Chlorogenic acid and rutin play a major role in the in vivo anti-diabetic activity of Morus alba leaf extract on type II diabetic rats

Abstract

The leaves of the white mulberry tree (Morus alba L.) are used worldwide in traditional medicine as anti-diabetics. Various constituents of mulberry leaves, such as iminosugars (i.e. 1-deoxynojirimicin), flavonoids and related compounds, polysaccharides, glycopeptides and ecdysteroids, have been reported to exert anti-diabetic activity, but knowledge about their contribution to the overall activity is limited. The objective of the present work was to determine the in vivo anti-diabetic activity of an extract of mulberry leaves (MA), and to examine to what extent three major constituents, chlorogenic acid, rutin and isoquercitrin, might contribute to the observed activity. Quantities of the three constituents of interest in the extract were determined by using HPLC-DAD. Activity was determined by using a type II diabetic rat model. After 11 days of per os administration of 250 or 750 mg/kg of MA or the corresponding amounts of each individual compound, a dose dependent decrease of non-fasting blood glucose levels were found for MA, chlorogenic acid and rutin, but not for isoquercitrin. Based on our results, chlorogenic acid and rutin might account for as much as half the observed anti-diabetic activity of MA, hence they can be considered as excellent markers for the quality control of mulberry products.

Figure 1. Structures of chlorogenic acid (1), rutin (2) and isoquercitrin (3).

Figure 2. DAD fingerprint of an analyzed sample of mulberry leaf extract together with the chromatograms at λ = 326.3 nm (used for determination of 1) and λ = 353.0 nm (used for determination of 2 and 3) and UV spectra of each compound (Fig. 2A), and results of the peak purity testing for compounds 1–3 (Fig. 2B).

In Fig. 2B , peak areas where purity was found over 99.9% (green) are represented as mean of percentages ± SD; n = 3.

Figure 3. Calibration curves and corresponding data for the three compounds of interest, chlorogenic acid (Fig. 3A), rutin (Fig. 3B) and isoquercitrin (Fig. 3C).

Inj: injected amount, PA: peak area, ±Diff(%): difference between the actual value of PA/inj and the averages of each such value in the table, which difference should be less than 5%. Dashed frames in each data table represent ranges where corresponding peak areas of the extracts were found when tested.

Figure 4. Plasma glucose levels after 11 days of treatment, where significant differences to the control group were found.

Results are shown as mean ± SEM, G: glibenclamide; for further details see Table 1 legend.