Introduction

Diabetes mellitus is a complex endocrine disorder characterized by abnormalities in insulin secretion and/or insulin action that leading to the progressive deterioration of glucose tolerance and causes hyperglycemia (Pereira et al. 2011). DM is a major and growing public health problem throughout the world, affecting about 171 million people and most of them have type 2 diabetes (Gershell 2005). This increasing trend in type 2 DM has become a serious medical concern worldwide. It accounts for 9 % of deaths; thus, great efforts are being made in the search for new therapeutic agents to stem its progress (Kumar et al. 2011). One therapeutic approach for decreasing postprandial hyperglycemia is to retard the absorption of glucose via the inhibition of carbohydrate-hydrolyzing enzymes, such as α-glucosidases (i.e. sucrase, maltase, isomaltase, glucoamylase, and lactase inhibition) in the intestine (Holman et al. 1999).

In recent years, much effort has been made to identify effective α-glucosidase inhibitors from natural sources in order to develop a physiologic functional food or to identify compounds for use against diabetes. Many α-glucosidase inhibitors have been isolated from plants, including flavonoids, alkaloids, terpenoids, anthocyanins, glycosides, and phenolic compounds (Kumar et al. 2011). On the other hand, medicinal plants were widely used for treatment of diabetes throughout the world as they are effective, nontoxic, and less or no side effects (Clifford and Caroline 1989). More than 1,200 plants have been used to treat diabetes in folk medicine (Marles and Farnsworth 1995), and 136 plants clearly showed the anti-diabetic effects (Kavishankar et al. 2011). Thus, many researchers are trying to search for more effectively anti-diabetic compounds from medicinal plants.

Rosa rugosa Thunb. ex Murray (Rosaceae) is a deciduous tree that grows in Asia, Europe, North Africa, and North America (Li et al. 2013a; Yu et al. 1985). R. rugosa is a 1–2 m tall shrub characterized by vegetative reproduction using root suckers. Its single large red-pink flowers, which are 6–12 cm in diameter, occur in clusters of 3–6 (Sulborska et al. 2012). The plant has been used in traditional medicine to treat stomach-ache, diarrhea, and women’s diseases. Recent studies have also shown that R. rugosa exhibited anti-HIV, antitumor activity, anti-nociceptive, anti-tobacco, and anti-inflammatory activities (An et al. 2011; Fu et al. 2006). In northern Japan, where this wild rose occurs naturally, the dried petals have been used as antidiarrheal and hemostatic agents. The dried petals of R. rugosa have been used in China for preparation of rose tea because of their sweet fragrance; moreover, the tea is believed to provide nourishment (Hshidoko 1996). In Korea, the plant has been used for the treatment of diabetes, chronic inflammatory diseases, pain, and cancer (Lee et al. 2008).

Previous phytochemical researches on the R. rugosa have shown that tannins, flavonoids, and terpenoids are the major components of this plant (An et al. 2011; Fu et al. 2006; Hashidoko et al. 1993; Ochir et al. 2010). In the course of our search for biologically active compounds from plant resources, the n-butanol-soluble part of a 95 % methanol extract of R. rugosa roots was found to inhibit rat intestinal sucrase. We herein focused on the sucrase inhibitory effect of the extracts and 13 triterpenoid saponins (1–13) from the roots of this plant (Table 1; Fig. 1).

Table 1 Rat intestinal sucrase inhibitory activity of the extracts and isolated compounds 1–13
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Fig. 1
figure 1

Structures of triterpene saponins 1–13 from R. rugosa roots

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Materials and methods

Plant material

The samples of the roots of R. rugosa Thunb. were purchased from an herbal market at Kumsan, Chungnam, Republic of Korea, in August 2011. The plant material was identified by Professor Young Ho Kim, and a voucher specimen (CNU-11104) was deposited at the Herbarium of College of Pharmacy, Chungnam National University.

