Identification of Compounds from the Water Soluble Extract of Cinnamomum cassia Barks and Their Inhibitory Effects against High-Glucose-Induced Mesangial Cells

The difficulty of diabetic nephropathy (DN) treatment makes prevention the best choice. Cinnamomum cassia barks, known as Chinese cinnamon or Chinese cassia, is one of the most popular natural spices and flavoring agents in many parts of the World. Since previous reports indicated that Chinese cinnamon extract could be used for the treatment of diabetes, we proposed that this spice may be beneficial for the prevention of DN. However, the responsible compounds need to be further identified. In this study, we isolated three new phenolic glycosides, cinnacassosides A–C (1-3), together with fifteen known compounds from the water soluble extract of Chinese cinnamon. The structures of the new compounds were identified by comprehensive spectroscopic evidence. Eleven compounds (6-9, 11, 13-18) were isolated from this spice for the first time, despite extensive research on this species in the past, which added new facets for the chemical profiling of this spice. These isolates were purposely evaluated for their inhibitory effects on IL-6 and extracellular matrix production in mesangial cells which are definitely implicated in DN. The results showed that compounds 4-8 could inhibit over secretion of IL-6, collagen IV and fibronectin against high-glucose-induced mesangial cells at 10 µM, suggesting that Chinese cinnamon could be used as a functional food against DN.


