Black rice and anthocyanins induce inhibition of cholesterol absorption in vitro

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ISSN 2042-6496

Food & FunctionLinking the chemistry and physics of food with health and nutrition

www.rsc.org/foodfunction Volume 2 | Number 5 | May 2011 | Pages 215–280

COVER ARTICLESchini-Kerth et al.Evaluation of diff erent fruit juices and purees and optimization of a red fruit juice blend 2042-6496(2011)2:5;1-7

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1

Black Rice and Anthocyanins Induce Inhibition of Cholesterol 1

Absorption in Vitro 2

Shu-Long Yao1, Ying Xu

1, Yan-Yan Zhang

2, Yan-Hua Lu*

1 3

1State Key Laboratory of Bioreactor Engineering, East China University of Science 4

and Technology, Shanghai 200237, China 5

2College of Tourism and Food Science, Shanghai Business School, Shanghai 200235, 6

China 7

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*Corresponding author 15

Dr. Yan-hua Lu 16

State Key Laboratory of Bioreactor Engineering 17

East China University of Science and Technology 18

Box 283#, 130 Meilong Road, Shanghai 200237, PR China 19

E-mail: [email protected] 20

Phone: +86-21-64251185 21

Fax: +86-21-64251185 22

Page 1 of 32 Food & Function

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View Article OnlineDOI: 10.1039/C3FO60196J


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Abstract 1

Black rice (Oryza sativa L.) is often associated with blood lipids control. This 2

study systematically assessed the inhibition of cholesterol absorption in vitro by black 3

rice and explored cholesterol-lowering compounds of this rice. Our results indicated 4

that black rice extracts (BRE), which were aqueous, ethanol extracts and a fraction of 5

macroporous resin, caused the reduction of cholesterol absorption by inhibiting 6

pancreatic lipase, decreasing the micellar cholesterol solubility, and suppressing 7

cholesterol uptake in Caco-2 cells. The inhibitory activity was positively associated 8

with anthocyanins (cyanidin-3-glucoside (Cy-3-G) and peonidin-3-glucoside (Pn-3-G)) 9

contents of the extracts. Therefore, the cholesterol absorption inhibiting properties 10

of anthocyanins were further explored. The IC50 values of Cy-3-G and Pn-3-G against 11

pancreatic lipase were 42.53 ± 4.45, 18.13 ± 4.22 µg/mL, respectively. Kinetic 12

analysis suggested that the enzymatic inhibitory mode of Cy-3-G and Pn-3-G 13

belonged to the competitive type. In mixed micelles, Cy-3-G and Pn-3-G dose 14

dependently reduced the solubility of cholesterol. Meanwhile, a potential mechanism 15

of cholesterol reduction by anthocyanins was investigated. Results showed that 16

anthocyanins led to precipitation of cholesterol from micellar solution, which may 17

induce the reduction of cholesterol. In Caco-2 cells, Cy-3-G and Pn-3-G (40 µg/mL), 18

exhibited a significant reduction in cholesterol uptake, and the degree of this 19

reduction was almost the same as that observed in the group treated with Ezetimibe at 20

the same concentration. These findings provide important evidence that anthocyanins 21

may partly contribute to the inhibitory effects of black rice on cholesterol absorption, 22

Page 2 of 32Food & Function

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and thus may be applied for the prevention and treatment of hypercholesterolaemia. 1

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Introduction 1

Hypercholesterolaemia is a group of metabolic disorders characterized by the 2

elevated levels of cholesterol in the blood. Although many drugs are available to 3

manage hypercholesterolemia, the first strategy in the reduction of 4

hypercholesterolaemia is by diet and lifestyle changes 1

. However, lifestyle changes 5

are not easily achieved and sustained by most people. As a result, healthy diet, 6

especially in forms of dietary supplements, continues to be attractive and 7

cost-effective treatments. In recent years, it has suggested that natural components 8

from food and/or herbal medicines could be recommended as cholesterol-lowering 9

agents 2-8

. 10

Cholesterol absorption, an effective way of lowering cholesterol levels, provides 11

multiple therapeutic targets in the management of hypercholesterolemia 9,10

. 12

Cholesterol absorption is a complex process that involves multiple pathways. 13

Cholesterol is initially solubilized into the mixed micelles formed by bile salts and 14

lipids catalysed by pancreatic lipases and then presented as a micelle to the brush 15

border of mucosal enterocytes for uptake 11

. After uptake by enterocytes, cholesterol 16

occurs in intracellular transport and packaging of cholesterol into chylomicrons for 17

secretion to the lymphatic system 12

. It is well known that delaying triglyceride 18

digestion with the inhibition of pancreatic lipase may suppress cholesterol absorption 19

