Evaluation of the Anti-diarrhoeal, Anti-inflammatory, and Analgesic Effects of the Polyherbal Formulation, Aju Mbaise in Experimental Rats.

doi:10.21203/rs.3.rs-2877604/v1

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Evaluation of the Anti-diarrhoeal, Anti-inflammatory, and Analgesic Effects of the Polyherbal Formulation, Aju Mbaise in Experimental Rats.

https://doi.org/10.21203/rs.3.rs-2877604/v1

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Background:

Aju Mbaise is a combination of herbs that is widely used in the southeast part of Nigeria for the purpose of managing postpartum complications and alleviating menstrual pain. The study aimed at assessing the analgesic, anti-inflammatory, and anti-diarrheal properties of Aju mbaise extract in rats.

Methods:

The extract was subjected to phytochemical analysis and acute toxicity testing. Subsequently, the anti-diarrheal effects of the extract were evaluated by administering charcoal as a meal and then using the castor oil-induced models. The evaluation of its anti-inflammatory effects was conducted through the carrageenan-induced paw oedema model, while its analgesic properties were assessed using the acetic acid-induced pain model.

Results:

Alkaloids, tannins, phenols, steroids, cardiac glycosides, terpenoids, flavonoids, and saponins were among the components that the phytochemical study revealed to be present. The acute toxicity value was also greater than 5000 mg/kg body weight. In animal studies, the extract displayed significant inhibitory effects on intestinal motility, while also reducing the wet stool frequency. The castor oil-induced model exhibited a decrease in the weight of intestinal contents and an elevation in serum electrolyte levels. Additionally, in the anti-inflammatory study, the extract was observed to suppress paw oedema, and in the analgesic study, it demonstrated a reduction in acetic acid-induced pain in rats.

Conclusions:

The Aju mbaise polyherbal extract possesses anti-diarrhoeal, anti-inflammatory, and analgesic properties, making it a potentially safe option for managing diarrhoea, inflammation, and pain. These therapeutic effects may be attributed to the extract's composition of alkaloids, tannins, phenols, steroids, cardiac glycosides, terpenoids, flavonoids, and saponins. Nonetheless, additional research is necessary to establish the extract's ideal dosages and potential adverse effects.

Aju mbaise polyherbal extract

analgesic

castor oil

diarrhoea

inflammation

motility

Medicinal plants as complementary forms of treatment is now accepted on a global scale (1). Global population currently depend on medicinal plants to treat various illnesses and in fact, it is envisaged that the acceptability of medicinal plants as means of treatment would experience a further upsurge due to the availability, accessibility, cheapness, and reported effectiveness of these medicinal plants in alleviating wide range of health complications (2). The meltdown in global economies, coupled with the poor socio-economic status of most individuals, especially across developing countries of Africa and Asia, also appears to have contributed to the current growing interest in herbal medicine (3). This is because most individuals living in these regions of the world find it difficult to cope with the demand for sophisticated treatments(4). Aju mbaise polyherbal is only one of the herbal preparations currently used against diseases (5).

Aju mbaise is a polyherbal that generally comprise a combination of various parts of medicinal plants, including stems, bark, leaves, and roots. A study reported that the polyherbal formulation comprises different plants they identified as Barteria fistulosa, Napoleona vogelii, Euphorbia convolvuloides, Spondias mombin, Uvaria chamae and Ceiba pentandra (6). The researchers also reported notable levels of alkaloids, flavonoids, saponins, tannins, terpenoids, steroids, phenols, and cardiac glycosides within the extract. (6). Although reports on the medicinal uses of the medicinal plant appear scanty, its constituent plants have been used to manage diarrhoea and other forms of gastrointestinal diseases (7). Aju mbaise extract improves lipid profile, electrolyte balance, hematological profile, male fertility indices and prevents oxidative stress and hepatic injury in rats(7).

The passage of frequent watery or unformed stools characterizes diarrhoea, which is prevalently implicated in the death of children aged 1–5 worldwide and a significant contributor to the current global disease burden, especially in developing countries with limited access to good water supply, and poor breastfeeding practices (8). Major causes of diarrhoea are gastrointestinal infection of bacterial, viral, and parasitic origin, contaminated food or drinking water, poor hygiene, and motility disorders of the gastrointestinal tract (9). Current management strategies, including oral rehydration and the use of drugs, have not achieved desired outcomes due to a lack of funds on the part of many affected individuals and also a lack of compliance on the part of those who can afford the drugs (10, 11). Medicinal plants have proven to be viable alternative treatment sources over the years due to their availability, accessibility, and effectiveness (12). Several medicinal plants have shown significant anti-diarrhoeal activities (13). Due to their vast phytochemical contents, medicinal plants can play a beneficial role as, antimicrobial, anti-inflammatory and anti-cytotoxic agents while reducing inflammation and protecting from cellular damage (14, 15). The fact that pains and inflammations have been associated with diarrhoea (8, 16, 17) makes the evaluation of a strategy for managing all three symptoms with a single agent an area of interest. Therefore, the study assessed in rats anti-diarrheal, anti-inflammatory, and analgesic effects of the polyherbal extract of Aju mbaise.