Introduction
Diabetic nephropathy (DN) is a major complication of diabetes and a leading cause of end-stage renal disease (ESRD) [1].For ESRD, the ultimate treatment is renal transplantation.However, due to the insufficient supply of kidneys, the patients who can benefit from renal transplantation are much less than those who should throughout the World.Quite a number of ESRD patients die during the surgery waiting period.Since DN is major factor of ESRD, retarding the progression of DN may be an alternative way to solve or alleviate the gap between kidney supply and demand.Unfortunately, the lack of efficient drugs or advanced technologies for DN in clinical practice makes it quite hard for DN patients to receive ideal treatment.Since it is very difficult to treat DN patients, earlier prevention appears to be more important to slow the progression of DN.Increasing clinical evidences showed that chronic inflammation, overproduction of extracellular matrix and reactive oxygen species in renal cells were implicated in the progression of DN [2,3].Therefore, research targeting on these pathogenic factors may contribute to the prevention of DN.
For the prevention and control of chronic disease, functional foods will be the prior choice because they would be able to avoid adverse effects when taken regularly.Spices and a part of traditional herbs are considered to be important sources of functional foods.Spices have been used as flavoring agents across the World for thousands of years, indicating that spices are both common and safe food adjuncts [4].Cinnamon, as a well-known spice comprising the types Ceylon cinnamon and cassia cinnamon (C.cassia) [5], is reported to be the second most important spice sold in the United States and European markets [6].Due to the wide use of cinnamon, it has attracted much attention in the past [7].Among these studies, many investigations on cinnamon have focused on type 2 diabetes.Polyphenols from cinnamon were reported to have in vitro and in vivo insulin-enhancing biological activity [8,9].A human study showed that consuming cinnamon is beneficial for the health of patients with type 2 diabetes [10].C. cassia (Lauraceae) is an evergreen tree originating in southern China (Guangdong, Guangxi and Yunnan Provinces), and now is widely cultivated there and southern and eastern Asia (Taiwan, Laos, Thailand, Vietnam, India, Indonesia and Malaysia) [11].The dried stem bark of C. cassia, known as Chinese cinnamon or Chinese cassia is sold under the label cinnamon in the United States [12].In China, it has been used as a spice, flavoring agent and preservative in food industry such as confectionery, desserts, pastries, and meat for a long history.Extensive phytochemical studies have been well conducted onC.cassia which revealed the presence of volatile oil, diterpenes [13,14], sesquiterpenoids [15], phenylpropanoids [16], flavonoids [17], and lignans [18].The biological effects of C. cassia components, including antimicrobial [19], antioxidant [20], antiulcerogenic [21], anti-diabetic [22], and analgesic effects [23], are also well documented.We hypothesize that C. cassia could be very important for the prevention of chronic kidney diseases including DN according to its warm, spicy properties.Cinnamic aldehyde, the main constituent of C. cassia volatile oil, has been reported to be used to ameliorate metabolic disorder and relieve renal damage induced by diabetes via targeting Nrf2 activation [24], however, whether the other components, especially water soluble compounds inC.cassia, are beneficial for DN still remains unclear.As part of our continuous studies on the prevention and therapy of diabetic nephropathy, the water soluble fraction of the stem bark of C. cassia was investigated.Two new lignan glycosides (compounds 1 and 2), a new phenolic glycoside 3 (Figure 1), were obtained, together with 15 known compounds 4-18, and their inhibitory effects on DN were investigated using high-glucose-induced mesangial cells.1) revealed six aromatic protons [δ H 6.74 (1H, d, J = 1.5 Hz, H-2), 6.66 (1H, d, J = 8.0 Hz, H-5), and 6.57 (1H, dd, J = 8.0, 1.5 Hz, H-6); 6.84 (1H, s, H-2′), and 6.70 (2H, d, J = 8.0 Hz, H-5′, H-6′)] and two methoxyl [δ H 3.74, 3.73 (6H, s, 3-OCH 3 , 3′-OCH 3 )] signals.The 13 C-NMR spectrum (Table 1) of 1 displayed 26 carbon signals, including one glucopyranosyl moiety [δ C 103.2 (C-1′′), 73.6 (C-2′′), 76.9 (C-3′′), 70.1 (C-4′′), 77.0 (C-5′′), and 61.2 (C-6′′)], two benzene ring, two methoxyl, three methylene (two oxygenated), and three methine (one oxygenated) groups.The 1 H-and 13 C-NMR data of 1 were thus characteristic of a lignan glycoside, and was highly similar to those of 7-hydroxy-9′-β-glucopyranosyloxyl secoisolariciresinol [25].The two compounds have the same molecular formula, indicating they were isomers, the only difference was that the hydroxyl group at C-7 in 7-hydroxy-9′-β-glucopyranosyloxyl secoisolariciresinol was moved to C-7′ in compound 1, which was established by the HMBC correlations (Figure 2) from H-7′ (δ  and C-9′.The 1 H-1 H COSY correlations (Figure 2) of H-7′/H-8′, H-8′/H-9′, H-8′/H-8, H-8/H-7, and H-8/H-9 further confirmed this assumption.The HMBC correlation from H-1′′ (δ H 4.17) to C-9′ confirmed the glucopyranosyl moiety was connected to C-9′.The presence of D-glucose was confirmed by TLC and optical rotation analyses.The configuration of glycosidic linkage of the glucopyranoside moiety in 1 was determined to be β on the basis of the J value of the anomeric proton signal at δ H 4.17 (1H, d, J = 7.8 Hz, H-1′′).Although the ROESY correlations of H-8′/H-7a and H-7′/H 2 -7 were observed, however, it is not helpful for determining the relative configurations at C-8 and C-8′ because of the free rotation of the chain.Thus, the structure of 1 was established to be 7′-hydroxy-9′-β-glucopyranosyloxyl secoisolariciresinol, and the new compound was named cinnacassoside A.  1) showed an ABX coupling system [δ H 6.95 (1H, d, J = 2.0 Hz, H-2′), 6.82 (1H, dd, J = 8.0, 2.0 Hz, H-6′), and 6.77 (1H, d, J = 8.0 Hz, H-5′)], two singlet aromatic protons [δ H 6.76 (1H, s, H-2) and 6.75 (1H, s, H-6)], and two methoxyl [δ H 3.86 (3H, s, 3-OCH 3 ), 3.82 (3H, s, 3′-OCH 3 )] signals.The 1 H-and 13 C-NMR data (Table 1) of 2 showed 31 carbon signals, and were similar to those of symplolignanoside A [26], indicating a lignan glucoside with the same aglycon and saccharide moieties as the latter.Inspection of the 13  2), and C-9′, along with the 1 H-1 H COSY correlations of H-7/H-8, H-8/H-9, H-7′/H-8′, and H-8′/H-9′ further confirmed the linkages of the two C 3 units in compound 2 were the same as those in symplolignanoside A. The two methoxyl groups was deduced to be located at C-3 (δ C 145.3) and C-3′ (149.2) by the HMBC correlations from 3-OCH 3 to C-3 and from 3′-OCH 3 to C-3′, respectively.The 3 J value of H-7′ was 6.3 Hz, along with the ROESY correlation of H-7′/H-9′ indicated the trans-configuration of H-7 and H-8 [27,28].The sugar moieties were assigned as D-glucose and D-apiose by TLC and optical rotation analyses.Thus, the structure of compound 2 was assigned as dihydrodehydrodiconiferyl alcohol-9-β-D-apiofuranosyl (1→6)-β-D-glucopyranoside, and it was named cinnacassoside B.