13,14. Recent reports have indicated that reducing cholesterol solubility is a new target 20

strategy for the reduction of cholesterol absorption and the treatment of 21

hyperlipidaemia 15,16

. Moreover, cholesterol absorption is a multistep process that is 22


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regulated by multiple genes in enterocytes. Cholesterol absorption might therefore be 1

suppressed by inhibiting pancreatic lipase activity, deceasing micellar solubilization 2

of cholesterol or reducing cholesterol uptake in enterocytes. 3

Black rice (Oryza sativa L.) has been regarded as a health-promoting food and 4

widely consumed in China and other Eastern Asian countries. Many studies have 5

indicated this rice to have a wide range of beneficial properties including 6

hepatoprotective 17

, anticancer 18

, antioxidant 19,20

, anti-inflammatory 21

. Recent 7

studies on chemical and biological properties of black rice were identified as two 8

major anthocyaninis: cyanidin-3-glucoside (Cy-3-G) and peonidin-3-glucoside 9

(Pn-3-G) 22,23

. In recent years, several studies have shown that supplementation of 10

black rice extracts (BRE) rich in anthocyanins or anthocyanins resulted in a decrease 11

in serum triglyceride, total cholesterol, non-HDL cholesterol in different animal 12

models 24,25

. These studies indicate that BRE or anthocyanins possess the capacity to 13

prevent or treat hypercholesterolaemia. However, available results mainly focused on 14

the effects of BRE or anthocyanins on cholesterol homeostasis or lipid metabolism in 15

the whole body. Existing studies have neither researched the effects of BRE on 16

cholesterol absorption nor investigated whether anthocyanins could contribute to the 17

effects. 18

The present study therefore aimed to investigate the effects of BRE on (1) 19

pancreatic lipase activity, and (2) cholesterol solubilization in mixed micelles, and (3) 20

cholesterol uptake in Caco-2 cells, and to confirm that the actions of BRE may be 21

partly attributed to anthocyanins. 22


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Materials and methods 1

Chemicals and reagents 2

Lipase from porcine pancreas type II, 4-methylumbelliferyl Oleate (4-MU), 3

phosphatidylchline, sodium taurocholate were purchased from Sigma-Aldrich 4

(Shanghai, China). Ezetimibe was purchsed from Pichemicals (Zhang Jiang Hi-Tech 5

Park, Shanghai , China) 6

22-(N(-7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3-ol 7

(NBD-Cholesterol) was obtained from Invitrogen (Eugene, Oregon, USA). Tris, 8

hydrochloric acid (HCl) were obtained from Sinopharm Chemical Reagent Co., Ltd. 9

(Shanghai, China). Anthocysnin standards Cy-3-G and Pn-3-G were purchased from 10

Tokiwa Phytochemical Co., Ltd. (Tokyo, Japan). The other reagents were of analytical 11

grade. 12

Sample preparation 13

Black rice was purchased from the Shanxi Province Rice Research Institute, 14

China. An aqueous extract (EH2O) was prepared as the procedure described previously 15

19 with some modifications. Briefly, the whole rice was ground to powder with a 16

laboratory mill and passed through a 60 mesh screen sieve to obtain the aleurone layer, 17

and then one hundred grams of the aleurone layer was mixed with 700 mL of distilled 18

water for 2.5 h at 40 °C, and vacuum filtered through Whatman filter paper. Similarly, 19

an ethanol extract (EEtOH) was prepared with a minor modification as described above. 20

Ethanol was replaced with distilled water to ethanol/water/HCl (70:30:1, v/v/v) of the 21

solid - liquid ratio 1:10 and other conditions are the same as described above. The 22


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filtrates were subjected to vacuum evaporation to remove ethanol. To prepare a 1

fraction of macroporous resin (EEnr), part of ethanol filtrates was loaded onto a D101 2

resin. D101 resin was washed with distilled water, and subsequently adsorbed 3

anthocyanins were eluted with 80% ethanol. The ethanol eluent was collected and 4

vacuum evaporation to remove ethanol. BRE were stored at -20 °C after 5

lyophilization. 6

Quantification of anthocyanins in BRE 7

Anthocyanins in BRE were identified using a Shimadzu LC-20A HPLC equipped 8

with a diode array detector by comparing their retention time and UV spectrum. The 9

