Elsevier

Nutrition

Volume 30, Issues 7–8, Supplement, July–August 2014, Pages S4-S10
Nutrition

Methods development
Artemisia dracunculus L. polyphenols complexed to soy protein show enhanced bioavailability and hypoglycemic activity in C57BL/6 mice

https://doi.org/10.1016/j.nut.2014.03.009Get rights and content

Abstract

Objective

Scientifically validated food-based interventions are a practical means of addressing the epidemic of metabolic syndrome. An ethanolic extract of Artemisia dracunculus L. (PMI-5011) containing bioactive polyphenols, such as 2′, 4′–dihydroxy-4-methoxydihydrochalcone (DMC-2), improved insulin resistance in vitro and in vivo. Plant polyphenols are concentrated and stabilized when complexed to protein-rich matrices, such as soy protein isolate (SPI), which act as effective food-based delivery vehicles. The aim of this study was to compare the bioaccessibility, bioavailability, and efficacy of polyphenols extracted from A. dracunculus and delivered as PMI-5011 (ethanolic extract alone), formulated with the non-food excipient Gelucire®, (5011- Gelucire), or sorbed to SPI (5011-Nutrasorb®).

Methods

PMI-5011, 5011-Gelucire or 5011-Nutrasorb each containing 162 μg of DMC-2 was delivered to the TNO intestinal model-1 of the human upper gastrointestinal tract to compare the effect of delivery vehicle on DMC-2 bioaccessibility. C57BL6/J mice were orally administered 5011-Nutrasorb or PMI-5011 to compare effects of polyphenol–protein complexation on acute hypoglycemic activity and bioavailability of DMC-2 in serum.

Results

At 500 mg/kg, 5011-Nutrasorb and PMI-5011 had similar hypoglycemic activity in a high-fat diet-induced diabetes mouse model despite the fact that 5011-Nutrasorb delivered 15 times less DMC-2 (40 versus 600 μg/kg). This can be partially explained by eight times greater DMC-2 absorption into serum from 5011-Nutrasorb than from PMI-5011. TNO intestinal model-1 experiments confirmed higher total bioaccessibility of DMC-2 in vitro when delivered in 5011-Nutrasorb (50.2%) or Gelucire-5011 (44.4%) compared with PMI-5011 (27.1%; P = 0.08).

Conclusion

Complexation with soy protein makes antidiabetic A. dracunculus polyphenols more bioavailable and bioaccessible.

Introduction

Polyphenols found in plant-derived foods have demonstrated therapeutic activities in chronic disorders, such as metabolic syndrome and type 2 diabetes [1], [2], [3]. Artemisia dracunculus L. (commonly called Russian tarragon) is a perennial herb with a long history of culinary and medicinal uses [4]. An ethanolic extract of A. dracunculus (called PMI-5011) has been evaluated for its antidiabetic properties. PMI-5011 enhanced insulin receptor signaling in skeletal muscle of KK-A(y) mice [5] and primary human skeletal muscle culture [6], reduced blood glucose in diabetic rodent models, and decreased PEPCK gene expression in rat muscle tissue, a key regulator of hepatic gluconeogenesis [7]. Bioactivity-guided fractionation of PMI-5011 led to isolation of six polyphenols that contribute to the hypoglycemic activity observed in vivo [8], [9], [10]. Evidence indicate that chalcone 2′, 4′-dihydroxy-4-methoxydihydrochalcone (DMC-2; Fig. 1), the most abundant polyphenol in PMI-5011, is also its main active component, at least partially responsible for down-regulation of PEPCK gene expression and decreased glucose production in H4IIE hepatocytes [10].

Many bioactive plant polyphenols are poorly bioavailable because of low absorption and/or rapid metabolism and elimination, which can limit their therapeutic activity [11]. Lipid-based excipients are used to improve dissolution and enhance bioavailability of orally administered compounds with poor water solubility and absorption, often resulting in superior efficacy [12], [13]. Examples include Labrasol®, approved for drug formulations, and Gelucire® 44/14, approved for drug and dietary supplement formulations [14], [15], [16], [17]. Oral administration of PMI-5011 formulated with Labrasol had significantly better hypoglycemic activity in high-fat diet (HFD)-fed mice compared with PMI-5011 alone [18]. Furthermore, compounds in PMI-5011 formulated with Labrasol showed increased bioavailability in serum, comparable to gavage with pure DMC-2 [18]. These bioavailability-enhancing excipients are, however, not approved or compatible for use in foods.

A food-compatible method of improving bioavailability of dietary polyphenols would be useful for developing scientifically based and efficacious functional foods. Nutrasorb technology leverages the natural affinity of polyphenols for protein [19], and uses edible protein-based matrices, such as soy protein isolate (SPI) or soy flour, to naturally sorb, concentrate, and stabilize polyphenols contained in plant extracts while excluding excess water, sugar, and lipid components [20]. Juiced or extracted plant materials, including fruits, spices, and pomace, have been used as a source of polyphenols, such as anthocyanins and proanthocyanidins, to produce polyphenol–protein complexes that have antidiabetic, antibacterial, and antienzymatic activities [20], [21], [22], [23], [24], [25], [26]. In a recent clinical trial, metabolomic profiling of blood serum collected from athletes who consumed blueberry and green tea polyphenols complexed to SPI for 17 d, rather than SPI alone, showed elevated 3-hydroxybutyrate and acetoacetate indicative of enhanced ketogenesis and fatty acid oxidation during recovery from 3 d of exercise [27].

