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Antihyperglycemic activity of Tarralin™, an ethanolic extract of Artemisia dracunculus L.
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Phytomedicine 13 (2006) 550–557 www.elsevier.de/phymed
Antihyperglycemic activity of TarralinTM, an ethanolic extract of Artemisia dracunculus L.$ D.M. Ribnickya,, A. Pouleva, M. Watfordb, W.T. Cefaluc, I. Raskina a
Biotech Center, Cook College, Rutgers University, 59 Dudley Road, New Brunswick, NJ 08901-8520, USA Nutritional Sciences, Cook College, Rutgers University, 59 Dudley Road, New Brunswick, NJ 08901-8520, USA c Pennington Biomedical Research Center/Louisiana State University, 6400 Perkins Road, Baton Rouge, LA 70808, USA b
Abstract The studies reported here were undertaken to examine the antihyperglycemic activity of an ethanolic extract of Artemisia dracunculus L., called TarralinTM in diabetic and non-diabetic animals. In genetically diabetic KK-Ag mice, TarralinTM treatment by gavage (500 mg/kg body wt./day for 7 days) lowered elevated blood glucose levels by 24% from 479725 to 352716 mg/dl relative to control animals. In comparison, treatment with the known antidiabetic drugs, troglitazone (30 mg/kg body wt./day) and metformin (300 mg/kg body wt./day), decreased blood glucose concentrations by 28% and 41%, respectively. Blood insulin concentrations were reduced in the KK-Ag mice by 33% with TarralinTM, 48% with troglitazone and 52% with metformin. In (STZ)-induced diabetic mice, TarralinTM treatment, (500 mg/kg body wt./day for 7 days), also significantly lowered blood glucose concentrations, by 20%, from 429741 to 376758 mg/dl relative to control. As a possible mechanism, TarralinTM was shown to significantly decrease phosphoenolpyruvate carboxykinase (PEPCK) mRNA expression by 28% in STZ-induced diabetic rats. In nondiabetic animals, treatment with TarralinTM did not significantly alter PEPCK expression, blood glucose or insulin concentrations. The extract was also shown to increase the binding of glucagon-like peptide (GLP-1) to its receptor in vitro. These results indicate that TarralinTM has antihyperglycemic activity and a potential role in the management of diabetic states. r 2005 Elsevier GmbH. All rights reserved. Keywords: Artemisia dracunculus; Diabetes; Hyperglycemia; Hypoglycemia; GLP-1; PEPCK; Russian tarragon; Blood glucose; Antidiabetic
Introduction The incidence of type-2 diabetes has increased globally to epidemic proportions, affecting approxi$ The STZ and KK Ag animal studies and the insulin and GLP-1 binding studies were performed by MDS Pharma Services. TarralinTM is a registered trademark of Degussa Food Ingredients GmbH. Corresponding author. Tel.: +732 932 8165x225; fax: +732 932 6535. E-mail address: [email protected] (D.M. Ribnicky).
0944-7113/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2005.09.007
mately 16 million persons in the US, and is projected to become more prevalent over the coming decade (National Diabetes Fact Sheet, 2003). Before the discovery of insulin in 1922, the only treatment options for diabetes were those based on traditional practices. Ethnobotanical knowledge played a particularly important role in historical diabetes therapies, with over 1200 species of medicinal plants recognized throughout the world for their ability to treat diabetic indications (Marles and Farnsworth, 1995). Recently, attention to natural products has increased once again, but there is a
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need for thoroughly controlled studies on the effectiveness and potential risks of treatment with such products. The plant genus Artemisia, comprised of over 1500 diverse species, has been a rich source of herbal remedies and conventional drugs (Tan et al., 1998; Watson et al., 2002). Diverse medicinal applications, often anecdotally ascribed, exist for a variety of species of the genus Artemisia. For example, artemisinin from Artemisia annua (annual wormwood) is now being developed as a commercial drug for the treatment of malaria (Mueller et al., 2000). Some reports suggest that extracts of other Artemisia species could be beneficial to people with diabetes. Tea made from Artemisia herba-alba has been used in Iraq for treating diabetes, with no reported side effects (AlWaili Dawood, 1986). Artemisia pallens was used as a folk remedy for diabetes in southern India, and alcoholic extracts of this species were shown to lower blood glucose concentrations in diabetic rats (Subramonium et al., 1996). In Turkish folk medicine, Artemisia santonicum has been used for the treatment of diabetes, and its ability to lower blood glucose was validated in diabetic rabbits (Korkmaz and Gurdal, 2002). An unspecified cultivar of tarragon was considered to be a traditional treatment for diabetes in the United Kingdom (Swanston-Flatt et al., 1989, 1991). In most cases, however, the physiological effects of Artemisia species have not been substantiated with significant scientific or clinical research. In this study, we evaluated the ability of an alcoholic extract of Russian tarragon (Artemisia dracunculus), referred to as TarralinTM, to decrease blood glucose concentrations in vivo, as well as potential mechanisms in vitro by which the extract may exert its effects.
Materials and methods Preparation of TarralinTM The extract was produced as described previously (Ribnicky et al., 2004). The seed for A. dracunculus L. was purchased from Sheffield’s Seed Co., Inc. (Locke, New York). The herb was cultivated hydroponically and harvested as the total herb above the root mass. The harvested herb was stored frozen at 20 1C. For extraction, 3 kg of frozen herb was heated with 15 l of 80% ethanol (v/v) to 80 1C for 2 h and allowed to continue to extract for an additional 10 hours at 20 1C. The extract was then filtered through cheesecloth to remove particulates. The extract was reduced and the ethanol removed by rotary evaporation to less than 1 l of final extract. The aqueous extract was then freeze-dried from 48 to 20 1C in a glass tray. The dried extract was scraped from the glass tray and homogenized using a mortar and pestle.
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Analysis of TarralinTM TarralinTM was dissolved in 60% ethanol (10 mg/ml), separated and analyzed using the Waters (Milford, MA) LC-MS IntegrityTM system consisting of a W616 pump and W600S controller, W717plus auto-sampler, W996 PDA detector and Waters TMD ThermabeamTM electron impact single quadrupole mass detector. Data were collected and analyzed with Waters Millenniums v. 3.2 software, linked with the 6th edition of the Wiley Registry of Mass Spectral Data. Substances were separated on a Phenomenexs Luna C-8 reverse phase column, size 150 2 mm, particle size 3 mm, pore size ( equipped with a Phenomenexs SecurityGuardTM 100 A, pre-column. The mobile phase consisted of 2 components: Solvent A (0.5% ACS grade acetic acid in double distilled, de-ionized water, pH 3–3.5), and Solvent B (100% acetonitrile). The mobile phase flow was adjusted at 0.25 ml/min with a gradient from 5% B to 95% B over 35 min.
