Journal of Ethnopharmacology 190 (2016) 13–21

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The effect of methanol rhizome extract of Nymphaea lotus Linn.

(Nymphaeaceae) in animal models of diarrhoea
Fatima Hauwa Bello n, Bilkisu B. Maiha, Joseph A. Anuka
Department of Pharmacology, Faculty of Pharmaceutical Sciences, Ahmadu Bello University, Zaria, Kaduna State, Nigeria

art ic l e i nf o a b s t r a c t

Article history: Ethnopharmacological relevance: Nymphaea lotus, which is widely distributed throughout tropical Africa,
Received 22 November 2015 enjoys a number of ethnomedical uses in Nigeria. Traditionally, the rhizomes of N. lotus are used to cure
Received in revised form diarrhoea.
22 February 2016
Aim of study: This study aims to evaluate the antidiarrhoeal activity of the methanol rhizome extract of
Accepted 16 May 2016
Available online 20 May 2016
N. lotus plant in laboratory animals.
Materials and methods: The extract was screened for activity against castor oil-induced diarrhoea and
Keywords: magnesium sulphate-induced diarrhoea as well as effect on gastric transit time in mice. The effect of me-
Nymphaea lotus thanol rhizome extract of Nymphaea lotus on the perfused isolated tissue preparation was also determined.
Diarrhoea
Results: For castor oil-induced diarrhoea, the extract at doses of 200, 400 and 800 mg/kg produced sig-
Castor oil-induced diarrhoea
nificant reduction in the frequency of diarrhoea (at po0.001, po0.001 and po0.01 respectively). The
Magnesium sulphate-induced diarrhoea
Gastric transit time extract at 800 mg/kg produced a significant delay in onset of diarrhoea (po0.05) comparable to loperamide
Isolated tissue (3 mg/kg). The frequency of magnesium sulphate-induced diarrhoea was also significantly reduced in the
groups treated with 200, 400 and 800 mg/kg of the extract at po0.001, po0.001 and po0.01 respectively.
At doses of 200 mg/kg and 400 mg/kg, the protection produced was comparable to loperamide, 3 mg/kg.
All treated groups produced significant reduction in the transit of charcoal meal along the intestinal tract
at po0.001. The extract at low concentration (4
10  4–6.4
10  2 mg/ml) had contractile effect on the tone
of contraction of the rabbit jejunum while at higher concentrations (8
10  2–512
10  2 mg/ml) produced
significant reduction in the tone and rate of spontaneous contraction of rabbit jejunum. The extract at lower
concentrations (4
10  4–2
10  2 mg/ml) has no effect on contraction of the guinea pig ileum while higher
concentrations (4
10  2–512
10  2 mg/ml) produced significant relaxant activity on guinea pig ileum.
Conclusion: This study has shown that the methanol rhizome extract of N. lotus has antidiarrhoeal properties
thus justifying its use by the local population for this purpose.
& 2016 Elsevier Ireland Ltd. All rights reserved.

1. Introduction tropical Africa, enjoys a number of ethnomedical uses in Nigeria

(Burkill, 1997). Nymphaea lotus rhizome finds applications in the
Diarrhoeal disease account for nearly 1.3 million deaths a year management of circulatory system disorders, digestive system dis-
among children under-five years of age, making it the second most orders such as diarrhoea, dysentery; genitourinary system disorders
common cause of child deaths worldwide (UNICEF, 2012). In Nigeria, as diuretics, mental disorders as insanity, hyposomnia, leprosy, in-
it is the number one fatal outcome disease among children under fections and inflammations (Burkill, 1997). Therefore, it is important
five years of age (Ahmed et al., 2007). Up to 80% of the population in to establish the scientific basis for the ethnomedical claim of N. lotus
Africa uses traditional medicine to help meet their health care needs as an antidiarrhoeal agent. This may serve as a source for developing
more effective antidiarrhoeal drug.
(WHO, 2002). It is, therefore, important to identify and evaluate
traditional medicine that can be used as alternatives to commonly
used antidiarrhoeal drugs, which are often accompanied by adverse
2. Materials
effects such as addiction (e.g. diphenoxylate) and constipation (e.g.
loperamide) (Hardman and Limbird, 2010). Among these plants,
2.1. Plant material
Nymphaea lotus Linn, which is widely distributed throughout
The rhizomes of Nymphaea lotus was obtained from Dogarawa,
n
Corresponding author. Zaria Kaduna state. The botanical identification and authentication
E-mail address: tbells116@[Link] (F.H. Bello). of the plant was carried out by Mallam Musa Muhammad at the