Cy-3-G and Pn-3-G contents of BRE were determined by their standard curves. An 10

Aglient Eclipse Plus C18 column (250 mm * 4.6 mm, particle size of 5 µm, Agilent 11

Technologies Inc., St. Clara, CA) used at 30 °C was employed with a linear gradient 12

mobile phase containing solvent A (phosphate buffered saline, PBS, pH 2.5) and 13

solvent B (PBS, pH 2.5:acetonitrile = 20:80, v/v) at a flow rate of 1.0 mL/min. 14

Gradient elution was performed with 10-45% B from 0 to 15 min, and then 45-100% 15

B was performed from 15 to 30 min. Injection volume was 20 µL and the wavelength 16

was set at 520 nm. 17

Determination of pancreatic lipase activity 18

Pancreatic lipase activity was measured by using 4-MU as a substrate, as 19

described by You, Q and his co-workers 26

with slight modifications. Lipase from 20

porcine pancreas was dissolved in distilled water (0.5 mg/mL) and then centrifuged at 21

6000 rpm for 10 min to remove insoluble components. BRE, Cy-3-G and Pn-3-G 22


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were diluted in methanol at specified concentrations. An aliquot of 5 µL of diluted 1

sample solution was added into an aliquot of 25 µL of pancreatic lipase solution in 2

Tris-HCl buffer solution (pH 8.0) and mixed with 50 µL of a 0.5 mM 4-MU solution 3

dissolved in the above buffer in a 96-well microplate to start the enzyme reaction. The 4

plate was immediately placed at 37 °C. After incubation for 30 min, the amount of 5

4-methylumbelliferone released by lipase was measured with a fluorescence reader at 6

an excitation wavelength of 340 nm and an emission wavelength of 460 nm. Orlistat 7

was used as a positive control. The IC50 values of the test sample was obtained from 8

the least-squares regression line of the plots of the logarithm of the sample 9

concentration (log) versus the pancreatic lipase activity (%). 10

To identify the inhibition mode of Cy-3-G and Pn-3-G against lipase, the 4-MU 11

solutions at 100, 125, 200, 250, 500 µM for the pancreatic lipase activity were used to 12

react with the methanol (control), Cy-3-G and Pn-3-G to make the Lineweaver-Burk 13

plot from the Michaelis-Menten equations. 14

The solubility of cholesterol in mixed micelles 15

NBD-Cholesterol was used to explore the effects of BRE, Cy-3-G and Pn-3-G on 16

the solubility of cholesterol in mixed micelles which was prepared by the method 17

described by Ikeda et al. 27

. A mixed micellar solution containing 0.6 mM 18

phosphatidylcholine, 0.5 mM NBD-Cholesterol, 6.6 mM sodium taurocholate, 132 19

mM NaCl and 15 mM sodium phosphate at pH 7.4 was prepared by sonication and 20

kept at 37 °C for 12 h. An aliquot of 20 µL solution of various concentrations of BRE, 21

Cy-3-G or Pn-3-G was added into an aliquot of 180 µL solution of mixed micells. 22


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Immediately after the addition of the sample, the mixture was placed at 37 °C for 30 1

min and centrifuged at 13400 x g for 20 min. The supernatant was transferred to a 2

96-well fluorescent plate to measure the fluorescence at an excitation wavelength of 3

462 nm and an emission wavelength of 536 nm. PBS was used as a control. In the 4

Cy-3-G experiment, Cy-3-G was added to the micellar solution and its concentration 5

in the supernatant was measured by the method described above. 6

Cholesterol uptake by Caco-2 cells 7

Cholesterol uptake by Caco-2 cells was carried out as described previously 28

. 8

Colon adenocarcinoma (Caco-2) cells from the Shanghai Institutes for Biological 9

Sciences, Cas (Shanghai, China) were maintained in Dulbecco’s Modified Eagle’s 10

Medium (DMEM) containing 20% fetal bovine serum, 1% penicillin/streptomycin, 1% 11

nonessential amino acids. Cells were cultured at 37 °C in a humidified incubator with 12

