In vitro study of the effects of viscous soluble dietary fibers of Abelmoschus esculentus L in lowering intestinal glucose absorption




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In vitro study of the effects of viscous soluble dietary fibers of Abelmoschus esculentus L in lowering intestinal glucose absorption

Mst. Hajera Khatun1, Md. Ajijur Rahman2, Mohitosh Biswas3 and Md. Anwar Ul Islam2*



1Department of Pharmacy, Atish Dipankar University of Science and Technology, Dhaka, Bangladesh.

2Deaprtment of Pharmacy, University of Rajshahi, Rajshahi-6205, Bangladesh.

3Deaprtment of Pharmacy, The University of Asia Pacific, Dhaka-1209, Bangladesh.
Corresponding Author:

Md. Anwar Ul Islam, Ph.D

Department of Pharmacy,

University of Rajshahi, Rajshahi-6205, Bangladesh.

E-mail: profanwarulislam@yahoo.com

Tele: +88-0721-750041 (Ext. 4110)




In vitro study of the effects of viscous soluble dietary fibers of Abelmoschus esculentus L in lowering intestinal glucose absorption

Mst. Hajera Khatun1, Md. Ajijur Rahman2 Mohitosh Biswas3 and Md. Anwar Ul Islam2*



1Department of Pharmacy, Atish Dipankar University of Science and Technology, Dhaka, Bangladesh.

2Deaprtment of Pharmacy, University of Rajshahi, Rajshahi-6205, Bangladesh.

3Deaprtment of Pharmacy, The University of Asia Pacific, Dhaka-1209, Bangladesh.

Abstract:

The edible green fruits or pods of Abelmoschus esculentus L (vendi/lady’s finger) are popular all over the world for possessing valuable nutrients including fibers, vitamins, minerals, proteins and carbohydrates, nearly half of which is soluble fiber in the form of gums and pectins. Traditionally, they has been used in lowering serum cholesterol, relieving constipation and healing ulcers.The objective of the present study was to investigate the effects of Na-Carboxymethylcellulose (Na-CMC) and viscous soluble dietary fibers (VSDF) of the fruits of Abelmoschus esculentus L on intestinal glucose absorption using in-vitro model. Different diffusion systems were prepared using different concentrations of viscous water soluble portion of the fruits of Abelmoschus esculentus L and Na-CMC solution with fixed concentration of glucose solution (0.1 g/ml) and the diffusion of glucose from these systems into outer solution through the ultra-fine membrane was measured. Substantial reductions of diffusion of glucose from water soluble portion of the pods of Abelmoschus esculentus L and Na-CMC diffusion systems were observed compared to control in a concentration-dependant manner (P<0.05) which implicates a possible potential role of VSDF of fruits of Abelmoschus esculentus L in lowering postprandial serum glucose.



Key words: Viscous Soluble Dietary Fiber (VSDF), Abelmoschus esculentus L, Glucose diffusion, Diabetes.
Introduction:

Consumption of a diet high in soluble fiber has been suggested as a strategy to reduce the risk factors for development of obesity through the regulation of satiety and energy intake (Pereira et al., 2001). In addition, beneficial long-term effects of soluble viscous dietary fibers on blood glucose and lipids have been observed in diabetic subjects (Aro et al., 1981 and Ray et al., 1983). Glucose tolerance and insulin sensitivity are the key issues involved in the development of non-insulin dependent diabetes mellitus (NIDDM) in humans and soluble dietary fibers (SDF) have been shown to improve glucose tolerance and insulin sensitivity (Smith et al., 1997 and Ellis et al., 1996). Several studies suggest that these fibers exert such an effect by reducing the glucose absorption rate (Vaugelade et al., 2000). Moreover, viscous water-soluble dietary fibers have the effects of hampering the diffusion of glucose and postponing the absorption and digestion of carbohydrates, thus resulting in lower postprandial blood glucose (Yokoyama et al., 1997).