[Link]
0378-8741/& 2016 Elsevier Ireland Ltd. All rights reserved.
14 F.H. Bello et al. / Journal of Ethnopharmacology 190 (2016) 13–21

herbarium section of the Department of Biological Sciences, Ah- 3.3. Acute toxicity studies
madu Bello University, Zaria where an identification number (894)
was obtained. The median lethal dose (LD50) of the extract was determined by
Lorke's method (1983). The study was carried out in two phases and
mice were deprived of food for 12 h prior to administration of the
2.2. Experimental animals
extract. In phase 1, three groups of three mice per group were used.
The extract was administered orally in three graded doses (10, 100
Four male New Zealand rabbits (2–3 kg) and four male guinea
and 1000 mg/kg). The treated mice were observed for 4 h post
pigs (300–500 g) obtained from the Department of Pharmacology
administration for signs of toxicity. After 24 h, no death was re-
and Therapeutics, Ahmadu Bello University, Zaria as well as ninety
corded, thus phase 2 was initiated. Based on the result in phase 1,
adult Swiss albino mice (20–25 g) of both sexes obtained from
three mice were given the extract orally in doses of 1600 mg/kg,
National Institute for Trypanosomiasis (and Onchocerciasis) Re-
2900 mg/kg and 5000 mg/kg respectively. The mice were then ob-
search (NITR), Kaduna were used for the experiments. The animals
served for signs of toxicity for the first 4 h and mortality after 24 h.
were maintained under normal laboratory conditions of humidity,
temperature and light for two weeks prior to the experiment so as
3.4. In vitro studies
to allow for their acclimatization. They were provided with com-
mercial rodent diet and water ad libitum. The study was con- 3.4.1. Effect of methanol rhizome extract of Nymphaea lotus on the
ducted according to ethical guidelines on laboratory animal use isolated rabbit jejunum
and care policy, which is in compliance with Ahmadu Bello Uni- A New Zealand adult male rabbit used for the study was starved
versity Research Policy (Revised 2010). of feed for about 18 h. It was then sacrificed by cervical dislocation.
The abdomen was cut open and segments of the jejunum (about
2.3. Equipment and other laboratory materials 3 cm long) removed and dissected free of adhering mesentery. The
tissue was then suspended in a 25 ml organ bath containing tyrode
Microdynamometer (Ugo Basile, Italy); Soxhlet apparatus solution and allowed to stabilize for 30 min (period of acclimati-
(Quickfit, England); Water bath (HH-S Digital thermostatic water zation). The effects of acetylcholine (8
10  7–512
10  7 mg/ml)
bath, China); Weighing balance (Lab tech. BL 20,001 and Mettler and the methanol rhizome extract of N. lotus (4
10  4–
P162, USA); Cages (locally made for mice); Dissecting kit (Gold 512
10  2 mg/ml) were then tested on the jejunum. The contact
Cross Dissecting Set, Malaysia); Data Capsule (Ugo Basile, Italy); time for each concentration was 30 s, which was followed by
Filter papers (Whattman filter papers size 1); Glass wares such as washing three times with tyrode solution. The tissue was allowed a
Pipette, Test tubes (Pyrex, France), Syringes (1 ml, 2 ml, 5 ml and resting period before addition of the next concentration. The re-
10 ml); Porcelain pestle and mortar; Stop watch. sponses were recorded isometrically using a microdynamometer
set at a sensitivity of 3.0 mV and a speed of 24 mm/min.