5% CO2. Caco-2 cells were seeded on 24-well plate at a cell density of 50,000 cells 13

per well and cultured for 14 days to allow them to differentiate. During this period, 14

the cells were fed with fresh medium for every 2 days. After 14 days, cells were 15

plated in 96-well plates at a density of 2 × 104 cells/well for experiments. After 16

culture for 3 days, various concentrations of test samples, the positive control 17

(Ezetimibe) and 2 µg/mL of NBD-Cholesterol were added to the well to incubate for 18

4 h at 37 °C. The reaction was stopped by removal of the medium. Cells were washed 19

three times with ice-cold PBS and the fluorescence was determined at an excitation 20

wavelength of 462 nm and an emission wavelength of 536 nm. Cells were then 21

disrupted with 1 M NaOH for 12 h at 4 °C to determine protein concentration by the 22


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Bradford assay. 1

Data analysis 2

Data from each assay are means ± standard deviation (SD). Statistical analyses 3

were performed using one-way analysis of variance (ANOVA) followed by Tukey 4

adjustment. All analyses were performed using GraphPad Prism 5.0 software Inc. 5

Differences were considered to be significant when the p value is lower than 0.05. 6

Results 7

Quantification of anthocyanins in BRE 8

The anthocyanin extracts of black rice were directly analyzed by HPLC as shown 9

in Figure 1. As can be seen, peak 1 and 2 were assumed to the Cy-3-G and Pn-3-G, 10

respectively. Other peaks could not be identified because of lack of reference 11

standards. The total content of Cy-3-G and Pn-3-G in EH2O, EEtOH, EEnr was 2.10±12

0.08, 9.01±0.30, 37.35±0.27%, respectively. 13

In vitro inhibitory effects of BRE on cholesterol absorption 14

Inhibitory activities (IC50) listed in Table 1 were utilized to measure the inhibitory 15

effects of BRE on pancreatic lipase. Table 1 shows that EEnr is a stronger inhibitor 16

than EH2O as well as EEtOH. However, the IC50 values of BRE were much larger than 17

those of orlistat. In micellar solution, the effect of BRE on cholesterol solubility was 18

shown in Figure 2. The solubility of cholesterol in mixed lipid micelles appeared to be 19

dose dependently decreased in the presence of BRE. At selected concentrations 20

(0.05-1.0mg/mL), the cholesterol reduction by BRE was in the following order: EEnr > 21

EEtOH > EH2O. In Caco-2 cells, cholesterol uptake was depressed in a dose dependent 22


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manner by BRE. EEnr exhibited the value of reduction was almost the same as that 1

observed in the group treated with the positive control at 50 µg/mL (Table 2). 2

On the basis of results provided above, it hypothesizes that anthocyanins 3

contribute to the actions of BRE. Thus, we further analyzed the effects of 4

anthocyanins on cholesterol absorption. 5

Competitive inhibition of anthocyanins against pancreatic lipase 6

It has shown that both Cy-3-G and Pn-3-G dose dependently inhibited the activity 7

of lipase in Figure 3. The IC50 values of Cy-3-G and Pn-3-G were 42.53 ± 4.45, 18.13 8

± 4.22 µg/mL, respectively. Furthermore, the inhibitory mode of anthocyanin was 9

characterized by the kinetic method. It has shown their Lineweaver-Burk plots in 10

Figure 4. The L-B plots deduced inhibition linear equations for the control as y = 11

3.9482x + 0.0266 (R2

= 0.9947), for Cy-3-G as y = 4.7941x + 0.0264 (R2 = 0.9959) 12

and for Pn-3-G as y = 5.8596x + 0.0266 (R2 = 0.9952). Although mathematical 13

equations for all of the inhibitors and control differed in slopes, their y-intercepts were 14

nearly the same, indicating their enzymatic inhibitory mode belonged to the 15

competitive type. 16

Reduction of cholesterol solubility by anthocyanins 17

As shown in Figure 5, after addition of Cy-3-G or Pn-3-G, the solubility of 18

cholesterol was markedly decreased. At 100 µg/mL, Cy-3-G and Pn-3-G 19

approximately prevented half cholesterol solubilized into micellar solution. In order to 20

explore possible mechanisms of cholesterol reduction by anthocyaninis, the 21

concentration of Cy-3-G in the supernatant was determined. The amount of Cy-3-G 22