The green edible pods of Abelmoschus esculentus L. (Family: Malvaceae) is extensively used globally as a vegetable for its nutritional and health benefits (Begum et al., 2005). In Bangladesh, it is called vendi and one of the most popular vegetables because of its nutritional benefits, availability, and low cost. It constitutes minerals, vitamins, proteins, carbohydrates, enzymes and very high quantities of mucilage which are rich in soluble dietary fiber as pectin, guar gum, carboxymethylcellulose etc. (Alam et al., 2007). Traditionally it is believed that it is useful in the treatment of inflammatory disorders, constipation, retention of urine, lowering blood cholesterol and blood glucose level.

The previous studies reported that vendi possess hypo-lipidemic effect (Trinh et al., 2009). However, the effect of vendi on long term glycemic control is less clear. This study reveals the effects of viscous water-soluble portion of the pods of Abelmoschus esculentus L (vendi) and Na-CMC on intestinal glucose absorption using in vitro model.


Materials and Methods:
Preparation of viscous water soluble portion of Abelmoschus esculentus L:
500gm of fresh pods or fruits of Abelmoschus esculentus L were collected in the month of May, 2009 from a local market of Rajshahi, Bangladesh. Then, the pods were thoroughly washed with distilled water, cut into small slices (Fig-1) by a sharp knife and 15, 30, 45, 60 and 75 gm of the sliced pods were immersed into 150 ml distilled water in different beakers. The mixture was then stirred gently for 10 to 15 minutes with a glass rod. After 24 hours the contents were filtered using a thin layer of cotton to remove the insoluble matters. The viscous water soluble portions (VWSP) that contain the soluble dietary fibers were passed through the filter and filtrate was collected. The amount of filtrates were measured and numbered as solution A to E from low to high concentration (Table-1). In 50 ml beakers 1 milliliter of each solution was taken and dried in an oven at 85C for 12 hours and the weighed to measure the amount of dried constituents (Table-2).

1% w/v Na-CMC solution was prepared was prepared by warming the solution at 30°C in a water bath for 5 minutes and then vortexing at 4000 r.p.m for 5 minutes. The Na-CMC used in this study was obtained from Fluka, Switzerland. It had a dynamic viscosity of 1000 mPa.s (Pa.s= Pascal second) in 2% w/v water solution. The pH was 6.5-8.0.


Preparation of diffusion systems of glucose with Na-CMC and VWSP of the fruits of Abelmoschus esculentus L: Different diffusion systems were prepared according to the proportion mentioned in Table-3:
Determination of diffusion of glucose from the different systems:

To observe the effects of dietary fibers on intestinal glucose and drug absorption, in vitro model using dialysis method has been reported (Yokoyama et al., 1997). But due to limited facilities in our laboratory we have fabricated a process to simulate the effect of dialysis using Whatman ultra-fine membrane filter (retention down to 0.7 μm). The test tubes containing different systems were covered with the filters and fastened well (Fig 2). Then each test tube was inverted and soaked in the distilled water (50 ml) containing in glass beakers (Fig 3). Then the beakers were stirred at 35 r.p.m in a thermostat shaker at 37.50C (Fig 4). After 20, 40, 60, 80, 100 and 120 min 1ml of solution from each beaker was taken out and 1ml of Fehling solution was added, heated at 85C for 30 minutes, cooled. Then, 1ml of arsenic molybdate solution and 7ml of distilled H2O were added and the absorbance of each solution was measured in UV spectrophotometer at 520 nm. The diffusion of glucose into the solution was determined using standard glucose curve (Fig 5).


Statistical Analysis:

Significance of differences between the mean values were determined by analysis of variance (ANOVA), followed by Dunnett’s test. Results were considered significant when the p values were less than 0.05. Statistical calculations and the graphs were prepared using GraphPad Prism version 4.00 for Windows.


