Antioxidant and antidiarrheal activities of ethanol extract of Ardisia elliptica fruits

Abstract

Context: Ardisia elliptica Thunb Lam. (Myrsinaceae) is widely used traditionally in the treatment of diarrhea related health disorders in Bangladesh.

Objective: The crude ethanol extract of Ardisia elliptica fruits (EFA) was evaluated for its antioxidant and antidiarrhoeal activities.

Materials and methods: DPPH radical scavenging, nitric oxide scavenging, reducing power and Fe⁺⁺ ion chelating ability were used for determining antioxidant activities and animal models were used for antidiarrheal activities such as the castor oil and magnesium sulfate-induced diarrhea, enteropooling induced by the administration of castor oil and magnesium sulfate at the doses of 250 and 500 mg/kg.

Results: The extract possessed a significant DPPH free radical scavenging activity with an IC₅₀ value of 30.75 μg/ml compared to ascorbic acid (IC₅₀: 7.89 μg/ml). The IC₅₀ values of the extract and ascorbic acid were 51.72 and 38.68 μg/ml, respectively, in nitric oxide scavenging assay. The IC₅₀ value of the extract for Fe⁺⁺ ion chelating ability (41.30 μg/ml) was also found to be significant compared to the IC₅₀ value of EDTA (22.57 μg/ml). The EFA also showed a significant protection (p < 0.001) against experimentally induced diarrhea by castor oil and magnesium sulfate as evidenced by a decrease in the number of defecation with respect to control. The diarrhea induced by castor oil and magnesium sulfate enteropooling was prevented by all the tested doses.

Conclusion: Therefore, the obtained results confirm the antioxidant and antidiarrheal activity of EFA and thus support the traditional uses of this plant as a modality for antioxidant and antidiarrheal activity.

• Castor oil
• DPPH
• enteropooling
• nitric oxide
• magnesium sulphate

Introduction

Ardisia elliptica Thunb (Myrsinaceae) is an evergreen, glabrous shrub or small tree to 5 m tall, with smooth stems and new foliage often reddish. Leaves are 20 cm long, oblong to oval, flowers are in axillary clusters, star shaped, 13 mm wide and fruit is a rounded drupe, 6 mm wide, red turning to black when ripe, with white juicy flesh. It is available in India, China, Southeast Asia and Bangladesh. 5-Pentadecylbenzene-l, 3-diol or 5-pentadecylresorcinol, α-amyrin and taraxerone have been isolated from the leaves of A. elliptica (Jalil et al., 2004). Syringic acid, isorhamnetin, β-amyrin and quercetin have been isolated from fruits of A. elliptica (Jianhong et al., 2010; Methin & Omboon, 2006). The fruit extracts of A. elliptica have antibacterial activity (Methin & Omboon, 2006).

There is no scientific proof of the traditional use of A. elliptica fruits in the treatment of diarrhea and antioxidant activity. The present study was designed to provide scientific evidence for its use as a traditional folk remedy by investigating the above pharmacological and antioxidant potential of the EFA. It also confirms its use as a folk remedy for diarrhea and other pathological conditions where free radicals are implicated.

Materials and methods

Collection and identification of plant materials

The fresh fruits of A. elliptica were collected from Chittagong, Bangladesh in April 2011, during the daytime. The samples were identified by Sarder Nasir Uddin, Senior Scientific Officer, Bangladesh National Herbarium, Mirpur, Dhaka, Bangladesh. A voucher specimen (DACB: 35112) has been deposited in the Herbarium for further reference.

Preparation of ethanol extract

The fruits of A. elliptica were freed from all foreign materials. Then, the plant materials were chopped and air-dried under shade temperature followed by drying in an electric oven at 40 °C. The dried fruits of A. elliptica were ground into a coarse powder with the help of a suitable grinder (Capacitor start motor, Wuhu Motor Factory, China). The powdered sample was stored in an airtight container and kept in a cool, dark and dry place until analysis commenced. About 350 g of powered material was placed in a clean, flat-bottomed glass container and soaked in 1000 ml ethanol. The container along with its contents was sealed and kept for 10 d at 25 ± 2 °C with occasional shaking or stirring. The whole mixture then underwent a coarse filtration by piece of clean, white cotton material. It was then filtered through Whatman filter paper (Bibby RE200, Sterilin Ltd., Gwent, UK). The filtrate was concentrated by using a rotary vacuum evaporator (R-205, Buchi, Switzerland) at reduced pressure and dried. It rendered 43 g of a gummy concentrate (12.24%) and was designated as crude ethanol extract for antioxidant and antidiarrheal studies.