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precipitated from micellar solution increased with an increase in Cy-3-G added to the 1

micelles (Figure 6). Moreover, the amount of Cy-3-G was almost linearly correlated 2

with precipitated cholesterol (R2= 0.96). 3

Effects of anthocyanins on cholesterol uptake in Caco-2 cells 4

To examine the effect of anthocyanins on cholesterol uptake in enterocytes, 5

Caco-2 cells were used as an in vitro model. In this study, Ezetimibe, an inhibitor 6

cholesterol across the gut wall, was used as a positive control, and it caused a 7

substantial depression of cholesterol uptake. In Figure 7, it showed that both Cy-3-G 8

and Pn-3-G significantly and in a dose dependent manner suppressed cholesterol 9

uptake. At 40 µg/mL, Pn-3-G possessed virtually equivalent reduction of cholesterol 10

uptake compared with Ezetimibe. 11

Discussion 12

Xia et al. reported that supplementation of an anthocyanin-rich extract improved 13

the lipid profile by decreasing serum triglyceride, total cholesterol, and non-HDL 14

cholesterol in apolipoprotein E-deficient mice 25

. It was also found that 15

supplementation of atherogenic experimental diets with a black rice extract effectively 16

decreased lipid levels in hypercholesterolemic rats 29

. These results suggested that 17

black rice possesses beneficial effects on lipid profiles. However, these studies mainly 18

focused on the effects of black rice on lipid profiles in the whole body. Few studies 19

investigated the effects of black rice on cholesterol absorption which plays an 20

important role in maintaining total body cholesterol balance. The present study for the 21

first time showed that black rice induced inhibition of cholesterol absorption in vitro. 22


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Recently, it has been reported that berry consumption increased serum HDL 1

concentrations by 5.2% 30

. In another report, Zern and colleagues found that grape 2

powder modestly lowered plasma LDL cholesterol in women 31

. Moreover, it has been 3

found that moderate red wine consumption for 4 wk was associated with an increase 4

of 11-16% in HDL cholesterol in healthy human subjects 32

. These effects were 5

considered to be attributed to anthocyanins, flavans, quercetin, myricetin, kaempferol, 6

and resveratrol or other phytochemicals. Our results firstly indicated that pure 7

anthocyanins (Cy-3-G, Pn-3-G) resulted in a reduction of cholesterol absorption in 8

vitro, suggesting that anthocyanins may partly contribute to the actions of black rice. 9

Pancreatic lipase is a key enzyme in hydrolyzing triacylglycerol into 10

monoacylglycerols and non-esterified fatty acids, which may form micelles that serve 11

as necessary intermediates for cholesterol uptake in enterocytes 33

. Our results 12

indicate that BRE possess inhibitory activity against lipase (Table 1). Also, the 13

inhibitory ability of BRE is correlated with the content of anthocyanins, which 14

suggest that the anthocyanins in black rice may perform the inhibitory activity. 15

Cy-3-G and Pn-3-G also showed inhibitory activities against the pancreatic lipase 16

(Figure 3). Although their potencies are weaker than orlistat, anthocyanins might 17

provide a sufficient amount to inhibit pancreatic lipase because of their natural 18

resources. Recently, most of researches only reported the inhibitory activity of the test 19

samples against lipase within the range of concentrations instead of inhibitory mode. 20

In this study, inhibitory mode was determined from the Lineweavere-Burk plots of 21

Cy-3-G and Pn-3-G compared with the control. The result shown in Figure 4 is 22


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similar to You, Q and his co-workers’ report that cyanindin-3,5-diglucoside and 1

cyanidin fell into the competitive inhibition mode against the pancreatic lipase 26

. 2

Micellarization of cholesterol is necessary to facilitate its absorption into 3

intestinal mucosal cells in vivo. Direct evidence documenting the role of black rice in 4

the solubility of cholesterol is limited. Our data firstly suggest that micellarization of 5

cholesterol is inhibited by BRE (Figure 2). Also, several phytochemicals have been 6

proved to decrease cholesterol solubility, particularly phytosterols and tea catechins 7

