Comparison of pancreatic lipase inhibitory isoflavonoids from unripe and ripe fruits of Cudrania tricuspidata

The composition and content of the active constituents and their biological activity vary according to diverse factors including their maturation stages. A previous study showed that the fruits of Cudrania tricuspidata inhibited pancreatic lipase activity, a key enzyme in fat absorption. In this study, we investigated the chemical composition and pancreatic lipase inhibitory activity of unripe and ripe fruits of C. tricuspidata. Unripe fruits of C. tricuspidata have a higher content of total phenolic and flavonoids and exhibited stronger pancreatic lipase inhibition compared to ripe fruits. HPLC analysis revealed the different chemical compositions of the unripe and ripe fruits. Further fractionation resulted in the isolation of 30 compounds including two new isoflavonoids. Analysis of the chemical constituents of the unripe and ripe fruits revealed that a 2,2-dimethylpyran ring, a cyclized prenyl, was the predominant side chain in the unripe fruits, whereas it was a linear prenyl group in the ripe fruits. In addition, a new isoflavonoid (19) from the unripe fruits showed the most potent inhibition on pancreatic lipase. Taken together, the maturation stage is an important factor for maximum efficacy and that unripe fruits of C. tricuspidata are a good source of new bioactive constituents for the regulation of obesity


Introduction
A global rise in obesity has become a widespread issue due to its association with diverse pathological disorders, including atherosclerosis, diabetes, hypertension, and cancer [1,2]. High consumption of saturated fats in Western diets is suggested as one of the main contributors to obesity, as demonstrated by the correlation between the amount of dietary fat and obesity in epidemiological study. Fat is an ester of three fatty acid and glycerol and absorbed after digestion into monoglyceride and fatty acids by lipase. Lipase is a key enzyme in lipid absorption and pancreatic lipase, a main lipase of the human, is responsible for the hydrolysis of 50-70% of total dietary fats. Therefore, a reduction in fat absorption by the inhibition of pancreatic lipase is suggested to be beneficial for the regulation of obesity [3][4]. A specific pancreatic a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 water. Then, 1 ml of EtOAc was added to each mixture and mixed vigorously. After centrifugation of mixture for 30 s, EtOAc fraction and water fraction were collected, respectively.

Measurement of total flavonoid content
The total flavonoid content was measured with an aluminum chloride colorimetric assay. Samples were added to a 96-well plate, followed by the addition of 5% NaNO 3 . After 5 min incubation, 10% AlCl 3 was added to the reaction mixture. After incubation with gentle shaking, 1 N NaOH and H 2 O were added to the reaction mixture. The absorbance was measured at 510 nm with a microplate reader. The total flavonoid content was expressed as catechin equivalent (NE) using catechin as a standard.

Measurement of total phenolic content
The total phenolic content was measured with a Folin-Ciocalteau assay. Folin-Ciocalteau's phenol reagent was added to the 96-well plate containing the test samples. After 5 min of incubation with gentle shaking, 7% Na 2 CO 3 was added to the reaction mixture. The reaction mixture was left in the dark for 90 min at room temperature. The absorbance was measured at 630 nm with a microplate reader. The total phenolic content was expressed as gallic acid equivalent (GAE) using gallic acid as a standard.

HPLC analysis
Unripe and ripe fruit samples were prepared in methanol at a concentration of 10.0 mg/ml. Each sample solution was filtered through a 0.45 μm membrane filter before HPLC analysis. HPLC analysis was performed using a Waters HPLC system equipped with Waters 600 Qpumps, a 996 photodiode array detector, and Waters Empower software using Phenomenex Gemini-NX 3μ C18 110A (150 x 4.60 mm) for quantitation. Chromatographic separation was accomplished using acetonitrile-water (60:40) at a flow rate of 1.0 mL/min. The wavelengths for the detection and retention time were set at 254 nm and 16 min, respectively.