In vivo assessment To assess biological activity, TarralinTM was tested for its ability to modulate blood glucose concentrations in mice models of type-1 diabetes (STZ-treated) and type-2 diabetes (KK-Ag), and in non-diabetic ICR mice.
STZ-induced diabetes Groups of 5 ICR-derived mice weighing approximately 22 g had diabetes induced with the intravenous administration of STZ (Sigma, USA) at 160 mg/kg body wt. After 48 h and confirmation of the diabetic state by measuring blood glucose levels and glucosuria, TarralinTM was administered to mice once daily by gavage for 7 consecutive days at a dose of 500 mg/kg body wt. in a vehicle of 2% Tween 80 (Wako, Japan) in distilled water with a final volume of 20 ml/kg. As a positive control, a single dose of insulin (1 IU/kg) (Sigma, USA) was administered subcutaneously to a group of animals on the day corresponding to the seventh day of treatment with TarralinTM. Serum glucose was determined using enzymatic methods (Sigma, St Louis) from orbital sinus blood samples obtained 5 min before dosing with TarralinTM on the first day and 90 min after the administration of the final dose in the fasting state. The control group received the vehicle alone by gavage. The animals were maintained in a controlled environment (20–24 1C, 60–80% humidity) with a 12 h light/ dark cycle and fed standard lab chow for mice (Lab Diet Rodent Diet, PMI Nutrition International, USA) and water ad libitum.
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Spontaneous diabetes in KK-Ac mice Groups of 4, type-2 diabetic male mice (KK-Ag/ TaJcl) weighing approximately 50 g each (12–14 weeks of age) were treated orally with TarralinTM by gavage once daily for 7 days at a dose of 500 mg/kg body wt. in a vehicle of 0.9% NaCl, similar to methods described elsewhere (Johnson et al., 1993). As a positive control, metformin (300 mg/kg body wt./day) and troglitazone (30 mg/kg body wt./day) were similarly administered to separate groups of 4 mice each. Serum blood glucose, insulin and body weight were measured on day 0, 5 min before treatment, and days 3 and 7, 90 min after treatment. Glucose concentrations were determined as described above. Insulin measurements were made using an ELISA rat insulin enzyme immunoassay kit (SPIBIO, France). Non-diabetic ICR mice Groups of 4 male, ICR-derived mice weighing approximately 22 g each received the same treatment as described above for the KK-Ag mice. Each of the animal studies described above was performed by MDS Pharma Services (Bothell, WA) in general accordance with the International Guiding Principles for Biomedical Research Involving Animals.
Regulation of hepatic PEPCK mRNA expression by TarralinTM Eight adult male Sprague-Dawley rats (Taconic Farms, Germantown NY) received injections of STZ (75 mg/kg body wt.) via a tail vein and 8 were used as non-diabetic controls. Hyperglycemia was confirmed by the presence of glucosuria. After 7 days, the diabetic rats were assigned to 2 of 4 groups. Groups 1 and 2 received water and groups 3 and 4 received TarralinTM (1000 mg/ kg body wt.) by gavage daily for 7 days. The rats had free access to standard rat chow for the duration of the study. On the 14th day, the animals were killed by CO2 inhalation 24 h after the final dose. Livers were removed and RNA extracted according to a method reported previously (Chomzcynski and Sacchi, 1987). The abundance of phosphoenolpyruvate carboxykinase (PEPCK) mRNA was assessed by northern blotting using pck 20 cDNA as a probe (Yoo-Warren et al., 1983) with correction for differential loading of the gels by normalization to the 18S ethidiumbromide signal (Watford and Smith, 1990). This study was approved by the Animal Care and Facilities Committee of the Office of Research and Sponsored Programs at Rutgers University. These experiments were carried out at the Cook Animal Facility (Cook College, Rutgers University), an AAALAC International-accredited facility. Animals were cared for in accordance with public health guidelines for the care and use of laboratory animals.
Modulation of GLP-1 binding To evaluate the potential mechanism of action of the extract, the modulation of the glucagon-like peptide (GLP-1) (7–36 amide) binding to its human GLP-1 receptor was measured by an in vitro method described previously (Fehmann et al., 1995). CHO-K1 cells stably transfected with a plasmid encoding the human GLP-1 receptor were used to prepare membranes in modified Tris HCl pH 7.4 buffer. A 14-mg aliquot of membrane was incubated with 0.03 nM [125I]GLP (7–36) for 90 min at 37 1C. Non-specific binding was estimated in the presence of 0.1 M GLP (7–36) amide. Membranes were filtered and washed 3 times and the filters were counted to determine [125I]GLP (7–36) amide specifically bound. TarralinTM was used at 1000 and 100 mg/ml and each measurement was made in duplicate.
Results HPLC-MS analysis of TarralinTM TarralinTM was analyzed by LC-MS (Fig. 1) as a means to characterize the extract as well as to provide a basis for standardization of the extract. The extract was standardized on the basis of the peak areas from both the total ion current electron impact MS chromatograms and the PDA chromatograms measured at 254 nm (Fig. 1). These peaks are consistent between the batches of extract produced. Qualitative analysis of the peaks in the MS chromatograms by mass spectral
Fig. 1. UV and MS chromatographic profiles of TarralinTM. Panel A is an HPLC chromatogram measured by photodiodearray detection at 254 nm. Panel B is a total-ion current chromatogram as measured by electron impact mass spectrometry following separation by HPLC.
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Table 1.
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Activity of TarralinTM in hyperglycemic STZ-induced diabetic ICR mice after 7 days of oral feeding
Treatment
Vehicle TarralinTM Insulin
Dose
20 ml/kg/body wt./day 500 mg/kg body wt./day 1 IU/kg body wt.
N
5 5 5
Serum glucosea Pre-treated (mg/dl)
Post-treated (mg/dl)
Average % of 0 time
% Reduction relative to vehicle
460726 429741 428741
496738 376758 270751
107 86 62
— 20b 42b
A positive control insulin treatment was given only on day 7. a Measurements made 90 min after final treatment; values7s.e.m. b Considered significant at po0:05 relative to control (Student’s t test).
matching with known mass spectra from commercially available databases revealed that the constituents of the extract consisted primarily of flavonoids, sesquiterpene lactones and coumarin derivatives, for which the plant is well known.
