ANTINOCICEPTIVE EFFECTS OF TRECULIA AFRICANA DECNE (AFRICAN BREADFRUIT) SEED LECTIN IN WISTAR RATS

The aim. The use of synthetic compounds to treat many diseases must be strictly controlled due to their potential health hazards. Hence, there is a need to search for natural products to serve as safe alternatives to synthetic products. This study investigated the antinociceptive effects and anti-inflammatory activities of Treculia africana seed lectin. Materials and methods. Lectins were purified from Treculia africana seeds using ion exchange and size-exclusion chromatography. The antinociceptive activity of the lectin was assessed in Wistar rats using abdominal writhing and paw-licking tests induced by acetic acid and formalin, respectively. Anti-inflammatory activity was assessed using carrageenan-induced paw oedema. Results . Treculia africana seed lectins at 10 mg/kg (p.o.) produced sedation, reduced ambulation, reduced response to touch, analgesia, and decreased defecation in experimental animals. Administration of Treculia africana seed lectin (1 mg/kg and 10 mg/kg) in experimental animals significantly reduced (P<0.05) acetic acid-induced muscular writhing in a dose-dependent manner with 23.88 and 36.80 per cent inhibition, respectively. Both early and late phases of formalin-induced nociception were significantly inhibited (P<0.001) by the lectin at all doses (0.1, 1.0 and 10.0 mg/kg), comparably with the standard drug, diclofenac sodium. At 10 mg/kg, T. africana lectin caused a 69.12 % and 65.55 % reduction in both early and late phases of formalin-induced paw licking. Treculia africana lectin also significantly brought about a reduction (P<0.05) in inflammation induced by sub-plantar injection of carrageenan as measured by a decrease in paw swollenness. Conclusion. The study showed that Treculia africana lectin possesses antinociceptive and anti-inflammatory properties and can potentially be employed therapeutics to ameliorate pain and inflammation

Pain serves physiological functions, including notification of hazardous stimuli [11].Pain response is necessary for tissue healing and regeneration [11].In his hypothesis, Omoigui [12] summarised that all pain originates from inflammation and associated response.Inflammation is a defensive process in animal cells that usually occurs in response to injuries or pathogenic infections [13].Inflammatory response usually results in tissue repair; however, a prolonged inflammatory process may result in chronic diseases and organ damage [13,14].
Anti-inflammatory agents are essential in controlling inflammation and limiting its course.These agents work by inhibiting the overproduction of mediators of inflammation, especially pro-inflammatory cytokines, inducible nitric acid synthase (NOS) activity and cyclooxygenase (COX)-2.Anti-inflammatory drugs also act by inhibiting protein kinase C and mitogen-activated protein kinase by altering the DNA-binding capacities of transcription factors [15].Due to the adverse effects of synthetic anti-inflammatory drugs caused by prolonged usage, the search for anti-inflammatory agents from natural sources is of paramount interest.Plant lectins are recently being explored in the study of inflammation, and they have been discovered to act either as an anti-inflammatory or pro-inflammatory agent [14].
Treculia africana Decne, commonly known as African breadfruit, wild jackfruit and African boxwood, belong to Moraceae.It is commonly consumed as food and used for ethnomedicinal purposes in many countries in the tropics, such as West African countries, West Indies and Jamaica [16,17].In folk medicine, different parts of the T. africana plant are used to treat/manage various ailments such as diabetes, fever, helminth infections, indigestion, rheumatism, leprosy, whopping cough and malaria and swellings [18,19].
Earlier, Adeniran et al. [17] and Adeniran [20] purified the lectin from the seeds of the T. africana plant and investigated its physicochemical properties and toxicity in experimental rats.Shimokawa et al. [21] also described the purification of two jacalin-related lectins from the seeds.In 2010, Aderibigbe and Agboola [22] reported that the ethanolic extracts of T. africana stem bark had analgesic properties.
It is important to study and investigate the therapeutic properties of some of the active components of this plant in order to understand which ones the identified properties could be attributed to.This would aid future research into drug development and formulations.Thus, the present study aims to evaluate the unexplored antinociceptive and anti-inflammatory properties of T. africana seed lectin in experimental Wistar rat models of nociception and inflammation.

Planning (methodology) of research
The studied plant is highly regarded in traditional medicine due to its many health benefits, amongst which is to manage persons with pain and inflammation.Research has shown that different T. africana extracts are associated with various therapeutic properties.The protein lectin has been shown to possess many bioactivities.This study is intended to establish that the lectin isolated from this plant has analgesic properties.
Fig. 1 shows the graphical methodology followed for the research.