Chemicals and drugs

1,1-Diphenyl-2-picryl hydrazyl (DPPH), l-ascorbic acid, gallic acid, Folin-Ciocalteu phenol reagent, ferrozine and Griess reagent were obtained from Sigma Chemical Co. (St. Louis, MO). Trichloroacetic acid (TCA), phosphate buffer (pH 6.6), potassium ferricyanide [K₃Fe(CN)₆], FeCl₂, FeCl₃, sodium nitroprusside, sodium phosphate, EDTA, Tween 80, ammonium molybdate and sodium carbonate were of analytical grade and purchased from Merck (Darmstadt, Germany).

The standard drug loperamide was used for this study and purchased from Square Pharmaceuticals Ltd, Bangladesh. Ketamine was purchased from Popular Pharmaceuticals Ltd, Bangladesh.

Test animals

Swiss albino mice (20–25 g) and Wister rats (175–202 g) of both sexes were used for in vivo pharmacological screening. The mice and rats were collected from the Animal Research Branch of the International Centre for Diarrhea Disease and Research, Bangladesh (ICDDR, B). The animals were housed under standard laboratory conditions maintained at 25 ± 2 °C and under 12/12 h light/dark cycle and feed with standard diet and water ad libitum acclimatization period. The animals were fasted overnight before the experiments. All experimental protocols were in compliance with BCSIR Ethics Committee on Research in Animals as well as internationally accepted principles for laboratory animal use and care.

Phytochemical screening

The preliminary phytochemical screening of EFA was qualitatively tested for the presence of alkaloids, reducing sugars, steroids, saponins, flavonoids and tannins using standard procedures (Evans, 1989).

Total flavonoid content determination

Aluminium chloride colorimetric method was used for the determination of total flavonoid concentration of the ethanol extract (Chang et al., 2002). The extract (0.5 ml, 1:10 g/ml) in ethanol was separately mixed with 1.5 ml of methanol, 0.1 ml of 10% aluminum chloride, 0.1 ml of 1 M potassium acetate and 2.8 ml of distilled water. It was allowed to stand for 30 min at room temperature and the absorbance of the reaction mixture was measured at 415 nm with a double beam Analykjena UV/Visible spectrophotometer (Model 205, Jena, Germany). Total flavonoid content was determined as mg of quercetin equivalent per gram using the equation obtained from a standard quercetin calibration curve y = 4.7385x + 0.0355; R²= 0.9993.

Total tannin content determination

The tannins were determined using the Folin-Ciocalteu phenol reagent as reported by Amorim et al. (2008). Briefly, 0.1 ml of the sample extract was added with 7.5 ml of distilled water and added 0.5 ml of Folin-Ciocalteu phenol reagent, 1 ml of 35% sodium carbonate solution and dilute to 10 ml with distilled water. The mixture was shaken well, kept at room temperature for 30 min and absorbance was measured at 725 nm. Blank was prepared with water instead of the sample. A set of standard solutions of tannic acid is read against a blank. Total tannin content was determined as mg of tannic acid equivalent per gram using the equation obtained from a standard tannic acid calibration curve y = 4.5692x − 0.2538, R²= 0.9953.

Acute toxicity test

The acute toxicity of EFA was determined in rats according to the method of Hilaly et al. (2004) with slight modifications. Rats fasted for 16 h were randomly divided into five groups of five rats per group. Graded doses of the extract (200, 400, 800, 1600 and 3200 mg/kg, p.o.) were separately administered to the rats in each of the groups by means of bulbed steel needle. All the animals were then allowed free access to food and water and observed over a period of 72 h for signs of acute toxicity. The number of deaths within this period was recorded.