Activity of TarralinTM in vivo TarralinTM activity in STZ-mice The efficacy of TarralinTM to ameliorate hyperglycemia was initially evaluated in STZ-induced diabetic mice (Table 1). Orally administered (gavage) TarralinTM decreased blood glucose concentrations in STZ-treated mice by 20%, which was almost half the activity promoted by subcutaneously injected insulin (42%). TarralinTM activity in KK-Ac diabetic mice TarralinTM significantly reduced blood glucose concentrations in genetically diabetic KK-Ag mice, by 24%, whereas the hypoglycemic drugs, troglitazone and metformin, reduced blood glucose levels by 28% and 41%, respectively (Fig. 2). In addition to lowering blood glucose concentrations, TarralinTM treatment reduced hyperinsulinemia in the KK-Ag mice by 33%, while the oral hypoglycemic drugs troglitazone and metformin decreased blood insulin concentrations by 48% and 52%, respectively (Fig. 2). No significant differences in body weight change were observed between any of the treatment groups (data not shown). TarralinTM activity in non-diabetic ICR mice The potential of TarralinTM to cause hypoglycemia was investigated in non-diabetic ICR mice. Following 7 days of treatment, TarralinTM did not significantly affect blood glucose or insulin concentrations in the non-diabetic animals (Fig. 3). The decrease in blood glucose concentration in the TarralinTM-treated mice was not statistically or biologically significant, while blood glucose concentration of control mice remain unchanged. The decreases in blood insulin concentra-
Fig. 2. Blood glucose and insulin concentrations in KK-Ag mice (n ¼ 4) over 7 days of treatment with TarralinTM (500 mg/kg body wt./day, long dashed line with squares), troglitazone (30 mg/kg body wt./day, short dashed and dotted line with triangles) and metformin (300 mg/kg body wt./day, long dashed and dotted line with Xs) compared to vehicletreated control mice (solid line with diamonds). The decrease in blood glucose and insulin concentrations in each of the treatment groups was significant at po0:05, po0:01 relative to control animals (Student’s t test).
tions in both the untreated control and test mice were within the normal range of variation for those mice. Effect of TarralinTM on hepatic PEPCK expression PEPCK is the rate-limiting enzyme of gluconeogenesis, the metabolic pathway leading to the release of glucose from the liver (Hanson and Reshef, 1997). It is commonly accepted that the expression of PEPCK is used as an indicator of hepatic glucose output (Hanson
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Activity of TarralinTM in vitro GLP-1 binding to the GLP-1 receptor Several reports indicate that GLP-1 may be effective for the treatment of diabetes and GLP-1 receptor agonists are considered leads for antidiabetic drugs (Gutniak et al., 1992, 1996). Thus, we have evaluated the ability of TarralinTM to influence GLP-1 binding to its receptor in vitro. TarralinTM enhanced the binding of GLP-1 by 74% at 1000 mg/ml and by 45% at 100 mg/ml. This dose-dependent modulation of GLP-1 binding to its receptor may suggest an additional mechanism by which TarralinTM decreased blood glucose in diabetic animals.
Discussion
Fig. 3. Blood-glucose and insulin concentrations in nondiabetic ICR mice (n ¼ 4) over 7 days of treatment with TarralinTM (500 mg/kg body wt./day, solid line with diamonds) compared to untreated control mice (long dashed line with diamonds). Animals were fasted overnight prior to blood sampling. Any differences in blood glucose or insulin concentrations within each of the groups were not considered significant.
Table 2. The effect of TarralinTM on PEPCK mRNA expression in liver tissue from STZ-induced and control male Sprague-Dawley rats (n ¼ 4) gavaged with either TarralinTM (1000 mg/kg body wt.) or vehicle Animal
Treatment
PEPCK mRNA abundancea
STZ diabetic rats
TarralinTM Vehicle
1064371177b 1473171722b
Control rats
TarralinTM Vehicle
44687729 44217797
a Values are arbitrary units adjusted for equal loading, results are means7s.e.m. b Significant at po0:05 relative to controls (Student’s t test).
and Patel, 1994; Chan et al., 2003). PEPCK mRNA levels were elevated nearly 3-fold in diabetic animals, as compared to non-diabetic animals, but were significantly lower in diabetic animals treated with TarralinTM (Table 2). TarralinTM did not, however, affect PEPCK expression in non-diabetic animals.
The plant genus Artemisia is comprised of numerous diverse species, many of which are medicinal plants used to alleviate human conditions, including diabetes (Tan et al., 1998; Mueller et al., 2000; Al-Waili Dawood, 1986; Subramonium et al., 1996). Based on earlier sporadic reports of the hypoglycemic activity of some Artemisia species (Al-Waili Dawood, 1986; Subramonium et al., 1996; Korkmaz and Gurdal, 2002), we evaluated an extract of A. dracunculus (Russian tarragon) as a potential food ingredient (dietary supplement) for the management of hyperglycemia. TarralinTM significantly lowered blood glucose in STZ-induced diabetic mice that experienced hyperglycemia as a result of diminished or impaired insulin production (Table 1). Because TarralinTM was active in animals that were insulin-deficient and unable to produce insulin, it is not likely that its mode of action involved the stimulation of insulin production, like the sulfonylurea-type drugs. Therefore, the ability of TarralinTM to lower blood glucose concentrations in STZdiabetic mice was likely based on a mechanism that promoted an insulin effect in peripheral tissues (i.e., liver, muscle or adipose), independently of insulin. Alternatively, TarralinTM may have caused an insulinsparing effect if the animals were not completely insulindeficient. TarralinTM was also effective at lowering blood glucose concentrations in genetically diabetic KK-Ag mice that spontaneously develop diabetes as they age and are characterized by high blood-glucose and insulin concentrations indicative of insulin resistance (Suto et al., 1998; Shimura et al., 1997). TarralinTM was tested in this animal model at 500 mg/kg body wt. while troglitazone was tested at 30 mg/kg body wt. and metformin at 300 mg/kg body wt. (Fig. 2). In the KKAg mice, troglitazone showed the highest activity based on dose, but due to the severe hepatotoxicity that it