2.4. Chemicals and drugs 3.4.2. Effect of methanol rhizome extract of Nymphaea lotus on
isolated guinea pig ileum
Acacia powder (Evans Medical Lt Speke, Liverpool); Acet- Similar protocol as for that of the effect of methanol rhizome
ylcholine (Sigma-Aldrich Inc., 3050 Spruce Street, St. Louis, USA); extract of N. lotus on isolated rabbit jejunum was followed. The
Atropine sulphate (Gland Pharma, India); Castor oil (Bell, Sons and effects of histamine (2
10  6–128
10  6 mg/ml) and the me-
Co Ltd, Southport PR9 9 AL, England); Histamine (Sigma-Aldrich thanol rhizome extract of N. lotus (4
10  4–512
10  2 mg/ml)
Inc., 3050 Spruce Street, St. Louis, USA); Loperamide (Imodiums – were then tested on guinea pig ileum.
Janssen Pharmaceutical, Pakistan); Magnesium sulphate (BDH
Chemical Ltd, Poole England); Medicinal charcoal (Ultracarbons 3.5. Antidiarrhoeal studies
tablets – Merck KGaA, Darmstadt, Germany).
3.5.1. Castor oil induced diarrhoea in mice
The method described by Shoba and Thomas (2001) was used
3. Methods for this study. The mice were fasted for 12 h and divided into five
groups of six mice each. Group I received deionized water at a dose
3.1. Preparation of the plant extract of 10 ml/kg p.o. (negative control), Group II received the standard
drug loperamide (3 mg/kg p.o.) and served as the positive control
The rhizomes of Nymphaea lotus were cleaned and air dried while Groups III, IV and V received the methanol rhizome extract
under shade and reduced into a fine powder using mortar and of Nymphaea lotus at the doses of 200, 400 and 800 mg/kg p.o.,
pestle. The powdered rhizome weighing 1.2 kg was extracted respectively. One hour after administration, all mice received
using Soxhlet apparatus employing 80% aqueous methanol. The 0.5 ml of castor oil p.o and then were placed individually in cages
extract was later subjected to drying in flask evaporator under whose floors were lined with pre-weighed white filter paper.
reduced pressure and controlled temperature (40 °C) over a water During the observation period of 4 h, the time of onset of diar-
bath. It was then stored in a levelled air tight container for future rhoea, frequency of defecation and weight of faeces excreted by
use. The weight of the extract obtained was 44.5 g. the animals were recorded. The percentage protection against
diarrhoea was calculated with respect to the number of wet faeces
using the formula below:
3.2. Preliminary phytochemical screening
% inhibition = ⎡⎣ ( Number of WFC–Number of WFT) /
The extract was subjected to preliminary phytochemical Number of WFC⎤⎦ × 100%
screening tests for the presence of carbohydrates, anthraquinones,
flavonoids, alkaloids, saponins, steroids, tannins, triterpenes and where;
cardiac glycosides using standard test procedures (Evans, 2002; WFC ¼wet faeces in negative control group
Sofowora, 1993; Silva et al., 2003). WFT ¼wet faeces in test group.
 F.H. Bello et al. / Journal of Ethnopharmacology 190 (2016) 13–21 15

3.5.2. Magnesium sulphate induced diarrhoea in mice The positive control and test groups were compared with the
The method described by Afroz et al. (2006) (similar protocol negative control.
as for castor oil-induced diarrhoea) was used. The 30 mice
were fasted for 12 h and divided into five groups of six mice each.
Group I received deionized water at a dose of 10 ml/kg p.o. 5. Results
(negative control), Group II received the standard drug loperamide
(3 mg/kg p.o.) and served a positive control, while Groups III, 5.1. Percentage yield of crude rhizome extract of Nymphaea lotus
IV and V received the methanol rhizome extract of N. lotus at
the doses of 200, 400 and 800 mg/kg p.o. respectively. Diarrhoea The weight of the extract obtained when 1.2 kg (1200 g) of the
was induced in the mice by oral administration of magnesium dried rhizome of Nymphaea lotus was extracted with methanol
sulphate at the dose of 2 g/kg 60 min after pretreatment with using Soxhlet's extraction technique was 44.5 g, representing a
the extract and loperamide. The mice were then individually percentage yield of 3.7%.
placed in cages whose floors were lined with pre-weighed white
filter paper. During an observation period of 4 h, the time of 5.2. Phytochemical constituents of methanol rhizome extract of
onset of diarrhoea, the total number of faecal output (frequency of Nymphaea lotus
defecation) and weight of faeces excreted by the animals
were recorded. The percent protection against diarrhoea was cal- Preliminary phytochemical screening of the methanol rhizome
culated with respect to the number of wet faeces using the for- extract revealed the presence of alkaloids, anthraquinones, car-
mula below: bohydrates, cardiac glycosides, flavonoids, saponins, steroids,
tannins and triterpenes as shown in Table 1.
% inhibition = ⎡⎣ ( Number of WFC–Number of WFT) /
Number of WFC⎤⎦ × 100% 5.3. Acute toxicity study (LD50)