General experimental procedures

Optical rotations were determined using a Jasco DIP-370 digital polarimeter. The FT-IR spectra were measured using a Jasco Report-100 infrared spectrometer. ESI mass spectra were obtained using an Agilent 1200 LC-MSD Trap spectrometer. The NMR spectra were recorded on a JEOL ECA 600 spectrometer using TMS as an internal standard. TLC was performed on Kieselgel 60 F254 (1.05715; Merck, Germany) or RP-18 F254s (Merck) plates. Spots were visualized by spraying with 10 % aqueous in H2SO4 solution, followed by heating for 3–5 min. Column chromatography (CC) was performed on silica gel (Kieselgel 60, 70–230 mesh and 230–400 mesh, Merck) and YMC RP-18 resins (1.15685.0001, Merck, Germany). Automated flash chromatography was performed on a Teledyne CombiFlash Rf200 using C-18 RediSep columns.

Extraction and isolation

Dried R. rugosa roots (1.45 kg) were extracted with MeOH using ultrasonic maceration (2 × 3 h) at 30 °C. The resulted solutions were filtered, combined, and concentrated under reduced pressure to give a MeOH extract (A, 118 g). This extract was suspended in H2O and successively partitioned with CH2Cl2, EtOAc, and n-BuOH to give CH2Cl2 (B, 21.3 g), EtOAc (C, 12.4 g), and n-BuOH (D, 31.5 g) fractions, respectively. Bioactivity-guided fractionation of the n-butanol-soluble fractions was carried out using in vitro rat intestinal sucrase assay. The n-butanol fraction showed potent rat intestinal sucrase inhibitory activity with a value of 87.62 ± 5.84 % inhibition.

Fraction D was chromatographed over silica gel CC eluting with MeOH in CH2Cl2 (from 30 to 100 %, stepwise), yielding four subfractions (D-1 to D-4). Subfraction D-1 (5.6 g) was chromatographed over silica gel CC eluting with CH2Cl2–MeOH (10:1) to yield three smaller subfractions (D-1.1 to D-1.3). Subfraction D-1.1 (0.82 g) was chromatographed over silica gel CC eluting with EtOAc–MeOH (17:1) and then further purified by YMC RP-18 CC using MeOH–H2O (1:1) as the eluent to afford 13 (11.2 mg). Next, subfraction D-1.2 (1.1 g) was chromatographed over silica gel CC and eluted with CH2Cl2–MeOH (10:1) to obtain 11 (32.8 mg) and 12 (18.5 mg). Fraction D-2 (3.5 g) was chromatographed on a column of highly porous polymer (Diaion® HP-20), using stepwise eluent of MeOH–H2O (0/10, 2.5/10, v/v) to give three smaller fractions (D-2.1 to C-2.3) after removal of fraction eluted with H2O 100 %. Subfraction D-2.1 (0.73 g) was separated by YMC RP-18 CC using MeOH–H2O (1:2) as the eluent to afford 5 (12.2 mg) and 7 (14.5 mg). Similarly, subfraction D-2.3 (0.84 g) was separated by YMC RP-18 CC, using MeOH–H2O (1:3) as eluent, and further purified by silica gel CC, with CH2Cl2–MeOH–H2O (5:1:0.1), to obtain 8 (17.3 mg). Fraction D-3 (10.1 g) was separated by normal-phase medium-pressure liquid chromatography (MPLC) using CH2Cl2–MeOH (8:1) as the eluent to afford 4 (14.8 mg), 9 (15.4 mg), and a crude mixture of triterpenoid saponins. The latter was further separated by Sephadex LH-20 CC using H2O–MeOH (1:1) as the mobile phase, followed by YMC RP-18 CC using acetone–H2O (1.5:2.5) to afford 2 (13.5 mg) and 6 (18.7 mg). Next, fraction D-4 (12.2 g) was separated by YMC RP-18 CC eluting with acetone–H2O (1.2:3) to provide three smaller fractions (D-4.1 to D-4.3). Subfraction D-4.2 (0.8 g) was separated by YMC RP-18 chromatography using MeOH–H2O (1:2) as the eluent and further purified by silica gel CC, with CH2Cl2–MeOH–H2O (2:1:0.2), to obtain 3 (14.4 mg) and 10 (11.3 mg). And finally, subfraction D-4.3 (0.51 g) was subjected to silica gel CC with CH2Cl2–MeOH–H2O (1.5:10.2) and further separated by YMC RP-18 CC using acetone–H2O (1.2:1.5) as the eluent to afford 1 (20.1 mg).

