Mulberry leaf extract decreases digestion and absorption of starch in healthy subjects—A randomized, placebo-controlled, crossover study

Advances in Medical Sciences 62 (2017) 302–306

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Original research article

Mulberry leaf extract decreases digestion and absorption of starch in healthy subjects—A randomized, placebo-controlled, crossover study Jan Józefczuka , Klaudia Malikowskab , Aleksandra Glapab ,  ska-Witoszyn  skac, Jan Krzysztof Nowakb , Joanna Bajerskad , Barbara Stawin b Aleksandra Lisowska , Jarosław Walkowiakb,* a

Pediatric Division, City Hospital, 39-360 Nowa Deba, Poland Department of Pediatric Gastroenterology and Metabolic Diseases, Poznan University of Medical Sciences, Poznan, Poland c Department of Epidemiology, Chair of Social Medicine, Poznan University of Medical Sciences, Poznan, Poland d Department of Human Nutrition and Hygiene, Poznan University of Life Sciences, Poznan, Poland b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 3 October 2016 Accepted 8 March 2017 Available online xxx

Purpose: Mulberry (Morus alba L.) leaf tea has recently received much attention as a dietary supplement due to the wide range of putative health benefits, such as antidiabetic effects. Nevertheless, data evaluating its influence on carbohydrate metabolism in humans are scarce. The present study aims to investigate the effect of mulberry leaf extract supplementation on starch digestion and absorption in humans. Materials and methods: The study comprised of 25 healthy subjects, aged 19–27 years. In all subjects, a starch 13C breath test was performed twice in a crossover and single blind design. Subjects were initially randomized to ingest naturally 13C-abundant cornflakes (50 g cornflakes + 100 ml low fat milk) either with the mulberry leaf extract (36 mg of active component-1-deoxynojirimycin) or the placebo and each subject received the opposite preparation one week later. Results: The cumulative percentage dose recovery was lower for the mulberry leaf extract test than for the placebo test (median [quartile distribution]: 13.9% [9.9–17.4] vs. 17.2% [13.3–20.6]; p = 0.015). A significant decrease was detectable from minute 120 after the ingestion. Conclusions: A single dose of mulberry leaf extract taken with a test meal decreases starch digestion and absorption. These findings could possibly be translated into everyday practice for improvement of postprandial glycemic control. © 2017 Medical University of Bialystok. Published by Elsevier B.V. All rights reserved.

Keywords: Breath test Carbohydrate Diabetes Nutritional trial Obesity

1. Introduction Obesity is considered an epidemic and may contribute to the pathophysiology of various disorders such as postprandial hyperglycemia, diabetes, glycosphingolipid storage diseases and diabetic cardiovascular complications [1,2]. Therefore much attention has been focused on the improvement of postprandial glycemic control [3]. Materials that delay or inhibit digestion or absorption of carbohydrates could be helpful in this respect [4,5]. Although the effectiveness of digestive enzymes inhibitors is undoubtable, many pharmaceutics of this group cause serious side

* Corresponding author at: Department of Pediatric Gastroenterology and Metabolic Diseases, Poznan University of Medical Sciences, Szpitalna Street 27/ 33, 60-572 Poznan, Poland. E-mail address: [email protected] (J. Walkowiak).

effects, such as diarrhea or abdominal pain [6,7]. For this reason, the use of well-tolerated, natural dietary supplements could be considered as complementary and supportive treatment for obesity and associated carbohydrate-mediated disorders [8–10]. Mulberry leaf has recently received much interest as a herbal supplement due to the wide range of its biological activity, which includes antioxidant, neuroprotective, anti-inflammatory, and antimicrobial actions [11]. The worldwide known antidiabetic application of mulberry leaf extract has been ascertained to be the effect of a complex combination of various constituents, such as flavonoids, soluble dietary fiber, and iminosugars, that may act through alpha-glucosidase inhibition [12–14]. Mulberry leaves are rich in amino group-containing sugar analogs – termed iminosugars – in which the most abundant (accounting for 50%) iminosugar – 1-deoxynojirimycin (DNJ) – is believed to be the most bioactive agent [14–16]. Many studies show that mulberry preparations decrease glucose absorption in vivo [13,17–20]. In the

http://dx.doi.org/10.1016/j.advms.2017.03.002 1896-1126/© 2017 Medical University of Bialystok. Published by Elsevier B.V. All rights reserved.