Antioxidant activities

DPPH free radical scavenging activity

The method of Chang et al. (2001) was used for performing the DPPH radical scavenging activity. A stock solution of EFA (5 mg/ml) was prepared in respective solvent systems. A serial dilutions were the carried out to obtain concentrations of 5, 10, 20, 40, 60, 80 and 100 µg/ml. An equal amount of sample solution was mixed with an equal amount of 0.1 mM methanol solution of DPPH. The mixture was vortex and allowed to stand at the dark at 25 °C for 30 min. After a 30 min incubation, the absorbance of the mixture was read against a blank at 517 nm using a double beam Analykjena UV/Visible spectrophotometer (Model 205, Jena, Germany). The DPPH free radical scavenging activity was expressed as the inhibition percentage (I%) and calculated as per the following equation: where A_blank is the absorbance of the control (containing all reagents except the test compound), and A_sample is the absorbance of the experimental sample with all reagents. IC₅₀ value (the concentration of sample required to scavenge 50% DPPH free radical) was calculated from the plot of inhibition (%) against the concentration of the extract. All determinations were carried out in triplicate and the average of the results was noted. Ascorbic acid was used as a standard for this study.

Nitric oxide (NO) scavenging activity

Nitric oxide scavenging activity was measured spectrophotometrically (Govindarajan et al., 2003). Sodium nitroprusside (5 mmol) in phosphate buffered saline was mixed with different concentrations of EFA (5–100 µg/ml) dissolved in methanol and incubated at 25 °C for 30 min. A control without the test compound but with an equivalent amount of methanol was taken. After 30 min, 1.5 ml of the incubation solution was removed and diluted with 1.5 ml of Griess reagent (1% sulphanilamide, 2% phosphoric acid and 0.1% naphthylethylenediamine dihydrochloride). The absorbance of the chromophore formed during diazotization of the nitrite with sulphanilamide and subsequent coupling with naphthylethylene diamine dihydrochloride was measured at 546 nm. The nitric oxide (NO) radical scavenging activity was expressed as the inhibition percentage (I%) and calculated as per the equation: where A_blank is the absorbance of the control reaction (containing all reagents except the test compound), and A_sample is the absorbance of the test compound with all reagents. The IC₅₀ value is the concentration of sample required to scavenge 50% nitric oxide free radical and was calculated from the plot of inhibition (%) against extract concentration. All the tests were carried out in triplicate and the average of the absorptions was noted. Ascorbic acid was used as a positive control standard for this study.

Reducing power assay

The reducing power of EFA was determined according to method followed by Dehpour et al. (2009). Different concentrations of EFA (5–100 µg/ml) in 1 ml of distilled water were mixed with phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and potassium ferricyanide [K₃Fe(CN)₆] (2.5 ml, 1%). The mixture was incubated at 50 °C for 20 min. A 10% solution of trichloroacetic acid (2.5 ml) was added to the mixture, which was then centrifuged at 3000 rpm for 10 min. The upper layer of the solution (2.5 ml) was mixed with distilled water (2.5 ml) and FeCl₃ (0.5 ml, 0.1%) and the absorbance of the mixture was measured at 700 nm with spectrophotometer. Increased absorbance of the reaction mixture indicated increased reducing power. All the tests were carried out in triplicate and the average of the absorptions was recorded. Ascorbic acid was used as the standard reference compound.

Ferrous ion chelating ability

The ferrous ions chelating activity of ethanol extract and standards were investigated according to the method of Dinis et al. (1994). Briefly, different concentrations of the extract (5–100 µg/ml) were added to a 0.1 ml solution of 2 mM ferrous chloride (FeCl₂). Then, the reaction was initiated by the addition of 0.2 ml of 5 mM ferrozine and the mixture was shaken vigorously and left standing at room temperature for 10 min. After the mixture had reached equilibrium, the absorbance was measured at 562 nm in a spectrophotometer, wherein the Fe⁺² chelating ability of the extracts were monitored by measuring the ferrous ion-ferrozine complex. The percentage of inhibition of ferrozine-Fe⁺² complex formations was given in the formula below: where A₀ is the absorbance of the control solution (containing all reagents except extract); A is the absorbance in the presence of the sample of plant extracts. All the tests were carried out in triplicate and EDTA was used as a standard.

Antidiarrheal activities

Castor oil-induced diarrhea

Antidiarrheal activity of EFA was tested by using the castor oil-induced method in mice (Nwodo & Alumanah, 1991). Twenty Swiss albino mice were randomly divided into four groups (n = 5). The control group received only 1% Tween 80 in water (2 ml/mice), the positive control group received loperamide 3 mg/kg body weight as standard, and test groups received the extracts at doses of 250 and 500 mg/kg body weight. Mice were housed in separate cages having paper placed below for collection of fecal matter. Diarrhea was induced in the mice by oral administration of castor oil (1.0 ml/mice). Extract and drugs were given orally 1 h before the administration of castor oil. The time for first excretion of feces and the total number of fecal output by the animals were recorded. Normal stool was considered as numerical value 1 and watery stool as numerical value 2. Percent inhibition of defecation in mice was calculated by using the following equation: where Mo = Mean defecation of control and M = Mean defecation of test sample.