2.1. Chemicals and Drugs

Drugs used for these studies include Loperamide, Castor oil, and Aspirin (Emzor Drugs, Nigeria), while chemicals include Ethanol and activated charcoal (BDH Company, UK). Reagents include electrolytes test kits for sodium, potassium, chloride, and bicarbonate (Randox Laboratories, UK).

2.2. Gathering of plant material and preparation of extract

Aju mbaise polyherbal was obtained Ahiara Mbaise Local Government Area of Imo State, Nigeria, and were allowed to dry and this was done using laboratory bench for a period of 21 days. The dried sample was pulverized into a coarse powder in a locally made manual blender. Two hundred and fifty (250) grams of the pulverized material was immersed in 1500 millimeter of ethanol within a duration of 48 hours and intermittent stirring every 2 hours before being filtered to obtain a filtrate containing the extract in solution. Subsequently, filtrate was subjected to drying in a hot air oven at 40 ℃, resulting in a greenish paste extract that weighed 16.90 g, equating to a percentage yield of 6.76%. Extract was prepared and subsequently stored at very low temperatures in a refrigerator until required, and it is now identified as Aju mbaise polyherbal extract (APE).

2.3. Phytochemical analysis of Aju mbaise polyherbal extract

The identification and quantification of phytochemicals in Aju Mbaise polyherbal extract were duly carried out according to the protocols used by Orieke et al., (2019) (18). The methods outlined were used to test the extract's presence and amounts of alkaloids, phenols, cardiac glycosides, saponins, steroids, flavonoids, terpenoids and tannins.

2.4. Acute toxicity (LD₅₀) Evaluation of APE

Orieke et al. (2019) utilized a modified version of the Lorke's method to evaluate acute toxicity. The test involved two stages, with the first stage using 9 Wistar rats that were separated into 3 different groups of A, B, and C which consisted of 3 rats each. The groups received 10mg/kg, 100mg/kg, and 1000 mg/kg of the extract, respectively(18). The animals were observed for toxicity signs and death within 24 hours, and no deaths were recorded across the groups. Consequently, the study progressed to the subsequent phase, which involved another set of 9 rats divided into 3 groups (A-C) and administered single oral doses of the extract at 1600 mg/kg, 2900 mg/kg, and 5000 mg/kg, respectively. The animals were again checked for signs of toxicity and death within 24 hours, with no deaths recorded across the groups. As a confirmatory test, the highest dose used (5000 mg/kg) was repeated on different set of 3 rats.

Acute toxicity values calculated using Lorke’s formular stated as:

LD₅₀ =$\sqrt{A x B}$

A = Maximum dose that did not result in any deaths

B = Minimum dose that caused mortality in all animals within a group.

2.5. Experimental Animals

The study involved 146 adult Wistar rats, where 21 rats were used for acute toxicity evaluation, 75 for different anti-diarrhoea models, and the remaining 50 for anti-inflammatory and analgesic studies. The rats were procured from the Michael Okpara University of Agriculture Umudike, Abia State, Nigeria, laboratory animal house of the Department of Zoology and Environmental Biology. The rats stayed in well-ventilated cages with ad libitum access to clean water and finisher mash (Chikun Feeds, Nigeria). Before each experiment, the rats were starved for 24 hours. All experimentations were conducted according to international guidelines for laboratory animal care and use and were approved by the ethics committee of the Michael Okpara University of Agriculture, Umudike, with ethics number MOUAU/VPP/EC/2017/007.

2.6. Acute toxicity evaluation of Aju mbaise polyherbal extract

A modified method based on Orieke et al. (2019), consisting of two test stages, was employed. The first stage involved treating 9 rats, assigned to 3 groups (A, B and C) with 10 mg/kg, 100 mg/kg and 1000 mg/kg of the extract respectively. The animals were observed for signs of toxicity and deaths within 24 hours, with no mortality recorded. Second stage: 1600 mg/kg, 2900 mg/kg, and 5000 mg/kg of single oral extract doses were administered to nine additional rats in three groups (A-C). Within 24 hours, the animals were once more checked for poisoning indications and deaths, but there were none. The highest dose, 5000mg/kg, was administered once more to a new group of three rats in the test stage with the objective of validating the preceding findings.

Acute toxicity values calculated using Lorke’s formular stated as:

LD₅₀ =$\sqrt{A x B}$

A = Maximum dose that did not result in any deaths

B = Minimum dose that caused mortality in all animals within a group.

