Evaluation of the antidiarrheal and antioxidant properties of Justicia hypocrateriformis

Abstract

Content: Justicia hypocrateriformis Vahl (Acanthaceae) is used as an herbal remedy for diarrhea in Cameroon folk medicine.

Objective: This study evaluates the antidiarrheal and antioxidant properties of the aqueous extract of J. hypocrateriformis (JH).

Materials and methods: Preliminary phytochemical screening and an acute toxicity testing of the extract were carried out. The antidiarrheal activity of JH extract (100, 250, and 500 mg/kg) was assessed at curative and preventive levels in castor oil-induced diarrhea in mice. The antioxidant activity was measured by ferric reducing antioxidant power (FRAP), total phenolic content, and radical scavenging activity.

Results: A high lethal dose (LD₅₀) of 14.35 g/kg obtained in acute toxicity implies the extract is not toxic. Phytochemical screening revealed the presence of phenols, tannins, flavonoids, saponins, anthraquinones, and anthocyanins. JH showed a significant protection against castor oil-induced diarrhea as evidenced by a decrease in the number of defecation and wet stool. JH (100–500 mg/kg, p.o.) produced a non-significant dose-dependent decrease in castor oil-induced intestinal transit in the preventive study. In the curative and in healthy mice study, the decrease was only significant at 500 mg/kg. JH possessed a radical scavenging activity with an IC₅₀ of 9.93 mg/ml compared to 4.90 mg/ml for catechin. JH FRAP of 2703.77 ± 0 mg/g (catechin equiv) and phenolic concentration of 14 169.99 ± 612.39 mg/g (catechin equiv) were also obtained.

Conclusion: Justicia hypocrateriformis extract possesses antidiarrheal activity supported by its antioxidant potential and phytochemical constituents.

• Acute toxicity
• castor oil
• ferric reducing antioxidant power
• intestinal transit
• phytochemical screening
• radical scavenging activity
• total phenolic content

Introduction

Diarrhea is characterized by increased fluidity, frequency, or volume of bowel movements and may be acute or chronic. It has been reported as a major cause of infant mortality and morbidity and responsible for mortal death of elderly people in developing countries (Organisation Mondiale de la Santé, 1993; United Nations Children's Fund, 2009). In infants and elderly people, diarrhea can be very serious causing potentially fatal dehydration (Yadzi & Chang, 1993). Diarrhea is estimated to cause 5 million deaths in children below 5 years of age per year (Carlos & Saniel, 1990). In many African countries, the use of herbal drugs in the treatment of diarrhea is a common practice (Agunu et al., 2005). Many people in the developing countries still have strong belief on traditional healing practices and medicinal plants for their daily healthcare needs despite the immense development in technology and advancement in Western medicine (Ojewole, 2004).

Many studies have demonstrated the antidiarrheal properties of medicinal plants. These medicinal plants have the ability to stimulate water absorption, to reduce electrolytes secretion (Na⁺, K⁺, , etc.), intestinal motility (Agbor et al., 2004; Kambaska et al., 2006; Oben et al., 2006), and stimulate antispasmodic effects (Lozoya et al., 2002). Other studies have shown that different antidiarrheal medicinal plant extracts possess antibacterial, antioxidant, and antiinflammatory activities (Tzung-Hsun et al., 2005). The strength of a plant as medicinal lies on what secondary metabolites they contained. Medicinal plants possessing tannins, alkaloids, saponins, flavonoids, steroids, and terpenoids have been reported to possess antidiarrheal activity (Brijesh et al., 2009; Havagiray et al., 2004; Yadav & Tangpu, 2007). Flavonoids inhibit intestinal motility and hydroelectrolytic secretions (Venkatesan et al., 2005), tannins denature proteins in the intestinal mucosa by forming protein tannates which may reduce secretion and also reduced intracellular Ca²⁺ inward current or activation of the calcium pumping system, hence inducing muscle relaxation (Belemtougri et al., 2006).