Magnesium sulphate-induced diarrhea

Antidiarrheal activity of EFA was tested by using the magnesium sulphate-induced diarrhea method in mice (Afroz et al., 2006). Twenty mice were fasted for 18 h and randomly divided in to four groups (n = 5). The control group received only distilled water 2 ml/mouse, positive control group received loperamide 3 mg/kg body weight as standard and test groups received the extracts at the doses of 250 and 500 mg/kg body weight. After 60 min of drug treatment, the animals in each group received magnesium sulphate (2 g/kg) orally. The time for first excretion of feces and the total number of fecal output by the animals were recorded.

Castor oil-induced enteropooling

Antidiarrheal activity of EFA was tested by using the castor oil-induced enteropooling method in rats (Robert et al., 1976). Twenty Wistar rats were fasted for 18 h and randomly divided in to four groups (n = 5). The control group received only 1% Tween 80 in water (2 ml/rat), positive control group received loperamide 3 mg/kg body weight as standard and test groups received the extracts at doses of 250 and 500 mg/kg body weight orally. Sixty minutes after the administration of drug and extract, 1 ml of castor oil was administered to all animals including the control or vehicle treated group. After 30 min, following administration of castor oil, all rats were sacrificed by an overdose of ketamine, whole length of the intestine from pylorus to caecum was dissected out, its content collected in measuring cylinder and volume measured.

Magnesium sulphate-induced enteropooling

Antidiarrheal activity of EFA was tested by using the magnesium sulphate-induced enteropooling method in rats (Kouitcheu et al., 2006). Twenty Wistar rats were fasted for 18 h and randomly divided into four groups (n = 5). The control group received only distilled water 2 ml/rat, positive control group received loperamide 3 mg/kg body weight as standard and test groups received the extracts at the doses of 250 and 500 mg/kg body weight orally. Sixty minutes after the administration of drug and extract, 10% w/v aqueous solution of magnesium sulphate was administered to all animals including the control or vehicle treated group. After 30 min, following administration of magnesium sulphate, all rats were sacrificed by an overdose of ketamine, whole length of the intestine from pylorus to caecum was dissected out, its content collected in a measuring cylinder and volume measured.

Statistical analysis

The results are presented as mean ± SEM. The statistical analysis of the results was performed using one way analysis of variance (ANOVA) followed by Dunnett’s test using SPSS 11.5 software (Chicago, IL). Differences between groups were considered significant at a level of p < 0.05.

Results

Phytochemical analysis

Preliminary phytochemical screening of the EFA revealed the presence of alkaloids, steroids, carbohydrates, flavonoids, saponins and tannins.

Total flavonoids and tannins content

The amount of total tannins and flavonoids content was calculated as quite high in EFA (256.20 mg of tannic acid and 135.55 mg of quercetin equivalent per g of dry extract, respectively).

Acute toxicity test

In acute toxicity study, oral administration of graded doses (200, 400, 800, 1600 and 3200 mg/kg, p.o.) of the EFA to rats did not produce any significant changes in behavior, breathing, cutaneous effects, sensory nervous system responses or gastrointestinal effects during the observation period. No mortality or any toxic reaction was recorded in any group after 48 h of administering the extract to the animals. Ardisia elliptica was safe up to a dose level of 3200 mg/kg of body weight.

Antioxidant activities

DPPH free radical scavenging activity

DPPH free radical scavenging activity of the EFA was found to be increased with the increase of concentration of the extract (Table 1). The extract exhibited 84.74 ± 0.25% radical inhibition at 100 µg/ml whereas at the same concentration the standard ascorbic acid produced 96.46 ± 0.05% inhibition, respectively. The IC₅₀ value of the extract was found to be significant (30.75 ± 0.14 μg/ml) compared to the IC₅₀ value of the reference compound ascorbic acid (7.89 ± 0.09 μg/ml).

Table 1. DPPH radical scavenging activity of the EFA and standard.