34-36. It is hypothesized that the presence of anthocyanins may prevent micelles 8

information or cholesterol incorporation into micelles because the structures of 9

anthocyanins are similar to those of catechins. Our results indicate that cholesterol 10

solubilized in mixed bile salt micelles is markedly eliminated by the addition of 11

anthocyanins (Figure 5). Since anthocyanins added to the micellar solution 12

precipitated with cholesterol and since a positive correlation between the 13

concentration of precipitated cholesterol and anthocyanins is observed (Figure 6), it is 14

likely that anthocyanins form insoluble precipitates with cholesterol in mixed micellar 15

solution. Moreover, apart from cholesterol, bile acid is also an essential component in 16

mixed micelles. We did not determine the effect of anthocyanins on bile acid in this 17

study. Thereby the possibility cannot be ruled out that the water soluble complex of 18

anthocyanins and bile acid formed disrupts the micelles and consequently causes the 19

precipitation of cholesterol. Therefore, more detailed studies are necessary for the 20

reason of binding between cholesterol and anthocyanins. 21

Enterocytes play an important role in cholesterol absorption. In this study, Caco-2 22


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cells, a well-established model system for the investigation of transport across the 1

small intestinal epithelium, were selected as an in vitro model to research cholesterol 2

uptake 37

. Our results firstly demonstrate that BRE can potently reduce cholesterol 3

uptake in Caco-2 cells (Table 2). Cy-3-G and Pn-3-G also significantly induce 4

inhibition of cholesterol uptake in enterocytes (Figure 7). Therefore, it is considered 5

that the action of BRE may be partly due to anthocyanins. This reduction of 6

cholesterol uptake was not due to influences of samples on cell viability, since the cell 7

viability was not significantly affected by samples at the tested concentration (data not 8

shown). To our knowledge, no previous reports demonstrate that cholesterol uptake 9

(in vitro) is inhibited by anthocyanins in Caco-2 cells. As the first and a highly potent 10

antihyperlipidemic drug, Ezetimibe inhibits the function of NPC1L1, the transporter 11

of cholesterol in enterocytes, to limit the uptake of cholesterol 11

. Recently, a critical 12

role of the ATP-binding cassette transporter family (i.e. ABCA1, ABCG5, ABCG8) in 13

the regulation of cholesterol absorption has been suggested 38,39

. It is probable that 14

anthocyanins may influence the uptake of cholesterol in enterocytes through 15

interaction with transporters, particularly those exposed to the intestinal lumen. 16

Therefore, effects of anthocyanins on these transporters are required to be 17

investigated. 18

Anthocyanins are widely distributed in the human diet through crops, fruits, and 19

vegetables 40

, indicating that we ingest considerable amounts of anthocyanin pigments 20

from daily diets. Cy-3-G and Pn-3-G are among anthocyanins that have higher 21

contents in plants. It is suggested that a American typical diet contributes 180-215 mg 22


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of daily intake of anthocyanins 41

. He and colleagues reported that anthocyanins 1

uptake by small intestinal tissue reached 7.5% of the administered dose 42

. Intact 2

Cy-3-G was also found in human plasma within 30 min after oral administration of 3

blueberry extract and about 1% of anthocyanins was incorporated into rat plasma after 4

oral supplementation 43

. Based on the reported data, in this paper we used high 5

concentrations of anthocyanins to obtain conspicuous results. Due to the presence in 6

the intestinal lumen, anthocyanins likely influence the initial luminal processes of 7

lipid hydrolysis, micelle formation, and the transfer of cholesterol to the enterocyte 8

for uptake. Thus, the relevance of the in vitro studies to the in vivo situation needs to 9

be further confirmed. 10

Conclusion 11

Our results for the first time support the notion that black rice could reduce 12

cholesterol absorption in vitro. Anthocyanins may partly contibute to the beneficial 13

effects of black rice, which reduces cholesterol absorption via inhibition of pancreatic 14

lipase activity, decrease of cholesterol solubility in micelles and suppression of 15

cholesterol uptake in enterocytes. Overall, black rice can be regarded as an ideal 16

natural source to induce anti-hypercholesterolemia effects. 17

Acknowledgments 18

This work was supported by “the Fundamental Research Funds for the Central 19

Universities”, and partially supported by Shanghai Leading Academic Discipline 20

Project (B505), the National Special Fund for State Key Laboratory of Bioreactor 21