where;
There were no lethality or toxic reactions observed at any of the
WFC¼wet faeces in negative control group
doses of methanol rhizome extract of Nymphaea lotus used in the
WFT¼wet faeces in test group.
study upon oral administration. The oral median lethal dose of the
rhizome extract was therefore estimated to be greater than
3.5.3. Effect of methanol rhizome extract on gastric transit time in
5000 mg/kg in mice.
mice
Intestinal motility test was done according to the method of
Tafesse and Mekonnen (2012). The mice (30) were fasted for 12 h 5.4. In vitro studies
and divided into five groups of six mice each. Group I received
5.4.1. Effect of methanol rhizome extract of Nymphaea lotus on
deionized water at a dose of 10 ml/kg p.o. (negative control),
isolated rabbit jejunum
Group II received the standard drug atropine sulphate (5 mg/kg
The methanol rhizome extract of N. lotus (4
10  4–
p.o.) which served as positive control while Groups III, IV and V
6.4
10  2 mg/ml) produced contractile effect on the tone of
received the methanol rhizome extract of Nymphaea lotus at the
contraction of the rabbit jejunum, while at concentrations of
doses of 200, 400 and 800 mg/kg p.o., respectively. A charcoal
8
10  2–512
10  2 mg/ml, it produced a concentration depen-
meal (10 ml) was prepared by grinding 4 tablets of activated
dent significant reduction in the tone and rate of spontaneous
charcoal (250 mg of activated charcoal each) with 0.5 g of acacia.
contraction of rabbit jejunum (Plate 1) indicating a biphasic
To this, 10 ml of distilled water was added gradually with con-
tinuous stirring to give a slurry, charcoal meal (10% w/v charcoal
Table 1
suspension in 5% w/v suspension of acacia). This was then trans- Phytochemical constituents of methanol rhizome extract of Nymphaea lotus Linn.
ferred into a bottle for final use. Each of the animals in Groups I–V
was given 0.3 ml charcoal meal as a marker by the oral route Phytochemical constituents Tests Inferences
30 min after drug treatments. All mice were sacrificed after 30 min
Alkaloids Dragendoff þ
of the charcoal meal by cervical dislocation. The stomach and Wagners þ
small intestine were then removed and extended on a clean sur-
face. All care was taken to prevent any damage to the tissue. The
Anthraquinones Bontragers þ
distance moved by the charcoal meal from the pylorus was mea- Modified Bontragers þ
sured and then expressed as a percentage of the total distance
from the pylorus to the caecum as follows:
Carbohydrates Fehlings þ
%Movement of charcoal meal = ( C/D) × 100% Molisch þ

where: Cardiac glycosides Kadde þ

C: distance travelled by charcoal meal Kelle-Killiani þ
D: distance from the pylorus to the caecum.
Flavonoids Shinoda þ
Sodium hydroxide þ
4. Data analysis Saponins Frothing þ
Steroids Liberman Buchard þ

The data collected were expressed as mean 7SEM. Results

were presented as graphs and tables where appropriate. Data were Tannins Lead acetate þ
Ferric chloride þ
compared using one way analysis of variance (ANOVA) followed by
Triterpenes Liberman Buchard þ
Dunnett's post hoc test using SPSS Version 20 software, where
differences of p r0.05 were considered statistically significant. Keyþ present
16 F.H. Bello et al. / Journal of Ethnopharmacology 190 (2016) 13–21

Plate 1. Spasmogenic and spasmolytic effects of methanol rhizome extract of Nymphaea lotus (NL) 4
10  4  512
10  2 mg/ml on isolated rabbit jejunum.

Plate 2. Effect of methanol rhizome extract of Nymphaea lotus (128
10  2 mg/ml) when interacted with acetylcholine (3.2
10  6 mg/ml) on isolated rabbit jejunum.
 F.H. Bello et al. / Journal of Ethnopharmacology 190 (2016) 13–21 17

Plate 3. Effect of methanol rhizome extract of Nymphaea lotus (2
10  2–512
10  2) on isolated guinea pig ileum.