	% Inhibition of ethanol extract and Standards at different concentrations
Concentration (µg/ml)	EFA	Ascorbic acid (Standard)
5	6.20 ± 0.13	30.86 ± 0.10
10	25.87 ± 0.17	60.03 ± 0.08
20	41.09 ± 0.12	72.77 ± 0.13
40	58.43 ± 0.15	86.10 ± 0.10
60	77.42 ± 0.21	95.16 ± 0.12
80	83.29 ± 0.25	96.27 ± 0.18
100	84.74 ± 0.25	96.46 ± 0.05
IC50 (µg/ml)	30.75 ± 0.14	7.89 ± 0.09

EFA = Ethanol fruit extract of Ardisia elliptica. The values are expressed as mean ± standard deviation (n = 3).

Nitric oxide (NO) scavenging assay

The scavenging of NO by the EFA was also increased in a dose dependent manner. A significant decrease in the NO radical due to the scavenging ability of the extract and ascorbic acid was observed. The ethanol extract showed maximum scavenging activity of 76.44 ± 0.41% at 100 µg/ml, whereas ascorbic acid at the same concentration exhibited 87.75 ± 0.33% inhibition (Table 2). The IC₅₀ value for the ethanol extract was significant (51.72 ± 0.17 μg/ml) compared to the IC₅₀ value of the reference standard ascorbic acid (38.68 ± 0.22 μg/ml).

Table 2. Nitric oxide radical scavenging activity of the EFA and standard.

	% NO inhibition of the extract and standards at different concentrations
Concentration (µg/ml)	EFA	Ascorbic acid (Standard)
5	11.05 ± 0.40	13.71 ± 0.33
10	20.44 ± 0.34	28.64 ± 0.27
20	38.29 ± 0.29	38.10 ± 0.29
40	45.59 ± 0.13	51.24 ± 0.29
60	55.11 ± 0.22	67.05 ± 0.66
80	66.67 ± 0.29	82.54 ± 0.49
100	76.44 ± 0.41	87.75 ± 0.33
IC50 (µg/ml)	51.72 ± 0.17	38.68 ± 0.22

EFA = Ethanol fruit extract of Ardisia elliptica. The values are expressed as mean ± standard deviation (n = 3).

Reducing power assay

The reducing power of EFA was also determined using ascorbic acid as a positive control (Table 3). The maximum absorbance for EFA was 1.656 ± 0.009 at 100 μg/ml compared to 2.103 ± 0.006 for standard ascorbic acid, at the same concentration.

Table 3. Reducing power assay of the EFA and standards.

	Average absorbance at 700 nm of extract and standards at different concentrations
Concentration (µg/ml)	EFA	Ascorbic acid (Standard)
5	0.425 ± 0.005	0.579 ± 0.013
10	0.660 ± 0.008	0.821 ± 0.008
20	0.767 ± 0.009	1.128 ± 0.007
40	1.077 ± 0.004	1.466 ± 0.008
60	1.183 ± 0.012	1.633 ± 0.005
80	1.419 ± 0.009	1.843 ± 0.010
100	1.656 ± 0.009	2.103 ± 0.006

EFA = Ethanol fruit extract of Ardisia elliptica. The values are expressed as mean ± standard deviation (n = 3).

Fe⁺² ion chelating ability

Fe⁺² ion chelating ability of the ethanol extract is shown in Table 4. The extract showed 83.65 ± 0.11% Fe⁺² ion chelating ability at 100 µg/ml whereas the standard EDTA showed 96.99 ± 0.14% at the same concentration. The IC₅₀ value of the extract was also found significant (41.30 ±0.12 μg/ml) compared to the IC₅₀ value of the reference standard EDTA (22.57 ± 0.11 μg/ml).

Table 4. Fe²⁺ ion chelating ability of EFA and EDTA (standard).

	% Chelating Ability of differentsolvent extract and standard
Concentration (µg/ml)	EFA	Na2EDTA (standard)
5	12.95 ± 0.07	26.18 ± 0.14
10	19.94 ± 0.11	39.29 ± 0.11
20	28.58 ± 0.08	47.88 ± 0.14
40	49.16 ± 0.18	64.55 ± 0.08
60	65.83 ± 0.14	76.42 ± 0.10
80	80.14 ± 0.17	95.44 ± 0.07
100	83.65 ± 0.11	96.92 ± 0.14
IC50 (µg/ml)	41.30 ± 0.12	22.57 ± 0.11

EFA = Ethanol fruit extract of Ardisia elliptica. The values are expressed as mean ± standard deviation (n = 3).