The TNO intestinal model-1 (TIM-1) of the human upper gastrointestinal (GI) tract simulates the in vivo conditions and kinetic events of the stomach and duodenum, jejunum and ileum compartments of the small intestine [28]. TIM-1 provides information about nutrient/compound transit, release, stability, and availability for intestinal absorption or bioaccessibility. Bioaccessibility is defined as the amount of compound (<5 kDa) released from a food matrix that can pass through membranes of the jejunal and ileal compartments of the TIM-1 system, which is an indicator of intestinal absorption or bioavailability in vivo. The ileal efflux represents what would theoretically be delivered to the colon for further metabolism. TIM-1 coupled with colorimetric quantification of total monomeric anthocyanins was used to compare intestinal absorption/bioaccessibility of blueberry anthocyanins from blueberry juice or blueberry polyphenol-enriched soy flour [29]. Compared with blueberry juice, ileal efflux samples collected after TIM-1 digestion of blueberry polyphenol–soy flour complex contained 2.8-fold more anthocyanins suggesting that the soy flour matrix protected anthocyanins during transit through upper digestive tract for subsequent colonic delivery and metabolism [29]. Although TIM-1 is useful for the preliminary assessment of different formulations or food matrices on compound bioaccessibility, in vivo systems must be used to establish whether improved bioavailability of compounds results in enhanced efficacy. This study evaluated the hypoglycemic activity and bioavailability (in C57BL/6 diet-induced diabetic mice) and bioaccessibility (in TIM model) of three different formulations of DMC-2 (PMI-5011, Gelucire-5011, and 5011-Nutrasorb). For all three measured end points, 5011-Nutrasorb provided the most effective formulation for delivering pharmacologically active doses of DMC-2.

Section snippets

Chemicals

Pepsin A from porcine stomach mucosa (2500–3500 units/mg, P-7012), trypsin from bovine pancreas (7500 N-α-benzoyl-l-arginine ethyl ester (BAEE) units/mg, T9201), and α-amylase type II-A from Bacillus species (1333 units/mg A-6380) were obtained from Sigma-Aldrich (Stockholm, Sweden). Fresh pig bile was obtained from TNO Zeist, Netherlands. Rhizopus lipase (150 000 units/mg F-AP-15) was from Amano Enzyme Inc. (Nagoya, Japan). Samples of Labrasol® and Gelucire® 44/14 were provided by Gattefossé

Biochemical profile of PMI-5011 and 5011-Nutrasorb

LC-MS analysis showed that 5011-Nutrasorb contained the same bioactive compounds previously identified in PMI-5011 [6], [9], [10], [18]; however, their abundance was 7 to 15 times lower in 5011-Nutrasorb (Fig. 1). The A. dracunculus compounds have limited water solubility, therefore less are extracted in water compared with ethanol. Based on previous stoichiometry experiments [20], the amount of extracted A. dracunculus polyphenols sorbed to the SPI matrix depends on the concentration of

Discussion

Plants used for food contain an abundance of therapeutically active, but poorly bioavailable polyphenols [31]. Furthermore, dietary polyphenols often coexist with excess sugars, oils, fibers, and water that can dilute and/or counteract their health benefits [8], [32]. Although PMI-5011 provides concentrated delivery of DMC-2 and other polyphenols from A. dracunculus, it has low water solubility. PMI-5011 has a tarlike consistency and formulation with lipid-based excipients, such as Gelucire or

Conclusions

We suggest that polyphenol–protein complexes, such as 5011-Nutrasorb, provide a food-compatible solution for improving the bioavailability and efficacy of dietary polyphenols, such as DMC-2, and may be useful in formulating health-promoting functional foods.

Acknowledgments

The authors acknowledge Rob Havenaar, Susann Bellman, Mans Minekus, and Mark Jelier from TNO (Zeist, Netherlands) for providing the TIM-1 unit and support, Gattefossé (Paramus, NJ, USA) for providing Labrasol and Gelucire, and Archer Daniels Midland Company (Decatur, IL, USA) for supplying soy protein isolate.

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This project was supported by P50AT002776-01 from the National Center for Complementary and Alternative Medicine and the Office of Dietary Supplements, which funds the Botanical Research Center and the New Jersey Agricultural Experiment Station at Rutgers University. WTC is also supported in part by NIH grant 1 U54 GM104940. IR and DER have equity in Nutrasorb, LLC. DMR designed experiments. DER and DMR drafted the manuscript. AP conducted LC-MS analysis. PK conducted animal experiments. DER performed statistical analysis. AO prepared plant extracts, ran TIM-1 experiments and prepared TIM-1 and serum samples for analysis. WTC and IR provided experimental guidance and editorial comments.

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