Engineering (2060204). 22


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Abbreviations 1

Cy-3-G, cyanidin-3-glucoside 2

Pn-3-G, peonidin-3-glucoside 3

BRE, black rice extracts 4

NBD-Cholesterol, 5

22-(N(-7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3-ol; 6

4-MU, 4-methylumbelliferyl 7

HPLC, high-performance liquid chromatography 8

Caco-2, colon adenocarcinoma cells 9

DMEM, Dulbecco’s Modified Eagle’s Medium 10

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Figure captions 1

Figure 1 HPLC profile of BRE at 520 nm. (A) EH2O, (B) EEtOH, (C) EEnr. Peak 2

identification: (1) Cy-3-G, (2) Pn-3-G 3

Figure 2 Effects of BRE on micellar solubility of cholesterol in vitro. Values are 4

expressed as the mean ± SD of at least three independent experiments 5

Figure 3 Inhibition curve of pancreatic lipase activity by Cy-3-G and Pn-3-G. Values 6

are expressed as the mean ± SD of at least three independent experiments 7

Figure 4 Lineweaver – Burk plots of control, Cy-3-G and Pn-3-G for the pancreatic 8

lipase inhibitory activity 9

Figure 5 Effects of Cy-3-G and Pn-3-G on micellar solubility of cholesterol in vitro. 10

Values are expressed as mean ± SD of at least three independent experiments 11

Figure 6 Concentration of Cy-3-G and cholesterol coprecipitated from mixed micellar 12

solution. Values are expressed as the mean ± SD of at least three independent 13

experiments. An amount of cholesterol precipitated was calculated from the data in 14

Figure. 5 15

Figure 7 Effects of Cy-3-G and Pn-3-G on cholesterol uptake in Caco-2 cells. Values 16

are expressed as mean ± SD from five to six experiments. 17

a Significantly different from control, p < 0.05 18

b Significantly different from control, p < 0.01 19

20


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Table 1 IC50 on pancreatic lipase for BRE 1

Inhibitor EH2O EEtOH EEnr Orlistat

IC50(µg/mL) 63.19 ± 3.08 31.70 ± 3.75 19.70 ± 1.62 0.057 ± 0.017

Values are expressed as mean ± SD from three experiments 2

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Table 2 Effects of BRE on cholesterol uptake in Caco-2 cells 1

BRE

Inhibition Rate (%)

6.25 µg/mL 25 µg/mL 50 µg/mL

EH2O 11.75 ± 4.51 18.91 ± 0.13a 32.73 ± 8.39

b

EEtOH 16.19 ± 7.78a 28.28 ± 2.20

b 41.87 ± 7.97

b

EEnr 17.67 ± 1.07a 38.67 ± 12.68

b 57.78 ± 5.08

b

Ezetimibe 57.80 ± 1.14b

Values are expressed as mean ± SD from five to six experiments 2



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1

Figure 1 HPLC profile of BRE at 520 nm. (A) EH2O, (B) EEtOH, (C) EEnr. Peak 2

identification: (1) Cy-3-G, (2) Pn-3-G 3

4

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1

Figure 2 Effects of BRE on micellar solubility of cholesterol in vitro. Values are 2

expressed as the mean ± SD of at least three independent experiments 3

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1

Figure 3 Inhibition curve of pancreatic lipase activity by Cy-3-G and Pn-3-G. Values 2

are expressed as the mean ± SD of at least three independent experiments 3

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1

Figure 4 Lineweaver – Burk plots of control, Cy-3-G and Pn-3-G for the pancreatic 2

lipase inhibitory activity 3

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1

Figure 5 Effects of Cy-3-G and Pn-3-G on micellar solubility of cholesterol in vitro. 2

Values are expressed as mean ± SD of at least three independent experiments 3

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1

Figure 6 Concentration of Cy-3-G and cholesterol coprecipitated from mixed micellar 2

solution. Values are expressed as the mean ± SD of at least three independent 3

experiments. An amount of cholesterol precipitated was calculated from the data in 4

Figure. 5 5

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1

Figure 7 Effects of Cy-3-G and Pn-3-G on cholesterol uptake in Caco-2 cells. Values 2

are expressed as mean ± SD from five to six experiments. 3



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Table of contents entry 1

2

Black rice (Oryza sativa L.) and anthocyaninis (Cy-3-G, Pn-3-G) inhibit cholesterol 3

absorption in vitro by inhibiting pancreatic lipase, reducing cholesterol solubility in 4

mixed micells and suppressing cholesterol uptake in the enterocytes. 5


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Documents

Black rice and anthocyanins induce inhibition of cholesterol absorption in vitro