Antidiarrheal activities

Castor oil-induced diarrhea

Table 5 shows the effect of the EFA on the castor oil-induced diarrheal method in mice. The result show that the extract reduces the mean number of defecation which was 35.63 and 62.07% (p < 0.001) at the doses of 250 and 500 mg/kg, respectively. The latent period for the EFA on the treated group was (p < 0.05 and p < 0.01) increased as compared to the control group.

Table 5. Antidiarrheal activity EFA on castor oil-induced diarrheal test method on mice.

		Mean ± SE
Sample	Dose (mg/kg, p.o.)	Latent period	Defecation	% Inhibition
Distilled water	2 ml/mouse, p.o.	1.15 ± 0.28	17.40 ± 0.87	–
Loperamide	3 mg/kg, p.o.	2.70 ± 0.13***	4.20 ± 0.37***	75.86
EFA	250 mg/kg, p.o.	1.70 ± 0.23*	11.20 ± 0.97***	35.63
EFA	500 mg/kg, p.o.	2.23 ± 0.17**	6.60 ± 0.51***	62.07

Values are expressed as mean ± SEM (Standard Error Mean); EFA = Ethanol fruit extract of Ardisia elliptica. One-way ANOVA followed by Dunnett’s test as compared to control; n = Number of mice; p.o.: per oral.

*p < 0.05; **p < 0.01; ***p < 0.001.

Magnesium sulphate-induced diarrhea

All the mice in control group produced diarrhea after magnesium sulphate administration during the observation period. Pretreatment of mice with both doses of EFA reduced the mean number of defecation 39.06 and 65.63% at the dose of 250 and 500 mg/kg, respectively (p < 0.001). The latent period for the EFA on treated group was (p < 0.01 and p < 0.001) increased as compared to the control group as shown on Table 6.

Table 6. Antidiarrheal activity of EFA in magnesium-induced diarrheal test method on mice.

		Mean ± SE
Sample	Dose	Latent period	Defecation	% Inhibition
Distilled water	2 ml/mouse, p.o.	1.15 ± 0.28	12.80 ± 0.37	–
Loperamide	3 mg/kg, p.o.	3.05 ± 0.19**	2.40 ± 0.40**	81.25
EFA	250 mg/kg, p.o.	2.17 ± 0.23*	7.80 ± 0.37**	39.06
EFA	500 mg/kg, p.o.	2.55 ± 0.24**	4.40 ± 0.40**	65.63

Values are expressed as mean ± SEM (Standard Error Mean); EFA = Ethanol fruit extract of Ardisia elliptica. One-way ANOVA followed by Dunnett’s test as compared to control; n = Number of mice; p.o.: per oral.

*p < 0.01; **p < 0.001.

Castor oil-induced enteropooling

Castor oil caused accumulation of water and electrolytes in the intestinal loop of the rat. EFA at 250 and 500 mg/kg, p.o. dose produced 37.36 and 51.08% inhibition of weight of intestinal content, respectively, with significance (p < 0.05 and p < 0.001). The volume of intestinal content was also reduced significantly at both doses (Table 7).

Table 7. Antidiarrheal activity of EFA in castor oil-induced enteropooling test method on rat.

		Mean ± SE
Sample	Dose	Volume of fluid (ml)	Weight of intestinal content (gm)	% Inhibition
Distilled water	2 ml/rat, p.o.	2.26 ± 0.30	3.06 ± 0.30
Loperamide	3 mg/kg, p.o.	0.58 ± 0.14***	0.83 ± 0.17***	72.96
EFA	250 mg/kg, p.o.	1.64 ± 0.23*	1.92 ± 0.28*	37.36
EFA	500 mg/kg, p.o.	1.25 ± 0.20**	1.50 ± 0.19***	51.08

Values are expressed as mean ± SEM (Standard Error Mean); EFA = Ethanol fruit extract of Ardisia elliptica. One-way ANOVA followed by Dunnett’s test as compared to control; n = Number of mice; p.o.: per oral.

*p < 0.05; **p < 0.01; ***p < 0.001.

Magnesium sulfate-induced enteropooling

Magnesium Sulfate caused accumulation of water and electrolytes in the intestinal loop of the rat. EFA at 250 and 500 mg/kg, p.o. dose produced 27.69 and 47.29% inhibition of weight of intestinal content, respectively, with significance (p < 0.001). The volume of intestinal content was also reduced significantly at both doses (Table 8).

Table 8. Antidiarrheal activity of EFA in magnesium sulphate-induced enteropooling test method on rat.