J. Józefczuk et al. / Advances in Medical Sciences 62 (2017) 302–306

few randomized controlled trials, postprandial glucose levels decreased after mulberry ingestion [13,21–24]. Nevertheless, data evaluating its influence on carbohydrate digestion and absorption in healthy humans is scarce. Zhong et al. examined the effect of a mixture of black, green and mulberry teas on breath hydrogen concentrations after a rice containing test meal comparing with placebo [23]. In the study by Mudra et al., the effect of mulberry leaf extract (MLE) on blood glucose and breath hydrogen response was measured after saccharose loading in type 2 diabetic patients and control subjects [24]. However, there are no data on the influence of mulberry on starch digestion. In order to assess starch digestion and absorption we used a reliable method of non-invasive 13C breath test [25]. In the present study, we used cornflakes abundant in starch as a test meal. Starch is digested to monosaccharides such as glucose, which in excess may contribute to elevated post-prandial hyperglycemia. During the test, the 13C isotope naturally present in maize is digested in the small intestine then absorbed and metabolized in the liver and transported to lungs. Exhaled CO2 is labelled with the 13C isotope and measured as a cumulative percentage dose recovery values (CPDR). Different studies have evaluated the various effects of mulberry leaf on humans. The primary outcome of this study is to measure the effect of mulberry leaf extract on carbohydrate digestion and absorption after a starch test meal. The ingredient of the extract responsible for the decrease in starch digestion and absorption is 1- deoxynojirimycin which has been shown to inhibit a-glucosidase activity in vitro and in animal studies [12–14]. In the present study, we aimed to assess the influence of MLE on starch digestion and absorption in healthy human subjects with the use of a noninvasive 13C breath test. 2. Materials and methods

any food naturally abundant in 13C, such as products made of maize, cane sugar, pineapple, or kiwi fruit for 5 days preceding the examination. Fifty-one subjects were assessed for eligibility, of whom 26 were excluded (lactose intolerance n = 17; not meeting inclusion criteria n = 9) (Fig. 1). Eventually, 25 subjects were randomized and assigned to the first intervention: 13 subjects in the placebo arm and 12 in the MLE arm. One week later, 12 subjects received placebo and 13 subjects received MLE. No participants left the study or were excluded after the randomization had taken place. The protocol of the study was conducted according to the Declaration of Helsinki and approved by the Bioethical Committee of Poznan University of Medical Sciences, Poland (decisions 605/12 and 752/13). 2.2. Randomization and blinding The protocol of starch 13C breath test was applied as described previously [28]. The order of the tests (MLE/placebo test first) was determined according to the randomization list prepared by a qualified dietician. One week later, the second test was performed in a crossover manner: the subjects took the opposite preparation from that ingested initially. In this way, subjects served as selfcontrols to themselves. The trial was single-blinded. Only the qualified dietician was aware of the subject allocation. The allocation ratio was 1:1. 2.3. Intervention After collection of baseline breath samples for 13CO2 analysis, subjects took MLE or placebo and then ingested naturally 13C -abundant cornflakes (50 g cornflakes + 100 ml low fat milk). The ingestion of the test meal took consistently less than 10 min. The use of cornflakes as a test meal proved to be useful in evaluating the digestibility of carbohydrates [25,28,29]. The total energy load

2.1. Study population The study comprised of 25 healthy subjects (10 men and 15 women) (Table 1). Subjects were recruited from the Vocational Technical High School for Computer Science in Nakło nad Notecia˛, Poland over May 2013–May 2014. As subjects were expected to ingest milk, those suffering from lactose malabsorption were excluded via a hydrogen-methane breath test. The eligibility criterion was age 18 years. Inclusion criteria were as follows: willingness to participate in the study (confirmed by informed written consent) and good health status defined as no physical complaints in the month preceding the study, no acute or chronic disease, no current pharmacotherapy, no past hospitalizations for gastroenterological indications, and good nutritional status (weight and height within normal reference values and BMI cut-off points within 18.50–24.99 kg/m2). Exclusion criteria comprised of: celiac disease, exocrine pancreatic insufficiency [26,27] and other gastrointestinal diseases, medication that might affect digestion or absorption of carbohydrates, antibiotic therapy within the preceding month, and the use of mulberry preparations within the preceding month. Prior to the test, all subjects fasted for 12 h. The subjects were also informed not to eat Table 1 Basic data of study subjects (n = 25). Parameter

Median

1st–3rd quartile

Age [years] Body weight [kg] Height [cm] a BMI [kg/m2]

24 63.0 170 22.0

19–27 58.2–76.5 165–182 20.1–23.2

a

Body Mass Index (BMI).