activity. The extract (128
10  2 mg/ml) when interacted with reduction in onset and frequency of diarrhoea as well as weight of
acetylcholine (3.2
10  2 mg/ml) completely blocked the effect of wet faeces was observed in the group treated with loperamide
the latter (Plate 2). (3 mg/kg) at p o 0.05, p o0.001 and p o0.05 respectively. There
was also a significant reduction in frequency of diarrhoea in
5.4.2. Effect of methanol rhizome extract of Nymphaea lotus on groups treated with 200 mg/kg, 400 mg/kg and 800 mg/kg of the
isolated guinea pig ileum extract at p o0.05, p o0.01 and p o0.001 respectively as shown in
At lower concentrations (4
10  4–2
10  2 mg/ml), the me- Table 2. No significant decrease in the weight of faeces was ob-
thanol rhizome extract of Nymphaea lotus has no effect on the tone served in any of the groups treated with the extract.
of contraction of the guinea pig ileum while higher concentrations
(4
10  2–512
10  2 mg/ml) produced a concentration depen- 5.4.4. Magnesium sulphate-induced diarrhoea in mice
dent significant reduction in the tone of contraction of guinea pig Approximately 2 h after administration of magnesium sulphate,
ileum (Plate 3). When Nymphaea lotus extract (256
10  2 mg/ml) most of the mice in control group produced significant wet faeces
without mucus. The methanol rhizome extract produced sig-
was interacted with histamine (64
10  2 mg/ml), the effect of
nificant reduction in the frequency of diarrhoea with the lower
histamine was completely blocked with the extract still eliciting its
doses producing better effect. A statistically significant reduction
relaxant activity (Plate 4).
in frequency of diarrhoea was observed in the group treatment
with loperamide (3 mg/kg) at p o0.001. There was also statisti-
5.4.3. Castor oil-induced diarrhoea in mice
cally significant reduction in frequency of diarrhoea in the groups
An hour after castor oil administration, most mice in the con-
treated with 200, 400 and 800 mg/kg of the extract at p o 0.01,
trol group produced significant amount of wet mucoid faeces.
p o0.001 and p o0.001 respectively as shown in Table 3. At doses
Pretreatment of mice with methanol extract of N. lotus (800 mg/
of 200 mg/kg (76.5%) and 400 mg/kg (72.6%), the protection pro-
kg) significantly delayed the onset of diarrhoea (p o0.05). The duced was comparable to that of the positive control, loperamide
methanol rhizome extract of N. lotus decreased the frequency of 3 mg/kg (70.6%).
diarrhoea in a dose dependent manner. A statistically significant
5.4.5. Effect of methanol rhizome extract of Nymphaea lotus on
gastric transit time in mice
All treated groups produced statistically significant reduction in
the propulsive movement and transit of charcoal meal in the
gastrointestinal tract (p o0.001). The standard antidiarrhoeal
drug, atropine (5 mg/kg) produced greater antimotility effect
(56.83%) compared to the extract, as shown in Table 4.

6. Discussion

Plate 4. Effect of methanol rhizome extract of Nymphaea lotus (128
10  2 mg/ml) To restore personal comfort and convenience, many patients
when interacted with acetylcholine (3.2
10  6 mg/ml) on isolated rabbit jejunum. require antidiarrhoeal therapy and treatment is carried out to
18 F.H. Bello et al. / Journal of Ethnopharmacology 190 (2016) 13–21

Table 2
Effect of methanol rhizome extract of Nymphaea lotus on castor oil induced-diarrhoea in mice.

Group (mg/ Mean onset of diarrhoea Mean number of dry Mean frequency of Mean total wt of Mean total wt of wet % Protection
kg) 7 SEM (m) faeces 7 SEM diarrhoea7SEM faeces7 SEM (g) faeces7 SEM (g)

DW 10 ml/ 0.34 7 0.05 –

kg 77.167 7.48 2.66 70.88 6.83 7 0.79 0.317 0.05
Lop 3 0.23 7 0.07
160.25 7 18.50* 2.50 7 0.62 1.83 7 0.65*** 0.127 0.06* 73.20
NL 200 0.36 7 0.03
133.17 718.88 1.667 0.80 4.007 0.51* 0.33 7 0.02 41.43
NL 400 0.28 7 0.02
132.177 6.96 2.007 0.89 3.50 7 0.42** 0.247 0.03 48.76
NL 800 0.247 0.05
* ***
154.60 7 26.98 3.83 7 0.31 2.167 0.70 0.167 0.05 68.37

Values are represented as Mean 7SEM; n ¼ 6

– One-way Anova followed by Dunnet's post hoc test NL- Nymphaea lotus rhizome extract; DW – Distilled water; Lop – Loperamide; g – gram; m – minutes; wt – weight
*
p r 0.05.
***
p r 0.001.
**
p r0.01.

