Acylated phenylethanoid glycosides, echinacoside, and acteoside from Cistanche tubulosa improve glucose tolerance in mice
Mar 16, 2022
for more information:ali.ma@wecistanche.com
Abstract
phenylethanoid glycosides, echinacoside (1) and acteoside (2), principal constituents in stems of Cistanche tubulosa (Orobanchaceae), inhibited the increase in postprandial blood glucose levels in starchloaded mice at doses of 250–500 mg/kg p.o. These compounds (1 and 2) also significantly improved glucose tolerance in starch-loaded mice after 2 weeks of continuous administration at doses of 125 and/or 250 mg/kg/day p.o. without producing significant changes in body weight or food intake. In addition, several constituents from Cistanche tubulosa, including 1 (IC50 = 3.1 lM), 2 (1.2 lM), isoacteoside (3, 4.6 lM), 20 -acetylacteoside (4, 0.071 lM), tubulosides A (5, 8.8 lM) and B (9, 4.0 lM), syringalide A 3-O-a-L-rhamnopyranoside (10, 1.1 lM), campneoside I (13, 0.53 lM), and kankanoside J1 (14, 9.3 lM), demonstrated potent rat lens aldose reductase inhibitory activity. In particular, the potency of compound 4 was similar to that of epalrestat (0.072 lM), a clinical aldose reductase inhibitor.
Keywords Echinacoside, Acteoside, Glucose tolerance improvement effect, Aldose reductase inhibitor, Cistanche tubulosa
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Introduction
Phenylethanoid glycosides (PhGs) are a type of phenolic compound characterized by a b-glucopyranoside structure bearing a hydroxyphenylethyl moiety as the aglycone. These compounds often comprise a number of substituents such as aromatic acids (e.g., cinnamic acid, coumaric acid, caffeic acid, ferulic acid, isoferulic acid, etc.) and/or various sugars (e.g., rhamnose, xylose, apiose, arabinose, etc.) attached to the glucose residue through ester or glycosidic linkages, respectively. Although PhGs are widely distributed in the plant kingdom, the majority have been found in Scrophulariaceae, Oleaceae, Plantaginaceae, Lamiaceae, and Orobanchaceae families [1–3]. Echinacoside (1) [4, 5] and acteoside (2, also called verbascoside, kusaginin, and orobanchin) [6–9] are representatives of the well-studied PhGs and have been reported to possess a number of important bioactive properties such as antioxidative, neuroprotective, nitric oxide radical scavenging, antihepatotoxic, and antiosteoporotic activities [1–3, 10–14].
Previously, we reported the identification and biological properties of constituents from the stems of Cistanche tubulosa (Schrenk) R. Wight (Orobanchaceae) [15–19], the major constituents being 1 and 2. We revealed that these PhGs demonstrated a vasorelaxant activity in isolated rat thoracic aorta contracted by noradrenaline [15]. The stem part of Cistanche tubulosa (Kanka-nikujuyou in Japanese) has traditionally been used in Tibetan areas to promote blood circulation [20, 21], and this finding provides scientific evidence for the traditional use of Cistanche tubulosa.
Continuing studies on the bioactive properties of this plant material revealed that these principal PhGs possess hypoglycemic activity and improve glucose tolerance in starch-loaded mice. Furthermore, some of the PhGs isolated from Cistanche tubulosa (1–18) were found to have potent aldose reductase inhibitory activity.
Echinacoside in Cistanche tubulosa
Results and discussion
Previously [15], we reported the isolation and bioactive properties of constituents of the methanol extracts of dried stems from Cistanche tubulosa, including 1, 2, isoacteoside (3), 20 - acetylacteoside (4), tubulosides A (5), and B (9), and salidroside (18). Wiedemanninoside C (6), kankanosides H1 (7) and H2 (8) syringalide A 30 -O-a-L-rhamnopyranoside (10), kankanoside I (11), cistantubuloside A (12), campneoside I (13), and kankanosides J1 (14), J2 (15), K1 (16), and K2 (17), were additionally isolated from the fresh stems of the same material [16, 17] (Fig. 1).
First, the effects of the principal PhGs (1 and 2) on the postprandial increase in blood glucose levels in starch-loaded mice were examined. The activity of both 1 and 2 at doses of 250–500 mg/kg p.o. was significant (Table 1). In this experiment, an intestinal a-glucosidase inhibitor, acarbose, which was employed as a positive control, showed reasonable inhibition (Table 1).
To examine the effects of 1 and 2 on glucose tolerance, blood glucose level elevation was examined in starch-loaded mice after 2 weeks of continuous administration. At doses of 125 and/or 250 mg/kg/day p.o. both 1 and 2 significantly improved glucose tolerance (Table 2) without significantly changing either the body weight or food intake (data not shown). These results suggested that 1 and 2 were effective in both inhibiting postprandial glucose elevation and improving glucose tolerance.