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causes, it has been removed from the market. The moderate but significant reduction of blood-glucose and insulin levels caused by TarralinTM suggests its safe and effective use as nutritional support for blood-glucose management. TarralinTM exhibited 2 distinct activities, as it was able to lower blood glucose concentrations in insulin resistant animals and in animals lacking insulin. The TarralinTM-induced decrease in the insulin concentration of the KK-Ag mice likely resulted from an increase in the effectiveness of the insulin (decrease in insulin resistance), thereby enhancing glucose uptake into peripheral tissues (i.e., muscle or adipose) and decreasing glucose output. This suggests potentially multiple modes of action for TarralinTM, as is also known for some drugs such as troglitazone and metformin, which increase insulin sensitivity rather than insulin concentration (Stumvoll and Haring, 2002). Significant reductions in insulin and/or glucose concentrations in normal animals, however, could cause hypoglycemia, a common side effect of diabetic therapy (Davidson, 1998). It is therefore particularly noteworthy that TarralinTM did not significantly affect bloodglucose levels or insulin concentrations in normal ICR mice (Fig. 3). This observation was confirmed in a 90day subchronic toxicity evaluation of TarralinTM in rats gavaged with daily doses as high as 1000 mg/kg body wt. (Ribnicky et al., 2004). TarralinTM appears to be a modulator of blood glucose and insulin concentrations only in diabetic animals. PEPCK is a rate-controlling enzyme of gluconeogenesis in the liver and plays a key role in the process of glucose homeostasis (Hanson and Reshef, 1997). The expression of PEPCK mRNA is used as an indicator of hepatic glucose output (Hanson and Patel, 1994; Chan et al., 2003) since PEPCK activity is regulated at the level of gene transcription (Hanson and Reshef, 1997). Interventions into the signaling events that regulate the expression of PEPCK (key gluconeogenic enzyme) are regarded as a potential strategy for the improvement of hyperglycemia (Barthel and Schmoll, 2003). PEPCK expression in STZ diabetic animals was decreased significantly by TarralinTM treatment (Table 2). PEPCK mRNA levels are enhanced in STZ diabetic animals because they lack the insulin signaling cascade that serves as the cue to decrease PEPCK expression. In addition, elevated PEPCK expression is associated with type-2 diabetes since it causes enhanced glucose release from the liver and may be a leading cause of insulin resistance (Valera et al., 1994). Since TarralinTM effectively decreases PEPCK expression in diabetic animals, it may decrease hepatic glucose output as a mechanism to alleviate hyperglycemia. TarralinTM application did not, however, have any effect on PEPCK expression in non-diabetic animals. TarralinTM,
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therefore, had an insulin-like effect on PEPCK expression, selectively, in the diabetic animals. In order to further understand the mechanisms by which TarralinTM lowered blood-glucose concentrations in animal models, we studied additional effects of this plant extract in vitro. The fact that TarralinTM did not interfere with insulin binding to its receptor (data not shown) suggests that no structural similarities between compounds of the extract and insulin-related peptides exist. On the other hand, hormone–receptor interactions can also be modulated by mechanisms not requiring structural similarity of the interfering active substance as observed by the effects of TarralinTM on GLP-1 binding to its receptor. As tested in vitro, TarralinTM enhanced GLP-1 binding to its receptor in a dose-dependent manner. GLP-1 is the transient signal released by the small intestine in response to the presence of digestion products, and its receptors are expressed in various tissues. GLP-1 has multiple functions including the enhancement of insulin secretion, stimulation of proinsulin gene expression and suppression of glucagon secretion and gastric emptying (Drucker, 1998). GLP-1 may also increase insulin sensitivity, as well as stimulate pancreatic b-cell proliferation (Sandhu et al., 1999; Wang et al., 1999). Thus, at least part of TarralinTM’s hypoglycemic effect may be explained by the stimulation of GLP-1 binding, thus potentiating/prolonging its physiological effect. The extract may contain several compounds that act as a GLP-1 binding modulator or GLP-1 stabilizer, which enhance its effectiveness. In conclusion, TarralinTM, an ethanolic extract of A. dracunculus L., decreases hyperglycemia in rodents with chemically induced diabetes, as well as in genetically diabetic rodents showing insulin resistance. Blood glucose concentrations, blood insulin levels and PEPCK expression in healthy animals were not effected. Thus, TarralinTM’s hypoglycemic effects are restricted to the diabetic state. The plant extract was found safe in a 90day subchronic feeding study in rats (Ribnicky et al., 2004). The observation that TarralinTM has several distinct modes of action useful for the improvement of complications associated with diabetes suggests the presence of more than 1 active component in the extract. This is likely because according to the literature for several compounds found in the aerial part of the plant in vitro or in vivo antidiabetic activities have been reported. The investigated include flavonoids, such as luteolin and apigenin which are known constituents of A. dracunculus (Duke, 1992) that have well documented antidiabetic activity (Matsuda et al., 1995; Asgary et al., 2002), coumarins such as scopoletin which is a known constituent of A. dracunculus (Duke, 1992) for which antidiabetic activity has been reported (Okada et al, 1995; Huang et al., 1993), sesquiterpenoid lactones, such as costunolide which is a known constituent of
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A. dracunculus (Duke, 1992) with antidiabetic activity, (Fang et al., 2005) and cinnamates, present in A. dracunculus (Duke, 1992) with reported antidiabetic activity (Arlt et al, 2004; Liu et al., 2003; Neogi et al., 2003). Whether some individual compounds may together have a synergistic effect for the antidiabetic activity, is currently under thorough investigation. Our data indicate that the beneficial effects of TarralinTM on blood glucose levels in rodent models of type-1 and type-2 diabetes, together with further studies and clinical development, may make TarralinTM a useful supplement for the management of blood glucose levels and insulin resistance.
Acknowledgments The authors wish to thank Drs. Ralf Ja¨ger and Heike tom Dieck for a critical reading of the manuscript and Cook College of Rutgers University, The Agricultural Experiment Station, Degussa Food Ingredients GmbH and Phytomedics, Inc., for funding this research. This work was also supported by the NIH Center for Dietary Supplements Research: Botanicals (Botanicals and Metabolic Syndrome).