Justicia hypocrateriformis Vahl (Acanthaceae) is a shrub with scattered growth measuring above 1 m in height and is used as an ornamental plant. The flowers are tubular measuring approximately 3.75 cm long and are generally yellow or red in color (Burch & Demmy, 1986). In Cameroon traditional medicine, Justicia is used for the management of anemia and diarrhea (Adjanohoun et al., 1996). The present study investigated the antidiarrheal and antioxidant activities of J. hypocrateriformis against experimentally induced diarrhea. A preliminary phytochemical screen was carried out to determine the presence of bioactive component of the leaf extract.

Materials and methods

Chemicals

Loperamide (imodium, standard reference antidiarrheal drug), castor oil (laxative agent), charcoal meal (marker diet, 10% activated charcoal in 5% gum acacia), DPPH (diphenyl-1,2-picryl hydrazyl) (Sigma Chemical Co., St Louis, MO), Folin-Ciocalteu’s phenol reagent (Sigma Chemical Co., St. Louis, MO), hydrochloric acid (MERCK KGaA, Darmstadt, Germany), sodium acetate (MERCK KGaA, Darmstadt, Germany), acetic acid (LABOSI rue de Javel Paris 15), ferric chloride (MERCK KGaA, Darmstadt, Germany), 2,3,6-tripydyl-S-triazine (TPTZ) (Sigma Chemical Co., St. Louis, MO), catechin (Sigma Chemical Co., St. Louis, MO). All reagents were of analytical grade.

Animals

Male mice (20–30 g) bred in the Laboratory of Pharmacology, Institute of Medical Research and Medicinal Plants Studies, Yaoundé, Cameroon. Animals were maintained under standard environmental conditions (temperature: 24.0 ± 1.0 °C, relative humidity: 55–65% and 12 h light/dark cycle) and had free access to feed and water ad libitum. The trial was run in accordance with the practice and principles of the Institute of Medical Research and Medicinal Plants Studies Ethical Review Board that approved the study and the 1996 Guide for the Care and Use of Laboratory Animals.

Plant material

Fresh leaves of J. hypocrateriformis were collected in the morning hours of 20–25 September 2011 in the Yaoundé environ, Cameroon, in the month of September. The plant was identified and authenticated by a botanist in the National Herbarium Yaoundé, Cameroon, as a synonym to Ruspolia hypocrateriformis (Vahl) Milne-Redh assigned number 37822HNC. The freshly collected leaves were rinsed with tap water and air-dried at room temperature for 14 d. The dried leaves were then ground into powder.

Preparation of extract

The powdered sample (400 g) was soaked in 6 L hot water at 60–70 °C for 24 h. The mixture was then filtered and the residue was then subjected to the same procedure to maximize extraction. The resulting filtrate was then concentrated using a rotary evaporator at 60 °C with the aid of a vacuum pump, and further dried in an oven at 40 °C.

Phytochemical screening of plant extract

The crude extract of J. hypocrateriformis was qualitatively tested for the presence of secondary metabolites (alkaloids, glycosides, saponins, tannins, anthocyanins, emodines, polyphenols, anthraquinones, and flavonoids) using standard procedures as earlier described (Trease & Evans, 1983).

Acute toxicity testing

The method of Miller and Tainter (1944) was used for the acute toxicity test of J. hypocrateriformis. Oral acute toxicity testing was carried out as follows: eight groups of six mice each were fasted for 18 h and then given orally (using oral intubator) 1, 2, 4, 8, 10, 12, 14, and 16 g extract/kg body weight. A group administered distilled water was used as a control. The mice were then observed closely for 7 h for changes in physical activities such as shivering, hair loss, dizziness, feeding habit, somatic functions, secretions, muscle tone, and death. Observation was then continued after every 10 h for 72 h. The results were used to calculate the dose that resulted to 50% death (LD₅₀).

