen         0.376 [+ or -] 0.026
Lungs          0.478 [+ or -] 0.012
Kidney left    0.736 [+ or -] 0.029
Kidney right   0.727 [+ or -] 0.041

Female

Heart          0.378 [+ or -] 0.034
Liver          3.885 [+ or -] 0.282 *
Spleen         0.487 [+ or -] 0.046
Lungs          0.474 [+ or -] 0.017
Kidney left    0.557 [+ or -] 0.061
Kidney right   0.570 [+ or -] 0.051

Values are expressed as mean [+ or -] standard deviation, n = 10.

(a) Group given 140 mg/kg BW of AA double dose orally 1 day and
observed for 14 days.

* p < 0.05 compared with the normal control.

Table 6
CHOI, TG, LDL-C, HDL-C levels in rats serum at the end
of the experiment.

                 Normal                  Model

CHOI (mmol/l)    2.16 [+ or -] 0.33      3.30 [+ or -] 0.43 (++)
TG (mmol/l)      0.58 [+ or -] 0.10      0.38 [+ or -] 0.12 (++)
HDL-C (mmol/l)   1.61 [+ or -] 0.24      2.44 [+ or -] 0.41 (++)
LDL-C (mmol/l)   0.51 [+ or -] 0.11      0.62 [+ or -] 0.06 (+)

                 Nateglinide             AA ig

CHOI (mmol/l)    2.92 [+ or -] 0.27 *    3.10 [+ or -] 0.66
TG (mmol/l)      0.54 [+ or -] 0.13 **   0.31 [+ or -] 0.04
HDL-C (mmol/l)   2.25 [+ or -] 0.43      2.45 [+ or -] 0.53
LDL-C (mmol/l)   0.71 [+ or -] 0.17      0.58 [+ or -] 0.07

The data are expressed as mean [+ or -] S.E.M.; 2 way ANOVA; n = 10.

CHOI: cholesterol; TG: triglyceride; HDL-C: high-density lipoprotein;
LDL-C: low-density lipoprotein.

(++) p < 0.01, (+) p < 0.05 compared with the normal control.

** p < 0.01, * p < 0.05 compared with the model control.

Table 7
Comparison of diabetic characteristics in
GK rats and human diabetic patients.

Features                      GK rats     Human

Insulin response to glucose   Decreased   Decreased
Insulin response to GLP-1     Increased   Not determined
Glucose oxidation             Decreased   Decreased
GPDH activity                 Decreased   Decreased
ATP/ADP ratio                 Decreased   Decreased
GLUT2                         Decreased   Decreased
Glucokinase                   Decreased   Decreased
IRS-2                         Decreased   Decreased
Pro-insulin/insulin ratio     Increased   Increased
Markers of systemic           Increased   Increased
  inflammation
Lipid profiles                Increased   Increased

GK: Goto-Kakizaki; GLP-1: glucagon-like peptide-1;
CPDH: glycerol-3-phosphate dehydrogenase;
ATP: adenosine triphosphate; ADP: adenosine diphosphate;
GLUT2: glucose transporter; IRS-2: insulin receptor
substrate-2. Data were obtained from Akash et al. (2013).
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Author:Xua, Zhaohui; Gu, Chenchen; Wang, Kai; Ju, Jiaxing; Wang, Haiying; Ruan, Kefeng; Feng, Yi
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Jan 15, 2015
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