achieve, among other objectives, increased resistance to flow Table 4

(segmental contraction, decreased propulsion and persistalsis) and Effect of methanol rhizome extract of Nymphaea lotus on charcoal meal induced
hyperperistalsis in mice.
increased mucosal absorption or decreased secretion (Akindele
and Adeyemi, 2006). The most common test of acute (short-term) Test group Mean length of Mean move- Mean % % Protection
toxicity is the median lethal dose, LD50 test. The oral LD50 of the (mg/kg) intestine7 SEM ment of char- movement
methanol rhizome extract of Nymphaea lotus in mice was greater (cm) coal 7SEM (cm)
than 5000 mg/kg body weight. This suggests that the extract is
Deionized 53.50 7 0.65 45.667 1.68 85.34 72.96 –
non-toxic orally according to Lorke's classification of chemicals water
(Lorke, 1983). (10 ml/
Despite the multiplicity of aetiologies of diarrhoea, the four kg)
Atropine 5 52.43 7 1.45 19.03 7 2.66*** 36.84 75.52*** 56.83
major mechanisms responsible for the pathophysiology in water
NL 200 56.40 7 0.91 26.137 2.50*** 46.36 74.45*** 45.67
and electrolytes transport are increased luminal osmolarity (os- NL 400 52.43 7 1.75 25.56 7 2.79*** 48.32 73.98*** 43.37
motic diarrhoea), increased electrolytes secretion (secretory diar- NL 800 56.03 7 2.68 27.05 7 1.71*** 48.67 73.60*** 42.97
rhoea), decreased electrolytes absorption, and deranged intestinal
Values are represented as Mean 7 SEM; n ¼ 6
motility causing a decreased transit time (Umer et al., 2013). The
– One-way Anova followed by Dunnet's post hoc test; NL - Nymphaea lotus rhizome
antidiarrhoeal activity of methanol rhizome extract of N. lotus was extract; cm – centimeter; SEM – standard error of mean
evaluated by employing castor oil induced and magnesium sul- ***
p r 0.001.
phate-induced animal models of diarrhoea in addition to gastro-
intestinal motility test. and the active agent, ricinoleic acid (Semwal et al., 2014). Ricino-
The result of the phytochemical screening is in agreement with leic acid induces diarrhoea by hypersecretory response (Almeida
other studies such as analysis of the ethanol leaf extract of N. lotus
et al., 1995). Due to the polar nature of ricinoleic acid, it is poorly
by Akinjogunla et al. (2010) and Afolayan et al. (2013). Analysis of
absorbed and its presence in the small intestine results in changes
ethanolic extract of N. lotus by Dadi-Mamud and Musa (2013) in all
in permeability of the intestinal mucosa to electrolytes and sti-
the different parts of the plant analysed is in agreement with the
mulates peristaltic activity in the intestine which in turn produces
phytochemical analysis except for tannins that were absent in the
flowers extract. hypersecretion and fluid accumulation occur (Yoshio et al., 1999;
The effects of castor oil are mediated by ricinoleic acid, a hy- Dash et al., 2013). The ricinoleic acid produces irritating and in-
droxylated fatty acid released from castor oil by intestinal lipases flammatory actions on the intestinal mucosa leading to the release
(Yoshio et al., 1999). The triglyceride present in castor oil is hy- of prostaglandins (Yoshio et al., 1999) by colonic cells (Awouters
drolyzed in the small bowel by the action of lipases into glycerol et al., 1978) or stimulates adenyl cyclase in the intestinal epithelial

Table 3
Effect of methanol rhizome extract of Nymphaea lotus on magnesium sulphate induced-diarrhoea in mice.

Test group Mean onset of diarrhoea Mean number of dry Mean frequency of diar- Mean total weight of Mean weight of wet % Protection
(mg/kg) 7 SEM (min) feaces7 SEM rhoea 7 SEM faeces7 SEM (g) faeces 7 SEM (g)

DW10 (ml/kg) 115.83 7 16.22 6.83 7 1.16 8.50 7 0.92 0.157 0.01 0.08 70.02 –
Lop 3 186.677 12.16** 4.50 7 0.62 2.50 7 0.67*** 0.107 0.09 0.05 70.01 70.6
NL 200 156.60 7 11.60 3.16 7 0.48* 2.007 0.58*** 0.067 0.01* 0.03 70.01 76.5
NL 400 161.25 7 13.09 6.50 7 0.99 2.337 0.76*** 0.09 7 0.03 0.05 70.02 72.6
NL 800 159.25 7 18.69 7.337 1.35 3.50 7 1.48** 0.117 0.03 0.04 70.02 58.8