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Swanston-Flatt, S.K., Day, C., Bailey, C.J., Flatt, P.R., 1989. Evaluation of traditional plant treatments for diabetes: studies in Streprozoticin diabetic mice. Acta Diabetol. Lat. 26, 51–55. Swanston-Flatt, S.K., Flatt, P.R., Day, C., Bailey, C.J., 1991. Traditional dietary adjuncts for the treatment of diabetes mellitus. Proc. Nutr. Soc. 50, 641–651. Tan, R.X., Zheng, W.F., Tang, H.Q., 1998. Biologically active substances from the genus Artemisia. Planta Med. 64 (4), 295–302. Valera, A., Pujol, A., Pelegrin, M., Bosch, F., 1994. Transgenic mice overexpressing phosphoenolpyruvate carboxykinase develop non-insulin-dependent diabetes mellitus. Proc. Natl. Acad. Sci. USA 91, 9151–9154. Wang, X., Cahill, C.M., Pineyro, M.A., Zhou, J., Doyle, M.E., Egan, J.M., 1999. Glucagon-like peptide-1 regulates the beta cell transcription factor, PDX-1, in insulinoma cells. Endocrinology 140 (10), 4904–4907. Watford, M., Smith, E.M., 1990. Distribution of hepatic glutaminase activity and mRNA in perivenous and periportal hepatocytes. Biochem. J. 267, 265–267. Watson, L.E., Bates, P.L., Evans, T.M., Unwin, M.M., Estes, J.R., 2002. Molecular phylogeny of Subtribe Artemisiinae (Asteraceae), including Artemisia and its allied and segregate genera. BMC Evol. Biol. 2, 17. Yoo-Warren, H., Monahan, J.E., Short, J., Short, H., Bruzel, A., Wynshaw-Boris, A., Meisner, H.M., Samols, D., Hanson, R.W., 1983. Isolation and characterization of the gene encoding for cytosolic phosphoenolpyruvate carboxykinase (GTP) from the rat. Proc. Natl. Acad. Sci. USA 80, 3656–3660.
Phytomedicine 13 (2006) 550–557 www.elsevier.de/phymed
Antihyperglycemic activity of TarralinTM, an ethanolic extract of Artemisia dracunculus L.$ D.M. Ribnickya,, A. Pouleva, M. Watfordb, W.T. Cefaluc, I. Raskina a
Biotech Center, Cook College, Rutgers University, 59 Dudley Road, New Brunswick, NJ 08901-8520, USA Nutritional Sciences, Cook College, Rutgers University, 59 Dudley Road, New Brunswick, NJ 08901-8520, USA c Pennington Biomedical Research Center/Louisiana State University, 6400 Perkins Road, Baton Rouge, LA 70808, USA b
Abstract The studies reported here were undertaken to examine the antihyperglycemic activity of an ethanolic extract of Artemisia dracunculus L., called TarralinTM in diabetic and non-diabetic animals. In genetically diabetic KK-Ag mice, TarralinTM treatment by gavage (500 mg/kg body wt./day for 7 days) lowered elevated blood glucose levels by 24% from 479725 to 352716 mg/dl relative to control animals. In comparison, treatment with the known antidiabetic drugs, troglitazone (30 mg/kg body wt./day) and metformin (300 mg/kg body wt./day), decreased blood glucose concentrations by 28% and 41%, respectively. Blood insulin concentrations were reduced in the KK-Ag mice by 33% with TarralinTM, 48% with troglitazone and 52% with metformin. In (STZ)-induced diabetic mice, TarralinTM treatment, (500 mg/kg body wt./day for 7 days), also significantly lowered blood glucose concentrations, by 20%, from 429741 to 376758 mg/dl relative to control. As a possible mechanism, TarralinTM was shown to significantly decrease phosphoenolpyruvate carboxykinase (PEPCK) mRNA expression by 28% in STZ-induced diabetic rats. In nondiabetic animals, treatment with TarralinTM did not significantly alter PEPCK expression, blood glucose or insulin concentrations. The extract was also shown to increase the binding of glucagon-like peptide (GLP-1) to its receptor in vitro. These results indicate that TarralinTM has antihyperglycemic activity and a potential role in the management of diabetic states. r 2005 Elsevier GmbH. All rights reserved. Keywords: Artemisia dracunculus; Diabetes; Hyperglycemia; Hypoglycemia; GLP-1; PEPCK; Russian tarragon; Blood glucose; Antidiabetic
Introduction The incidence of type-2 diabetes has increased globally to epidemic proportions, affecting approxi$ The STZ and KK Ag animal studies and the insulin and GLP-1 binding studies were performed by MDS Pharma Services. TarralinTM is a registered trademark of Degussa Food Ingredients GmbH. Corresponding author. Tel.: +732 932 8165x225; fax: +732 932 6535. E-mail address: [email protected] (D.M. Ribnicky).
0944-7113/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2005.09.007
mately 16 million persons in the US, and is projected to become more prevalent over the coming decade (National Diabetes Fact Sheet, 2003). Before the discovery of insulin in 1922, the only treatment options for diabetes were those based on traditional practices. Ethnobotanical knowledge played a particularly important role in historical diabetes therapies, with over 1200 species of medicinal plants recognized throughout the world for their ability to treat diabetic indications (Marles and Farnsworth, 1995). Recently, attention to natural products has increased once again, but there is a
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need for thoroughly controlled studies on the effectiveness and potential risks of treatment with such products. The plant genus Artemisia, comprised of over 1500 diverse species, has been a rich source of herbal remedies and conventional drugs (Tan et al., 1998; Watson et al., 2002). Diverse medicinal applications, often anecdotally ascribed, exist for a variety of species of the genus Artemisia. For example, artemisinin from Artemisia annua (annual wormwood) is now being developed as a commercial drug for the treatment of malaria (Mueller et al., 2000). Some reports suggest that extracts of other Artemisia species could be beneficial to people with diabetes. Tea made from Artemisia herba-alba has been used in Iraq for treating diabetes, with no reported side effects (AlWaili Dawood, 1986). Artemisia pallens was used as a folk remedy for diabetes in southern India, and alcoholic extracts of this species were shown to lower blood glucose concentrations in diabetic rats (Subramonium et al., 1996). In Turkish folk medicine, Artemisia santonicum has been used for the treatment of diabetes, and its ability to lower blood glucose was validated in diabetic rabbits (Korkmaz and Gurdal, 2002). An unspecified cultivar of tarragon was considered to be a traditional treatment for diabetes in the United Kingdom (Swanston-Flatt et al., 1989, 1991). In most cases, however, the physiological effects of Artemisia species have not been substantiated with significant scientific or clinical research. In this study, we evaluated the ability of an alcoholic extract of Russian tarragon (Artemisia dracunculus), referred to as TarralinTM, to decrease blood glucose concentrations in vivo, as well as potential mechanisms in vitro by which the extract may exert its effects.
Materials and methods Preparation of TarralinTM The extract was produced as described previously (Ribnicky et al., 2004). The seed for A. dracunculus L. was purchased from Sheffield’s Seed Co., Inc. (Locke, New York). The herb was cultivated hydroponically and harvested as the total herb above the root mass. The harvested herb was stored frozen at 20 1C. For extraction, 3 kg of frozen herb was heated with 15 l of 80% ethanol (v/v) to 80 1C for 2 h and allowed to continue to extract for an additional 10 hours at 20 1C. The extract was then filtered through cheesecloth to remove particulates. The extract was reduced and the ethanol removed by rotary evaporation to less than 1 l of final extract. The aqueous extract was then freeze-dried from 48 to 20 1C in a glass tray. The dried extract was scraped from the glass tray and homogenized using a mortar and pestle.