		Mean ± SE
Sample	Dose	Volume of fluid (ml)	Weight of intestinal content (gm)	% Inhibition
Distilled water	2 ml/rat, p.o.	6.36 ± 0.54	12.25 ± 0.36	–
Loperamide	3 mg/kg, p.o.	3.45 ± 0.46***	4.85 ± 0.42***	60.35
EFA	250 mg/kg, p.o.	5.24 ± 0.32*	8.86 ± 0.72***	27.69
EFA	500 mg/kg, p.o.	4.44 ± 0.24**	6.45 ± 0.65***	47.29

Values are expressed as mean ± SEM (Standard Error Mean); EFA = Ethanol fruit extract of Ardisia elliptica. One-way ANOVA followed by Dunnett’s test as compared to control; n = Number of mice; p.o.: per oral.

*p < 0.05; **p < 0.01; ***p < 0.001.

Discussion

EFA was subjected to screens for its possible antioxidant activities. Five complementary test systems, namely DPPH free radical scavenging, nitric oxide scavenging activity, reducing power and ferrous ion chelating ability, were determined for this analysis.

DPPH is a relatively stable free radical scavenger which converts the unpaired electrons to paired ones by hydrogen proton donation. Scavenging of DPPH radical in this study indicates the potency of the plant extracts in donating hydrogen proton to the lone pair electron of the radicals. Because the inhibition was more at a higher concentration in all the solvent extracts, it could be suggested that the plant extracts contain compounds capable of donating protons to the free radicals. The methods have proven the effectiveness of the extracts in a concentration-dependent manner (Mondal et al., 2005) The antioxidant activity observed in the DPPH radical scavenging assay may be as a result of the of phytoconstituent content in the plant extracts.

In vitro inhibition of nitric oxide radical is a measure of antioxidant activity of plant drugs. Nitric oxide is a free radical that plays an important role in the pathogenesis of pain, inflammation, etc. Scavenging of nitric oxide radical is based on the generation of nitric oxide from sodium nitroprusside in buffered saline, which reacts with oxygen to produce nitrite ions that can be measured by using Griess reagent (Marcocci et al., 1994). The absorbance of the chromophore is measured at 546 nm in the presence of the fractions. All the fractions of EFA decreased the amount of nitrite generated from the decomposition of sodium nitroprusside in vitro. This may be due to the antioxidant principles in the fractions which compete with oxygen to react with NO• thereby inhibiting the generation of nitrite.

The transformation of Fe³⁺ into Fe²⁺ in the presence of various fractions was measured to determine the reducing power ability. The reducing ability of a compound generally depends on the presence of reductones (antioxidants), which exert the antioxidant activity by breaking the free radical chain by donating a hydrogen atom (Meir et al., 1995). The antioxidant principles present in the fractions of EFA caused the reduction of Fe³⁺/ferricyanide complex to the ferrous form, and thus proved the reducing power ability.

The metal chelating ability of the fractions of EFA was measured by the formation of ferrous ion ferrozine complex. Ferrozine combines with ferrous ions forming a red-colored complex that absorbs at 562 nm (Yamaguchi et al., 2000). It was reported that the chelating agents, which form σ bond with a metal, are effective as secondary antioxidants, because they reduce the redox potential thereby stabilizing the oxidized form of the metal ion (Duh et al., 1999). The results of our study demonstrate that the fractions have an effective capacity for iron binding, suggesting its antioxidant potential. In addition, the metal chelating ability of the fractions demonstrated that they reduce the concentration of the catalyzing transition metal involved in the peroxidation of lipids.

The reported compound quercetin from this plant is also responsible for antioxidant activity (Urmila et al., 2011). Again it has been reported that saponins, tannins and certain flavonoids are potential free-radical scavengers, and that their activity against the DPPH radical is closely associated with their chemical structure (Ponou et al., 2010; Takako et al., 1998). Tannins have also ferrous ions (Fe²⁺) chelating activity (Ilhami et al., 2010). Again, flavonoids are very potent nitric oxide radical (NO.) scavengers (Van et al., 1995). So it has been reported that the antioxidant activity of the EFA might be due to presence of a significant amount of flavonoids and tannins in the extract. Flavonoids and tannins, commonly found in plants, have been reported to have significant antioxidant activity (Vinson et al., 1995).

