Antidiabetic Effect of the Chrysobalanus icaco L. Aqueous Extract in Rats

Abstract

Chrysobalanus icaco L. is a medicinal plant popularly known in Brazil as “Grageru” or “Abageru.” It is used in African and American continents as medicinal food in the treatment of several diseases, including diabetes. This study used phytochemical screening to determine the antioxidant and α-amylase inhibitor activities of the aqueous extract (AECI) of C. icaco, and evaluated its antidiabetic potential in rodents. Phytochemical screening was performed using colorimetric tests with specific reagents. The in vitro antioxidant activity was evaluated by the scavenging activity of 2,2-diphenyl-1-picril-hydrazyl. The lethality test and behavioral screening was performed using an oral administration of 5 g/kg of AECI. The antidiabetic potential of AECI was evaluated through the oral glucose tolerance test (OGTT) and chronic hypoglycemic test at the doses of 100, 200, and 400 mg/kg (orally). Metformin was used as a reference drug in all tests. Diabetes was induced by injection of alloxan (40 mg/kg; intravenously). Phytochemical screening showed the presence of various compounds, including tannins, flavones, triterpenoids, steroids, saponins, and alkaloids. The in vitro antioxidant test demonstrated that AECI presented potent antioxidant activity. The lethality test and behavioral screening did not show lethality signs. In the OGTT test, AECI administration was not able to inhibit the elevation of glycemia. However, chronically administrated, it was able to cause a significant (P<.05) reduction of glycemia from 335±27 up to 197±15 mg/dL. These results demonstrate that the AECI presents a potential beneficial effect for diabetes.

Introduction

D iabetes Mellitus (DM) is a very common endocrine disease and affects about 10% of the world population.¹ It is reported that DM may present symptoms such as thirst, polyuria, and weight loss, and usually leads to increase in production of free radicals and microvascular complications.²

The available therapies for diabetes include insulin and oral antidiabetic agents such as sulfonylureas, biguanides, and α-glucosidase inhibitors. However, many of these oral antidiabetic agents have a number of serious adverse effects.^3,4 Thus, the management of diabetes without any side effects is still a challenge.³ For these reasons, the popularity of complementary medicine, such as medicinal food therapy, has increased in recent years.⁵

The use of plants as medicinal food, based on cultural tradition, has become widespread in folk medicine of several developed or developing countries. Chrysobalanus icaco (Chrysobalanaceae) is a medicinal plant popularly known as “Grageru” or “Abageru” and characterized as a medium-sized shrub.⁶ It is consumed in Brazil as an antidiabetic agent in the form of tea.^7,8 In Nigeria, the seeds of C. icaco are consumed in the form of soup.⁹ Its fruits are edible, and in many countries the fruit is commonly sweet-pickled.¹⁰ These plants have been used as an alternative treatment for various diseases, including DM.¹¹

The aims of this study were to perform the phytochemical screening and to evaluate the antioxidant and antidiabetic activities of the aqueous extract of C. icaco (AECI) in rodents.

Materials and Methods

Plant material and preparation of the aqueous extract

C. icaco leaves were collected from the village Jatoba (coordinates: 10°47′50′′ S and 36°50′44′′ W), Sergipe State, Brazil, in February 2008, and were identified by the botanist Dr. Ana Paula Prata (Department of Biology, Anatomy, and Botany, Federal University of Sergipe). A voucher specimen (ASE 11855) is deposited at the herbarium of the Federal University of Sergipe.

After collection, the leaves were dried in an oven (Marconi, MA–035/5) at 40°C for 72 h and triturated in an electric mill. This powder was extracted with distilled water at 100°C for 15 min. Then, the extract was concentrated under reduced pressure in a rotavapor, lyophilized, and stored at 4–8°C. At the time of use, the extract was dissolved in water at the desired concentration.

Animals

Male Wistar rats (Rattus norvegicus), weighing between 150 and 180 g, and Swiss mice (Mus musculus) of both sexes, weighing between 25 and 35 g, were used in this study. They were obtained from the Central Biotery at the Federal University of Sergipe. The animals were kept under controlled conditions of temperature (22°C±2°C), 12-h light–12-h dark cycle, and minimum noise, and were housed in polypropylene cages with food and water ad libitum. Experimental protocols were approved by the Committee on Animal Research at the Federal University of Sergipe (no. 67/2009) and were in compliance with the Helsinki Declaration of 1975, as revised in 2008.