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Analysis of TarralinTM TarralinTM was dissolved in 60% ethanol (10 mg/ml), separated and analyzed using the Waters (Milford, MA) LC-MS IntegrityTM system consisting of a W616 pump and W600S controller, W717plus auto-sampler, W996 PDA detector and Waters TMD ThermabeamTM electron impact single quadrupole mass detector. Data were collected and analyzed with Waters Millenniums v. 3.2 software, linked with the 6th edition of the Wiley Registry of Mass Spectral Data. Substances were separated on a Phenomenexs Luna C-8 reverse phase column, size 150 2 mm, particle size 3 mm, pore size ( equipped with a Phenomenexs SecurityGuardTM 100 A, pre-column. The mobile phase consisted of 2 components: Solvent A (0.5% ACS grade acetic acid in double distilled, de-ionized water, pH 3–3.5), and Solvent B (100% acetonitrile). The mobile phase flow was adjusted at 0.25 ml/min with a gradient from 5% B to 95% B over 35 min.
In vivo assessment To assess biological activity, TarralinTM was tested for its ability to modulate blood glucose concentrations in mice models of type-1 diabetes (STZ-treated) and type-2 diabetes (KK-Ag), and in non-diabetic ICR mice.
STZ-induced diabetes Groups of 5 ICR-derived mice weighing approximately 22 g had diabetes induced with the intravenous administration of STZ (Sigma, USA) at 160 mg/kg body wt. After 48 h and confirmation of the diabetic state by measuring blood glucose levels and glucosuria, TarralinTM was administered to mice once daily by gavage for 7 consecutive days at a dose of 500 mg/kg body wt. in a vehicle of 2% Tween 80 (Wako, Japan) in distilled water with a final volume of 20 ml/kg. As a positive control, a single dose of insulin (1 IU/kg) (Sigma, USA) was administered subcutaneously to a group of animals on the day corresponding to the seventh day of treatment with TarralinTM. Serum glucose was determined using enzymatic methods (Sigma, St Louis) from orbital sinus blood samples obtained 5 min before dosing with TarralinTM on the first day and 90 min after the administration of the final dose in the fasting state. The control group received the vehicle alone by gavage. The animals were maintained in a controlled environment (20–24 1C, 60–80% humidity) with a 12 h light/ dark cycle and fed standard lab chow for mice (Lab Diet Rodent Diet, PMI Nutrition International, USA) and water ad libitum.
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Spontaneous diabetes in KK-Ac mice Groups of 4, type-2 diabetic male mice (KK-Ag/ TaJcl) weighing approximately 50 g each (12–14 weeks of age) were treated orally with TarralinTM by gavage once daily for 7 days at a dose of 500 mg/kg body wt. in a vehicle of 0.9% NaCl, similar to methods described elsewhere (Johnson et al., 1993). As a positive control, metformin (300 mg/kg body wt./day) and troglitazone (30 mg/kg body wt./day) were similarly administered to separate groups of 4 mice each. Serum blood glucose, insulin and body weight were measured on day 0, 5 min before treatment, and days 3 and 7, 90 min after treatment. Glucose concentrations were determined as described above. Insulin measurements were made using an ELISA rat insulin enzyme immunoassay kit (SPIBIO, France). Non-diabetic ICR mice Groups of 4 male, ICR-derived mice weighing approximately 22 g each received the same treatment as described above for the KK-Ag mice. Each of the animal studies described above was performed by MDS Pharma Services (Bothell, WA) in general accordance with the International Guiding Principles for Biomedical Research Involving Animals.
Regulation of hepatic PEPCK mRNA expression by TarralinTM Eight adult male Sprague-Dawley rats (Taconic Farms, Germantown NY) received injections of STZ (75 mg/kg body wt.) via a tail vein and 8 were used as non-diabetic controls. Hyperglycemia was confirmed by the presence of glucosuria. After 7 days, the diabetic rats were assigned to 2 of 4 groups. Groups 1 and 2 received water and groups 3 and 4 received TarralinTM (1000 mg/ kg body wt.) by gavage daily for 7 days. The rats had free access to standard rat chow for the duration of the study. On the 14th day, the animals were killed by CO2 inhalation 24 h after the final dose. Livers were removed and RNA extracted according to a method reported previously (Chomzcynski and Sacchi, 1987). The abundance of phosphoenolpyruvate carboxykinase (PEPCK) mRNA was assessed by northern blotting using pck 20 cDNA as a probe (Yoo-Warren et al., 1983) with correction for differential loading of the gels by normalization to the 18S ethidiumbromide signal (Watford and Smith, 1990). This study was approved by the Animal Care and Facilities Committee of the Office of Research and Sponsored Programs at Rutgers University. These experiments were carried out at the Cook Animal Facility (Cook College, Rutgers University), an AAALAC International-accredited facility. Animals were cared for in accordance with public health guidelines for the care and use of laboratory animals.
Modulation of GLP-1 binding To evaluate the potential mechanism of action of the extract, the modulation of the glucagon-like peptide (GLP-1) (7–36 amide) binding to its human GLP-1 receptor was measured by an in vitro method described previously (Fehmann et al., 1995). CHO-K1 cells stably transfected with a plasmid encoding the human GLP-1 receptor were used to prepare membranes in modified Tris HCl pH 7.4 buffer. A 14-mg aliquot of membrane was incubated with 0.03 nM [125I]GLP (7–36) for 90 min at 37 1C. Non-specific binding was estimated in the presence of 0.1 M GLP (7–36) amide. Membranes were filtered and washed 3 times and the filters were counted to determine [125I]GLP (7–36) amide specifically bound. TarralinTM was used at 1000 and 100 mg/ml and each measurement was made in duplicate.
Results HPLC-MS analysis of TarralinTM TarralinTM was analyzed by LC-MS (Fig. 1) as a means to characterize the extract as well as to provide a basis for standardization of the extract. The extract was standardized on the basis of the peak areas from both the total ion current electron impact MS chromatograms and the PDA chromatograms measured at 254 nm (Fig. 1). These peaks are consistent between the batches of extract produced. Qualitative analysis of the peaks in the MS chromatograms by mass spectral
Fig. 1. UV and MS chromatographic profiles of TarralinTM. Panel A is an HPLC chromatogram measured by photodiodearray detection at 254 nm. Panel B is a total-ion current chromatogram as measured by electron impact mass spectrometry following separation by HPLC.
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Table 1.