Antidiarrheal testing

Inhibition of castor oil-induced diarrhea

The method proposed by Galvez et al. (1993) modified by Oben et al. (2006) was used for experimental needs. Adult mice fasted for 22 h were divided into six groups (groups I–VI) of six each and treated as follows:

• Group I (normal control): distilled water (0.2 ml).

• Group II (standard control): loperamide (0.25 mg/kg body weight).

• Group III: 100 mg/kg body weight J. hypocrateriformis extract.

• Group IV: 250 mg/kg body weight J. hypocrateriformis extract.

• Group V: 500 mg/kg body weight J. hypocrateriformis extract.

• Group VI (diarrheal control): distilled water (0.2 ml).

In groups III, IV, and V, the extracts were prepared in 0.2 ml of distilled water.

Thirty minutes later, castor oil (0.2 ml) was administered by intubation to groups II, III, IV, V, and VI. The animals were then placed in separate wired-meshed cages and the total number of stool and wet stool were counted. The percentage diarrhea inhibition was then calculated as a function of the diarrheal control.

Inhibition of upper gastrointestinal transit (preventive studies)

The method proposed by Aye-Than et al. (1989) was used in groups of six mice each, using a “charcoal meal” as a marker diet. The mice were given increasing doses of the plant extract in 0.2 ml of distilled water by intubation and 30 min later castor oil was administered, followed by the marker diet after another 30 min. The mice were sacrificed by cervical dislocation 30 min after the market diet, i.e., 1 h after castor oil administration. The intestinal transit of the marker diet was calculated as a percentage of the distance traveled by the charcoal meal compared to the length of the small intestine.

Inhibition of upper gastrointestinal transit (curative studies)

The method proposed by Aye-Than et al. (1989) was used in groups of six mice each, using a “charcoal meal” as a marker diet. The mice were given castor oil and 30 min later was administered increasing doses of the plant extract in 0.2 ml of distilled water by intubation, followed by the marker diet after another 30 min. The mice were sacrificed by cervical dislocation 30 min after the market diet. The intestinal transit of the marker diet was calculated as a percentage of the distance traveled by the charcoal meal compared to the length of the small intestine.

Inhibition of upper gastrointestinal transit (effect in normal mice)

The experimental mice were used in groups of six each; a “charcoal meal” was used as a marker diet. The mice were given increasing doses of the plant extract in 0.2 ml of distilled water by oral intubation followed by the marker diet 30 min later. The mice were sacrificed by cervical dislocation 60 min after the market diet. The intestinal transit of the marker diet was calculated as a percentage of the distance traveled by the charcoal meal compared to the length of the small intestine.

Antioxidant capacity of J. hypocrateriformis

The following concentrations of the plant extract 1, 2, 4, 6, 8, and 10 mg/ml were prepared for the in vitro antioxidant capacity testing. Each test was performed in triplicate and catechin was used as the standard.

Total phenolic content (TPC)

Folin–Ciocalteu’s phenol reagent was used to determine the total phenolic concentration as a measure of antioxidant potential. Folin–Ciocalteu’s phenol reagent (2 N) was diluted 10 times with distilled water before used, as described by Vinson et al. (1998). To 1 ml of the 10-fold diluted Folin–Ciocalteu’s phenol reagent in a cuvette, 40 μl of extract was added and mixed by inversion. The mixture was allowed to stand for 15 min and the absorbance was read at 750 nm in a UV spectrophotometer against a reagent blank. A standard curve of catechin (10–100 μM) was prepared from a 1 mM catechin stock standard and the regression line of the standard curve was used to determine the total phenolic concentration in the plant extract.

