Hyperglycemia, when sustained over a long time in diabetes mellitus (DM), leads to biochemical and cellular abnormalities, primarily through the formation of advanced glycation end-products (AGEs). In the treatment of diabetes, beside blood-sugar-lowering medications, a consumption of herbal products that can inhibit the AGEs’ formation is recommended. This study investigated the in vitro antiglycoxidative potential of extracts and fractions from the rhizomes of Japanese, Giant, and Bohemian knotweeds (Reynoutria japonica (Houtt.), R. sachalinensis (F. Schmidt) Nakai, and R.× bohemica Chrtek et Chrtkova). Their effects on glycooxidation of bovine and human serum albumin were evaluated by incubation of the proteins with a mixture of glucose and fructose (0.5 M) and 150 µg/mL of extract for 28 days at 37 °C, followed by measuring early and late glycation products, albumin oxidation (carbonyl and free thiol groups), and amyloid-β aggregation (thioflavin T and Congo red assays). The highest antiglycoxidative activity, comparable or stronger than the reference drug (aminoguanidine), was observed for ethyl acetate and diethyl ether fractions, enriched in polyphenols (stilbenes, phenylpropanoid disaccharide esters, and free and oligomeric flavan-3-ols). In conclusion, the antiglycoxidative compounds from these three species should be further studied for potential use in the prevention and complementary treatment of DM.
Global prevalence of diabetes mellitus (DM) was 9.3% (463 million people) in 2019, and it is estimated to affect 10.2% (578 million) of the population by 2030 and 10.9% (700 million) by 2045. The incidence of DM is higher in urban (10.8%) than rural (7.2%) areas, and it is more common in high-income areas (10.4%) than in low-income countries (4.0%). Every second person with DM does not know about their disease [1]. Uncontrolled diabetes is associated with other diseases—cardiomyopathy, atherosclerosis, Alzheimer’s disease, neuropathy, nephropathy, and retinopathy. DM belongs to metabolic diseases. Diabetics have impaired sugar metabolism and insulin deficiency and/or decreased cellular sensitivity to insulin. Long-term hyperglycemia causes an increase in metabolic disorders and biochemical abnormalities; it results in increased protein glycation and accelerated development of complications: micro- and macroangiopathies [2,3,4]. The Maillard reaction is a biochemical transformation leading to non-enzymatic protein glycation and, consequently, to the loss or modification of their original role when occurring in the body [5,6,7]. The Maillard process is divided into three stages: early, intermediate, and late.
The early products of protein glycation include Schiff’s bases and the Amadori products. This reaction begins with the attachment of a reducing sugar carbonyl group (e.g., glucose, pentose, mannose, fructose, and galactose) to a free amino group of proteins, nucleic acids, or lipids to form an unstable compound called Schiff’s base (aldimine). This process is reversible [3,5]. This is followed by a slow rearrangement of the Schiff base, which gives rise to a stable ketoamine—the Amadori product. In vivo, Amadori products reach equilibrium after about 28 days, and by binding irreversibly to, inter alia, proteins, they begin to accumulate in cells, tissues, and organs. In an intermediate stage, the early products of the Maillard reaction are degraded. Through the oxidation and dehydration of Amadori products, various carbonyl compounds are formed (including glyoxal, methylglyoxal, 3-deoxyglucosone, N ε-(carboxymethyl)-lysine, and N ε-(carboxyethyl)-lysine). These products act as reaction promoters, reacting again with free amino groups, e.g., proteins. The last stage of glycation involves oxidation, dehydration, polymerization, cyclization, and condensation reactions with other amino groups. As a result, advanced glycation products, AGEs, are created [5,6]. These products are often fluorescent, insoluble, and irreversible compounds. In healthy people, modified proteins resulting from glycation are degraded and eliminated from the body in a physiological manner. It is worth mentioning that with the aging of the body or in the case of chronic diseases such as diabetes, atherosclerosis, and kidney disease, the glycation process is intensified, and the elimination of glycated proteins is significantly weakened. AGEs most often accumulate in the eyes, kidneys, atherosclerotic plaque, central nervous system, and brain, leading to disruption of the proper functioning of these organs and to micro- and macroangiopathy [3,5,6]. The glycation process occurs more intensively along with the oxidative process; both processes affect each other. It is the mechanism of the “vicious circle,” as a result of which the serious and negative consequences in the body worsen. Both of these accompanying processes are known as glycoxidation. Moreover, it has been shown that the glycation products that are formed during the Maillard reaction can be supplied with food. The content of glycated products depends on the degree of processing of the food and the way the meal is prepared. These products are also significantly associated with chronic diseases and their complications [3,5,6,7]
