ANTI-INFLAMMATORY, ANTIMICROBIAL, ANTIDIARRHEA AND TOXIC EFFECTS OF THE AQUEOUS AND METHANOLIC LEAF AND FRUIT EXTRACTS OF CUCUMIS DIPSACEUS IN WINSTAR RATS AND NEW ZEALAND WHITE RABBITS

Inflammation, diarrhea, and microbial infections produce high morbidity and mortality globally, contributing to a high burden of disease in developing countries. Despite the utilisation of conventional drugs to treat inflammation, diarrhea, and microbial infections, their inaccessibility, unaffordability and their side effects hinder the successful treatment and alleviation of disease. The emergence of antimicrobial-resistant bacteria and fungi strains have compromised the efficiency of antimicrobial chemotherapy, which leads to undesirable sequalae. There is an urgent need to search for alternative therapies, which are efficacious, affordable, accessible and safe in order to alleviate human suffering and promote the quality of life. Medicinal plants present a feasible alternative source of potent pharmacological molecules, which are affordable, easily accessible and safe due to the many biologically active phytochemicals they have. Although medicinal plants have been used by humans for ages as medicines and food, only a few have been empirically investigated. Cucumis dipsaceus has a long history of ethnomedicinal usage in treating inflammation, diarrhea, and microbial infections, in Kenya and other countries. However, there is scanty empirical data to validate its efficacy and safety. The anti-inflammatory antidiarrhea, antimicrobial, and toxic effects of methanolic and aqueous leaf and fruit (methanolic and aqueous leaf and fruit) extracts of C. dipsaceus were investigated. The in vivo anti-inflammatory activities of the methanolic and aqueous leaf and fruit extracts of C. dipsaceus were studied using the formalin-induced paw oedema technique in Wistar rats. The castor oil-induced diarrhea method was used to determine the antidiarrhea activity of the methanolic and aqueous leaf and fruit extracts of the studied plant in Wistar rats. Isolated rabbit ileum was used to determine the effects of the C. dipsaceus extracts on the gastrointestinal motility. Antimicrobial activity of the methanolic and aqueous leaf and fruit extracts was determined using the disk diffusion and broth microdilution techniques described previously. The acute oral and dermal toxicity effects of the methanolic and aqueous leaf and fruit extracts of C. dipsaceus were studied in rabbits and rats, respectively, using appropriate guidelines. Data was analysed using GraphPad Prism statistical software version 9.1. The results indicated significant reductions in formalin-induced paw oedema in the Wistar rats by methanolic and aqueous leaf and fruit extracts of C. dipsaceus, in a dose- and time-dependent manner (P<0.05), with percentage inhibition of between 1.56% and 97.59%. Methanolic and aqueous leaf and fruit extracts of C. dipsaceus significantly (P<0.05) inhibited diarrhea and intestinal motility in Wistar rats and rabbits respectively, in a dose-dependent manner, thereby depicting their antidiarrhea effects. Furthermore, the methanolic and aqueous leaf and fruit extracts of C. dipsaceus significantly inhibited the growth of P. aeruginosa, S. enteriditis, E. coli, C. albicans, and B. subtilis in varying degrees as depicted by the different growth inhibition zones of between 6 mm and 20 mm and the Minimum Inhibitory Concentrations of

3.125 µg/ml. The observed anti-inflammatory, antidiarrhea, and antimicrobial activities of the plant extracts were attributed to various phytochemicals extractable by water and methanol, which exerted pharmacological efficacy via various mechanisms. Moreover, aqueous and the methanolic and aqueous leaf and fruit extracts of C. dipsaceus showed no observable acute dermal toxicity effects on the abraded and intact skins of New Zealand White rabbits. Similarly, the methanolic and aqueous leaf and fruit extracts of C. dipsaceus showed no observable acute oral toxicity during the 14-day experimental period. In conclusion, the extracts were considered safe according to the OECD Guidelines. Further pharmacological and toxicological investigations of the tested plant extracts using more advanced techniques should be done in order to elucidate and optimise bioactive molecules for treating inflammation, diarrhea, and microbial infections.