2.7. Anti-diarrhoeal activity of Aju mbaise polyherbal extract

Model 1: Effect of the polyherbal extract on intestinal transit

The study adopted the method of Ibe et al., (2021)(19), which involved dividing 25 rats into five groups of five rats each, after a 24-hour starvation period. The standard drug group (group 2) received Loperamide (0.5 mg/kg body weight), while the control group (group 1) received 0.2 ml of ordinary saline. Using the oral method, groups 3, 4, and 5 received 250 milligrammes, 500 milligrammes, and 1000 milligrammes per kilogramme of the polyherbal extract, respectively. Each rat received 5 ml/kg body weight of a 10% activated charcoal solution after 30 minutes, and after another 30 minutes, it was sacrificed by cervical dislocation. The small intestine’s length was measured, and the distance covered by the charcoal meal was calculated as a proportion of the entire length. Utilising the relationship, it was determined what percentage of intestinal motility was inhibited:

$$\% \text{i}\text{n}\text{h}\text{i}\text{b}\text{i}\text{t}\text{i}\text{o}\text{n}= \frac{\text{D}\text{T} \text{i}\text{n} \text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l} – \text{D}\text{T} \text{i}\text{n} \text{t}\text{e}\text{s}\text{t} \text{x}100}{\text{D}\text{T} \text{i}\text{n} \text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}$$

Where DT = Distance travelled by charcoal meal

Model 2: Effect of the polyherbal extract on castor oil induced diarrhoea in rats

For model 2, a separate group of 25 adult rats were fasted for 24 hours and divided into 5 groups of 5 rats each. The rats received the same treatment as in model 1 and, 30 minutes later, were given castor oil (1 ml) orally before being housed in individual cages coated with absorbent paper for three hours to monitor episodes of diarrhoea. Wet and dry stools were counted, and their weights were recorded, as well as when the diarrhoea first started (the latent phase). At the conclusion of each period, the absorbent papers were changed, and the average total of stools passed by the treatment groups and the control group were compared. Average number of diarrheal faeces passed in the control group was calculated to be a hundred percent (100%).

The inhibition percentage of wet faeces and stool frequency induced by the extract was determined by calculating the variance between the number of faeces in the control group (NC) and the amount of faeces in the treated group (NT), divided by NC and multiplied by 100.

% Inhibition of bowel movement = [(N_C - N_T)/N_C] x 100

Where: N_{C =} Number of wet faeces/stools of control group

N_T- Number of wet faeces/stools of treated group.

Model 3: Effect of the polyherbal extract on castor oil induced gastrointestinal fluid accumulation and serum electrolytes concentration in rats.

25 extra rats were given the same treatments as model 2 using the same protocols. In this instance, six groups of five adult albino rats each were divided into thirty total. After an hour of administering castor oil, the rats were euthanized, and their small intestines were removed from the pyloric region to the caecum, after ligating both ends. Each intestine's weight with its contents within was determined before the contents were expelled out, and the empty intestine was then weighed again. It was then determined by how much heavier the full intestine was than the empty one.

Percentage anti diarrhoeal activity was evaluated using the relationship:

$$\% \text{A}\text{c}\text{t}\text{i}\text{v}\text{i}\text{t}\text{y}= \frac{\text{W}\text{I}\text{C} \text{i}\text{n} \text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l} – \text{W}\text{I}\text{C} \text{i}\text{n} \text{t}\text{e}\text{s}\text{t} \text{x}100}{\text{W}\text{I}\text{C} \text{i}\text{n} \text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}$$

Where WIC = weight of intestinal content.

The concentrations of serum electrolytes were also determined on blood samples collected from each rat. Randox commercial test kit for electrolytes including sodium, potassium, chloride and bicarbonate were used according to standard protocols prescribed by the manufacturer, Randox Laboratories, UK.

2.8. Evaluation of the anti-inflammatory effects of the polyherbal extract

A different group of 25 mature rats were split into 5 groups with each group having 5 rats. They were subjected to treatments as described in section 2.6 above. However, in group 2, Loperamide was replaced with a standard anti-inflammatory drug (100 mg/kg Aspirin). The animals' paw circumferences (PC) were measured and recorded before these treatments. After a period of thirty minutes following treatment, the rats were subjected to acute inflammation (paw edema) by means of a sub-plantar injection of 0.1ml of 1% λ-carrageenan (in 0.9% saline solution) into the right hind paw of each rat. After that, PC of the treated rats and control were measured and recorded at different time intervals (30mins, 1 hour, and 2 hours) following induction. The percentage inhibition of oedema was estimated using the following relationship, and the degree of oedema was determined as the difference between the initial and final PC values:

$$\text{P}\text{e}\text{r}\text{c}\text{e}\text{n}\text{t}\text{a}\text{g}\text{e} \text{i}\text{n}\text{h}\text{i}\text{b}\text{i}\text{t}\text{i}\text{o}\text{n} \text{o}\text{f} \text{o}\text{e}\text{d}\text{e}\text{m}\text{a}=\frac{\text{P}\text{C} \text{i}\text{n} \text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l} – \text{P}\text{C} \text{i}\text{n} \text{t}\text{e}\text{s}\text{t} \text{x} 100}{\text{P}\text{C} \text{i}\text{n} \text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l} }$$