Diarrhea results from an imbalance between the absorptive and secretory mechanisms in the intestinal tract resulting in an excess loss of fluid in the feces. The EFA showed significant inhibitory activity against castor oil and magnesium sulfate-induced diarrhea and castor oil and magnesium sulfate-induced enteropooling. At doses of 250 and 500 mg/kg, the EFA showed significant antidiarrheal activity against castor oil-induced diarrhea as compared with the control group in a dose dependant manner. Several mechanisms have been supposed to be involved in the diarrheal effect of castor oil (Robert et al., 1976). These include castor oil decreases fluid absorption, increases secretion in the small intestine and colon, and affects smooth muscle contractility in the intestine. Castor oil produces diarrhea due to its active component of recinoleic acid (Dicarlo et al., 1994), inhibition of intestinal Na⁺,K⁺-ATPase activity to reduce normal fluid absorption (Mascolo et al., 1994), activation of adenylyl cyclase (Mascolo et al., 1994), stimulation of prostaglandin formation (Phillips et al., 1965), and platelet activating factor. Recently nitric oxide was found to contribute to the diarrhea effect of castor oil (Nell & Rummel, 1984). Prostaglandin contributes to pathophysiological functions in gastrointestinal tract (Sanders, 1984). Inhibitors of prostaglandin biosynthesis delay castor oil-induced diarrhea (Awouters et al., 1978). The antidiarrheal activity of the plant extract was closely comparable to the standard drug, loperamide, which at present is one of the most efficacious and widely employed antidiarrheal drugs. Loperamide effectively antagonizes diarrheal activity induced by castor oil (Karim & Adaikan, 1977). Since the EFA successfully inhibited castor oil-induced diarrhea, it can be assumed that the antidiarrheal action was exerted by an antisecretory mechanism. This was also evident from reduction of the total number of wet feces in the test groups in the experiment. Again, flavonoids present in the plant extract are reported to inhibiting release of autacoids and prostaglandins, thereby inhibit motility and secretion induced by castor oil (Veiga et al., 2001). The antidiarrheal activity of the extract may also be due to denatured proteins forming protein tannates that make intestinal mucosa more resistant and reduce secretion.

Magnesium sulfate similarly causes an increase in the electrolyte secretion by creating an osmotic imbalance (Inayathulla et al., 2010). The extract sufficiently counteracted the increase in electrolyte secretion by means of an anti-electrolyte permeability action. EFA may act beyond any one of the mechanism. It is also noted that EFA significantly inhibited castor oil-induced intestinal fluid accumulation and the volume of intestinal content. Secretory diarrhea is associated with an activation of Cl⁻ channels, causing Cl⁻ efflux from the cell, the efflux of Cl⁻ results in massive secretion of water into the intestinal lumen and profuse watery diarrhea (Ammon & Soergel, 1985). The involvement of muscarinic receptor effect was confirmed by increased production of both gastric secretion and intraluminal fluid accumulation induced by castor oil. EFA may inhibit the secretion of water into the intestinal lumen and this effect is partly mediated by both α2-adrenoceptor and muscarinic receptor systems. The significant inhibition of the castor oil-induced enteropooling in mice suggests that the EFA produced relief in diarrhea by spasmolytic activity in vivo and antienteropooling effects (Nwodo & Alumanah, 1991).

It has been reported that the isolated compound (quercetin) from this plant is responsible for antidiarrheal activity (Gálvez et al., 1995). Furthermore, antiantidiarrheal properties of medicinal plants were found to be due to presence of tannins, alkaloids, saponins, flavonoids, sterols and/or triterpenoids (Brown & Taylor, 2000). Phytochemical analysis of EFA revealed the presence of significant amounts of flavonoids, and tannins in ethanol extract. These constituents may mediate the antidiarrheal properties of the EFA.

Conclusion

The potential of the extract of A. elliptica as antioxidant and antidiarrheal agents may be due to the presence of phytoconstituents like flavonoids and tannins and might be responsible for its activity. In conclusion, the present study has shown that the EFA is a potential therapeutic option in the effective management diarrhea, thus justifying its widespread use by the local population for these purposes. Concerted efforts are being made to fully investigate the mechanisms involved in the pharmacological activities of EFA and phytochemical studies are also in progress to isolate and characterize the active constituents of A. elliptica. The isolated compound may serve as useful prototypes of antioxidant and anti-diarrheal drugs of natural origin possessing the desired pharmacological activities while lacking certain untoward effects.

Declaration of interest

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