There are numerous studies searching for compounds that may reduce the concentration of AGEs and free radicals in the body by inhibiting their synthesis at different stages of the Maillard reaction [8,9,10]. The use of AGEs synthesis inhibitors is a promising therapeutic target. It was proved that both natural and synthetic compounds might act as inhibitors of AGEs, indicating different mechanisms of action [10]. Many synthetic compounds were tested for protein glycation inhibition. However, despite promising results, none of them were approved for the treatment of diabetic complications due to observed side effects [10]. Polyphenols, being naturally derived compounds found in plants, attract a large amount of attention from researchers because of their multidirectional biological activity, including significant antioxidant properties [8]. More and more studies on polyphenols demonstrate their beneficial anti-diabetic effects. Fernandez-Gomez et al. [11] conducted a study that sought new information concerning the inhibition of AGEs formation from extracts of coffee-roasting byproducts. It was concluded that the antiglycation activity of polyphenols contained in that extract might be related to their capacity to uptake carbonyl compounds and their capacity to react with the side chains of amino acid residues of proteins that block the glycation sites. Myrica gale extracts and rose extracts containing a high amount of polyphenols have a very high antioxidant capacity and significantly decreased α-amylase and α-glucosidase activities compared to acarbose [12,13]. Anthocyanidins and flavonols, which are the main components of berries and leaves, show a strong inhibitory effect on α-glucosidase, α-amylase, aldose reductase, and BSA glycation [12].
In the present study, we examined extracts from rhizomes of three species of large, invasive knotweeds from the genus Reynoutria Houtt. (Polygonaceae): R. japonica Houtt., R. sachalinensis (F.Schmidt) Nakai, and R. × bohemica Chrtek & Chrtková for their in vitro anti-glycoxidative activity. According to our previous research, these rhizomes are a rich source of polyphenols with high antioxidant capacity [14,15]. The R. japonica rhizomes (Huzhang in Chinese) are listed in the Chinese Pharmacopoeia, and they have been used for centuries in Chinese and Japanese traditional medicine for treating inflammation, jaundice, skin burns, scalds, and hyperlipidemia. Since 2017, under the synonym Polygoni cuspidati rhizoma, they have been included in the European Pharmacopoeia [15]. Despite not being a pharmacopoeial plant, another large knotweed—R. sachalinensis (Giant, or Sakhalin, knotweed)—has been, to some extent, used traditionally as an herbal medicine in East and North-East Asia for treatment of arthralgia, jaundice, amenorrhea, coughs, scalds and burns, traumatic injuries, carbuncles, and sores. In Europe, both species crossed and produced a hybrid called Bohemian knotweed (R. × bohemica). These plants are also occasionally used as wild spring vegetables both in their original range and in Europe [16].
Previous studies conducted on R. japonica leaf extracts indicate an antiglycation potential of these plants [17]. Moreover, studies in diabetic rats have shown that R. japonica rhizome extract reduced streptozotocin-induced early podocyte damage in the kidneys, and its active ingredient, emodin, inhibited methylglyoxal-mediated protein glycation [18]. Given the above, we have decided to investigate the antiglycoxidative potential of extracts and fractions from rhizomes of R. japonica, R. sachalinensis, and R. × bohemica [14,15]. BSA and HSA were used as model proteins for optimizing the method. Both BSA and HSA were the subject of glycation using a mixture of sugars (glucose and fructose), and the induced changes were analyzed in three steps: prevention of early and late formation of AGEs, albumin oxidation, and amyloid-β aggregation.