Ferric reducing antioxidant power (FRAP)

The FRAP was measured as described earlier by Benzie and Strain (1996). The FRAP reagent constituted a working reagent of 300 mM acetate buffer (pH 3.6), 10 mM TPTZ, and 20 mM ferric chloride in the ratio of 10:1:1, respectively. To 2000 μl of freshly prepared FRAP reagent, 75 μl of plant extract was added. The absorbance was read at 593 nm using a spectrophotometer after 15 min of incubation. A standard curve of catechin (50–800 μM) was prepared from a 1 mM catechin stock standard and the regression line of the standard curve was used to determine the FRAP of the plant extract.

DPPH Radical scavenging activity measured the ability of the extracts to scavenge free radical. This was estimated using the method described by Braca et al. (2001). Plant extract (0.1 ml) was added to 3 ml of a 0.004% methanol solution of DPPH and incubated in a dark room. Absorbance was read at 517 nm using a spectrophotometer after 30 min, and the percentage inhibition activity was calculated from [(A₀−A₁)/A₀] × 100, where A₀ is the absorbance of the control, and A₁ is the absorbance of the extract/standard. The percentage radical scavenging activity was then plotted against the sample extract concentration, and a linear regression curve was established in order to calculate the IC₅₀ (mg/ml), which is the amount of sample necessary to decrease the colorization of DPPH by 50%. This procedure was repeated with catechin standard to compare the reducibility activity of the plant extract.

Statistical analysis

Data are presented as mean ± standard deviation. The results were analyzed using a one-way ANOVA. Significance differences of group comparison were determined using Student–Newman–Keuls’ test (p < 0.05). The Sigmastat 3.01 (Systat Software, Richmond, CA) statistical package was used for this analysis.

Results and discussion

The percentage yield of the extraction process of J. hypocrateriformis was 14.02%. Phytochemical screening of J. hypocrateriformis revealed the presence of alkaloids, tannins, glycosides, flavonoids, anthocyanins, emodins, and polyphenols which may be important secondary metabolites (Table 1). Earlier studies have reported that tannins and flavonoids possess antidiarrheal activity (Galvez et al., 1991, 1993). Thus, tannins and flavonoids present in J. hypocrateriformis may be responsible for its antidiarrheal properties.

Table 1. Phytochemical constituents in Justicia hypocrateriformis leaf extract.

Substances	Reducing sugars	Saponins	Triterpenic glycosides	Tannins	Flavonoids	Anthocyanins
Test	−	++	++	+	+	++
Substances	Phenols	Emodins	Anthraquinones	Coumarins glycosides	Alkaloids	Oils
Test	+	+	++	++	+++	+

(−), absent; (+), present; (++), abundant; (+++), highly abundant.

The acute toxicity study of J. hypocrateriformis produced a LD₅₀ of 14.35 g/kg. According to Organization of Economic Cooperation and Development (OECD) Test guidelines on Acute Oral Toxicity (OECD, 2001) an LD₅₀ of 2000 mg/kg and above is categorized as unclassified. Hodge and Sterner (1943) on classifying toxicity based on acute toxicity stated that samples with LD₅₀ between 5 and 15 g/kg are nontoxic. Also, Ouedraogo et al. (2001) stated that LD₅₀ above 5 g/kg body weight are generally not considered as dose-related toxicity. Thus, these allow us to infer that the J. hypocrateriformis extract with a LD₅₀ of 14.35 g/kg is not toxic through the oral route.

