Identification of Bioactive Phytochemicals in Mulberries

Abstract

Mulberries are consumed either freshly or as processed fruits and are traditionally used to tackle several diseases, especially type II diabetes. Here, we investigated the metabolite compositions of ripe fruits of both white (Morus alba) and black (Morus nigra) mulberries, using reversed-phase HPLC coupled to high resolution mass spectrometry (LC-MS), and related these to their in vitro antioxidant and α-glucosidase inhibitory activities. Based on accurate masses, fragmentation data, UV/Vis light absorbance spectra and retention times, 35 metabolites, mainly comprising phenolic compounds and amino sugar acids, were identified. While the antioxidant activity was highest in M. nigra, the α-glucosidase inhibitory activities were similar between species. Both bioactivities were mostly resistant to in vitro gastrointestinal digestion. To identify the bioactive compounds, we combined LC-MS with 96-well-format fractionation followed by testing the individual fractions for α-glucosidase inhibition, while compounds responsible for the antioxidant activity were identified using HPLC with an online antioxidant detection system. We thus determined iminosugars and phenolic compounds in both M. alba and M. nigra, and anthocyanins in M. nigra as being the key α-glucosidase inhibitors, while anthocyanins in M. nigra and both phenylpropanoids and flavonols in M. alba were identified as key antioxidants in their ripe berries.

Keywords: antioxidant activity; high resolution mass spectrometry; in vitro gastrointestinal digestion; mulberry; α-glucosidase inhibitory activity.

Figure 1

α-glucosidase inhibitory activity of mulberry methanol extracts. The Y axis represents the α-glucosidase activity (increase in 415 nm absorbance per minute) and the X axis the sample type tested. (a) inhibitory activity of water extracts of Morus alba and Morus nigra compared to the negative control (water). (b) enzyme activity inhibition by acarbose (positive control) at increasing concentrations (mM) in the assay. Data represent means and standard deviations (n = 3 assays).

Figure 2

α-glucosidase inhibitory activity after in vitro gastrointestinal digestion. Inhibition activity of original Morus nigra (MN) and Morus alba (MA) fruit extracts, and after their in vitro stomach (Post Gastric, PG) digestion and in vitro gastrointestinal (GI) digestion. DC: digestion control, representing the digestion process, including all enzymes, without plant material; NC: negative control (NC), representing only water. Data represent means values and standard deviations (n = 3 measurements).

Figure 3

α-glucosidase inhibitory activity of 96-well LC-MS fractions of (A) M. alba and (B) M. nigra extracts. The Y axis shows the enzyme activity and the X axis the retention time corresponding to the LC-MS fraction. The vertical line at an enzyme activity of 0.17 indicates the average value in the water control. The wells considered bioactive are the ones below an enzyme activity value of 0.15.

Figure 4

Antioxidant activity. Overlay of representative antioxidant chromatograms of fruit of Morus alba (in blue) and Morus nigra (in black). Antioxidant profiles of fruit extracts were determined online, by a post column reaction with ABTS^·+ cation radicals after HPLC separation and PDA detection of compounds. The ABTS-radicals remaining after post-column reaction were recorded at 600 nm: negative peaks thus indicate antioxidant activity. The numbers refer to the main peaks identified (see Table 1): 6 cyanidin hexoside, 9 pelargonidin hexoside, 10 and 13 caffeoylquinic acid isomers, 15 dihydroquercetin hexoside, 26 quercetin hexose deoxyhexose, 28 quercetin hexoside, and 32 kaempferol hexoside.