The following chemicals were used in the study, all of which were of analytical grade if not mentioned otherwise: D (+)–glucose (C 6 H 12 O 6, GLC), D (−)–fructose (C 6 H 12 O 6, FRC): (both purchased from POCH Ltd. Gliwice, Poland); BSA: fraction V, >92% purity; M 66,430 Da, (Fluka, Buchs, Switzerland); HSA: fraction V, 98% purity, M 67,000 Da (this and all following reagents were from Sigma-Aldrich, St. Louis, MO, USA); trans-resveratrol (C 14 H 12 O 3); aminoguanidine hydrochloride >98% purity; sodium azide (NaN 3); dimethyl sulfoxide ((CH 3)2 SO, DMSO); 2,4-dinitrophenylhydrazine (DNPH); 5,5’-dithiobis (2-nitrobenzoic acid) (Ellman’s reagent, DTNB); Congo red; nitro blue tetrazolium chloride (NBT); thioflavin T; and trichloroacetic acid (TCA).
Plant extracts and fractions of Reynoutria rhizomes were prepared at the Department of Pharmaceutical Biology and Botany, Wroclaw Medical University, in accordance with the procedure described in the previous article [14]. The species were identified by Klemens Jakubowski from the Wroclaw Medical University Botanical Garden herbarium based on vegetative morphology and generative organs (according to available florae). All extracts and fractions were placed in glass bottles and stored until use at −80 °C. For testing, the extracts and standards (resveratrol and aminoguanidine) were dissolved in 50% DMSO at a stock concentration of 20 mg/mL [14,15].
In our study, we used bovine serum albumin (BSA) and human serum albumin (HSA). BSA is the standard used in in vitro testing. The structure of BSA is well known, which allows it to be widely used in scientific research as a model protein for endogenous and exogenous ligand binding. The results of BSA glycation can be easily compared with other studies. However, in this study we also used HSA to confront the results with BSA glycation. BSA or HSA solutions were glycated according to the procedure described by Münch et al. in our own modification [19]. A mixture (0.5 mL, 0.5 M) of sugars (0.25 M glucose and 0.25 M fructose) was added to the BSA solution or HSA solution (0.5 mL, 10 mg/mL). A sample in which phosphate-buffered saline (PBS) buffer (0.5 mL) was added to the BSA solution or HSA solution (0.5 mL, 10 mg/mL) was the control of the reaction. The mixture of sugars (0.5 mL, 0.5 M) and 7.5 µL of the extract or fraction in question (150 µg/uL) were then added to the BSA solution or HSA solution (0.5 mL, 10 mg/mL). A sample in which 0.5 mL of phosphate-buffered saline (PBS) and 7.5 µL of the extract or fraction in question (150 µg/uL) were added to the BSA solution or HSA solution (0.5 mL, 10 mg/mL) was the control. The samples with glycation control contained sodium azide at 0.02%. All samples were performed in triplicate. The glycation process was performed at 37 °C for 28 days on the Heidolph Polymax 1040 Platform (Heidolph, Schwabach, Germany) using the Heidolph Incubator 1000 at approximately 50 rpm. After the incubation period was over, the dialysis of all solutions was performed against PBS buffer at pH 7.4 for 48 h at 4 °C. After the dialysis was over, the samples were divided at 1 mL of the analyzed material and frozen at −20 °C until fluorescence analysis and other determinations were performed.
The method described by Johnson et al. was used for fructosamine measurement [20]. The glycated protein solution (20 µL) was added to 300 µM NBT solution (180 µL) prepared in sodium carbonate buffer (100 mM, pH 10.4). The solution was incubated for 30 min at room temperature. Absorbance was measured at 530 nm (µQuant UV/VIS spectrophotometer, Biotek, Winooski, VT, USA). Fructosamine levels were calculated using the standard 1-deoxy-1-morpholinofructose curve; the equations of the calibration curves and the R 2 coefficients were y = 0.3459 × with R 2 =0.9991 for BSA and y = 0.2516 × with R 2 = 0.9995 for HSA; the values were expressed in mM/L.
AGEs formation in glycated albumin samples was evaluated by the method previously reported by Münch et al. [19]. The fluorescence of glycated samples was measured at excitation and emission wavelengths of 370 nm and 440 nm, respectively, using the Perkin Elmer LS50B spectrofluorometer (Perkin-Elmer, Waltham, MA, USA). Results were expressed in arbitrary fluorescence units (AFU).
The method described by Rice-Evans et al. was