Results and Discussion:
Dialysis tubing technique is a simple model to evaluate the potential of soluble diatray fibers to retard the diffusion and movement of glucose in the intestinal tract (Adiotomre, et al., 1990). Our fabricated technique to test the diffusion of glucose perfectly simulates this model. In our fabricated technique, substantial reduction of diffusion of glucose from Na-CMC-glucose systems was found in a concentration dependant manner (Table 4). Increasing the concentration of Na-CMC decreased the diffusion of glucose from the systems. Diffused glucose from systems H to L (Na-CMC and glucose diffusion systems) was 0.30-0.35 mg/ml at 20 min and 75-85.07% of control, respectively. In the highest concentration of Na-CMC (10mg/ml) significant reduction of glucose diffusion (P<0.05) was found at 100 and 120 min (Fig 6). From the system L maximum reduction of diffusion was observed at 60 min (70% of control).
VWSP of vendi exerted the same effect as Na-CMC. From the system G to Q, reduction of the diffusion of glucose was observed in a concentration-dependant manner (P<0.05) (Fig 7, Table 5). The system Q contains maximum amount of mucilaginous constituents and the diffusion of glucose from this system was minimum. At 20 minute, the diffusion of glucose from the system M, N, O, P and Q were 0.34, 0.25, 0.20, 0.15 and 0.12 mg/ml, respectively. The diffusion of glucose from system Q was 30% of the control where there was no dietary fiber component. Thus, it is clear that glucose entrapping capacity of VWSP of vendi was significantly higher than that of Na-CMC.
Studies reported that consumption of viscous water-soluble dietary fibers reduced postprandial blood glucose by reducing the diffusion of glucose and postponing the absorption and digestion of carbohydrates (Yokoyama et al., 1997). It has also been reported that different types of dietary fibers (especially soluble) reduced the diffusion of glucose in vitro (Ou et al., 2001) and also in vivo (Lafrance et al., 1998 and Torsdottir et al., 1991). Our data are consistent with earlier studies. The decreased extent of diffusion of glucose by Na-CMC and VWSP of the pods of Abelmoschus esculentus L across the ultra fine membrane was almost similar to that reported by Ou, S. et al., 2001. Although the situation for absorption in the small intestine could not be completely judged by the results from results, we can assume the possible reason. It has been assumed by Ou et al., that the possible mechanism may be the increase of the viscosity of the systems, retard the diffusion of glucose and/or adsorption of glucose on the dietary fibers and prevent its diffusion. Further studies are required to evaluate the exact mechanism by which intestinal glucose absorption was reduced by soluble dietary fiber.
Conclusion:

In this study, we observed that viscous water-soluble portion of the fruits of Abelmoschus esculentus L has significant capacity to reduce the glucose diffusion form the dietary fiber- glucose systems. As the Abelmoschus esculentus L is widely distributed and the fruits commonly used as an edible item, it therefore represents a potentially dietary adjunct in diabetic people to control their blood glucose.


References:
Alam, M. S. and Khan, G. M. A. Chemical analysis of okra bast fiber (Abelmoschus esculentus) and the physico-chemical properties. Journal of Textile and Apparel, Technology and Management (JTATM). 5:4, 2007.
Aro, A., Uusitupa, M., Voutilainen, E., Hersio, K..,Korhonen, T. & Siitonen, O. Improved diabetic control and hypocholesterolemic effect induced by long-term dietary supplementation with guar gum in type II (insulin-independent) diabetes. Diabetologia21: 29-33, 1981.

Begum, M. Lokesh,, S. Ravishankar , R.V. Shailaja, M. D. Kumar and Shetty, S. H. Evaluation of certain storage conditions for okra (Abelmoscus esculentus L. Moench) seeds against potential fungal pathogens. International Journal of Agriculture and Biology. 4:550-554, 2005.


Cameron-Smith, D., Habito, R., Barnett, M., Collier, G. R. Dietary guar gum improves insulin sensitivity in streptozotocin-induced diabic rats. J. Nutr. 127:359-364, 1997.