303

Fig. 1. The flowchart of the study.

304

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of the test meal amounted to 234 kcal and consisted of 7.00 g of protein, 46.50 g of carbohydrates, and 2.25 g of fat. MLE was prepared in a powder form and was given in digestible starch wafers. Simple starch wafers constituted as placebo. In the current study we used 1800 mg of commercially available mulberry leaf product with a standardized 2% 1-DNJ content. Each subject ingested 36 mg of 1-DNJ. The dose used in the study was established with regard to previous investigations [30].

vs. 17.2% [13.3–20.6]; p = 0.015) (Table 2). The trend of the decrease in starch digestion and absorption was evident from the beginning of the test and the aforesaid effect persisted until the last measure. A significant decrease was detectable from minute 120 after the ingestion of MLE in comparison to placebo. The median value of DCPDR was 2.4 [ 8.2–( 0.4)] (Fig. 2). No side effects were reported from the participants during or after the study. 4. Discussion

2.4. Outcome measures Breath samples were obtained every 30 min for 4 h after the test meal. The samples were collected into sealed aluminum bags. The subjects were asked not to consume any additional food or beverages, not to smoke cigarettes, and not to perform any physical activity for 4 h. The results of the breath test were obtained by an independent researcher. Naturally 13C-abundant cornflakes were used as a source of stable 13C isotope: maize incorporates more 13C atoms into endogenous starch than C3 plants do (e.g. European grain, potatoes, rice). The isotope 13CO2/12CO2 enrichment in breath samples was measured with the use of isotope-selective nondispersive infrared spectrometry (IRIS Wagner Analysen Technik GmbH, Bremen, Germany) and showed by CPDR [31,32]. CPDR was considered to reflect digestion and absorption of dietary starch. The primary outcome of the study included the difference in CPDR values (DCPDR) between MLE and placebo interventions. No changes to the trial methods and outcomes were implemented after the trial had commenced. 2.5. Statistical analysis and power calculation The sample size (n = 25) was adequate to detect at least 2/3 of the effect on starch digestion and absorption obtained in our previous study employing identical methods [28], which corresponds to a decrease of 20%, assuming a standard deviation of 25%, a significance level of 0.05, and b of 80%. The statistical significance of differences between the MLE and placebo tests was determined with the use of the Wilcoxon rank test. Results are expressed as medians and interquartile ranges. The level of significance was set at p < 0.05. Statistical analysis was performed using STATISTICA 10.0 (StatSoft Inc., Tulsa, USA). 2.6. Study oversight Every subject signed an informed written consent to participate in the study. The nutritional intervention was carried out independently of any commercial entities. The study was designed and performed according to CONSORT [33] (Fig. 1). 3. Results CPDRs were significantly lower with the MLE ingestion than for the placebo test (median [quartile distribution]: 13.9% [9.9–17.4]

To date, several randomized placebo-controlled trials have evaluated the effect of mulberry preparations on healthy subjects [23,34,35]. However, the majority of data concern populations of Asian descent. The available data show that the metabolism of people of Asian descent is different in comparison to those of European origin (e.g. higher percentage of total body fat, higher accumulation of fat in truncal adipose tissue or higher insulin resistance) [36,37]. In this study, we are the first to evaluate the effect of mulberry in persons of European origin. Furthermore, our study focuses on the processes of carbohydrate digestion and absorption, whereas most of the data assess the impact of mulberry only on postprandial glucose levels [34,35]. This study uses non-invasive 13C breath test which is a reliable method to examine starch digestion and absorption. A major strength of the study is the trial design. Randomized, placebo controlled trials have been considered the most powerful tool in modern clinical research [38]. However, the present study has some limitations. We did not assess fasting or postprandial glucose levels, as some studies do [24,39]. Volunteers were not required to complete the dietary diary. Despite that, before the test, each subject had an appointment with our dietician, who had interviewed them about current eating habits. Moreover, subjects were instructed to avoid products naturally rich in 13C (maize, cane sugar, pineapple, kiwi fruit), consumption of which might affect the test results. Subjects were also frequently reminded to be consistent with the diet before and during the study. Previously, Zhong et al. have reported that in healthy subjects, a combination of black, green and mulberry teas cause carbohydrate malabsorption of 25% compared to placebo [23]. Despite similarities to our trial, the decrease in digestion was assessed by breath hydrogen analysis and the test meal consisted of rice. The exact effect of mulberry is also made unclear by the mixture of different teas. Similarly, Mudra et al. also measured the breath hydrogen response, but did so after a 75 g load of sucrose in healthy, as well as diabetic subjects [24]. Mulberry extract increased breath hydrogen response and additionally reduced blood glucose in this study. A similar effect with 30 g of sucrose was obtained in the study of Nakamura et al. [39]. Chung et al. examined the effect of different doses of mulberry leaf aqueous extract (MLAE) (1.25, 2.5 and 5 g) after a 75 g maltose load in healthy subjects [34]. Significant reduction of glucose concentration was seen after 2.5 g and 5 g of MLAE ingestion, that is 9 mg and 18 mg of DNJ, respectively. The area under the curve for plasma glucose after 5 g (18 mg DNJ) was diminished by 33.9% in