Values are represented as Mean 7 SEM; n ¼6

– One-way Anova followed by Dunnet's post hoc test
NL - Nymphaea lotus rhizome extract; Lop–Loperamide; g – gram; m – minutes; wt – weight
*
p r 0.05.
**
p r0.01.
***
p r 0.001.
 F.H. Bello et al. / Journal of Ethnopharmacology 190 (2016) 13–21 19

cells (Racusen and Binder, 1979). Prostaglandins thus released increasing the intestinal transit time probably due to its anti-
promote vasodilatation, smooth muscle contraction, and mucus cholinergic effect (Brown and Taylor, 2001). In this study, atropine
secretion in the small intestines and thereby producing diarrhoea. and different doses of the extract decreased the propulsive
Prostaglandins of the E series are considered to be good diar- movement in the gastric transit time study with the former being
rheogenic agents in experimental animals as well as in human more potent than the rhizome extract at the doses used. The
beings. The inhibitors of prostaglandins biosynthesis are therefore methanol rhizome extract of N. lotus significantly reduced the
considered to delay castor oil– induced diarrhoea (Brijesh et al., intestinal propulsive movement in the charcoal meal treated
2009; Sunilson et al., 2009). From the result obtained in this study, model at doses of 200, 400 and 800 mg/kg of body weight. The
the extract was capable of inhibiting the castor oil induced diar- effect was comparable to atropine sulphate (5 mg/kg). The results
rhoea in a dose dependent manner. N. lotus, therefore, may also show that the extract suppressed the propulsion of charcoal meal
have the ability to inhibit prostaglandin synthesis and possess (probably in the same way as atropine sulphate) and increased the
anti-inflammatory activity. The effect of the extract at 800 mg/kg time for absorption of water and electrolytes. The reduction in
is comparable to loperamide at the dose used. Loperamide, a well- percentage distance travelled can be a proof of gastrointestinal
known antidiarrhoeal agent (Hughes et al., 1982) is an analog of smooth muscle relaxation and antisecretory effect. This results in
meperidine, an opioid-receptor agonist. It acts on the μ-opioid increase in gastric transit time allowing more time for water and
receptors in the myenteric plexus of the large intestine. Similar to electrolyte absorption into the system. The smooth muscle re-
morphine, it works by decreasing the activity of the myenteric laxation may be responsible for the use of the plant ethnomedi-
plexus, which in turn decreases the tone of the longitudinal and cally for the treatment of diarrhoea. Several antidiarrhoeal medi-
circular smooth muscles of the intestinal wall. It activates pre- cations are well known for reduction of intestinal contractions and
synaptic opioid receptors in the enteric nervous system to inhibit thereby reducing the intestinal transit (Mathad et al., 2005). In the
acetylcholine release and decrease peristalsis (Lutterodt, 1989). peripheral nervous system, acetylcholine activates gastrointestinal
This increases the transit time allowing for more water to be ab- smooth muscles, and is a major neurotransmitter in the autonomic
sorbed from the faecal matter. Loperamide also decreases colonic nervous system. Acetylcholine, with increasing concentration,
mass movements and suppresses the gastrocolic reflex. The causes a dose dependent stimulation of rabbit jejunum contrac-
pharmacological effect of loperamide is due to its anti-motility and tion. This effect attributed to the muscarinic effect of the drug. In
anti-secretory properties (Karim and Adeikan, 1977). From the the spontaneously contracting rabbit jejunum preparation, the
result obtained, it is likely that the plant extract mediate its effect methanol rhizome extract of Nymphaea lotus exhibit spasmogenic
through similar mechanisms. Alkaloids, glycosides, quinone, ter- effect at lower concentration (4
10  4–2
10  2 mg/ml), followed
penoids and essential oils inhibit release of autocoids and pros- by concentration dependent spasmolytic effect at higher con-
taglandins (Tiwari et al., 2011; Yasmeen et al., 2010). Tannins de- centration (4
10  2–512
10  2 mg/ml) in contrast to concentra-
nature proteins by forming a complex (protein tannate) and this tion-dependent contractility caused by acetylcholine which in-
complex coats the intestinal mucosa and makes it more resistant dicates the presence of a combination of gut stimulant and in-
while simultaneously diminishing gastric secretions (Westendarp, hibitory constituents in the methanol rhizome extract of N. lotus.
2006). The extract blocked the effect of acetylcholine on the spontaneous
Magnesium sulphate produces diarrhoea by osmotic proper- contraction of the rabbit jejunum. The effect produced by the in-