To examine the mode of action of these antihyperglycemic activities, the effects of 1 and 2 on rat small intestinal a-glucosidases, maltase, and sucrase, were examined. Concurrently, the inhibitory effects of other constituents (3–18) from Cistanche tubulosa against these enzymes were also examined. We previously identified a thin sugar sulfonium salt, salacinol (one of the most potent naturally occurring a-glucosidase inhibitors; IC50 = 6.0 and 1.3 lM against maltase and sucrose, respectively) [22–24], which was also employed as a positive control in this study. 1 (IC50 = 149 and 174 lM) and 2 (188 and 152 lM) showed similar enzyme inhibitory activities (Table 3). However, their activities were far less than those of the positive controls, salacinol, and acarbose (IC50 = 6.0 and 1.3, and 1.7 and 1.5 lM, respectively). It is worth noting that among the isolated PhGs, 3 (IC50 = 70.4 and 152 lM) and 9 (88.2 and 175 lM), which possess a trans-caffeoyl moiety at the 60 -position of the inner glucopyranosyl part, moderately inhibited maltase and sucrase, whereas their regioisomers 2 and 4 ([300 lM), which possess the caffeoyl moiety at the 40 -position, showed less activity against these enzymes.
Acteroside in Cistanche tubulosa
The inhibitory activities of the PhGs against human intestinal maltase were also examined in the microsomal fraction. As a result, 1 (IC50 = 125 lM), 2 (154 lM), 3 (117 lM), 5 (63 lM), 9 (139 lM), and 15 (168 lM) were found to exhibit this activity. However, these compounds were far less active than acarbose (15.2 lM).
Previously, we identified several flavonoids [25–31], stilbenoids [25, 32], quinic acid derivatives [30], and terpenoids [33] as potent aldose reductase inhibitors. In this work, the inhibitory effects of the isolated PhGs (1–18) on rat lens aldose reductase were also examined. Aldose reductase is known to be a key enzyme that catalyzes the reduction of glucose to sorbitol in the polyol processing pathway. Sorbitol does not readily diffuse across cell membranes, and the intracellular accumulation of sorbitol has been implicated in the chronic complications of diabetes such as cataracts [25].
First, a methanol extract from fresh stems of Cistanche tubulosa was found to demonstrate potent inhibitory activity (IC50 = 3.8 lg/mL). Among the isolated PhGs, rat lens aldose reductase inhibitory activity was observed with 1 (IC50 = 3.1 lM), 2 (1.2 lM), 3 (4.6 lM), 4 (0.071 lM), 5 (8.8 lM), 9 (4.0 lM), 10 (1.1 lM), 13 (0.53 lM), and 14 (9.3 lM) (Table 3). Compound 4, particularly, showed the most potent activity, which was equivalent to that of epalrestat (0.0072 lM), a clinically used aldose reductase inhibitor.
In conclusion, echinacoside (1) and acteoside (2), the major PhGs from stems of Cistanche tubulosa, were found to inhibit the increase in postprandial blood glucose levels in starch-loaded mice at doses of 250–500 mg/kg p.o. These two PhGs also significantly inhibited blood glucose elevation in starch-loaded mice after 2 weeks of administration at doses of 125 and/or 250 mg/kg/day p.o., without producing significant changes in body weight or food intake. These results suggested that 1 and 2 were effective not only in inhibiting postprandial glucose elevation but also in improving glucose tolerance. However, their a-glucosidase inhibitory activities were unworthy of attention. Thus, inhibition of a-glucosidase barely contributed to their antihyperglycemic activity. The detailed model of action should be studied further. Among the isolated PhGs, 1–3, 5, 9, and 15 moderately inhibited rat and human intestinal a-glucosidases. In contrast to this moderate inhibition, PhGs 1–5, 9, 10, 13, and 14 effectively inhibited rat lens aldose reductase. In particular, the activity of compound 4 was of similar magnitude to that of epalrestat, a clinically used inhibitor.
Experimental method
Plant material
Stems of Cistanche tubulosa cultivated at Urumqi, Xinjiang Province, China were collected in September 2007. The plant material was identified by one of the authors (X. Jia, President of Xinjiang Institute of Chinese Materia Medica and Ethnodrug). A voucher specimen of this plant is on file in our laboratory.
Animals
Male ddY mice (6 or 10 weeks) were purchased from Kiwa Laboratory Animal Co., Ltd., Wakayama, Japan. The animals were housed at a constant temperature of 23 ± 2 ℃ and were then fed a standard laboratory chow (MF; Oriental Yeast Co., Ltd., Tokyo, Japan). The animals were fasted for 20–24 h prior to the beginning of the experiment but were allowed free access to tap water. All of the experiments were performed with conscious mice unless otherwise noted. The experimental protocol was approved by the Experimental Animal Research Committee at Kinki University.