According to George (1992), diarrhea may be characterized as the abnormally frequent expulsion of stool of low consistency which may be due to a disturbance in the transport of water and electrolytes in the intestine. Castor oil (ricinoleic acid) has been known to induce diarrhea by bringing about changes in electrolyte and water transport and increases peristaltic activity (Agbor et al., 2004; Capasso et al., 1986; Luderer et al., 1980). These changes have earlier been associated with prostaglandins release which is the main cause of arachidonic acid-induced diarrhea (Luderer et al., 1980). Loperamide is a standard antidiarrheal medication in Cameroon. Studies on loperamide showed that loperamide can effectively antagonized the diarrhea induced by castor oil (Niemegeers et al., 1974), prostaglandin (Karim & Adaikan, 1977), or cholera toxin (Farack et al., 1981) by stimulating antimotility and antisecretary activities (Couper, 1987). In the present study, we evaluated the antidiarrheal activity of J. hypocrateriformis using loperamide as the standard control drug in castor oil-induced diarrhea. The castor oil induced diarrhea which was characterized by an increase in percentage intestinal transit as well as an increase in the number of wet stool. Administration of the extract of J. hypocrateriformis significantly (p < 0.001) decreased the number of wet stool passed out by the experimental animals (Table 2). The effect of the extract was dose dependent (100 < 250 < 500 mg/kg extract) in the order of 7.69%, 28.20% and 46.15% inhibition, respectively. Loperamide (0.25 mg/kg) equally inhibited the castor oil-induced diarrhea by 89.74%. This was far more effective than the most effective plant extract (500 mg/kg).

Table 2. Effect of Justicia hypocrateriformis on castor oil-induced diarrhoea in mice.

Treatment	Total number of stool	Total number of wet stool	Inhibition (%)
Group 1: distilled water (0.2 ml)	20.43 ± 2.52	0
Group 2: castor oil (0.2 ml)	41.36 ± 3.35	39.00 ± 3.00	0
Group 3: loperamide (0.25 mg/kg)	27.52 ± 4.42	4.33 ± 0.58^ab	89.74
Group 4: extract (100 mg/kg)	49.14 ± 2.15	36.00 ± 6.00^c	7.69
Group 5: extract (250 mg/kg)	31.13 ± 4.24	28.67 ± 2.08^ad	28.20
Group 6: extract (500 mg/kg)	38.33 ± 3.25	21.33 ± 2.52^ade	46.15

Dose-dependent inhibitory effect of extract on castor oil-induced diarrhoea. Loperamide presented the highest inhibition, n=6.

^ap < 0.001, significantly lower compared to castor oil control.

^bp < 0.001, significantly lower compared to groups 4, 5, and 6.

^cp > 0.05, not significant difference compared to castor oil control.

^dp < 0.001, significantly lower compared to group 4.

^ep < 0.01, significantly lower compared to group 5.

Administration of castor oil increased the percentage gastrointestinal transit of the marker diet. In the curative study, castor oil administration had an intestinal transit of 66.36 ± 8.37 (Table 3). Administration of plant extract (100 and 250 mg/kg dose treatment) to diarrheal mice did not have any significant effect on the percentage gastrointestinal transit diarrheal mice (p > 0.5, Table 3). However, when diarrheal mice were administered 500 mg/kg of plant extract, the percentage gastrointestinal transit was significantly (p < 0.001) reduced. Loperamide also significantly (p < 0.001) decreased the percentage gastrointestinal transit of mice. The decreasing effect of the gastrointestinal transit on castor oil-induced gastrointestinal transit in mice measures the antidiarrheal curative effect. In a preventive study, the J. hypocrateriformis extract/loperamide was administered before the induction of diarrhea (administration of castor oil). The data for the preventive effect of J. hypocrateriformis on castor oil-induced gastrointestinal transit are presented in Table 4. All the three doses of plant extract (100, 250, and 500 mg/kg) significantly decreased (p < 0.001) the percentage gastrointestinal transit to 54.25 ± 2.10, 45.20 ± 7.02, and 41.72 ± 10.66%, respectively (Table 4), in a dose–response manner in which 500 mg/kg was the most effective. Loperamide (0.25 mg/kg) caused a significant (p < 0.001) reduction in the percentage gastrointestinal transit 2.85 ± 2.97% (Table 4). The effect of the standard drug loperamide was far better than the plant extract.

Table 3. Curative effect of Justicia hypocrateriformis on castor oil-induced gastrointestinal transit in rats.