Phytochemical screening

The aqueous extract was divided into different test tubes, and various chemical constituents were qualitatively analyzed according to methods described in Araújo et al. ¹² This analysis was conducted by observing colorimetric variation after the addition of specific reagents.

The different chemical constituents and reagents tested included flobabenic tannins, pirogallic tannins, anthocyanins, anthocyanidins, flavonoids, flavanonols, xanthones, flavones, chalcones, aurones, leucoanthocianidins, and catechins (acid alcohol/basic alcohol, solid magnesium, ferric chloride reagents); triterpenoids/steroids (Lieberman-Bouchard and Dragendorf reactions); saponin (frothing test); and alkaloids (Dragendorf and Mayer reactions).

Total phenolic content

A 100-μL aliquot of an AECI/methanol solution (1 mg/mL) was agitated with 500 μL of the Follin-Ciocalteu reagent and 6 mL distilled water for 1 min; after this time, 2 mL of 10% Na₂CO₃ were added to the mixture and agitated for 30 sec. Finally, the solution volume was corrected to 10 mL with distilled water.¹³

After 2 h of incubation at 25°C, the absorbance of the samples was measured by UV-Vis spectrophotometer at 750 nm, having as blank the methanol and all reagents, except the extract, and compared to a gallic acid calibration curve. The total phenolic content (TP) was determined as gallic acid equivalents (mg gallic acid/g extract), and the values are presented as the means of triplicate analyses.¹³

Evaluation of the in vitro antioxidant activity

The evaluation of the antioxidant activity of AECI (30 μg/mL) was determined by its ability to scavenge free radical 2,2-diphenyl-1-picryl-hydrazyl (DPPH), using the method described by Sousa et al. ¹³ Measurements of the decrease in absorbance of the solutions tested were made in a UV-Vis spectrophotometer at 515 nm, using as positive control the gallic acid and butylated hydroxytoluene (BHT). All tests were performed in triplicate.

Lethality and behavioral screening

The animals were distributed in four groups (n=12/group, six males and six females) and kept in adequately maintained cages. The animals received AECI at the dose of 5 g/kg orally (via gavage). Specific behaviors (sedation, ambulation, response to touch, analgesia, and defecation) and their intensities were observed according to Almeida et al. ¹⁴ at 1, 2, 3, and 4 h after gavage. Finally, the animals were observed daily for 14 days to verify lethality.¹²

Evaluation of acute antihyperglycemic effect in normoglycemic rats

The acute antihyperglycemic effect of AECI was evaluated by analysis of curves obtained from the oral glucose tolerance test (OGTT), according to the methods described by Pushparaj et al. ¹⁵ and Souza et al. ¹⁶

Thirty animals were divided into five groups with six animals each: the negative control group (CG), treated with saline (0.9%); the positive control group (MG), treated with metformin (500 mg/kg; EMS), a reference drug;¹⁵ and three experimental groups, treated with AECI at doses of 100, 200, and 400 mg/kg (G100, G200, and G400, respectively).

Before experiments, all animals were placed in fasting with water ad libitum for 12 h overnight. After this, the glucose levels were measured in fasting and defined as glucose level, time 0 (zero). Then, the animals received their respective treatments by oral administration (gavage) and, after 30 min, glucose was administered at the dose of 3 g/kg. Glucose levels were then measured at 30, 60, 90, and 120 min after glucose administration. The glycemia was measured by a blood drop collected from the tail of the animals and put on reagent strips (ACCU-CHEK Advantage II, Roche) coupled to a portable digital glucometer (ACCU-CHEK Advantage II). The results for each group were expressed as the mean of the differences between glucose levels at the times of observation and the fasting glucose level, and the area under curve (AUC).

Evaluation of chronic antidiabetic effect of AECI in diabetic rats

Type I diabetes induction and experimental design

The experimental DM was induced by the method described by Prince et al. ¹⁷ After 12 h of nonwater fasting, 75 animals received 40 mg/kg body weight of alloxan monohydrate (Inlab) diluted in a saline solution (0.9%) by a single intravenous injection (penian vein). One week after diabetes induction, the animals with blood glucose ≥200 mg/dL (40 animals only) were selected as diabetic. Blood glucose was measured as previously described.