Compounds

Thirteen compounds 1–13 were isolated and structurally elucidated from the roots of R. rugosa. Their purity (96–98 %) was determined by NMR and HPLC analyses. Stock solutions of tested compounds in DMSO were prepared, kept at −20 °C, and diluted to the desired final concentration in fresh medium before each experiment. To avoid influencing cell growth, the final DMSO concentration did not exceed 0.5 % in all experiments.

In vitro sucrase inhibition assay

Rat intestinal acetone powders were purchased from Sigma Chemical Co. (St. Louis, MO, USA). A slightly modified version of rat intestinal sucrase assay method developed by Kwon et al. was used (Kwon et al. 2006). A total of 1.0 g of the rat intestinal acetone powder was suspended in 3.0 mL of 0.9 % saline, and the suspension was sonicated 12 times for 30 s at 4 °C. After centrifugation (10,000×g, 30 min, 4 °C), the resulting supernatant was used for the assay. The sample solution (50.0 μL) and 0.1 M phosphate buffer (pH 6.9, 100 μL) containing sucrase solution (1.0 U/mL) was incubated at 25 °C for 10 min. After preincubation, 5.0 mM p-nitrophenyl-α-d-glucopyranoside solution (50.0 μL) in 0.1 M phosphate buffer (pH 6.9) was added to each well at timed intervals. The reaction mixtures were incubated at 25 °C for 5 min. Before and after incubation, the absorbance was read at 405 nm by a microplate reader Sunrise (Tecan, Salzburg, Austria) and compared to a control which had 50.0 μL of buffer solution in place of the extract or compound. Sucrase inhibitory activity was expressed as % inhibition and was calculated as follows:

$$\%\;{\text{inhibition}} = \left( {\left[ {\frac{{\Delta A_{405}^{Control} -\Delta A_{405}^{Extract} }}{{\left[ {\Delta A_{405}^{Control} } \right]}}} \right]} \right) \times 100$$

Statistical analysis

All experiments were performed in triplicate. Data is presented as the mean ± standard deviation (SD) the results were statistically analyzed by ANOVA and Duncan’s multiple range tests (GraphPad Prism version 6.02). Statistical significance was accepted at a level of *P < 0.05 and **P < 0.01 using the SPSS software package (SPSS Inc. Chicago, IL, USA version 10.0).

Results and discussion

Natural resources provide a huge and highly diversified chemical bank from which we can search for potential therapeutic agents using bioactivity-targeted screening. Given the development of the herbal industry and continued interest in herbal medicine (i.e. phytopharmaca), opportunities for the exploration of medicinal plants are widely available (Lam et al. 2008). As an economically important plant, R. rugosa has been widely cultivated in several areas of Korea for use as an ornamental flowers, food, and incense materials (Gao et al. 2013). In addition the legumes of this plant are used to treat various diseases (Guo et al. 2011; Horváth et al. 2012).

To our knowledge, there have been only a few reports on α-glucosidase inhibitory effects of R. rugosa (Feng et al. 2013; Li et al. 2014), but rat intestinal sucrase inhibition of the extracts and/or pure compounds from this plant have not been reported. A methanol extract prepared from the roots of R. rugosa was screened and found to exhibit potent rat intestinal sucrase inhibitory activity, with value of 81.91 ± 2.90 % inhibition. Subsequently, this extract was successively partitioned with dichloromethane and n-butanol. Bioassay-guided screening indicated that the n-butanol-soluble fraction (0.5 mg/mL) of the R. rugosa roots showed strong rat intestinal sucrase inhibitory activity, with inhibition percentage value of 87.62 ± 5.84 % (Table 1), relative to the positive control acarbose (0.02 mM), with value of 50.96 ± 2.97 % inhibition. Previously, acarbose isolated from Actinoplanes sp. is now used in the management of type 2 diabetes. A main drawback of using drugs such as acarbose is their side effects including abdominal distention, flatulence, meteorism, and possibly diarrhea (Kimura et al. 2004).