Ellis, P. R., Rayment, P., Wang, Q. A physicochemical perspective of plant polysaccharides in relation to glucose absorptionj, insulin secretion and the entero-insular axis. Proc. Nutr. Soc. 55:881-898, 1996.


Lafrance, L., Raabasa-Lhoret, R., Poisson, D., Ducros, F., Chiasson, J. L. Effects of different glycemic index foods and dietary fiber intake on glycemic control in type I diabetic patients on intensive insulin therapy. Diabetes Med. 15:972-978, 1998.
Liljeberg, H. G. M., Granfelt, W. E., Bjorck, M. E. Products based on a high fiber barley genotype, but not common barley or oats, lower postprandial glucose and insulin response in healthy man. J. Nutr. 126:458-461, 1996.
Ou, S., Kwok, K., Li, Y., and Fu, L. In-vitro study of possible role of dietary fiber in lowering postprandial serum glucose. J. Agric. Food Chem. 49:1026-1029, 2001.

Pereira, M. A., and Ludwig, D. S. Dietary fiber and body weight regulation: Observations and Mechanisms. Pediatr. Clin. N. Am. 48:969, 2001.


Ray, T. K., Mansell, K. M., Knight, L. C., Malmud, L. S., Owen, O. E. & Boden, G. Long-term effects of dietary fiber on glucose tolerance and gastric emptying in noninsulin-dependent diabetic patients. Am. J. Clin. Nutr. 37: 376-381,1983
Torsdottir, I., Alpsten, M., Holm, G., Sandberg, A. M., Tolli, J. A small dose of soluble alginate fiber affects postprandial glycemia and gastric emptying in humans with diabetes. J. Nutr. 121:795-799, 1991.
Trinh, H. N., Quynh, N. N., Anh, T. T. V. and Nguyen, V. P. Hypolipidemic effect of extracts from Abelmoschus esculentus L. – Malvaceae on tyloxapol-induced hyperlipidemia in mice. 13rd International Electronic Conference on Synthetic Organic Chemistry (ECSOC-13), 2009.
Vaugelade, P., Hoebler, C., Bernerd, F., Guillon, F., Lahaye, M., Duee, P. H, Vrillon, B. D. Non-starch polysaccharides extracted from seaweed can modulate intestinal absorption of glucose and insulin response in the pig. Repro. Nutr. Dev. 40:33-47, 2000.
Yokoyama, W. H., Hudson, C. A., Knuckles, B. E., Chiu, M. C. M, Sayre, R. N.; Turnlund, J. R.; Schneeman, B. O. Effect of barley beta-glucan in durum wheat pasta on human glycemic response. Cereal Chem. 74:293-296, 1997.

Table 1: Amount of VWSP of Abelmoschus esculentus L obtained after filtration


Serial No.

Amount of sliced Abelmoschus esculentus L

Amount of distilled water

Amount of water soluble portion obtained after filtration

1

15 gm

150 ml

135 ml (Solution A)

2

30 gm

150 ml

130 ml (Solution B)

3

45 gm

150 ml

110 ml (Solution C)

4

60 gm

150 ml

100 ml (Solution D)

5

75 gm

150 ml

90 ml (Solution E)

Table 2: Amount of dried constituents in 1 milliliter of filtrate


Solution

Amount of solution

Amount of dried constituents obtained

Solution A

1 ml

0.1 gm

Solution B

1 ml

0.2 gm

Solution C

1 ml

0.34 gm

Solution D

1 ml

0.52 gm

Solution E

1 ml

0.57 gm



Table 3: Formulation of different systems (Control Na-CMC solution: System G to L)


No. of Test Tube
Glucose Solution

(0.1gm/ml)
Na-CMC solution
(0.01gm/ml)
Water soluble portion of ladies finger

Distilled water
Designation of systems

No. (1)

10 ml

0 ml

0

10 ml

System G

No. (2)

10 ml

2 ml

0

8 ml

System H

No. (3)

10 ml

4 ml

0

6 ml

System I

No. (4)