The diabetic animals were divided into five groups of eight animals each: the diabetic-negative control group, treated with saline (0.9%) (DCG); the diabetic-positive control group, treated with metformin (500 mg/kg), a reference drug¹⁵ (DMG); and three experimental groups, treated with AECI at doses of 100, 200, and 400 mg/kg (DG100, DG200, and DG400, respectively). Eight healthy animals were randomly chosen to compose the nondiabetic control group (CG), treated with saline (0.9%). The treatments were performed by gavage (orally), at 8:00 A.M. daily for 28 consecutive days.

Experimental protocols

Water intake was measured on the last day of treatment before fasting, using bottles filled with known volumes, where the volume variation was observed over a 24 h period. After this period, the animals were weighed and blood samples were collected from the tail vein. Body weight gain was obtained by subtracting the initial weight from the final weight (day 28) and expressed as percentage of initial weight (day 1). Blood glucose was measured from blood samples using an enzymatic kit (Labtest) and a spectrophotometer (Autolab) and expressed as mg/dL of plasma.

Evaluation of the in vitro α-amylase inhibition

The possible α-amylase inhibition induced by AECI (20 mg/mL in distilled water) was assessed according to the chromogenic method described by Hasenah et al. ¹⁸ using Type VI-B porcine pancreatic α-amylase (from Sigma) and DNS color reagent solution (96 mM of 3,5-dinitrosalisylic acid plus 5.31 M of potassium sodium tartrate tetrahydrate in 2 M NaOH).

The α-amylase activity was determined by measuring the absorbance of the mixtures at 540 nm using ELISA (Uniscience). In the control incubations, representing a 100% enzyme activity, the plant extract was replaced with distilled water (40 μL). For blank incubations (to allow for absorbance produced by the plant extract), the enzyme solution was replaced with distilled water and the same procedure was carried out as above. Acarbose (6 mg/mL) (50 mg; EMS) was dissolved in distilled water and used as a positive control. All assays were performed with four replicate determinations.

Statistical analysis

Data are expressed as mean±standard error of mean or standard deviation. One- or two-way analyses of variance followed by the Newman–Keuls post-test were used to identify significantly different groups. For these procedures, the statistical software Graphpad Prism ™ version 3.02 was used.

Results

Phytochemical screening

Phytochemical screening of AECI showed the presence of flobabenic tannins, flavones, chalcones, aurones, leucoanthocianidins, catechins, triterpenoids, steroids, saponins, and alkaloids (Table 1).

Table 1.

Phytochemical Screening of Aqueous Extract of Chrysobalanus icaco

Compounds	Test/reagents	Results
Flobabenic tannins	Precipitation with FeCl₃	+
Pyrogallic tannins	Precipitation with FeCl₃	−
Anthocyanins and anthocyanidins	Acid and alkaline solution	−
Flavonoids, flavanonols, and xanthones	Solid magnesium; acid solution	+
Flavones	Solid magnesium; alkaline solution	+
Chalcones and aurones	Alkaline solution	+
Leucoanthocianidins	Alkaline solution; heating	+
Catechins	Acid solution; heating	+
Triterpenoids/steroids	Lieberman-Bouchard and Dragendorf reactions	+
Saponin	Frothing test	+
Alkaloids	Dragendorf and Mayer reactions	+

TP content and evaluation of the in vitro antioxidant activity

As shown in Table 2, the TP content present in 1 g of AECI was 276 mg gallic acid equivalent of phenols. Furthermore, these results show that AECI was able to scavenge DPPH radicals and its inhibition percentage (IP%) was similar to that of the positive controls. In regard to the effective concentration of samples required to reduce by 50% the initial DPPH (EC50), it can be noted that the EC50 of AECI was similar to the positive controls (gallic acid and BHT).

Table 2.

Total Phenolic Content and Antioxidant Activity of AECI and Samples of Gallic Acid and Butylated Hydroxytoluene

Samples	TP (mg GA/g AECI)	IP%	EC₅₀ (μg/mL)
Gallic acid	—	96.24	9.33±0.22
BHT	—	96.91	12.33±3.25
AECI	276±16.3	93.24	7.96±1.47

Percent of inhibition (IP) and effective concentration for half-maximal inhibition (EC₅₀) of the samples were calculated at 60 min. Values represent the mean±standard deviation. Data were analyzed by one-way analysis of variance (ANOVA) followed by the Newman–Keuls post-test.

TP, total phenolic content; GA, gallic acid; AECI, aqueous extract of Chrysobalanus icaco; BHT, butylated hydroxytoluene.