 

 

 

 

Table 4.1: Anti-inflammatory activity of aqueous and methanolic leaf and fruit extracts of C. dipsaceus in Wistar rats 30

Table 4.2: Antimicrobial effects of aqueous and methanolic leaf and fruit extracts of C. dipsaceus on S. enteriditis 31

Table 4.3: Antimicrobial effects of aqueous and methanolic leaf and fruit extracts of C. dipsaceus on E. coli 32

Table 4.4: Antimicrobial effects of aqueous and methanolic leaf and fruit extracts of C. dipsaceus on P. aeruginosa 33

Table 4.5: Antimicrobial effects of aqueous and methanolic leaf and fruit extracts of C. dipsaceus on B. subtilis 34

Table 4.6: Antimicrobial effects of aqueous and methanolic leaf and fruit extracts of C. dipsaceus on C. albicans 35

Table 4.7: Minimum Inhibitory Concentrations of aqueous and methanolic leaf and fruit extracts of C. dipsaceus 36

Table 4.8: Effects of methanolic leaf and fruit extracts of C. dipsaceus on gastrointestinal contraction frequency of isolated rabbit ileum 43

Table 4.9: Acute dermal toxicity effects of aqueous and methanolic leaf and fruit extracts of C. dipsaceus on New Zealand White rabbits 44

Table 4.10: Acute oral toxicity effects of the aqueous and methanolic leaf and fruit extracts of C. dipsaceus in Wistar rats 46

 

Figure 4.1: Percentage inhibition of diarrhea by the aqueous fruit extracts of C. dipsaceus in castor oil-induced diarrhea in Wistar rats 37

Figure 4.2: Percentage inhibition of diarrhea by the aqueous leaf extracts of C. dipsaceus in castor oil-induced diarrhea in Wistar rats 38

Figure 4.3: Percentage inhibition of diarrhea by the methanolic fruit extract of C. dipsaceus in castor oil-induced diarrhea in Wistar rats 39

Figure 4.4: Percentage inhibition of diarrhea by the methanolic fruit extract of C. dipsaceus in castor oil-induced diarrhea in Wistar rats 40

Figure 4.5: Comparison among the percentage inhibition of diarrhea exhibited by the aqueous and methanolic extracts of C. dipsaceus in castor oil-induced diarrhea in Wistar rats. 42

 

 

 

Traditional medicine has been practiced for ages, even before the emergence of modern medicine (Sofowora et al., 2013). Previously, herbalism was based on crude preparations in order to manage various diseases, however, with the advent of modern technology, various plant-derived drugs have been developed to present-day drugs such as creams, ointments, injections, capsules, and Tablets (Ozioma and Chinwe, 2019; Mahomoodally, 2013). Although conventional drugs were developed for various diseases, the usage of medicinal plants in order to treat various diseases is still dominant, due to their easy availability, accessibility, and relative affordability, especially in sub-Saharan Africa (James et al., 2018). The World Health Organization (WHO) reported that more than 80% of people, especially in the low and medium income countries, depend on traditional medicine with over 85 % of the medicines being derived from plant extracts (WHO, 2018). Traditional medicine is very common in Kenya, with various ethnic communities utilising plant-based remedies in order to prevent and treat diseases (Kigen et al., 2016, 2019; Kipkore et al., 2014). Herbal medicines are relatively cheap, accessible and cause minimal side effects (Nasri and Shirzad, 2013).Despite the prominence of herbal medicine, documented empirical data on their efficacy, phytochemical composition, mechanisms of action, and toxicity profile is still scarce.

Despite the extensive uses of medicinal plants for the alleviation of pain and inflammation management, they are excluded from conventional healthcare plans (Alemu et al., 2018). Phytochemicals can offer novel compounds of therapeutic value; hence, there is need for their empirical characterisation. 

Inflammation is an immune response, which is often non-specific, and is evoked after injury, pathogenic invasion, chemical irritation, or allergy (Ptaschinski and Lukacs, 2018). The process involves recognition of injury or pathogen, activation of mediators, recruitment of defence cells, breakdown of diseased tissues, and promotion of tissue repair. Sometimes, inflammation persists due to defects in the body’s defence system, thus exacerbating the associated syndrome’s sequelae (Chen et al., 2018; Hunter, 2012).

Antimicrobial molecules are crucial in lowering the burden of communicable diseases affecting the world population (Abbafati et al., 2020a; Christou, 2011). However, availability of fewer, or the lack of efficacious antimicrobial agents which can thwart resistant pathogenic strains poses a great public health problem worldwide (Dadgostar, 2019; Frieri et al., 2017). In the view of rapidly emerging isolates of resistant strains, development of effective agents is critical. Notably, the history of new antimicrobials is tainted with rapid emergence of resistance, further dimming the hope for long term effectiveness of the current therapeutic agents (Ayukekbong et al., 2017; Manandhar et al., 2019). Scientists need to continuously explore the derivates of medicinal plants that contain useful secondary metabolites such as alkaloids, tannins, flavonoids and phenolic compounds that contain anti-inflammatory, antioxidant and antimicrobial properties (Kathare et al., 2021a; Nyarang and Bonareri, 2021; Obey et al., 2016; Scalbert, 1991).