Pancreatic lipase activity
Pancreatic lipase activity was determined by measuring the hydrolysis of p-nitrophenyl butyrate (p-NPB) to p-nitrophenol using a method reported previously [18]. The 0.1 mg/ml of enzyme solution was prepared by reconstituting porcine pancreatic lipase using 0.1 M Tris-HCl buffer (pH 8). Then, 5 μl of test sample was mixed with 90 μl of enzyme buffer, and incubated for 15 min at 37˚C. After incubation, 5 μl of 10 mM p-nitrophenylbutyrate (p-NPB) was added to enzyme mixture and the reaction was allowed to proceed for further 15 min at 37˚C. After incubation, the absorbance was measured at 405 nm using a microplate reader. Relative pancreatic lipase activity (%) was calculated as [(the activity of the compound with the substrate-the activity of the compound without the substrate)/(activity without the compound and with the substrate-negative control without the compound and substrate)] x 100. Orlistat was used as a positive control.
Pancreatic lipase activity against triglyceride was also determined using Lipase Activity Assay Kit according to manufacturer's protocol (Sigma-Aldrich Co.). Relative pancreatic lipase activity (%) was calculated using glycerol as a standard.

Statistical analysis
The evaluation of statistical significance was determined by a one-way ANOVA test, with a value of p<0.05 or less considered to be statistically significant.

Effects of maturity
Division into unripe and ripe groups. The fruits of C. tricuspidata were collected at different maturation stages and divided into two groups, unripe (green) and ripe (red) fruits, by their color appearance and color-difference meter ( Table 1). The sizes of ripe fruits were much bigger than unripe ones, as determined by weight and diameter. The a Ã value which determines red (+) or green (-) were significantly different between two groups. The b Ã value which determines yellow (+) or blue (-) were also different between two groups, whereas little differences of the L Ã values, which indicated the lightness.
Effect on chemical composition and pancreatic lipase inhibition. To investigate the effect of the maturity of C. tricuspidata fruits on their chemical composition and biological activity, they were divided into unripe and ripe two groups"'( Fig 1A). First, we determined the pancreatic lipase inhibitory activity of unripe and ripe fruits. The inhibitory activity of the total extract of the unripe fruits was stronger than that of the ripe fruits at 100 μg/ml ( Fig 1B). Next, we compared the chemical composition of the unripe and ripe fruits. The water-soluble fraction of the ripe fruit extract was relatively higher than that of the unripe fruits extract (Fig 1C). The flavonoid and phenolic content was much higher in the unripe fruit extract (Fig 1D and  1E). Further HPLC analysis confirmed the difference in the major constituents of the unripe and ripe extract (Fig 2).
Taken together, there was a substantial difference in the chemical composition of the unripe and ripe fruits. The phenolic and flavonoid content was higher in the unripe fruit and lower in the ripe fruit (Fig 1D and 1E). The increase in the dry weight due of the accumulation of sugars and anthocyanins might explain this finding. The red color and the increase of water-soluble fraction in the ripe fruit extracts provide further support for this idea (Fig 1A and 1C). The  differences in the chemical composition affected the pancreatic lipase activity, with the unripe fruit extract showing stronger pancreatic lipase inhibition compared to that the ripe fruit extract ( Fig 1C). Therefore, the maturation stages of C. tricuspidata fruits were suggested to be an important factor for active resources.