Table 2 Starch digestion and absorption based on cumulative percentage 13C dose recovery (CDPR). Median values and interquartile ranges are presented. CPDR [%13C] Minutes

30

60

90

120

150

180

210

240

a

0 [0–0.2] 0.1 [0–0.3] 0.071

0.3 [0–1.1] 0.6 [0.2–1.5] 0.095

1.2 [0.4–2.9] 2.3 [1.5–3.7] 0.069

2.8 [1.7–5.0] 4.8 [3.8–6.7] 0.025

4.7 [3.9–7.5] 8.1 [6.8–10.4] 0.010

7.6 [6.2–10.6] 11.2 [9.4–13.9] 0.006

10.8 [8.1–14.1] 14.1 [11.5–17.1] 0.009

13.9 [9.9–17.4] 17.2 [13.3–20.6] 0.015

MLE

Placebo Statistical significance (p) a

Mulberry leaf extract.

J. Józefczuk et al. / Advances in Medical Sciences 62 (2017) 302–306

305

Fig. 2. Individual variations in CPDR changes (DCPDR) for each subject at minute 240 of the test.

comparison to placebo. The same dose of 18 mg DNJ was used in the study of Kim et al. that evaluated the effect of a 4 week MLAE supplementation among subjects with impaired fasting glucose. Supplementation of MLAE resulted in a decrease of glucose and insulin levels after a carbohydrate load compared with the control group [21]. At present, various mulberry leaf products are available on the market as food supplements; however, there have been few assessments of the effect of such products on starch digestion and absorption. Kimura et al. have proved that DNJ content in most commercially available mulberry leaf products is too low (approximately 0.1% of dry matter) to exert an alpha-glucosidase inhibitory effect [30,35]. Maize starch contained in cornflakes is processed by salivary and pancreatic a-amylase to maltose, maltotriose, and a number of a-(1-6) and a-(1-4) oligoglucans. Further digestion by a-glucosidases degrades them to glucose, which may cause elevated postprandial hyperglycemia, especially in diabetic patients. The 13C breath test measures CO2 in exhaled air that comes from metabolized glucose which has been previously hydrolyzed from starch [40]. Starch from corn is digested in the small intestine, absorbed, then metabolized in the liver, and transported to lungs. There are multiple studies showing that extracts of mulberry reduce blood glucose in animal models of diabetes [41,42] and in type 2 diabetic patients [23,24]. The purported mechanism of mulberry action is the carbohydrate-digesting enzyme inhibition [14,23,24,43]. To date, literature data are insufficient to relate starch 13C breath test differences such as those observed, to the amount of starch malabsorbed, therefore further studies are needed to elucidate the exact effect. The antidiabetic activity of mulberry is attributed to the complex mixture of various constituents such as flavonoids, soluble dietary fiber and iminosugars that constitute the mulberry extract. Total phenolic concentration of each component depends on various conditions, such as climate or extraction procedure [41]. In the present study, the total content of flavonoids amounted to 32.1%. DNJ has been reported to be the most active component of MLE. Due to structural similarity to D-glucose, DNJ has been shown to competitively inhibit a-glucosidases’ activity in the small intestine. Naowaboot et al. suggested another mulberry extract polyphenolic compound – gallic acid – to possess the antidiabetic activity as it decreases fasting blood glucose among diabetic rats [42]. Our results suggest that mulberry products decrease carbohydrate digestion and absorption in the healthy population. However, the results of the 13C breath test does not allow one to exclude that mulberry components may affect metabolism and glucose storage. The efficacy and safety profile of mulberry preparations may potentially be favorable in weight control and diabetes prevention. Further research is needed to determine the value of long-term

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