2. Sample collection and preparation
Treculia africana fruits were purchased from Oja-Oba Market, Ado-Ekiti, Nigeria.The plant was identified and authenticated by a taxonomist at the Department of Plant Science, Ekiti-State University.A copy of the plant sample was deposited in the departmental herbarium, and a voucher number UHAE2018076 was obtained.The seeds were removed, air-dried, ground to a fine powder and kept at four degrees Celsius until further use.

Experimental animals
Healthy adult Wistar rats of both genders weighing between 50-70 g were used for this study.The animals were procured from the animal house of Afe Babalola University, Ado-Ekiti and acclimatized for 14 days in a laboratory environment of 12 h light/dark phase.The rats were allowed free access to standard rat pellets and water ad libitum.They were grouped into five of five animals each and maintained at room temperature.Animals were subjected to an overnight fast before each experiment.This research was approved by Afe Babalola University's Animal Ethical Committee, Ado-Ekiti, Nigeria and assigned approval number: 17SCI031002.The ethical standards of experiments were in accordance with ARRIVE guidelines and conducted in accordance with the EU Directive 2010/63/EU on protecting laboratory animals.
The study was carried out between 2018 and 2019.

4. Lectin purification
Ground Treculia africana seeds were defatted with petroleum ether.The crude extract was obtained by suspending defatted T. africana seed powder in phosphate-buffered saline (PBS) (10 %, w/v) and stirring for three hours.The mixture was kept overnight at four degrees Celsius and centrifuged at 4000 rpm for 20 min.The lectin was obtained by purifying the crude extract on a DEAE-cellulose column, followed by purification on a Sephadex G-100 column, as earlier described by Adeniran et al.
[17], followed by lyophilization.Protein concentration was determined using the Lowry method described by Olson and Markwell [23] with 1 mg/ml bovine serum albumin (BSA) as standard.

5. Behavioural screening
This was used to detect the possible effects of T. africana lectin on the central nervous system of the study animals.Specific behaviours (sedation, ambulation, response to touch, analgesia, and defecation) were observed.Rats were treated with a single dose of saline (10 mL/kg, p.o) or the lectin isolated from T. africana seeds at doses (p.o) of 0.1, 1.0 and 10 mg/kg (normal saline was used as a vehicle for the animal study).Animals were observed for 4 h, and their behaviour was recorded at 30, 60, 120, 180, and 240 min after treatment.

6. Antinociceptive activity Acetic acid-induced writhing test
The acetic acid-induced abdominal writhing test was done according to the method described by Fonsêca et al.
[24] and Ajayi et al. [25] with slight modifications.Experimental rats were pretreated orally (p.o) with normal saline (0.9 %), graded doses of T. africana lectin (0.1, 1.0 and 10 mg/kg) or diclofenac sodium (10 mg/kg).This was followed by the administration of acetic acid (0.85 %, 0.25 ml) by intraperitoneal (i.p.) route to induce abdominal constrictions 60 min after.The number of writhing/muscular contractions (which includes the extension of lower limbs and elongation of the body) was counted for 15 min after injection.Formalin-induced paw-licking test.
Formalin-induced paw-licking test in rats was done using a slightly modified method of Hunskaar and Hole [26].Experimental Wistar rats were pretreated with T. africana seed lectin (0.1, 1.0 and 10 mg/kg, p.o), diclofenac sodium or vehicle (normal saline).The pain was induced in the rats after 30 min by administering formalin (2.5 %, 20 µL) under the skin of the dorsal surface of the right hind paw.Paw licking frequency was counted 0-5 min after administration of formalin (neurogenic phase) and 15-30 min after formalin (inflammatory phase) administration.

7. Anti-inflammatory activity
The anti-inflammatory activity of T. africana lectin was assessed using the carrageenan-induced paw oedema model, which was determined by injecting 0.1 mL of 1 % carrageenan suspension into the sub-plantar region of the left hind paw of the rats to induce oedema 60 min after pre-treatment with varying concentrations of T. africana lectin (0.1 mg/kg, 1.0 mg/kg and 10 mg/kg, p.o), vehicle (3 mL/kg), or diclofenac sodium (10 mg/kg) as described by Oladokun et al. [27].The sub-plantar region of the paw was measured before and at 2 and 5 h after induction of oedema using a micrometre screw gauge.An increase in paw thickness was measured by taking the difference in paw before induction of inflammation and at the respective experimental hours.Percentage inhibition of inflammation was calculated as (1) where V i and V f represent initial and final paw thickness, respectively.