Treatments	% Transit	Inhibition (%)
Group 1: distilled water (0.2 ml)	48.06 ± 4.49^a
Group 2: castor oil (0.2 ml)	66.36 ± 8.37	0
Group 3: loperamide (0.25 mg/kg)	36.42 ± 6.50^a	46.22
Group 4: extract (100 mg/kg)	52.57 ± 11.84^c	22.24
Group 5: extract (250 mg/kg)	51.88 ± 11.32^c	21.17
Group 6: extract (500 mg/kg)	45.36 ± 6.06^a	31.61

Results are means ± SD, n=6.

^ap < 0.05, significantly lower compared to castor oil control.

^cp > 0.05, no significant difference compared to castor oil control.

Table 4. Preventive effect of Justicia hypocrateriformis on castor oil-induced gastrointestinal transit in rats.

Treatments	% Transit	Inhibition (%)
Group 1: distilled water (0.2 ml)	52.81 ± 5.76
Group 2: castor oil (0.2 ml)	63.29 ± 9.48	0
Group 3: loperamide (0.25 mg/kg)	2.85 ± 2.97^a	95.51
Group 4: extract (100 mg/kg)	54.25 ± 2.10^ab	14.26
Group 5: extract (250 mg/kg)	45.20 ± 7.02^ab	28.56
Group 6: extract (500 mg/kg)	41.72 ± 10.66^ab	34.07

Results are means ± SD, n = 6.

^ap < 0.001, significantly lower compared to castor oil control.

^bp < 0.001, significantly higher than standard control.

Table 5 presents the effect of J. hypocrateriformis and loperamide on gastrointestinal transit of normal mice (healthy or non-diarrheal mice). It was observed that only the 500 mg/kg dose of J. hypocrateriformis and loperamide significantly reduced (p < 0.001) gastrointestinal transit at the levels of 39.17 ± 4.68% and 34.73 ± 6.57%, respectively. Hence, 100 and 250 mg/kg of extract did not have any significant effect on intestinal transit of normal mice. Earlier studies have reported the presence of alkaloids, saponins, tannins, sterols flavonoids, and reducing sugar to be responsible for the antidiarrheal activity of medicinal plants (Galvez et al., 1991, 1993; Longanga et al., 2000). All these with the exception of reducing sugars were detected in the aqueous extract of J. hypocrateriformis. Hence, these phytochemicals may be responsible for the antidiarrheal activity of J. hypocrateriformis.

Table 5. Effect of plant extract on upper gastrointestinal transit of standard charcoal meal in (healthy) normal mice.

Treatments	% Transit	Inhibition (%)
Group 1: distilled water (0.2 ml)	54.27 ± 5.75
Group 2: loperamide (0.23 mg/kg)	34.73 ± 6.57^a	36.00
Group 3: extract (100 mg/kg)	48.92 ± 10.48^c	9.87
Group 4: extract (250 mg/kg)	45.65 ± 12.12^c	15.88
Group 5: extract (500 mg/kg)	39.17 ± 4.68^a	27.84

Results are means ± SD, n = 6.

^ap < 0.05, significantly lower to normal control.

^cp > 0.05, no significant difference compared to normal control.

Reactive oxygen species (ROS) have been implicated in several pathological situations such as cell membrane disintegration, membrane protein damage, and DNA mutation, which can further initiate the development of many diseases, such as cancer, liver injury, and cardiovascular disease (Liao & Yin, 2000). Therefore, plants with antioxidant and free radical scavenging activities may have great relevance in the prevention and treatment of diseases associated with oxidants or free radicals. The antioxidant capacity of the aqueous extract of J. hypocrateriformis was characterized by the phenolic content (Folin–Ciocalteu method), FRAP, and radical scavenging activity (DPPH method) (Table 6). The Folin–Ciocalteu method has been in use for the determination of antioxidant capacity for a long time now. The Folin–Ciocalteu phenol reagent consists of a mixture of the heteropoly acids, phosphomolybdic, and phosphotungstic acids in which the molybdenum and the tungsten are in the 6⁺ oxidation state (Singleton & Rossi, 1965). This method is based on the ability of the plant extract to reduce the phosphomolybdic–tungstate chromogene in solution and produces a blue coloration with maximum absorbance at 760 nm. In the present study, the phenolic concentration increased with an increase in plant extract concentration but did not reach saturation even at 10 mg/ml.