2.9. Evaluation of analgesic effects of the polyherbal extract

A separate group of 25 adult rats was also subjected to the experimental procedures outlined in section 2.7. Thirty minutes after treatment, acute pain was inducted by intraperitoneal administration of 10 ml/kg body weight of 0.6% acetic acid solution. Thereafter, the number of writhes made by each rat in 30 minutes was ascertained for both the control and test groups. Percentage inhibition of pain (analgesic activity) was calculated using the relationship:

$$\text{%} \text{i}\text{n}\text{h}\text{i}\text{b}\text{i}\text{t}\text{i}\text{o}\text{n} \text{o}\text{f} \text{p}\text{a}\text{i}\text{n}=\frac{\text{N}\text{u}\text{m}\text{b}\text{e}\text{r} \text{o}\text{f} \text{w}\text{r}\text{i}\text{t}\text{h}\text{e}\text{s} \text{i}\text{n} \text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l} – \text{N}\text{u}\text{m}\text{b}\text{e}\text{r} \text{o}\text{f} \text{w}\text{r}\text{i}\text{t}\text{h}\text{e}\text{s} \text{i}\text{n} \text{t}\text{e}\text{s}\text{t} \text{x} 100}{\text{N}\text{u}\text{m}\text{b}\text{e}\text{r} \text{o}\text{f} \text{w}\text{r}\text{i}\text{t}\text{h}\text{e}\text{s} \text{i}\text{n} \text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}$$

2.10. Statistical analysis

SPSS Version 20.0, IBM SPSS Inc, Chicago, IL was utilized to analyse the data. ANOVA was used to obtain significant levels. The means of different doses and fractions were compared using the Duncan Multiple Range Test, and then Student's t-test was employed for post-hoc analysis. Values with P < 0.05 were regarded as being statistically significant and show a difference for the measured values between the control and test groups as well as within the test groups.

3.1. Phytochemical composition of Aju mbaise polyherbal extract

Phytochemicals identified in the extract and quantified include saponins, tannins, phenolics, flavonoids, steroids, terpenoids, glycosides and alkaloids, and aer presented in Table 1.

Table 1

Phytochemical composition of *Ajumbise* polyherbal extract
Parameters	Qualitative test	Qualitative test result (mg/100g)
Saponins	++	8.33 ± 0.12
Tanins	++	5.71 ± 0.16
Phenolics	+++	13.18 ± 0.30
Flavonoids	++	7.19 ± 0.22
Steroids	++	5.65 ± 0.14
Terpenoids	+	2.52 ± 0.11
Cardiac glycosides	+	3.63 ± 0.09
Alkaloids	+++	14.91 ± 0.32
Key
Present in high quantity +++
Present in moderate quantity ++
Present in low quantity +
Absent -

3.2. Acute toxicity (LD₅₀) evaluation of Aju mbaise polyherbal extract

No mortality occurred across all test groups and in all stages of acute toxicity evaluation of the extract, including the highest dose of 5000 mg/kg body weight. After treatment, the animals appeared physically stable, and all survived till the end of 24 hours and a further seven days of the LD₅₀ test, except those administered 5000 mg/kg, which at the initial moments after administration were calm with decreased physical activity. However, these animals all survived to the end of the test period as zero percent mortality was recorded. The LD₅₀ value for the extract was termed to be > 5000 mg/kg (Tables 1a&b).

Table 2

a: Phase 1 acute toxicity (LD₅₀) results
Group	Dose (mg/kg)	No. of death	Observation
1	10	0/3	Animals displayed normal physical activity and appeared to be in good health
2	100	0/3	Animals displayed normal physical activity and appeared to be in good health
3	1000	0/3	Animals displayed normal physical activity and appeared to be in good health

Table 2

b: Phase 2 acute toxicity (LD₅₀) results
Group	Dose (mg/kg)	No. of death	Observation
1	1600	0/3	Animals exhibited normal activity and were physically stable
2	2900	0/3	Animals exhibited normal activity and were physically stable
3	5000	3/3	No mortality. Animals were calm for up to 2 hours before regaining activity.
LD₅₀ > 5000 mg/kg

3.3. Anti-diarrhoeal activity of Aju mbaise polyherbal extract

Model 1: Effect of the polyherbal extract on intestinal transit.

The treated rats exhibited significantly inhibited intestinal motility (p < 0.05) when compared to the control group. The inhibition occurred in a dose-dependent manner, causing a decrease in charcoal meal transit in rats. The treatment was found to be comparable to Loperamide activity but demonstrated greater activity than Loperamide at the highest dose (100 mg/kg).). The table showing the inhibitory effect of Aju mbaise Polyherbal Extract on intestinal motility is presented in Table 3.