Lethality and behavioral screening

The lethality and behavioral screening study of AECI at the dose of 5 g/kg produced some changes, such as sedation, analgesia, palpebral ptosis, loss of ear reflex, and a decrease of defecation. AECI did not show signs of lethality in mice at the dose of 5 g/kg body weight orally administered.

Evaluation of acute antihyperglycemic effect in normoglycemic rats

As can be seen in Table 3, AECI was not able to induce a significant acute antihyperglycemic effect when compared with the control group. Metformin promoted a significant reduction of the hyperglycemia and consequently of the AUC.

Table 3.

Glucose Levels at 30, 60, 90, and 120 min After Oral Administration of 3 g/kg Glucose in Normoglycemic Animals Treated with Saline, Metformin, and Various Doses of AECI

	Time (min)
Groups	30	60	90	120	%MI	Area under curve
CG	58.5±5.7	52.5±7.4	44.2±6.0	21.7±6.3	—	4101±546
MG	29.2±2.8^***	24.0±1.3^**	24.2±4.4	24.5±6.0	54.3	2250±234^*
G100	63.5±5.8	58.5±6.1	49.3±6.8	37.3±4.4	—	4747±533
G200	60.3±4.4	56.5±3.9	48.5±4.7	44.0±6.5	—	4715±315
G400	60.2±2.6	55.8±5.3	41.2±5.7	34.0±3.4	6.8	4322±379

Data were analyzed by using two-way ANOVA followed by Newman-Keuls post-test. Values represent the mean±standard error of the mean, n=6. The increments in blood glucose are expressed in miligrams of glucose per dL of blood.

P<.05, ^** P<.01, ^*** P<.001 vs. CG.

%MI, maximum percentage inhibition of hyperglycemia; CG, negative control group; MG, metformin (500 mg/kg) group; G100, G200, G400, AECI groups dosed at 100, 200, and 400 mg/kg.

Evaluation of chronic antidiabetic effect of AECI in diabetic rats

As can be seen in Table 4, diabetic animals (DCG) showed a significant elevation of fasting blood glucose and water intake, and a significant reduction in weight gain was seen when compared with healthy animals (CG).

Table 4.

Glycemia, Water Intake, and Body Weight Gain in Normoglycemic and Diabetic Rats Treated with Saline and in Diabetic Rats Treated for 28 Days with Metformin or Various Doses of AECI

Groups	Glycemia (mg/dL)	Water intake (mL/24 h)	Body weight gain (%)
CG	104.0±6.4	36.9±3.0	87.1±12.6
DCG	335.4±27.0^***	121.5±5.6^***	34.6±4.5^*
DMG	201.4±14.2^†††	85.6±3.0^†††	51.9±2.8^†
DG100	205.1±12.8^†††	111.4±3.4	14.8±1.4^†
DG200	236.4±10.5^†††	90.8±3.4^†††	14.1±2.4^†
DG400	268.8±22.2^††	195.9±4.1^†††	2.4±0.5^†††

Data were analyzed by using two-way ANOVA followed by Newman-Keuls post-test. Values represent the mean±standard error of the mean, n=8.

P<.05, ^*** P<.001 vs. CG; ^† P<.05, ^†† P<.01, ^††† P<.001 vs. DCG.

DCG, diabetic control group; DMG, diabetic metformin (500 mg/kg) group; DG100, DG200, DG400, diabetic AECI groups dosed at 100, 200, and 400 mg/kg.

Treatment of diabetic animals with metformin (DMG) was able to significantly reduce fasting blood glucose and water intake, and to increase body weight gain when compared with the DCG animals.

Similar to metformin, AECI (100, 200, or 400 mg/kg) was able to reduce fasting blood glucose at all doses. However, body weight gain was reduced at all doses (Table 4).

Evaluation of the in vitro α-amylase inhibition

As demonstrated in Figure 1, acarbose was able to significantly reduce the concentration of maltose when compared with the vehicle. However, AECI (20 mg/mL) was not able to provide the same effect.

FIG. 1.

Maltose formation in the presence of acarbose and aqueous extract of Chrysobalanus icaco (AECI). Results expressed as mean±standard error of the mean (n=4). *P<.05 vs. control at same time point. Data were analyzed by one-way analysis of variance followed by the Newman–Keuls post-test.