Inhibitory effects of 1 and 2 on blood glucose levels in starch-loaded mice
A mixture of each test sample and a-starch (1 g/kg) suspended in 5 % (w/v) acacia solution (20 mL/kg) was administered orally to fasted mice (6 w). Blood samples (ca. 0.1 mL) were collected from the infraorbital venous plexus under ether anesthesia 0.5, 1, and 2 h after oral administration. The collected blood was immediately mixed with heparin sodium (5 units/tube). After centrifugation of the blood samples, the plasma glucose level was determined enzymatically by the Glucose CII test Wako (Wako Pure Chemical Industries Ltd., Osaka, Japan). Intestinal a-glucosidase inhibitor acarbose was used as a reference compound.
Improvement effects of glucose tolerance after 2 weeks administration of 1 and 2 in starch-loaded mice
Each test sample was administered once a day (10:00- 12:00) for 2 weeks to 10-week-old mice fed a standard laboratory chow (MF; Oriental Yeast Co., Ltd.). Bodyweight was measured every day before the administration of the test sample. After fasting for 20 h, a starch (1 g/kg) solution was orally administered to the mice at 20 mL/kg. Through the same procedure, blood samples (ca. 0.1 mL) were collected, and plasma glucose levels were determined as described above.
Inhibitory effects on rat intestinal a-glucosidases
The experiments were performed according to the method described in our previous reports with a slight modification [23, 24]. Thus, rat small intestinal brush border membrane vesicles were prepared and their suspensions in 0.1 M maleate buffer (pH 6.0) were used as small intestinal glucosidases of maltase and sucrase. A test sample was dissolved in dimethyl sulfoxide (DMSO), and the resulting solution was diluted with 0.1 M maleate buffer to prepare the test sample solution (concentration of DMSO 10 %). A substrate solution in the maleate buffer (maltose 74 mM, sucrose 74 mM, 50 lL), the test sample solution (25 lL), and the enzyme solution (25 lL) were mixed at 37 C for 30 min, and then immediately heated by boiling water for 2 min to stop the reaction. The glucose concentrations were determined by a glucose-oxidase method. The final concentration of DMSO in the test solution was 2.5 % and no influence of DMSO on the inhibitory activity was detected. The intestinal a-glucosidase inhibitors acarbose and salacinol were used as reference compounds.
Inhibitory effects on human intestinal maltase
A human small intestinal microsome (batch MIC318017, purchased from BIOPREDIC International, Rennes, France) in 0.1 M maleate buffer (pH 6.0) was used to determine the small intestinal a-glucosidase activity of maltase. Through a similar procedure, the effect of maltase inhibitory activity was measured as described above.
Inhibitory effects on rat lens aldose reductase
The experiments were performed according to the method described in our previous reports [25–33] with slight modifications. Thus, the supernatant fluid of a rat lens homogenate was used as a crude enzyme. The enzyme suspension was diluted to produce approximately 2 nmol/ well of nicotinamide adenine dinucleotide phosphate (NADP) in the following reaction. The incubation mixture contained phosphate buffer 135 mM (pH 7.0), Li2SO4 100 mM, reduced nicotinamide adenine dinucleotide phosphate (NADPH) 0.03 mM, DL-glyceraldehyde 1 mM as a substrate, and 20 lL of enzyme fraction, with a test sample, in a total volume of 100 lL. The reaction was initiated by the addition of NADPH at 30 C. After 30 min, the reaction was stopped by the addition of 30 lL of HCl 0.5 M. Then, 100 lL of NaOH 6 M containing imidazole 10 mM was added, and the solution was heated at 60 C for 20 min to convert NADP into a fluorescent product. Fluorescence was measured using a fluorescence microplate reader (SH-9000, CORONA) at an excitation wavelength of 360 nm and an emission wavelength of 460 nm. Each test sample was dissolved in DMSO. Measurements were performed in duplicate, and IC50 values were determined graphically. An aldose reductase inhibitor epalrestat was used as a reference compound.
Statistics
Values were expressed as mean ± SEM. For statistical analysis, a one-way analysis of variance followed by Dunnett’s test was used. Acknowledgments This work was supported in part by a Grant-in-Aid for Scientific Research by Japan Society for the Promotion of Science (JSPS) KAKENHI a Grant Number 24590153 and The Japan-China Medical Association for the financial support
From: 'Acylated phenylethanoid glycosides, echinacoside, and acteoside from Cistanche tubulosa improve glucose tolerance in mice' by Toshio Morikawa, et al
---J Nat Med (2014) 68:561–566 DOI 10.1007/s11418-014-0837-9
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