Inhibition of Fibronectin, Collagen IV, and IL-6 Secretion
Many pathogenic factors are involved in DN, and overproduction of extracellular matrix (such as fibronectin and collagen IV) and proinflammatory factors (such as IL-6) are distinctly implicated in DN [2,41].Considering the traditional medical applications of C. cassia, the isolated compounds were purposely evaluated for their inhibitory effects on fibronectin, collagen IV, and IL-6 overproduction against high-glucose-induced mesangial cells (Figures 3-5).The results showed that compounds 4−8 could inhibit overproduction of fibronectin, collagen IV, and IL-6 against high-glucose-induced mesangial cells at 10 μM, while all the other compounds were not active in this assay at the same concentration.Compound 4 is a novel natural hybrid with a unique geranylphenylacetate carbon skeleton which was previously isolated from the same material [29], however, its biological activities remained unknown yet.This study revealed its possible role in DN.We noted that compounds 3, 8-11 are structurally analogues, however, only 8 was found to be active in this assay.Although compounds 5 and 12 differed only from the presence of apiose or glucose, their bioactivity is different, 5 is active and 12 is inactive.Actually, compound 5 exhibited a broad range of biological effects such as on antidepressant, nootropic, anticancer, neurotropic, immunostimulating and hepatoprotective properties which have attracted great research interest in the past [42][43][44][45][46].Oxidative stress in also one of the major pathogenic factors of DN, in general, the other phenolic compounds should possess antioxidant activity which will be beneficial for DN, however, this need to be further investigated.

Plant Material
The C. cassia barks were collected at Yunnan Xianghui Biological Technology Co. Ltd. (YNXHBT) in July, 2010.The sample was identified by Mr. Fu-Shou Xie at YNXHBT.A voucher specimen (CHYX0173) was preserved at the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, China.

Inhibition of Fibronectin, Collagen IV, and IL-6 Secretion
Rat mesangial cells were grown in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA, USA) containing 5.6 mM D-glucose (pH 7.4; Sigma Chemical Co., St. Louis, MO, USA), supplemented with 20% fetal calf serum (FCS; Invitrogen), 100 U/mL penicillin, 100 μg/mL streptomycin, and 10 mM HEPES.After the mesangial cells reached 80% confluence, their growth was arrested in 0.5% FCS for 24 h.Exposure of the mesangial cells to medium containing high-concentration glucose induced the overproduction of fibronectin (FN), collagen IV (Col IV), and IL-6, as described in the previous reports [47,48].To determine whether the compounds inhibited the FN, collagen IV, and IL-6 overproduction triggered by high glucose, the mesangial cells were pretreated with 1 or 10 μM of each compound for 1 h and then stimulated with high glucose (25 mM) for 24 h.The levels of supernatant FN, collagen IV, and IL-6 were measured with a solid-phase quantitative sandwich enzyme-linked immunosorbent assay (ELISA) kit for FN, collagen IV, and IL-6 (Uscn Life Science Inc., Wuhan, China).