Pharmacy and Drug Development

Abstract

Background: Euphorbia grantii is widely used in ethnic medicine for the treatment of diseases such as syphilis, gonorrhoea and wound infections. This study aimed at isolating and evaluating antibiotic activity of E. grantii crude extracts. E. grantii plants samples were collected from Kirinyaga county and crude extracts obtained using methanol, hexane and distilled water. Sensitivity test against Shigella dysentriae (ATCC 13313), Salmonella typhi (6539), Klebsiella pneumoniae (70063), Staphylococcus aureus (25923) and Streptococcus epidermidis (12228) was carried out using agar well diffusion bio assay and minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) determined. Cytotoxicity of the crude extracts was established using brine shrimp lethality test. The crude extracts contained cardiac glycosides, saponins, tannins, anthroquinones, alkaloids, flavonoids and phenolics. The zones of inhibition of the test pathogens by the crude extracts from Euphorbia grantii varied significantly (F= 5.573 P=0.008197). The MIC varied significantly between hexane, methanol and distilled water crude extracts (F=6.6400 P=0.005). However, the MIC and MBC were not significantly different among the crude extracts (P=0.067). The toxicity of the crude extracts increased with increase in concentration. The crude extracts from E. grantii inhibited growth of the test pathogens. There is need to carry out structure elucidation of the crude extracts.

Introduction

Infectious diseases are a leading cause of morbidity and mortality. In developing countries, they account for 50% of all deaths (Kemboi et al., 2022). Globally, 5.8 million deaths each year in infants and children below 5 years are due to microbial infections (Wang et al., 2020). However, records for morbidity and mortality occurring as a result of microbial infections are scanty in most developing countries (Al-Snafi et al., 2017). According to Kausar et al. (2016) most pathogens that cause diseases have developed resistance to the commonly prescribed antibiotics. Bacterial resistance to treatment using antibiotics increases mortality, likelihood of hospitalization and the length of stay in the hospital (Edrees, 2019). Increased usage of antibiotics leads to increased levels of bacterial resistance (Tripathi et al., 2022).

Previous studies have pointed out that resistance spread among bacteria. This is attributed to the mobilization of drug resistance markers by a variety of agents encoded on plasmids, transposons and integrons (Mamun-Or-Rashid et al., 2022). Indeed, current studies indicate that it’s very hard to isolate bacteria that are susceptible to regularly used antibiotics. This clearly indicates that antibacterial therapy is a global problem (Silalahi, 2021).

The situation is even dire in developing world due to poor sanitation and ignorance of good hygienic practices which expose a large number of people to infectious agents (Tran et al., 2020). Bahadur et al. (2018) noted that E. coli, salmonella spp. Proteus spp., shigella spp., pseudomonas spp. and staphylococci are common infectious agents in developing countries. Majority of these bacteria are normal flora in human bodies, have virulent factors and colonize in a biofilm fashion, causing a variety of intestinal and extra intestinal diseases (He et al., 2020).

This calls for development of some newer, safer, effective and above all, cheaper antibiotics. The big millstones in drug discovery is attributed to continuous search for new drugs. Previous studies have pointed at plants as sources of antibiotics of high potency against microbial infections (Ribeiro et al., 2015). Traditionally, herbs have been used as sources of food and medicinal purposes for many years and the knowledge about their potency passed on from one generation

to the other (Ernst et al., 2015). This is particularly more common in rural areas where infectious diseases are endemic and modern health care facilities are few. Besides, the facilities are far apart leading to people turning towards herbs in nursing their ailments (Tedesse et al., 2016).

E. grantii is one of such herbs belonging to the family Euphorbiaceae. The herb is abundant in open waste spaces, grasslands, road sides, and pathways (Mwine & Van Damme 2015). The herb is native to central America but has spread throughout the tropics, especially in west, central and east Africa (Esposito et al., 2017). It’s an annual broad-leaved herb with a hairy stem with many branches from the base to the top. The stem and leaves produce a milky juice when cut. Numerous studies have attempted to establish the medicinal usefulness of the herb (Fridlender et al., 2015). Previous studies have shown that the plant contain triterpenoids, sterols, alkaloids, glycosides, flavonoids, tannins, phenols, choline and shikimic acid (Mahmood et al., 2013; Srivastava et al., 2014; Sultana, 2017).

The plant has previously been used as an antispasmodic, antiasthmatic, expectorant, anticatarrhal

and antisyphilitic (Taib et al., 2023). The bioactive activity of the plant is attributed to presence of choline, shikimic acid and the quercetin (Mekam et al., 2019). E. grantii is a very popular herb in traditional medicine (Sharma et al., 2014). The plant bears different local names such as nonon furchiya in Hausa, tepel in fulzfulde, Harvom in Kaka and Hammock sand mat (Kirbag et al., 2013).

Materials And Methods

Collection of plant samples

E. grantii plant samples were collected from Kirinyaga county and transported to Kirinyaga University biology laboratory. The identification of the plant was done using family keys. The samples were air dried at room temperature for 14 d.