8. Statistical analysis
Results were expressed as mean ± standard deviation.Statistical significance of differences (set at P<0.05) among groups were analyzed using one-way analysis of variance (ANOVA) followed by Tukey post-hoc test.Data analysis was done using Graph pad statistical package (Prism 5).

1. Purification of Treculia Africana lectin
A mannose-binding lectin, which agglutinated human blood groups A, B and O non-specifically, was purified from Treculia africana seeds by ion-exchange chromatography followed by gel filtration chromatography (Fig. 2, a, b).
control group mean test group mean 100, control group mean

2. Behavioural results on oral administration of Treculia Africana seed lectin
Lectin isolated from Treculia africana seeds at a dose of 10 mg/kg (p.o.) produced sedation, reduced ambulation, reduced response to touch, analgesia, and a decrease in defecation.However, at doses of 0.1 mg/kg and 1.0 mg/kg, no observable effects on motor coordination were recorded.Thus, the highest dose of Treculia africana seed lectin indicated depressive activity in the central nervous system.

3. Acetic acid-induced muscular writhing
Treculia africana seed lectin (0.1, 1.0 and 10.0 mg/ kg) administration (p.o) produced a significant and dose-dependent inhibition of acetic acid-induced muscular writhing compared to rats in the control group (5.66 %, 23.38 % and 36.80 %, respectively).However, inhibition of writhing response in experimental rats by the standard drug diclofenac sodium at 10 mg/kg was significantly higher (P<0.05)than those produced by T. africana lectin at 0.1 and 1.0 mg/kg but not significantly different from the response elicited at 10 mg/kg of lectin (Table 1).

Formalin-induced paw licking
Injection of formalin into the paw of rats caused an increase in the number of times spent paw licking in the two phases of formalin-induced nociception (early phase: 22.67±2.52 and late phase: 80.33±5.01).However, administration of T. africana seed lectin caused a significant reduction in time spent licking formalin-injected paw in both phases (Table 2) (P<0.0001).At 10 mg/kg, T. africana lectin caused a reduction in paw licking by 69.12 % and 65.55 % in the early and late phases, respectively which was not significantly different from the response elicited by diclofenac sodium.