Table 3

*Aju mbaise* Polyherbal Extract's inhibitory effects on rat intestinal motility.
Treatments	Length of intestine (cm)	Distance travelled by charcoal meal (cm)	Percentage charcoal meal movement (%)	Percentage inhibition of motility (%)
Control	92.60 ± 2.07^a	75.60 ± 2.23^c	81.00 ± 6.92^c	0.00 ± 0.00^a
Loperamide 0.5 mg/kg	90.00 ± 1.58^a	56.80 ± 3.56^b	63.20 ± 3.70^b	25.40 ± 1.82^b
Extract 250 mg/kg	91.20 ± 2.95^a	57.80 ± 2.28^b	63.40 ± 2.70^b	24.20 ± 2.28^b
Extract 500 mg/kg	91.60 ± 4.83^a	54.20 ± 2.17^b	59.20 ± 3.03^a,b	27.40 ± 3.29^b
Extract 1000 mg/kg	91.00 ± 6.21^a	45.00 ± 3.77^a	52.60 ± 3.50^a	33.00 ± 4.47^c

The mean and standard deviation of the values are shown (n = 5). The means of the identical columns with various letter superscripts differ considerably (P < 0.05).

Model 2: Effect of the polyherbal extract on castor oil induced diarrhoea in rats

In the rat model of castor oil-induced diarrhea, the extract exhibited a significant inhibitory effect on the induction of diarrhea at doses of 250, 500, and 1000 mg/kg, comparable to Loperamide. Specifically, the extract reduced the incidence of diarrhea by 57.29 ± 2.93%, 66.66 ± 2.95%, and 84.77 ± 2.73%, respectively, when in comparison to the control (p < 0.05). The extract treatment groups also showed a significant decrease in the number and weight of wet stools with longer latent periods compared to the control group (refer to Table 4 for details). In addition, the extract significantly reduced the intestinal weight contents in the enteropooling version of the castor oil-induced model, comparable to Loperamide (refer to Table 5 for details). Serum electrolyte composition analysis revealed a significant increase in sodium and potassium ions in the extract-treated groups, while the levels of chloride and bicarbonate remained unchanged when in comparison to the diarrhea control group (Fig. 1).

Table 4

Inhibitory effect of *Aju mbaise* Polyherbal Extract on castor oil induced diarrhoea in rats.
Treatments	Latent period (mins)	No wet stool	Weight of wet stool (g)	No of Dry stool	Weight of dry stool (g)	% inhibition of diarrhoea
Control	30.60 ± 5.86^a	7.20 ± 0.84^c	4.82 ± 0.18^d	1.40 ± 0.55^a	1.41 ± 0.28^a,b	0.00 ± 0.00^a
Loperamide 0.5 mg/kg	56.80 ± 1.64^c	0.80 ± 0.05^a	0.71 ± 0.09^a	2.60 ± 0.48^b	2.62 ± 0.33^c	89.10 ± 6.22^d
Aju mbaise 250 mg/kg	48.00 ± 1.23^b	2.60 ± 0.55^b	1.87 ± 0.23^c	1.20 ± 0.08^a	1.10 ± 0.07^a	57.29 ± 2.93^b
Aju mbaise 500 mg/kg	70.00 ± 2.45^d	1.60 ± 0.48^a	1.26 ± 0.37^b	2.80 ± 0.47^b	2.57 ± 0.42^c	66.66 ± 2.95^c
Aju mbaise 1000 mg/kg	73.80 ± 0.84^d	0.80 ± 0.04^a	0.56 ± 0.06^a	2.20 ± 0.44^b	1.97 ± 0.21^b	84.77 ± 2.73^d

The mean and standard deviation of the values are shown (n = 5). The means of the identical columns with various letter superscripts differ considerably (P 0.05).

Table 5

Inhibitory effect of *Aju mbaise* polyherbal extract on weight of intestinal contents in castor oil induced diarrhoea model in rats.
Treatments	Weight of filled intestine (cm)	Weight of empty intestine (g)	Weight of intestine content (g)	% Activity
Control	15.59 ± 0.51^d	4.58 ± 0.23^a	11.01 ± 0.41^d	0.00 ± 0.00^a
Loperamide 0.5 mg/kg	12.34 ± 0.20^b	4.73 ± 0.36^a,b	7.60 ± 0.31^b	58.60 ± 0.71^e
Aju mbaise 250 mg/kg	13.93 ± 0.20^c	4.93 ± 0.09^b	9.01 ± 0.22^c	17.20 ± 0.53^b
Aju mbaise 500 mg/kg	12.17 ± 0.24^b	4.94 ± 0.04^b	7.23 ± 0.28^b	32.85 ± 2.57^c
Aju mbaise 1000 mg/kg	11.48 ± 0.38^a	5.01 ± 0.07^b	6.48 ± 0.29^a	43.65 ± 1.42^d

The data is displayed as mean standard deviation (n = 5). With different letter superscripts, all of the means in the same column are substantially different (P 0.05).

3.4. Anti-inflammatory effect of Aju mbaise polyherbal extract in carrageenan induced paw oedema in rats.

The extract, similar to Aspirin, significantly reduced the degree of paw oedema in rats at all administered doses when in comparison to the control (p < 0.05). The extract's inhibitory effect on carrageenan-induced paw edema was 47.83 ± 1.90%, 77.35 ± 3.97%, and 85.05 ± 1.09% for 250, 500, and 1000 mg/kg doses, respectively, comparable to the effect of Aspirin at 56.93 ± 1.59%. Comparing the extract-treated groups to the control groups, inflammation-related indicators showed a significant change. The levels of Interleukin 1b and prostaglandin E2 were lower in the extract-treated groups compared to the control (refer to Tables 6 and 7).