Therefore, the n-butanol-soluble fraction was selected for further separation using various chromatographic methods, which resulted in the isolation of 13 secondary metabolites 1–13 (see “Materials and methods” section). These compounds were identified as pulsatilloside F (1) (Li et al. 2013b), hederacolchiside F (2) (Kang 1989), cernuoside B (3) (Zhang et al. 2000), kalopanaxsaponin B (4) (Sano et al. 1991), 3-O-{[β-d-glucopyranosyl(1→4)]-[α-l-rhamnopyranosyl-(1→2)]-α-l-arabinopyranosyl} oleanolic acid (5) (Ekabo and Farnsworth 1996), patrinia saponin H3 (6) (Kang and Kim 1997), 3-O-β-d-glucopyranosyl(1→3)-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranosyl oleanolic acid (7) (Saito et al. 1990), 3-O-[β-d-glucopyranosyl(1→3)-α-l-rhamnopyranosyl-(1→2)]-β-d-glucopyranosyl(1→4)]-α-l-arabinopyranosyl oleanolic acid (8) (Schenkel et al. 1991), hederasaponin B (9) (Sano et al. 1991), cussosaponin C (10) (Mimaki et al. 2001), 3β,19α,23α-trihydroxy-2-oxo-12-ursen-28-oic acid β-d-glucopyranosyl ester (11) (Jia et al. 1993), kaji-ichigoside F1 (12) (Liang and Cao 1992), and rosamultin (13) (Liang and Cao 1992) by comparison of their spectroscopic data with the literature values. This is the first integrated chemical investigation of triterpenoid saponins from R. rugosa roots.

Subsequently, all isolated compounds were tested for their sucrase inhibitory activity. Among them, compounds 11–13 (1.0 mM) showed significant sucrase inhibitory activity, with values of 41.17 ± 3.52, 46.80 ± 4.00, and 39.39 ± 4.19 % inhibition, respectively. This is in agreement with recent reports suggesting that the sucrase inhibitory activity increased significantly as compared to the other compounds when a sugar unit attached at C-28 position of the aglycone (Benalla et al. 2010; Jabeen et al. 2013; Kang et al. 2011; Lai et al. 2012; Wei et al. 2012). Moreover, compounds 2–6, 8, and 10 (with additional the sugar units attached at C-3 position) showed mild sucrase inhibitory activity, with percentage inhibition values ranging from 13.26 ± 7.00 to 32.08 ± 6.04 % at a same concentration. The other compounds did not exert significant inhibitory effects on the enzyme (less than 10 % inhibition, Table 1).

Consideration of the structure–activity relationship (SAR) of these triterpenoid saponins suggested that the presence of the sugar units at C-3 and/or C-28 of the aglycone might play an important role in the sucrase inhibitory activity of these compounds. Interestingly, rat intestinal sucrase inhibitory activity of the methanol extract and n-butanol fraction was stronger than that of the isolated triterpenoid saponins. This evidence suggested that rat intestinal sucrase inhibitory activity of methanol extract and n-butanol fraction might arise from the synergy of many compounds but not of each individual.

In conclusion, the enzyme inhibition of the extract/fractions and isolated constituents was less than that of the reference inhibitor acarbose. However, keeping in mind with the multiple health benefits of herbal medicines, this report provides a basis for further evaluation and/or utilization of R. rugosa roots alone or in combination with other agents for the treatment or management of diabetic complications. The research on the R. rugosa Thunb. provides support for the ethnomedicinal use of the plant in the treatment of diabetes and also partly defines the mechanism underlying the anti-diabetic properties.