Discussion
Pain can be said to be an uncomfortable feeling triggered by disease or injury.Pain has three elements: 1. Nociception, that is, activation of heat, pressure or chemical stimuli (all termed noxious stimuli) receptors, which in turn initiate a signal that is transmitted to the brain.
2. The feeling of the pain.
3. Behavioural changes in response to the pain.The primary goal of pain management is blockage of the inbound signals before they can elicit their effects [28].Even though conventional pharmacological therapies for pain are generally effective, they produce intolerable side effects such as gastrointestinal effects, kidney dysfunction, hypertension aggravation and deterioration of joints, and unwanted interactions with other drugs, hence the search for alternative approaches [29,30].Pain control hinders detrimental outcomes such as muscular, gastrointestinal and cardiovascular alterations [31,32].The abilities of any pain relieving agent to express their activities depend on the type of noxious stimuli used to elicit pain [33].
Naturally occurring bioactive agents, such as lectins, are reliable and have been an abundant resource for drug development.Lectins are ubiquitous and information mediators.Several lectins have been shown to play an essential role in pain and inflammation [30]; thus, the isolation, purification and exploration of medicinal properties of lectins from various sources, particularly plants, cannot be overemphasized.
Induction of abdominal writhing by acetic acid is a standard and simple test for assessing anti-nociception, particularly the nonspecific pain-relieving activity of a product [3,34].Acetic acid stimulates visceral and somatic nociceptive neurons by releasing endogenous chemo-sensitive inflammatory mediators such as prostaglandins and cytokines, causing an increase in peritoneal fluid, thereby contributing to the occurrence of nociception of inflammatory origin [3,[34][35][36].The antinociceptive activity shown by T. africana seed lectin may be due to the inhibition of these endogenous mediators in a similar manner to Canavalia grandiflora seed lectin [37] and Bauhinia monandra leaf lectin [3], which brought about a significant reduction in acetic acid-induced writhing.The neurotransmitter glutamate level is increased in the spine, and the antinociceptive effect of Treculia africana lectin could be possibly due to a reduction in the level of this excitatory amino acid in the cerebrospinal fluid [38].
Formalin-induced paw licking test, because of the two phases involved (neurogenic and inflammatory phase), is usually performed to differentiate between central and peripheral actions [24,26].Formalin acts by directly exciting sensory neurons, resulting in the opening of TRPA1 (transient receptor potential ion channel-1), a mediator of inflammatory pain [39].The first phase of inhibition indicates the blocking of direct nerve stimulation.In contrast, second-phase inhibition, dependent on the release of mediators from damaged cells that stimulate peripheral nociceptors, indicates anti-inflammatory activity [25].It has been reported that drugs/compounds that inhibit nociception in both phases of formalin-induced test act on the central and peripheral nervous system, while drugs that act only in the second phase are peripherally acting drugs [26,40].Treculia africana seed lectin, as revealed in the study, reduced the paw-licking time at both phases.This shows that T. africana seed lectin can act on and alleviate both neuropathic pain and inflammatory processes and thus possess both antinociceptive and anti-inflammatory properties.
Due to the carbohydrate-binding affinities of lectins, they can exert their medicinal applications through lectin domain interaction [41].Lectins could bind and block carbohydrate moieties on leukocytes, cause a reduction in intercellular adhesion molecule-1 expression, and prevent leukocytes from rolling and binding to the surface of endothelial cells, thus inhibiting the inflammatory process [30].Lectins can also act by inhibiting pro-inflammatory cytokines.Since there is a direct relationship between inflammation and pain development, the antinociceptive effects of T. africana lectin may be directly linked to the reduction of inflammation.
Carrageenan-induced paw oedema is a standard model for investigating the anti-inflammatory properties of pharmaceutical products.The extent of inflammation and pain in this model is majorly contributed by an increase in prostaglandin-E2 production, mediated by COX-2 in the central nervous system [3].The anti-inflammatory potential of T. africana seed lectin may be a result of the inhibitory effect of the lectin on the release of inflammatory mediators.Several lectins have shown similar anti-inflammatory properties as T. africana seed lectin on carrageenan-induced paw oedema, such as Bauhinia monandra leaf lectin (60 mg/kg, 60.5 % inhibition) [3], Tetracarpidium conophorum seed lectin (12 mg/kg, 62-79 % inhibition) [27], and Bryothamnion triquetrum lectin (10 mg/kg, 73.52-96.51%) [42].
To establish the underlying mechanism of the anti-inflammatory effects of Treculia africana lectin, there is a need for further investigations using several models of inflammation, immunohistology and gene expression.
Limitations of the study.Even though the molecular weight of Treculia africana lectin has been determined in earlier studies, it would have been appropriate to compare the molecular weight of the lectin purified in this study with the earlier one.
Prospects for further research This study established that Treculia africana lectin has analgesic (in formalin-induced paw licking and acetic acid-induced muscular writhing models of nociception) and anti-inflammatory (in carrageenan-induced paw oedema model) potentials.However, there is a need to investigate the interaction of Treculia africana seed lectin in several other models of nociception and inflammation and establish its mechanism of action.

Conclusion
From the results of this study, Treculia africana lectin is shown to be an effective protein in inhibiting/ reducing nociception both centrally and peripherally and inflammation in acetic acid-induced muscular writhing, formalin-induced paw licking and carrageenan-induced paw oedema models of nociception and inflammation.The inhibition of nociception and inflammation by the lectin compared favourably with the standard drug, diclofenac sodium.This could explain the use of T. africana in folk medicine.This study presented experimental corroboration for the efficacy of the use of the plant in managing diseases linked with pain and inflammation traditionally.T. africana lectin could thus be used as po-tential therapeutics in the management of nociception and inflammation.

Fig. 1 .
Fig. 1.General approaches in the planning of the research Fig. 1.General approaches in the planning of the research

Fig. 3 .
Fig. 3. Anti-inflammatory activity of Treculia africana seed lectin in carrageenan-induced paw oedema test.Percentage inhibition was calculated in reference to the control group (treated with normal saline).Values are expressed as mean±standard deviation (SD), n=5.Values with different superscripts along the column are significantly different (p<0.05)

Table 1
Antinociceptive activity of Treculia Africana seed lectin as evaluated by acetic acid-induced writhing test Note: values are expressed as mean ± standard deviation (SD), n=5.A significant difference in comparison to the negative control group: ***p < 0.0001, *p < 0.05

Table 2
Antinociceptive activity of the lectin isolated from Treculia Africana seeds evaluated by formalin -induced paw licking test in rats