Table 6

Anti-inflammatory effect of *Aju mbaise* polyherbal extract in rats.
Treatments	Initial paw circumference (cm)	0.5 h paw post circumference (cm)	1 h paw post circumference (cm)	2 h paw post circumference (cm)
Control	2.44 ± 0.17^a,1	3.82 ± 0.21^b,2	4.22 ± 0.20^d,3	4.20 ± 0.47^d,3
Aspirin 100 mg/kg	2.70 ± 0.10^b,1	3.26 ± 0.25^a,2	3.66 ± 0.13^b,2,3	3.42 ± 0.48^b,3
Aju mbaise 250 mg/kg	2.74 ± 0.21^b,1	3.20 ± 0.12^a,2	3.94 ± 0.27^c,3	3.66 ± 0.15^c,3
Aju mbaise 500 mg/kg	2.72 ± 0.08^b,1	3.04 ± 0.13^a,3	3.20 ± 0.11^a,2	3.00 ± 0.01^a,2
Aju mbaise 1000 mg/kg	2.58 ± 0.18^a,b,1	3.26 ± 0.06^a,4	3.06 ± 0.17^a,3	2.84 ± 0.23^a,2

The data are shown as mean SD (n = 5). A significant difference (P 0.05) is shown by values in the same row with distinct superscripts for the numbers, and by values in the same column with different superscripts for the letters.

Table 7

Anti-inflammatory effect of *Aju mbaise* polyherbal extract in rats (Cont’d.)
Treatments	Change in paw circumference after 2 h	% inhibition of inflammation	IL-1b ng/ml	PGE-2 ng/ml
Control	1.72 ± 0.30^c	0.00 ± 0.00^a	1.32 ± 0.03^d	73.26 ± 1.42^a
Aspirin 100 mg/kg	0.72 ± 0.16^b	56.93 ± 1.59^c	0.89 ± 0.03^b	64.94 ± 1.31^b
Aju mbaise 250 mg/kg	0.94 ± 0.05^b	47.83 ± 1.90^b	1.01 ± 0.02^c	71.66 ± 2.70^a
Aju mbaise 500 mg/kg	0.32 ± 0.06^a	77.35 ± 3.97^d	0.81 ± 0.02^a	68.84 ± 1.20^c
Aju mbaise 1000 mg/kg	0.24 ± 0.05^a	85.05 ± 1.09^d	0.84 ± 0.06^a	66.77 ± 2.34^c

The results are shown as mean ± standard deviation (n = 5). Different letters superscripts indicate significant differences (P < 0.05) among the means in the same column.

3.5. Analgesic effect of Aju mbaise polyherbal extract in acetic acid-induced pain in rats

The extract also showed significant analgesic activity similar to that of Aspirin when compared with the control (p < 0.05), as the extract-treated rats made fewer number of writhes than the control with 36.76 ± 3.80%, 66.01 ± 2.79% and 77.54 ± 3.01% analgesic activities for 250, 500 and 1000 mg/kg treatment dose levels respectively (Table 8).

Table 8

Anti-analgesic effect of *Aju mbaise* polyherbal extract in rats.
Treatment	% writhes after 30 min	% Analgesic activity
Control	59.80 ± 2.44^d	0.00 ± 0.00^a
Aspirin 100 mg/kg	14.00 ± 2.35^a	69.37 ± 1.13^c
Aju mbaise 250 mg/kg	33.80 ± 1.64^c	36.76 ± 3.80^b
Aju mbaise 500 mg/kg	18.80 ± 2.17^b	66.01 ± 2.79^c
Aju mbaise 1000 mg/kg	11.60 ± 1.95^a	77.54 ± 3.01^d

The data is displayed as mean standard deviation (n = 4). A different letter superscript in the same column (P 0.05) indicates significantly different means.

The findings of this research study indicate that the Aju mbaise polyherbal extract has an LD50 value exceeding 5000 mg/kg body weight, suggesting that it is likely safe for oral consumption and may not cause systemic toxicity in rats. These findings can potentially be extrapolated to humans and other animals. The non-toxic effect of Aju mbaise polyherbal extract had earlier been reported by (5), following acute and sub-acute in vivo toxicity studies, and has only been confirmed by the findings of the current study. International standards for acute toxicity studies indicate that a major outcome in testing toxic substances is mortality and that where no mortality occurs, the agent being investigated may be considered safe for acute use (20). The conclusion is consistent with this report by (21), who classified any substance with an LD50 value above 5000 mg/kg as non-toxic. In plant-based substances, toxicity usually results from the consumption of intolerable amounts of phytochemical components in plants (22). However, this study's findings have shown that the amount of the various phytochemical agents in Aju mbaise polyherbal extract was well tolerated in rats, hence translated into positive pharmacological effects which could be harnessed for the management of human health challenges. In this study, Aju mbaise polyherbal extract has shown significant anti diarrhoeal, anti-inflammatory, and analgesic effects in rats.