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Activity of TarralinTM in hyperglycemic STZ-induced diabetic ICR mice after 7 days of oral feeding
Treatment
Vehicle TarralinTM Insulin
Dose
20 ml/kg/body wt./day 500 mg/kg body wt./day 1 IU/kg body wt.
N
5 5 5
Serum glucosea Pre-treated (mg/dl)
Post-treated (mg/dl)
Average % of 0 time
% Reduction relative to vehicle
460726 429741 428741
496738 376758 270751
107 86 62
— 20b 42b
A positive control insulin treatment was given only on day 7. a Measurements made 90 min after final treatment; values7s.e.m. b Considered significant at po0:05 relative to control (Student’s t test).
matching with known mass spectra from commercially available databases revealed that the constituents of the extract consisted primarily of flavonoids, sesquiterpene lactones and coumarin derivatives, for which the plant is well known.
Activity of TarralinTM in vivo TarralinTM activity in STZ-mice The efficacy of TarralinTM to ameliorate hyperglycemia was initially evaluated in STZ-induced diabetic mice (Table 1). Orally administered (gavage) TarralinTM decreased blood glucose concentrations in STZ-treated mice by 20%, which was almost half the activity promoted by subcutaneously injected insulin (42%). TarralinTM activity in KK-Ac diabetic mice TarralinTM significantly reduced blood glucose concentrations in genetically diabetic KK-Ag mice, by 24%, whereas the hypoglycemic drugs, troglitazone and metformin, reduced blood glucose levels by 28% and 41%, respectively (Fig. 2). In addition to lowering blood glucose concentrations, TarralinTM treatment reduced hyperinsulinemia in the KK-Ag mice by 33%, while the oral hypoglycemic drugs troglitazone and metformin decreased blood insulin concentrations by 48% and 52%, respectively (Fig. 2). No significant differences in body weight change were observed between any of the treatment groups (data not shown). TarralinTM activity in non-diabetic ICR mice The potential of TarralinTM to cause hypoglycemia was investigated in non-diabetic ICR mice. Following 7 days of treatment, TarralinTM did not significantly affect blood glucose or insulin concentrations in the non-diabetic animals (Fig. 3). The decrease in blood glucose concentration in the TarralinTM-treated mice was not statistically or biologically significant, while blood glucose concentration of control mice remain unchanged. The decreases in blood insulin concentra-
Fig. 2. Blood glucose and insulin concentrations in KK-Ag mice (n ¼ 4) over 7 days of treatment with TarralinTM (500 mg/kg body wt./day, long dashed line with squares), troglitazone (30 mg/kg body wt./day, short dashed and dotted line with triangles) and metformin (300 mg/kg body wt./day, long dashed and dotted line with Xs) compared to vehicletreated control mice (solid line with diamonds). The decrease in blood glucose and insulin concentrations in each of the treatment groups was significant at po0:05, po0:01 relative to control animals (Student’s t test).
tions in both the untreated control and test mice were within the normal range of variation for those mice. Effect of TarralinTM on hepatic PEPCK expression PEPCK is the rate-limiting enzyme of gluconeogenesis, the metabolic pathway leading to the release of glucose from the liver (Hanson and Reshef, 1997). It is commonly accepted that the expression of PEPCK is used as an indicator of hepatic glucose output (Hanson
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Activity of TarralinTM in vitro GLP-1 binding to the GLP-1 receptor Several reports indicate that GLP-1 may be effective for the treatment of diabetes and GLP-1 receptor agonists are considered leads for antidiabetic drugs (Gutniak et al., 1992, 1996). Thus, we have evaluated the ability of TarralinTM to influence GLP-1 binding to its receptor in vitro. TarralinTM enhanced the binding of GLP-1 by 74% at 1000 mg/ml and by 45% at 100 mg/ml. This dose-dependent modulation of GLP-1 binding to its receptor may suggest an additional mechanism by which TarralinTM decreased blood glucose in diabetic animals.
Discussion
Fig. 3. Blood-glucose and insulin concentrations in nondiabetic ICR mice (n ¼ 4) over 7 days of treatment with TarralinTM (500 mg/kg body wt./day, solid line with diamonds) compared to untreated control mice (long dashed line with diamonds). Animals were fasted overnight prior to blood sampling. Any differences in blood glucose or insulin concentrations within each of the groups were not considered significant.
Table 2. The effect of TarralinTM on PEPCK mRNA expression in liver tissue from STZ-induced and control male Sprague-Dawley rats (n ¼ 4) gavaged with either TarralinTM (1000 mg/kg body wt.) or vehicle Animal
Treatment
PEPCK mRNA abundancea
STZ diabetic rats
TarralinTM Vehicle
1064371177b 1473171722b
Control rats
TarralinTM Vehicle
44687729 44217797
a Values are arbitrary units adjusted for equal loading, results are means7s.e.m. b Significant at po0:05 relative to controls (Student’s t test).
and Patel, 1994; Chan et al., 2003). PEPCK mRNA levels were elevated nearly 3-fold in diabetic animals, as compared to non-diabetic animals, but were significantly lower in diabetic animals treated with TarralinTM (Table 2). TarralinTM did not, however, affect PEPCK expression in non-diabetic animals.