10 ml

6 ml

0

4 ml

System J

No. (5)

10 ml

8 ml

0

2 ml

System K

No. (6)

10 ml

10 ml

0

0 ml

System L

No. (7)

10 ml

0

10 ml of Solution A

0

System M

No. (8)

10 ml

0

10 ml of Solution B

0

System N

No. (9)

10 ml

0

10 ml of Solution C

0

System O

No. (10)

10 ml

0

10 ml of Solution D

0

System P

No. (11)

10 ml

0

10 ml of Solution E

0

System Q

Table 4: Effect of Na-CMC-glucose diffusion systems on diffusion of glucose




Diffusion of glucose (mg/ml) in outer solution from systems

20 min

40 min

60 min

80 min

100 min

120 min

Solution G

0.40±0.2a

0.80±0.5

1.20±0.3

1.60±0.7

2.10±0.8

2.45±0.8

System H

0.35±0.6

0.70±0.8

1.10±0.1

1.45±0.5

1.85±0.2

2.20±0.1

System I

0.35±0.1

0.68±0.9

1.05±0.1

1.44±0.2

1.75±0.3

2.10±0.8

System J

0.34±0.6

0.65±0.2

0.95±0.1

1.28±0.2

1.64±0.5

1.95±0.1

System K

0.32±0.6

0.60±0.2

0.94±0.4

1.25±0.3

1.60±0.8

1.85±0.2

System L

0.30±0.3

0.58±0.3

0.90±0.4

1.22±0.8

1.55±0.2

1.84±0.5


aMean values ± standard deviation
Table 5: Effect of VWSP of Abelmoschus esculentus L. on diffusion of glucose




Diffusion of glucose (mg/ml) in outer solution from systems

20 min

40 min

60 min

80 min

100 min

120 min

Solution G (control)

0.40±0.4 a

0.80±0.3

1.20±0.6

1.60±0.8

2.10±0.8

2.45±0.5

System M

0.34±0.6

0.65±0.8

0.80±0.2

0.90±0.5

1.01±0.2

1.75±0.4

System N

0.25±0.6

0.62±0.7

0.70±0.9

0.80±0.2

0.90±0.6

1.10±0.8

System O

0.20±0.5

0.54±0.2

0.68±0.4

0.72±0.2

0.854±0.4

1.02±0.5

System P

0.15±0.6

0.48±0.3

0.55±0.4

0.65±0.8

0.82±0.8

0.98±0.2

System Q

0.12±0.3

0.36±0.9

0.50±0.4

0.60±0.8

0.75±0.2

0.90±0.5


aData are expressed as Mean values ± SEM



Fig 1: Slices of Abelmoschus esculentus




Figure 2: Fastened test tube Fig 3: Inverted test tube Fig 4: Shaking of systems

with ultra-fine filter paper in a beaker in a thermostat shaker



F
igure 5:
Standard Glucose Curve (5 concentrations of glucose solution were used: 0.5, 1.0, 1.5, 2.0 and 2.5 mg/ml)





Figure 6: Diffusion of glucose (mg/ml) from Na-CMC-glucose diffusion system where different systems contain different conc. of Na-CMC (system H, 2mg/ml; system I, 4mg/ml; system J, 6mg/ml; system K, 8mg/ml; system L, 10mg/ml) with fixed conc. of glucose solution (0.1g/ml). Data were presented as mean ± SEM; n=3, *p<0.05 compared to control (ANOVA followed by Dunnett's test)



Figure 7: Diffusion of glucose (mg/ml) from VWSP of Abelmoschus esculentus L.-glucose diffusion system where different systems contain different conc. of VWSP of Abelmoschus esculentus L (system M, 0.1g/ml; system N, 0.2g/ml; system O, 0.34g/ml; system P, 0.52g/ml; system Q, 0.57g/ml) with fixed conc. of glucose solution (0.1g/ml). Data were presented as mean ± SEM; n=3, *p<0.05 compared to control (ANOVA followed by Dunnett's test)


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