The three-model approach adopted in the investigation of the anti-diarrhoeal potential of Aju mbaise polyherbal extract all yielded positive outcomes as results obtained suggest that the extract may be a valuable agent for treating diarrhoea. In the charcoal meal transit model, the inhibition of both transit and intestinal motility observed suggest that the extract may contain bioactive compounds which act by inhibiting acetylcholine activity in the gastrointestinal tract. The crucial role played by acetylcholine in the initiation and maintenance of intestinal motility has been extensively studied (18, 23–25). Acetylcholine induces contractions of the gastrointestinal smooth muscle through cholinergic signalling, which is mediated by muscarinic acetylcholine receptors present on the surface of smooth muscle cells in the intestine (24). Inhibition of acetylcholine activity is a well-established mechanism for the anti-diarrheal effects of drugs (26). Additionally, castor oil-induced diarrhoea in rats was significantly lowered by the polyherbal extract. Typically released from castor oil by intestinal lipase, ricinoleic acid is a hydroxylated fatty acid that irritates and inflames the intestinal mucosa. This causes prostaglandins and nitric oxide to be released, which in turn increases gastrointestinal secretion, motility, and epithelial permeability and causes diarrhoea. (27). The extract may have inhibited the successful promotion of this cascade of activities by inhibiting either the release of ricinoleic acid from castor oil or interfering with intestinal lipase activity. The anticholinergic/spasmolytic activity of the extract in the charcoal meal transit study and anti-enteropooling and ability to remove fluid from the intestinal lumen in the castor oil models all support the claim that the extract may be a good anti- diarrhoeal agent. Agents with such effects have been employed in managing diarrhoea (27). These activities of the extract may not be unconnected with its combined effects of composite phytochemical agents. Tannins, alkaloids and saponins have been implicated in inhibiting intestinal motility and fluid accumulation in the gastrointestinal lumen (26). The presence of these phytochemical agents in Aju mbaise polyherbal extract was reported in a previous communication (5). Another mechanism for achieving antidiarrhoeal effect by agents like Loperamide is the antisecretory pathway. Loperamide alters intestinal absorption and inhibits its secretion by binding to the δ receptor via a cascade of biochemical activities reported by Tagne et al., (2019) (28) and culminating in the maintenance of a low and high intracellular concentrations of Na⁺ and K⁺ respectively and inhibition of the diarrhoea causing action of ricinoleic acid in castor oil. Aju mbaise polyherbal extract may have also achieved its antidiarrhoeal effect via this mechanism, having produced similar activity as Loperamide.

Like Aspirin, the extract significantly inhibited carrageenan induced paw oedema in rats. Carrageenan induces rat paw edema by first increasing histamine and serotonin activities in the tissues, increasing local synthesis of prostaglandin, and later by bradykinin, leukotrienes, and leukocyte infiltrations and also biosynthesis of prostaglandin by inducible cyclooxygenase (29). The extract, in exerting an anti-inflammatory effect may have interfered with or reasonably inhibited these biochemical processes leading to less inflammations in the extract-treated rats. The findings of the analgesic study indicate that the Aju mbaise polyherbal extract has a significant and dose-dependent analgesic effect in rats using the acetic acid pain-induced model. This model induces pain and inflammation in the abdominal cavity through the activation of nociceptors, and the writhings observed in the treated animals are a result of intense endogenous pain (30). The observed decrease in the levels of interleukin-1b and prostaglandin E2 in rats treated with the extract, compared to the control group, provides additional evidence supporting the argument in favor of the anti-inflammatory and analgesic effects of Aju mbaise polyherbal extract. (31). These inflammatory markers usually increase in inflammatory conditions and the opposite may indicate the presence of an anti-inflammatory agent, which in this study is Aju mbaise polyherbal extract.

Aju mbaise polyherbal extract inhibited diarrhoea by reducing intestinal motility/peristalsis, wet stool frequency, fluid accumulation in the gastrointestinal lumen and loss of electrolytes. These effects suggest that the extract may be a good anti diarrhoeal agent and but may be further dissected to characterize other useful phytochemicals eliciting these beneficial effects. Further studies may also provide more support for our current findings.

LD50 - Median Lethal Dose (a measure of acute toxicity)

APE - Aju mbaise polyherbal extract

DT - Distance travelled.

NC - Control group (referring to the number of faeces in the control group)

NT - Treated group (referring to the amount of faeces in the treated group)

WIC - Weight of intestinal content

PC - Paw circumferences

NIH - National Institutes of Health (a research agency in the United States)

CPCSEA - Committee for the Purpose of Control and Supervision of Experiments on Animals (an organization in India that oversees animal experimentation)

MOUAU - Michael Okpara University of Agriculture Umudike

Ethics approval.

The National Institutes of Health (NIH) and Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines were followed in this study's handling, care, and use of experimental animals. Additionally, the ethical committee of the Michael Okpara University of Agriculture, Umudike, authorised all studies in compliance with the international standards for the care and use of laboratory animals. The ethical number for the study is (MOUAU/VPP/EC/2017/007).