Discussion

C. icaco (Chrysobalanaceae) is a medicinal plant largely used in tropical African and South American countries as a food and antidiabetic agent in the form of tea or soups.^6,7 Our results provide phytochemical data and evidence that this plant present an antidiabetic potential.

Phytochemical screening of the AECI showed the presence of several groups of substances, such as flavonoids, triterpenoids, steroids, and saponins. In accordance with the literature, these chemical constituents present antidiabetic and antioxidant activities.^19,20

The antidiabetic potential was evaluated by acute (OGTT) and chronic in vivo tests, and antioxidant and alpha-amylase inhibitor in vitro tests. In the OGTT, which is a fast and low-cost technique, the elevation of postprandial glycemia after a glucose load, and the subsequent normalization to baseline levels after about 2 h, characterizes a normal function in glucose metabolism.¹⁶ In this set of experiments, AECI was unable to inhibit the elevation of postprandial glycemia. In contrast, the metformin, an oral hypoglycemic of the biguanide class,²¹ succeeded in inhibiting this elevation.

On the other hand, when chronically administered AECI was able to reduce fasting blood glucose to similar levels to the metformin. The results are in agreement with those found by Presta and Pereira,²² who showed that the tea leaves of C. icaco, 5% orally administered, for 33 days, reduced fasting glucose levels of alloxan-induced diabetic mice.

Interestingly, the reduction in fasting blood glucose at the dose of 400 mg/kg was directly accompanied by a reduction in body weight gain and conversely accompanied by an increase of water intake. It has been related in the literature that glycosuria and increased urine volume can further reduce weight gain.²³ Furthermore, the presence of flavonoids and saponins in the chemical composition of the plant is able to promote an increased urine output.^24,25 Therefore, it is possible to suggest that the reduction in weight gain observed in animals treated with 400 mg/kg of AECI may be associated not only with the antidiabetic effect, but also with an intense diuresis promoted by the extract. However, other studies are necessary to confirm this assumption.

To determine a possible mechanism of action of AECI, the ability of this extract to inhibit the α-amylase enzyme and potential antioxidant effect was also evaluated.

The inhibition of the α-amylase enzyme can significantly reduce the postprandial increase of blood glucose after a mixed carbohydrate diet and, therefore, can be an important strategy in the management of diabetic patients.²⁶ Therefore, new agents that control postprandial hyperglycemia have been developed. Among them, acarbose has received considerable attention in the past few decades.^26,27

A natural α-amylase inhibitor from plant sources also offers an attractive strategy for the control of hyperglycemia.²⁸ Several in vitro studies have been performed with medicinal plants, and some have proven to be potent α-amylase inhibitors, such as Cuscuta reflexa,²⁹ Phyllanthus amarus,¹⁸ and Euclea undulate.³⁰

Our results demonstrated that AECI, in the doses tested, was unable in inhibit the α-amylase enzyme.

Diabetes is usually accompanied by increased production of free radicals or impaired antioxidant defenses.³¹ Therefore, the antioxidant therapies could provide a potential means to treat conditions in which the formation of reactive oxygen species exceeds the capability of natural protective mechanisms.³² Among the various classes of naturally occurring antioxidants, phenolic compounds have received much attention in recent years, primarily for inhibiting lipid peroxidation and lipoxigenase in vitro.¹³ As noted in our results, the TP content present in 1 g of AECI was a value considered low when compared with the extracts of other species described in the literature.¹³ Nevertheless, AECI was able to scavenge DPPH radicals and its IP% and EC₅₀ were similar to that of the positive controls.

It is well accepted in the literature that some oral hypoglycemic drugs, such as sulfonylureas, produce hypoglycemia in normal animals by stimulating the pancreatic β cells to release more insulin. These drugs, however, do not decrease blood glucose in alloxan-diabetic animals.³³ On the other hand, other drugs produce hypoglycemia even in the absence of insulin, such as metformin. Taken together, our results provide evidence that the AECI hypoglycemic mechanism involves an insulin-like effect, probably increasing the peripheral glucose consumption such as previously identified for other natural products.^33
–35

Conclusion

AECI presents an antidiabetic activity when chronically administered. This effect can be related to its chemical constituents and its antioxidant potential effect. These results are in agreement with the popular use of this plant.

Footnotes

Acknowledgments

This research was supported by CAPES, CNPq, FAPITEC/SE, Ministério da Saúde, and SES/SE, Brazil.

Author Disclosure Statement

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

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