The plant genus Artemisia is comprised of numerous diverse species, many of which are medicinal plants used to alleviate human conditions, including diabetes (Tan et al., 1998; Mueller et al., 2000; Al-Waili Dawood, 1986; Subramonium et al., 1996). Based on earlier sporadic reports of the hypoglycemic activity of some Artemisia species (Al-Waili Dawood, 1986; Subramonium et al., 1996; Korkmaz and Gurdal, 2002), we evaluated an extract of A. dracunculus (Russian tarragon) as a potential food ingredient (dietary supplement) for the management of hyperglycemia. TarralinTM significantly lowered blood glucose in STZ-induced diabetic mice that experienced hyperglycemia as a result of diminished or impaired insulin production (Table 1). Because TarralinTM was active in animals that were insulin-deficient and unable to produce insulin, it is not likely that its mode of action involved the stimulation of insulin production, like the sulfonylurea-type drugs. Therefore, the ability of TarralinTM to lower blood glucose concentrations in STZdiabetic mice was likely based on a mechanism that promoted an insulin effect in peripheral tissues (i.e., liver, muscle or adipose), independently of insulin. Alternatively, TarralinTM may have caused an insulinsparing effect if the animals were not completely insulindeficient. TarralinTM was also effective at lowering blood glucose concentrations in genetically diabetic KK-Ag mice that spontaneously develop diabetes as they age and are characterized by high blood-glucose and insulin concentrations indicative of insulin resistance (Suto et al., 1998; Shimura et al., 1997). TarralinTM was tested in this animal model at 500 mg/kg body wt. while troglitazone was tested at 30 mg/kg body wt. and metformin at 300 mg/kg body wt. (Fig. 2). In the KKAg mice, troglitazone showed the highest activity based on dose, but due to the severe hepatotoxicity that it
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causes, it has been removed from the market. The moderate but significant reduction of blood-glucose and insulin levels caused by TarralinTM suggests its safe and effective use as nutritional support for blood-glucose management. TarralinTM exhibited 2 distinct activities, as it was able to lower blood glucose concentrations in insulin resistant animals and in animals lacking insulin. The TarralinTM-induced decrease in the insulin concentration of the KK-Ag mice likely resulted from an increase in the effectiveness of the insulin (decrease in insulin resistance), thereby enhancing glucose uptake into peripheral tissues (i.e., muscle or adipose) and decreasing glucose output. This suggests potentially multiple modes of action for TarralinTM, as is also known for some drugs such as troglitazone and metformin, which increase insulin sensitivity rather than insulin concentration (Stumvoll and Haring, 2002). Significant reductions in insulin and/or glucose concentrations in normal animals, however, could cause hypoglycemia, a common side effect of diabetic therapy (Davidson, 1998). It is therefore particularly noteworthy that TarralinTM did not significantly affect bloodglucose levels or insulin concentrations in normal ICR mice (Fig. 3). This observation was confirmed in a 90day subchronic toxicity evaluation of TarralinTM in rats gavaged with daily doses as high as 1000 mg/kg body wt. (Ribnicky et al., 2004). TarralinTM appears to be a modulator of blood glucose and insulin concentrations only in diabetic animals. PEPCK is a rate-controlling enzyme of gluconeogenesis in the liver and plays a key role in the process of glucose homeostasis (Hanson and Reshef, 1997). The expression of PEPCK mRNA is used as an indicator of hepatic glucose output (Hanson and Patel, 1994; Chan et al., 2003) since PEPCK activity is regulated at the level of gene transcription (Hanson and Reshef, 1997). Interventions into the signaling events that regulate the expression of PEPCK (key gluconeogenic enzyme) are regarded as a potential strategy for the improvement of hyperglycemia (Barthel and Schmoll, 2003). PEPCK expression in STZ diabetic animals was decreased significantly by TarralinTM treatment (Table 2). PEPCK mRNA levels are enhanced in STZ diabetic animals because they lack the insulin signaling cascade that serves as the cue to decrease PEPCK expression. In addition, elevated PEPCK expression is associated with type-2 diabetes since it causes enhanced glucose release from the liver and may be a leading cause of insulin resistance (Valera et al., 1994). Since TarralinTM effectively decreases PEPCK expression in diabetic animals, it may decrease hepatic glucose output as a mechanism to alleviate hyperglycemia. TarralinTM application did not, however, have any effect on PEPCK expression in non-diabetic animals. TarralinTM,
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therefore, had an insulin-like effect on PEPCK expression, selectively, in the diabetic animals. In order to further understand the mechanisms by which TarralinTM lowered blood-glucose concentrations in animal models, we studied additional effects of this plant extract in vitro. The fact that TarralinTM did not interfere with insulin binding to its receptor (data not shown) suggests that no structural similarities between compounds of the extract and insulin-related peptides exist. On the other hand, hormone–receptor interactions can also be modulated by mechanisms not requiring structural similarity of the interfering active substance as observed by the effects of TarralinTM on GLP-1 binding to its receptor. As tested in vitro, TarralinTM enhanced GLP-1 binding to its receptor in a dose-dependent manner. GLP-1 is the transient signal released by the small intestine in response to the presence of digestion products, and its receptors are expressed in various tissues. GLP-1 has multiple functions including the enhancement of insulin secretion, stimulation of proinsulin gene expression and suppression of glucagon secretion and gastric emptying (Drucker, 1998). GLP-1 may also increase insulin sensitivity, as well as stimulate pancreatic b-cell proliferation (Sandhu et al., 1999; Wang et al., 1999). Thus, at least part of TarralinTM’s hypoglycemic effect may be explained by the stimulation of GLP-1 binding, thus potentiating/prolonging its physiological effect. The extract may contain several compounds that act as a GLP-1 binding modulator or GLP-1 stabilizer, which enhance its effectiveness. In conclusion, TarralinTM, an ethanolic extract of A. dracunculus L., decreases hyperglycemia in rodents with chemically induced diabetes, as well as in genetically diabetic rodents showing insulin resistance. Blood glucose concentrations, blood insulin levels and PEPCK expression in healthy animals were not effected. Thus, TarralinTM’s hypoglycemic effects are restricted to the diabetic state. The plant extract was found safe in a 90day subchronic feeding study in rats (Ribnicky et al., 2004). The observation that TarralinTM has several distinct modes of action useful for the improvement of complications associated with diabetes suggests the presence of more than 1 active component in the extract. This is likely because according to the literature for several compounds found in the aerial part of the plant in vitro or in vivo antidiabetic activities have been reported. The investigated include flavonoids, such as luteolin and apigenin which are known constituents of A. dracunculus (Duke, 1992) that have well documented antidiabetic activity (Matsuda et al., 1995; Asgary et al., 2002), coumarins such as scopoletin which is a known constituent of A. dracunculus (Duke, 1992) for which antidiabetic activity has been reported (Okada et al, 1995; Huang et al., 1993), sesquiterpenoid lactones, such as costunolide which is a known constituent of
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A. dracunculus (Duke, 1992) with antidiabetic activity, (Fang et al., 2005) and cinnamates, present in A. dracunculus (Duke, 1992) with reported antidiabetic activity (Arlt et al, 2004; Liu et al., 2003; Neogi et al., 2003). Whether some individual compounds may together have a synergistic effect for the antidiabetic activity, is currently under thorough investigation. Our data indicate that the beneficial effects of TarralinTM on blood glucose levels in rodent models of type-1 and type-2 diabetes, together with further studies and clinical development, may make TarralinTM a useful supplement for the management of blood glucose levels and insulin resistance.
Acknowledgments The authors wish to thank Drs. Ralf Ja¨ger and Heike tom Dieck for a critical reading of the manuscript and Cook College of Rutgers University, The Agricultural Experiment Station, Degussa Food Ingredients GmbH and Phytomedics, Inc., for funding this research. This work was also supported by the NIH Center for Dietary Supplements Research: Botanicals (Botanicals and Metabolic Syndrome).
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