ties, preventing reabsorption of water molecules, thus leading to teraction is similar to that of atropine (a cholinergic blocker) when
increase in the volume of the intestinal content. It promotes the interacted with acetylcholine. This shows that the extract may
release of cholecytokinin from the duodenal mucosa, which in- have anticholinergic activity. Flavonoids inhibit gastrointestinal
creases the secretion and motility of small intestine thereby pre- release of acetylcholine and quinone (anthraquinone) inhibits
venting the reabsorption of sodium chloride and water (Galvez contractions caused by spasmogens (Tiwari et al., 2011).
et al., 1993; Parimala et al., 2002). Since the extract was capable of The co-existence of spasmogenic and spasmolytic activity is
inhibiting the magnesium sulphate-induced diarrhoea, N. lotus common in herbal remedies (Khan et al., 2012) such as Calendula
may also have the ability to inhibit production of diarrhoea by officinalis (Bashir et al., 2006), Ginger (Ghayur and Gilani, 2005),
osmotic properties. The lower doses 200 and 400 mg/kg produced Lavandula stoechas (Jabeen et al., 2007), Forsskalea tenacissima
response comparable to loperamide at the dose tested (3 mg/kg). (Shah et al., 2010), Plantago ovata (psyllium husk) (Mehmoud et al.,
This indicates that increase in the dose of the extract does not 2011), Amaranthus spinosus (Chaudhary et al., 2012), etc. These
cause an increase in activity (non-dose dependent effect). The herbs have spasmolytic and spasmogenic effect depending on the
non-dose dependent effect may possibly be that with increasing solvent used for extraction, the concentration of the herbal extract
concentration of the extract, the constituent(s) responsible for its or the type of isolated tissue used as well as the plant part used.
activity against magnesium sulphate-induced diarrhoea may act This effect may be mediated through muscarinic receptor blockade
differently with increasing dose. Quinone and tannins stimulate and muscarinic receptor activation respectively. This is probably
normalization of deranged water transport across the mucosal meant by nature not to allow the gut stimulant effect to go beyond
cells and reduction of the intestinal transit (Tiwari et al., 2011). a certain limit beyond which it could have been harmful, causing
Steroids enhance intestinal absorption of sodium and water (Ku- abdominal cramp. This is in line with the general perception that
mar et al., 2010). The methanol rhizome extract of Nymphaea lotus natural products possess “side effects neutralizing” combination
produced a dose dependent decrease in mean frequency of castor (Gilani and Atta-ur-Rahman, 2005). There is growing evidence
oil-induced diarrhoea and a non-dose dependent decrease in the supporting this concept (Kumar et al., 2015). The presence of this
mean frequency of magnesium sulphate-induced diarrhoea. effect in medicinal plants is a long established concept put forth by
The test of the methanol rhizome extract of N. lotus on gastric the Hippocrates and strengthened by Ibn Sina and others.
transit time is a method used to study the effect of drugs on the However, until now, this concept remained dormant and lacks
motility of intestine (peristaltic movement) (Shamkuwar et al., sufficient scientific evidence mainly due to scarcity of ethno-
2012). Activated charcoal is a non-absorbable agent that prevents pharmacologists with wider background (Gilani et al., 2000;
absorption of chemicals and drugs due to its adsorption properties. 2005). Food plants are considered relatively safe as they are likely
Atropine is a parasympatholytic drug which acts by blocking to contain synergistic and/or side effect neutralizing combinations
the actions of acetylcholine at muscarinic receptors. Atropine, as of activities (Gilani and Atta-ur-Rahman, 2005). The rhizome of
an antidiarrhoeal, possesses anti-motility effect and acts by Nymphaea lotus may be a good example of such plants.
20 F.H. Bello et al. / Journal of Ethnopharmacology 190 (2016) 13–21

Methanol rhizome extract of Nymphaea lotus at higher con- 7. Conclusion

centrations (4
10  2–512
10  2 mg/ml) exerted a dose depen-
dent relaxant effect on the guinea pig ileum but has no effect at This study has shown that the methanol rhizome extract of
lower concentrations (4
10  4–2
10  2 mg/ml). The methanol Nymphaea lotus contains pharmacologically active substances with
rhizome extract of N. lotus blocked the contractile effect of hista- antidiarrhoeal properties thus justifying its use by the local po-
mine upon interaction but this effect was not similar to when pulation for this purpose.
mepyramine was interacted with histamine. The extract produced
significant relaxation while mepyramine did not because it is a
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