Consent for publication

Not Applicable

Availability of data and material

Data generated or analysed during the course of this study are available upon request.

Competing interests

Concerning this study, the authors say they have no conflicts of interest.

Funding

The authors did not receive any funding to carry out the study.

Authors' contributions

Uroko Robert Ikechukwu (URI), IJIOMA Solomon Nnah (ISN), and Ogbonna Henry Nnaemeka (OHN) designed the experiments and performed the literature search. URI, OPN, ISN, Aguwamba Chinedu (AC), and Ugwuanyi Kingsley Chijioke (UKC) carried out the experimental analyses, data collection, interpretation, and discussions. The publication was drafted by URI, ISN and OHN; AC and UKC approved the final version of the manuscript for subsequent publication.

Acknowledgements

We sincerely thank the staff of the Department of Veterinary Medicine, MOUAU for their help with the animal study and for timely inputs. We acknowledge all the discussions from the Uroko lab members at MOUAU.

Authors' information

¹UROKO Robert Ikechukwu, ²IJIOMA Solomon Nnah, ³OGBONNA Henry Nnaemeka, ⁴AGUWAMBA Chinedu, ²UGWUANYI Kingsley Chijioke,

¹Department of Biochemistry, College of Natural Sciences, Michael Okpara University of Agriculture, Umudike, Abia State, Nigeria

²Department of Zoology and Environmental Biology, Michael Okpara University of Agriculture, Umudike, Abia State, Nigeria

³Graduate Biomedical Sciences, University of Alabama at Birmingham, USA

⁴Biochemistry Unit, Department of Chemical Sciences, Faculty of Sciences, Clifford University, Owerrinta, Abia State, Nigeria.

Corresponding author: OGBONNA Henry Nnaemeka

Corresponding author: [email protected]

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Evaluation of the Anti-diarrhoeal, Anti-inflammatory, and Analgesic Effects of the Polyherbal Formulation, Aju Mbaise in Experimental Rats.

Status:

Version 1

Abstract

Background:

Methods:

Results:

Conclusions:

Figures

1.0 Background

2.0 Methods

2.1. Chemicals and Drugs

2.2. Gathering of plant material and preparation of extract

2.3. Phytochemical analysis of Aju mbaise polyherbal extract

2.4. Acute toxicity (LD₅₀) Evaluation of APE

2.5. Experimental Animals

2.6. Acute toxicity evaluation of Aju mbaise polyherbal extract

2.7. Anti-diarrhoeal activity of Aju mbaise polyherbal extract

2.8. Evaluation of the anti-inflammatory effects of the polyherbal extract

2.9. Evaluation of analgesic effects of the polyherbal extract

3.0. RESULTS

3.1. Phytochemical composition of Aju mbaise polyherbal extract

3.2. Acute toxicity (LD₅₀) evaluation of Aju mbaise polyherbal extract

3.3. Anti-diarrhoeal activity of Aju mbaise polyherbal extract

3.4. Anti-inflammatory effect of Aju mbaise polyherbal extract in carrageenan induced paw oedema in rats.

3.5. Analgesic effect of Aju mbaise polyherbal extract in acetic acid-induced pain in rats

4.0. DISCUSSION

Conclusions

Abbreviations

Declarations

References

Status:

Version 1

Evaluation of the Anti-diarrhoeal, Anti-inflammatory, and Analgesic Effects of the Polyherbal Formulation, Aju Mbaise in Experimental Rats.

Status:

Version 1

Abstract

Background:

Methods:

Results:

Conclusions:

Figures

1.0 Background

2.0 Methods

2.1. Chemicals and Drugs

2.2. Gathering of plant material and preparation of extract

2.3. Phytochemical analysis of Aju mbaise polyherbal extract

2.4. Acute toxicity (LD50) Evaluation of APE

2.5. Experimental Animals

2.6. Acute toxicity evaluation of Aju mbaise polyherbal extract

2.7. Anti-diarrhoeal activity of Aju mbaise polyherbal extract

2.8. Evaluation of the anti-inflammatory effects of the polyherbal extract

2.9. Evaluation of analgesic effects of the polyherbal extract

3.0. RESULTS

3.1. Phytochemical composition of Aju mbaise polyherbal extract

3.2. Acute toxicity (LD50) evaluation of Aju mbaise polyherbal extract

3.3. Anti-diarrhoeal activity of Aju mbaise polyherbal extract

3.4. Anti-inflammatory effect of Aju mbaise polyherbal extract in carrageenan induced paw oedema in rats.

3.5. Analgesic effect of Aju mbaise polyherbal extract in acetic acid-induced pain in rats

4.0. DISCUSSION

Conclusions

Abbreviations

Declarations

References

Status:

Version 1

2.4. Acute toxicity (LD₅₀) Evaluation of APE

3.2. Acute toxicity (LD₅₀) evaluation of Aju mbaise polyherbal extract