Cucumis dipsaceus has been used widely to manage inflammation and pain, and in the treatment of bacterial infections, among other functions, including consumption as food (Kipkore et al., 2014; Mutie et al., 2020; Tefera and Kim, 2019). Previous research indicate that leaf extracts of C. dipsaceus have antimicrobial activity (Shivakoti et al., 2015), and various parts including fruits possess anti-inflammatory, analgesic, diuretic, anti-dysentery, among other properties (Kaur and Lata, 2019). In Kenya, a concoction of boiled roots is used to treat abdominal pains among the Keiyo residents (Kigen et al., 2014). The present study explored anti-inflammatory, antimicrobial, antidiarrhea, and toxic effects of the methanolic and aqueous leaf and fruit extracts of C. dipsaceus.

Inflammatory diseases cause debilitating physical, psychological, and economic effects, to the affected patients, globally, with the highest burden lying in the less developed Nations, especially in sub-Saharan Africa (Abbafati et al., 2020a, 2020b; Alema et al., 2020; Henschke et al., 2015; Jairath and Feagan, 2020). Microbial infections of the gastrointestinal tract, cause gastric irritation, inflammation, and impaired gastric functioning, manifesting in abdominal discomfort and diarrhea (CDC, 2015; Christou, 2011; Troeger et al., 2017). Diarrhea is among the leading causes of morbidity and mortality worldwide especially in the children, elderly, and immunocompromised persons (CDC, 2015).

Despite the availability of conventional anti-inflammatory, antimicrobial, and antidiarrhea drugs, inflammatory disorders, microbial infections, and diarrhea still continues to pose global Public health problem (Alatab et al., 2020; Centers for Disease Control and Prevention, 2015; Jairath and Feagan, 2020; Mathers, 2017). The available anti-inflammatory drugs are associated with adverse effects, following chronic use (Monteiro and Steagall, 2019). For instance, the commonest anti-inflammatories cause gastrointestinal bleeding, intestinal perforations, indigestion, constipation, hepatotoxicity, nephrotoxicity, cardiotoxicity, among other side effects (Felson, 2016; Harirforoosh et al., 2013; Moriasi et al., 2021).

The emergence of antimicrobial resistant strains of bacteria and fungi, has complicated successful use of chemotherapy (Ayukekbong et al., 2017).The anti-inflammatory agents used now, cause undesirable effects, including constipation, gastric irritation, cardiotoxicity, hepatotoxicity, nephrotoxicity, low efficacy among others (Caldwell and Cluff, 1974; Dadgostar, 2019; Fernebro, 2011; Mahmoud abd El-Baky, 2016; Mohsen et al., 2020; Shehab et al., 2016). Antidiarrhea drugs are associated with constipation, impaired gastric motility, among other life-threatening effects (WHO, 1990; Dosso et al., 2012; Niemegeers et al., 1981; Sisay et al., 2017).

Conventional anti-inflammatory, antimicrobial, and antidiarrhea drugs are costly, inaccessible, and unavailable, especially in rural areas of developing Countries, where over 80 % of the global burden of diseases lies, due to inadequate, and insufficient healthcare systems, and resources (Mathers, 2017; Murray and Lopez, 1996; WHO, 2020). Therefore, alternative drug agents, which are safe, efficacious, affordable, easily accessible are required to avert human suffering, are required especially in the developing Nations.

Medicinal plants present a viable source of potent therapies against inflammation, microbial infections, and diarrhea, among other diseases, due to their rich ethnomedical history of application, and the diverse array of bioactive principles they contain (Kathare et al., 2021; Moriasi et al., 2021). However, only few medicinal plants have been empirically investigated and validated for use against the ailments they are traditionally used to treat. Moreover, herbal medicines are preferred because of their affordability, availability, ease of usage, ability to treat multiple conditions, and their minimal adverse effects.

Despite the widespread applications of plant-based ethnomedicines to treat diseases, various safety concerns have been raised (George, 2011). This is due to the lack of standard preparation guidelines, specific dose regimens for each disease, appropriate labelling and marketing, empirical Pharmacological, Toxicological and safety profile data, and regulations governing the practice (Abdullahi, 2011; George, 2011; Kaur et al., 2013; Zhang et al., 2012). Therefore, empirical pharmacologic investigations and safety evaluations of herbals may foster the elucidation of efficacious and safe therapies, against various diseases, especially those which are associated with inflammation, microbial infections, and diarrhea, which negatively impact human health. 

Despite the utilisation of C. dipsaceus in traditional medicine in order to manage inflammatory, microbial, and diarrhea associated diseases (Kareru et al., 2007; Kigen et al., 2014; Mutie et al., 2020; Njoroge and Newton, 1994), there is scanty empirical evidence to back the claimed Pharmacologic efficacy and safety. This study investigated the anti- inflammatory, antimicrobial, antidiarrhea and toxic effects of methanolic and aqueous leaf and fruit extracts of C. dipsaceus to lay a framework towards validation of their usage, and the development of potent, safe, accessible, and affordable therapies.

The following were the specific objectives of the study:

This study was guided by the following research questions: