Since time immemorial, humans have been using plants for the management of various conditions. Many of such plants have well been scientifically proven to be effective through experimental bioassay and the active principle have isolated and developed into drugs used conventional orthodox medicine. Some of the plants used in ethno medicine which was not found effective are discouraged of their continual usage especially in acute and life threatening conditions such as diarrhoea.
Diarrhoea is ranked high among the serious health problems claiming lives of
children and immune compromised patients in the developing countries accounting
for more than 5 million deaths worldwide each year in infants and children of
less than 5 years. These could be attributed to lack of access to potable water
and constitute a medical emergency primary health care units are not readily
available (Shoba and Thomas, 2001). In order to reduce
the impact of diarrhoea in developing countries, international organizations
such as WHO have recommended the use of traditional remedies of proven efficacy
in remote communities as a tentative approach before accessing standardized
medical treatment (Atta and Mouneir, 2004).
This study attempts to determine the efficacy of the plant Detarium microcarpum stem bark in an attempt to further encourage their use if found effective.
MATERIALS AND METHODS
Plant material: The stem bark of D. microcarpum was collected in suburb of Yola town in Nigeria. It was identified and authenticated by a taxonomist in the Department of Biological Sciences, University of Maiduguri. It was then air dried to a constant weight, pulverized using mortar and pestle. The resulting powder was stored in an airtight container and kept in a cool, dark and dry place prior to extraction process.
Preparation of extracts: The plant was extracted using soxhlet extractor, where 250 mL of distilled water was added to 500 g of the powder. After 60 min, the mixture was cooled and decanted. The decoction was filtered with a filter and the filtrate was dried using digital heating drying oven (DHG-9030A) preset at 50°C. The air-dried extract was secured in air-tight material until use. A concentration of 200 mg mL-1 solution was constituted for use in the experiment.
Phytochemical analysis: The crude aqueous extract of D. microcarpum
stem-bark was subjected to qualitative chemical screening for the identification
of the various classes of active chemical constituents. The phytochemical analyses
were carried out according to standard methods described by Trease
and Evans (1997) and Sofowora, 2006. 80 mg mL-1
of the stem-bark extract was constituted for the analysis.
Test for carbohydrate
General test Molischs test: Two drops of Molischs reagent
was added to 2 mL of the aqueous solution of the extract in a test tube, then
a small volume of concentrated tetraoxosulphate VI acid was allowed to run down
the side of the inclined test tube to form a layer without shaking. The presence
of purple colour at the interface was indicative of the presence of carbohydrate.
General Test for Monosaccharide Barfoeds Test: One milliliter of the aqueous solution and 1 mL of Barfoeds reagent were mixed in a test tube and placed in a water bath for about 1 min. The presence of red precipitate was indicative of monosaccharide.
Standard test for free reducing sugar fehlings test: To 2 mL of the aqueous solution of the extract was added 5 mL mixture of equal volumes of Fehlings solutions I and II and boiled in a water bath for about 2 min. The brick red precipitate observed indicates the presence of reducing sugars.
Standard test for combined reducing sugars: One milliliter (1 mL) of the aqueous solution of the extract was hydrolysed by boiling with 5 mL of 10% hydrochloric acid solution. Then the above procedure for Fehlings test was carried out and the presence of brick red precipitate was indicative of combined reducing sugars.
Test for tannins
Ferric chloride test: To 2 mL of the aqueous solution of the extract
was added few drops of 10% ferric chloride solution. The occurrence of bluish
black colour indicates the presence of gallic tannins and greenish-black colour
indicates the presence of catechol tannins. Control tests were done by repeating
the procedure using distilled water and wild cherry bark separately in place
of the extract as standards.
Formaldehyde test: To 2 mL of the aqueous solution of the extract in a test tube was added a drop of 10% formaldehyde solution and 3 drops of 10% hydrochloric acid. The mixture was then observed for the presence of precipitate indicative of tannin. Control tests were done by repeating the procedure using distilled water and catechol separately in place of the extract as standards.
Test for saponins (froth test): Three milliliter of the aqueous solution of the extract was mixed with 10 mL of distilled water in a test tube. The tube was stoppered and shaken vigorously for about 5 min. It was then allowed to stand for 30 min and observed for honey comb froth for positive result.
Test of cardiac glycosides: To about 2 mL of the aqueous solution of the extract was added 3 drops of strong solution of lead acetate. This was mixed thoroughly and filtered. The filtrate was shaken with 5 mL of chloroform in a separating funnel. The chloroform layer was evaporated to dryness in a small evaporating dish. The residue was dissolved in glacial acetic acid containing a trace of ferric chloride. This was then transferred to the surface of 2 mL concentrated tetraoxosulphate (vi) acid in a test tube. The upper layer and interface of the two layers were observed for bluish-green and reddish-brown colouration respectively which are positive for cardiac glycosides. The control study was carried out in the absence of the extract.
Test for flavonoids: Two grams of the powdered stem bark was detanned with acetone. The residue was extracted in water after evaporating the acetone on a water bath. The mixture was filtered while hot and used for the following tests for flavonoids.
Lead acetate test: To 5 mL of detanned extract was added 10% lead acetate solution and observed for coloured precipitates indicative of flavonoid. Control test was run by repeating the procedure using distilled water in place of the extract.
Sodium hydroxide test: Five milliliters of 20% sodium hydroxide was added an equal volume of detanned extract and observed for yellow coloration which was indicative of flavonoid. Control test was run by repeating the procedure using distilled water in place of the extract.
Test for alkaloids: One gram (1 g) of the extract was dissolved in 5 mL of 10% ammonia solution and extracted with 15 mL of chloroform. The chloroform portion was evaporated to dryness and the resultant residue dissolved in 15 mL of dilute tatraoxosulphate (vi) acid. This was used for the following tests.
Mayers test: Three drops of Mayers reagent was added to 2 mL of the acidic solution in a test tube and observed for an opalescence or yellowish white precipitate which was indicative of the presence of alkaloids. Control test was carried out by repeating the procedure using water in placed of the extract.
Dragendorffs test: Two milliliters of acidic solution in a test tube was neutralized with 10% ammonia solution. Dragendorrffs reagent was added and turbidity or precipitate was observed indicative of presence of alkaloids. Control test was carried out by repeating the procedure using 10% ammonia in place of the extract as standard.
Test for anthraquinones
Borntragers reaction for free anthraquinones: One gram of powdered
stem bark was placed in a dry test tube and 20 mL of chloroform was added. It
was heated in a steam bath for five minutes. The extract was filtered while
hot and allowed to cool. To the filtrate was added equal volume of 10% ammonia
solution. It was then shaken and the upper layer was observed for bright pink
coloration indicative of anthraquinones. The control test was performed by adding
10 mL of 10% ammonia solution in 5 mL of chloroform in a test tube.
Test for combined form of anthraquinone: One gram of powdered stem bark was boiled with 10 mL of ferric chloride and 5 mL of dilute hydrochloric acid for 5 min. The mixture was filtered while hot, allowed to cool and the filtrate was shaken with equal volume of chloroform. The layers were allowed to separate in a separating funnel. The chloroform layer was transferred into another tube containing 5 mL of 10% ammonia solution and the upper aqueous layer was observed for a bright-pink colour. Control test was performed by adding 10 mL of 10% ammonia solution in 5 mL of chloroform in a test tube.
Experimental animals: Albino rats of both sexes weighing between 150-200
g were obtained from laboratory animal house of the Faculty of Pharmacy; University
of Maiduguri, Nigeria, was used for the study. They were allowed to acclimate
for two weeks in the Department of Veterinary Physiology Laboratory, University
of Maiduguri. The rats were fed with standard laboratory diet and allowed access
to water ad-libitum. Assessment of anti-diarrheal effect of D. microcarpum
by protection of induced diarrhoea.
Twenty-five rats divided into five groups of five rats each and are designated
as groups A to E. Group A was orally given distilled water, groups B and C were
given graded (400 and 600 mg kg-1) doses of the extract. Group D
serving as the positive control received the standard drug loperamide. After
1 h, 1 mL of castor oil was administered to all the rats to induce diarrhoea
and was then kept in individual cages with a clean white sheet of paper placed
at its bottom. Numbers of dry and wet faeces were counted after 4 hour and percentage
protection from diarrhoea was deduced. Group E were not administered castor
oil and they served as the negative control.
Assessment of anti-diarrheal effect of D. microcarpum by intestinal secretory inhibition: Total of twenty-five was used for studying the anti-diarrheal effect of the extract using castor induced enteropooling by administering 0.2 mL. of castor oil orally. The rats were divided into five groups of five animals per group designated as A, B C, D and E. Group A serve as negative control and were administered castor oil only while groups B, C and D were given graded doses (400, 600 and 800 mg kg-1) while group E served as the positive control which were given atropine only.
Two hours after administration of the castor oil, animals in each group were
sacrificed and volume of the content of the small intestine was measured by
milking it into graduated tube (Robert et al., 1976).
Assessment of antidiarrheal effect by intestinal motility: Twenty five
rats were fasted for 18 hours and grouped into five groups (A-E) of five rats
each. Group A served as the negative control and were given distilled water.
Groups B, C, D and E were first given castor oil an hour before treatment and
then administered 10% charcoal solution 1 hour after treatment. Treatments given
to group B was distilled water only, groups C and D were administered graded
doses (400 and 800 mg kg-1, respectively) of the extract while group
E served as the positive control and was administered standard drug, atropine.
Animals were sacrificed after 1 h and intestinal motility was assessed by measuring
the distance travelled by charcoal along the intestinal length with respect
to the total length of the intestine as described by Mascolo
et al. (1994).
Statistical analysis: Data obtained from this experiment was expressed
as Mean±standard deviation and were analysed using one way analysis of
variance. Statistical software, Graphpad InStat (2003)
was used for the analysis and p≤0.05 was considered significant.
Phytochemical analysis: The presence of carbohydrate, alkaloids, tannins and flavonoids were detected in the aqueous extract of D. microcarpum as shown in Table 1.
|| Phytochemical constituents of D. microcarpum aqueous
|+: Present, -: Not detected
||Effect of D. microcarpum aqueous extract on castor-oil
|Values are expressed as Mean±SD, N = 5, Values with
superscript a in the given column are significantly (p≤0.01) lower than
the values in control group
Protection from induced diarrhoea test: Inhibition of castor induced diarrhoea was presented in Table 2. It showed that, compared to the control that had mean total number of faeces of 13±2.0, groups treated with 400 and 600 mg/kg of the aqueous extract of D. microcarpum stem bark showed significantly (p≤0.05) lower count of 6.4±2.5 and 5.8±2.5, respectively. Similarly, out of these totals, wet faecal counts for groups treated with the extracts at 400 and 600 mg kg-11 were 4.4±1.8 and 4.2±2.7, respectively. These values were significantly (p≤0.05) low in comparison to control that had a value of 9.0±1.6. Positive control group that received a standard drug Loperamide showed remarkable resistance to castor-induced diarrhoea because the value of the mean number of wet faeces was 0.6±0.1 out of 0.8±0.2 mean total. Negative control which received distilled water had no wet faceces. The percentage protection offered by the extract to the castor-oil induced diarrhoea was deduced to be 51 and 53% for 400 and 600 mg kg-1 while a very high value of 92% was obtained for the group treated with loperamide. This result signifies that the protective effect of extract was not dose dependant.
Enteropooling test: Result of enteropooling test (Table 3) showed that in the groups which received the aqueous extract of D. microcarpum stem bark at a dose rate of 400, 600 and 800 mg kg-1 had a volume of intraluminal fluid content weighing 1.8±0.4, 1.3±0.7 and 1.1±0.3, respectively. When these values were compared with the control group that had 2.4±1.0, statistically significant (p<0.05) reduction was observed in 600 and 800 mg kg-1 extract treated group. However, in the lowest dose of 400 mg kg-1, the value obtained was 1.8±0.4 and there was no significant (p<0.05) difference compared to the control.
||Effect of D. microcarpum aqueous extract on castor-oil
induced enteropooling in albino rats
|Values are expressed as Mean±SD, Values with superscript,
a in the given column are significantly (p≤0.05) lower than the values
in control group, Values with superscript, b in the given column are significantly
(p≤0.01) lower than the values in control group
||Effect of D. microcarpum aqueous extract on charcoal
transit time in albino rats
|Values are expressed as Mean±SD, Values with superscript
ain the given column are significantly (p≤0.01) lower than
the values in control group
The calculated value of percentage fluid accumulation was lowest in atropine control group having 17% which was comparable to 18% recorded in the groups that received the extract at 800 mg kg-1. The values for percentage fluid accumulation for 400, 600 mg kg-1 were 26 and 27%, respectively.
Intestinal motility test: Results from the gastrointestinal motility tests (Table 4) showed that the average distance (58±3 cm) moved by the charcoal marker was greatest for the control (group A). These distances were least (28±5 cm) for the group which received atropine. In the groups treated with the aqueous extract of D. microcarpum stem bark at 400, 600 and 800 mg/kg, the values for distance travelled by charcoal were 48±5, 41±4 and 38±4 cm, respectively which were all significantly (p<0.05) lower compared to the negative control group.
In line with the objective of WHOs objectives (Sofowora,
2006) of ethno-medicinal research of evaluating the efficacy of herbs used
in managing disease condition and disease states to encourage or discourage
its use, D. microcarpum was evaluated for antidiarrheal efficacy in experimental
Ricinoleic acid released from castor oil when hydrolysed by pancreatic lipase
causes irritation and inflammation of the intestinal mucosa leading to the release
of prostaglandin that stimulate motility and secretion (Pierce
et al., 1971). This agent was used in inducing functional diarrhoea
in this experiment.
The presence of tannins in the extract may explain the antidiarrheal activity
of the as they are known to have astringent action. Tannins are referred to
as gastrointestinal modifiers which following ingestion and consequent hydrolysis
precipitate proteins and other large molecules, thereby altering fluidity of
the intestinal content and hence used as an antidiarrheal agent (Bone,
1998). Flavonoids have been ascribed to have antidiarrheal activity of due
to their ability to inhibit intestinal motility and hydro electrolytic secretion
(Di Carlo et al., 1993).
Modulators of intestinal motility such as antimuscarinics and opiates have
been categorized as antidiarrheal agent due to their ability to relax gastrointestinal
smooth muscle by inhibiting the effect of endogenous acetyl choline (Akubue,
2006). Atropine also inhibits secretion of intestinal mucosa. D. microcarpum
stem aqueous extract was also observed in this study to reduce significantly
the intestinal motility in rats that were fed with charcoal meal similar to
that obtained in atropine treated group.
In the pathophysiology of diarrhoea, there is an increase in frequency of defecation associated with watery stool. The significant reduction of frequency of defecation and number of wet faecal dropping observed in this study demonstrates the efficacy of D. microcarpum stem aqueous extract as antidiarrheal agent.
Gastrointestinal Protestants and astringent such as kaolin are also in symptomatic
control of diarrhoea because of their ability to coat the intestinal mucosa
thereby preventing irritation and erosion of potentially harmful substances
(Nicholas and McDonald, 2001). The percentage protection
offered by D. microcarpum stem aqueous extract to diarrhoea induced by
castor oil was about 55%. This signifies the extract possess some marginal coating
effect on the intestinal mucosa.
The claim by traditional herbalists on use of the plant D. microcarpum stem bark decoction in the management of diarrhoea can be said to be scientifically justified since in this study, its aqueous extract was found to contain tannins, a known astringent and was able to reduce intestinal motility and secretion. These properties are attributes of some conventional antidiarrheal agents of orthodox medicine and therefore its usage for this purpose in traditional medicine should be encouraged. Further studies need to be carried out to identify the active principle responsible for this activity as well as toxicological studies to determine its safety margin.