Identification of compounds
Isolation of compounds from unripe and ripe fruits. As the HPLC patterns of the unripe and ripe fruits extract of C. tricuspidata were very different, further separation of each unripe and ripe fruit extract was carried out to elucidate the difference in the constituents, depending on maturity.
Pancreatic lipase inhibitory effect. Pancreatic lipase inhibitory activity of isolated compounds was first determined employing in vitro assay system using porcine pancreatic lipase against p-NPB as a substrate. Among the compounds isolated from unripe and ripe fruits, compounds 1, 9, 12, 15, 16, 17 and 19 showed >50% inhibition at 100 μM (Fig 5). In particular, a new compound from unripe fruits (19) showed the most potent inhibition with an IC 50 value of 16.8 μM. Compound 19 also inhibited pancreatic lipase activity against triglyceride substrate with an IC 50 value of 41.8 μM. For the further characterization of the mechanism of the inhibitory effect of compound 19 on pancreatic lipase, Lineweaver-Burk analysis was performed. As the concentration of compound 19 increased, the value for the y-intercept in the equation for each curve increased, whereas the x-intercept remained at a fixed point ( Fig  6). These results suggest that compound 19 exerted an inhibitory effect on pancreatic lipase in a noncompetitive manner.
All the compounds isolated from C. tricuspidata fruits in this study belong to a class of flavonoids. They can be further subdivided according to the position of the phenyl moiety into flavonoids (2-phenylchroman) and isoflavonoids (3-phenylchroman). Compounds 1-22 are isoflavonoids and compounds 23-30 are flavonoids. Regarding structure-activity relationships, all the glycosides (4-8 and 25-30) showed weak activity, pointing to the importance of the absence of a glycoside moiety. The position and number of hydroxyl moiety also affected the inhibitory activity. The isoflavonoids that exhibited >50% inhibition (1, 9, 12, 15-17, and  19) have a free hydroxyl group at C-4'. The addition of a methoxyl group at C-4' (3, 10, and 18) resulted in lower inhibition (34.6, 39.8 and 22.5%, respectively) compared to that of corresponding isoflavonoids (1, 9 and 17), which had stronger inhibitory activity (60.7, 65.7 and 68.2%, respectively). Isoflavonoids containing a 2,2-dimethylpyran ring at C-4' instead of a free hydroxyl group (20)(21)(22) showed weak inhibition (<30%). The number of hydroxyl groups also affected the inhibitory activity, as was observed by the stronger inhibitory activity (92.8 and 83.1%, respectively) the isoflavonoids with two hydroxyl groups at C-3', 4' (12 and 19), compared to that of its corresponding isoflavonoid with a hydroxyl group at C-4' (11 and 17) (31.5 and 68.2%, respectively). The findings showed that the position and number of the Effect of maturation of Cudrania tricuspidata fruits on flavonoids hydroxyl moiety, together with the presence of a glycoside moiety, affected the pancreatic lipase inhibitory activity of isoflavonoids from C. tricuspidata fruits.
Comparison of the compounds in the unripe and ripe fruits. The phytochemical investigation of C. tricuspidata revealed that xanthones were major constituents of the roots, whereas flavonoids were the major constituents of the fruits. Both xanthones of roots and flavonoids of fruits commonly contained isoprenyl moiety as side chains. They existed in diverse forms such as linear, cyclized or oxidized. The HPLC analysis of the unripe and ripe fruits extract revealed that isoflavonoids, with linear isoprenyl moieties, were major constituents of ripe fruits (10 and 11), whereas isoflavonoids with cyclized isoprenyl moieties (13 and 17) were the major constituents of unripe fruits. Further fractionation of the fruits resulted in the isolation of 12 compounds from unripe fruits and 21 compounds from ripe fruits. To better understand the difference in chemical composition between unripe and ripe fruits, isolated compounds were classified into two classes, compounds isolated only from unripe and compounds only from ripe fruits. Consistent with HPLC analysis for the major isoflavonoids, isoprenyl moieties were cyclized in unripe fruits, whereas exist in linear in ripe fruits. In addition, flavonoid glycosides are more abundant in ripe fruits. Although a more comparison is needed to clarify the chemical composition of C. tricuspidata, we cautiously suggest that the chemical composition of C. tricuspidata fruits depends on the maturation stages (Fig 4).
We recently reported the pancreatic lipase inhibitory activity of 6,8-diprenylgenistein, a major isoflavonoid of ripe fruits of C. tricuspidata. This compounds also showed anti-obesity effect in high-fat diet-induced obese mice [19]. Flavonoids are also known as potent antioxidants against oxidative stress which leads to the development of obesity and related complications. Therefore, flavonoids with pancreatic lipase inhibitory effect can be used as anti-obesity therapeutics by the combinatory action, which needs to be clarified with human study.

Conclusions
We investigated the difference in the chemical composition of unripe and ripe fruits of C. tricuspidata. The unripe fruits had a higher content of total phenolics and flavonoids and exhibited stronger pancreatic lipase inhibition compared to the ripe fruits. HPLC analysis revealed the different chemical composition of the unripe and ripe fruits. Further fractionation resulted in the isolation of 30 compounds including two new isoflavonoids from the unripe fruits of C. tricuspidata. Analysis of the chemical composition of the unripe and ripe fruits revealed that 2,2-dimethylpyran ring, a cyclized prenyl was predominant in the unripe fruits, whereas a linear prenyl group in the ripe fruits. In addition, a new isoflavonoid from unripe fruits showed strong pancreatic lipase inhibition in a noncompetitive manner. Therefore, the maturation stage is an important factor in the maximum efficacy, and unripe fruits of C. tricuspidata are appeared to be a good source of new bioactive constituents for the regulation of obesity.