Table 6. Antioxidant capacity of aqueous extract of J. hypocrateriformis.

Extract concentration (mg/ml)	Percentage radical scavenging (%)	FRAP (mg/g catechin eqv)	Phenolic conc (mg/g catechin eqv)
1	16.01 ± 1.54	653.39 ± 40.36	5832.96 ± 891.82
2	17.01 ± 0.72	1283.44 ± 52.93	6558.56 ± 548.30
4	29.65 ± 1.60	2350.03 ± 56.70	9179.62 ± 1070.19
6	33.55 ± 7.31	2609.15 ± 0.00	9209.24 ± 734.27
8	35.36 ± 1.40	2683.34 ± 0.00	11 978.37 ± 362.27
10	56.68 ± 0.82	2703.76 ± 0.00	14 169.99 ± 612.39

Results are means ± SD. Each sample was tested trice.

The FRAP mechanism of action is basically electron transfer. The principle of this method is based on the fact that antioxidant substances reduce the ferric tripyridyltriazine to ferrous tripyridyltriazine yielding a blue coloration measurable at 593 nm (Benzie & Strains, 1996). The reaction detects compounds with redox potentials of <0.7 V (the redox potential of Fe³⁺–TPTZ). Thus, FRAP is a reasonable screen for the ability to maintain redox status in cells or tissues and the reducing power appears to be related to the degree of hydroxylation and extent of conjugation in polyphenols (Pulido et al., 2000). Just like in the phenolic content, the FRAP value of the extract increased with increased concentration of the plant extract. However, the values of phenolic content were higher than those of FRAP.

1,1-Diphenyl-2-picrylhydrazyl (α,α-diphenyl-β-picrylhydrazyl; DPPH) is a stable free radical because the spare electron is delocalized over the molecule as a whole. This prevents dimerization as is obtained with most other free radicals and maintain a deep violet color. On accepting, an electron or a hydrogen atom reduced form of DPPH with the loss of its violet color is obtained (Jun et al., 2004). The loss of the violet color depends on the strength of the antioxidant property of the electron or hydrogen atom donor. In situ, free radicals like polyaromatic hydrocarbon cations have been linked with carcinogenesis (Yen & Chen, 1995). Thus, products that will scavenge DPPH in vitro may also scavenge polyaromatic hydrocarbon cations in vivo. J hypocrateriformis was able to bleach the DPPH radical and the degree of bleaching increased towards yellow with increased concentration of plant extract. J. hypocrateriformis exhibited a radical scavenging activity with an IC₅₀ value of 9.93 g/ml compared with the IC₅₀ value of 4.03 mg/ml of reference antioxidant catechin (Figure 1). The antioxidant potential of J. hypocrateriformis may be contributed by the presence of flavonoids, anthocyanines, and polyphenols.

Figure 1. DPPH radical scavenging activity of the aqueous extracts Justicia hypocrateriformis and catechin standard. DPPH IC₅₀ of J. hypocrateriformis was found to be 9.93 mg/ml while the IC₅₀ for catechin was found to be 4.90 mg/ml.

Conclusion

From the results obtained, the aqueous extract of J. hypocrateriformis possesses antidiarrheal and antioxidant activities which were not comparable to loperamide (one of the most efficacious and widely employed antidiarrheal drugs in the market). The phytochemical screen revealed the presence of an array of secondary metabolites which may be responsible for both antioxidant and antidiarheal properties. However, bio-guided fractionation may improve on the antidiarrheal properties.

Declaration of interest

The authors report no declarations of interest.