Preparation of plant material

The plant samples were chopped into small pieces using a sterile blade and ground into coarse powder using pestle and mortar. The powder was further ground into fine powder using kenwood electric blender (Kenwood Limited, Harvant, United Kingdom). The powder was stored in airtight bottle at -4oC in a refrigerator awaiting further processing.

Preparation of the extracts

Briefly, 1000 g of the powder was separately placed in 100 mL methanol, distilled water and hexane. The mixture was incubated for 24 h at 40°C in shaking conditions at 200 rpm using an orbital shaker (Nkya et al., 2014). The mixture was filtered using a clean muslin cloth followed by Whatman No. 1 filter paper. The filtrate was concentrated to dryness using a rotary evaporator attached to a vacuum pump (Model type 349/2, Corning Limited). The percentage yield of the crude extracts was determined using the formula below (Awouafack et al., 2013);

Percentage yield=Dry weight of the sampleDry material weightx 100

The crude extracts were dissolved in their respective solvents diluted further to obtain 400, 200, 100, 50,25, 12.5, 6.25, 3.085 and 1.03 mg/mL and stored at 2 - 8°C awaiting further processing.

Test pathogenic microorganisms

The following pathogenic bacteria were used in the study; Shigella dysentriae (ATCC 13313), Salmonella typhi (6539), Klebsiella pneumoniae (70063), Staphylococcus aureus (25923) and Streptococcus epidermidis (12228). The bacteria were maintained at a temperature of 4oC ± 2. Standardization of culture was carried out as suggested by the National Committee for Clinical Laboratory Standards (NCCLS, 1990). Briefly, 18 h culture of bacteria was inoculated into sterile universal bottles containing nutrient broth. Normal saline was gradually added and turbidity compared with Mcfarland Standard of 0.5 which corresponds to approximately 1.0 x 108 cfu/mL.

Photochemical screening of the plant material

Phytochemical screening was carried out on the powdered plant extracts for the presence of bioactive components such as cardiac glycosides, alkaloids, flavonoids, tannins, phenols, anthroquinones, saponins and flavonoids (Yuet et al., 2012).

Determination of the antibiotic activity

Briefly, 1.0 mL of 18 h culture of each test bacterial pathogens adjusted to 1.0 x 10 8 cfu/mL was placed on a Petri dish containing sterile Mueller Hinton agar and spread using a L shaped glass rod in triplicate. Wells approximately 8 mm in diameter and 2.5 mm deep were made aseptically using a cork borer. Using a micropipette, 0.5 mL of each crude extract at a concentration of 100 mg/mL was pipetted in to one of the holes. Following this, 0.5 mL of pure solvent was pippeted into the second hole as negative control. An aqueous solution of 12.5 ug amoxicillin was used as positive control. The plates were allowed to stand for 1 h for prediffusion of the extracts to occur and incubated at 37°C for 24 h and the zones of inhibition determined.

Determination of (MIC) and MBC

Determination of the minimum inhibitory concentration (MIC) was carried out using the Broth dilution method (Sahm and Washington, 1990; Adesokan et al., 2007; Oyeleke et al., 2008). Briefly, 1.0 mL of each crude extract at a concentration of 200 mg/mL was placed in a test tube containing 1 mL of sterile broth so as to obtain a concentration of 100 mg/mL. Aseptically, 1 mL of this dilution was serially transferred to another test tube till the 7th test tube was reached.  In the 8th test tube a solution of pure solvent was placed to act as negative control. Briefly, 1 mL of an 18 h old pathogenic bacterial culture of each of the bacteria was placed into each tube and mixed using a vortex mixer and incubated at 37°C for 24 h. The test tube with the lowest dilution with no detectable growth presented the MIC. To determine the MBC, 0.10 mL of bacterial suspension from the MIC tubes was sub-cultured into Mueller Hinton agar plates and incubated at 37°C for 24 h. The concentration at which no visible growth was observed was recorded as the MBC.

Cytotoxicity bioassay

Brine shrimp eggs were hatched in Petri dishes having artificial brine with 9.5 g artificial sea salt dissolved in 250 mL distilled water and illuminated with a lamp. Briefly, 10 mg of accurately weighed crude extract was dissolved in 1 mL of dimethyl sulphoxide (DMSO). DMSO was added to achieve 60 to 2 μg/mL dilutions in vials. Brine shrimps and DMSO 0.6%v/v in artificial sea water acted as negative control. The vials were covered with aluminum foil and incubated at room temperature (25oC±2) for 24 h. The number of dead shrimps was determined. Tests were performed in triplicate. The LC50 in µg/mL was determined using probit analysis (Nasrullah et al., 2012)

Data analysis

Statistical package for social sciences (SPSS) version 2025 software was used in data analysis.  The analysis of data from yield determination, growth inhibition of the test pathogens, MIC and MBC was carried out using single factor ANOVA. Data on brine shrimp lethality test was analyzed using regression and probit analysis.

Results

Yield of the crude extracts of E. grantii

The percentage yield of crude extract was 0.0300 in methanol, hexane (0.0259) and water (0.0205) (Table 1).

Table 1: Percentage yield of the crude extracts of Euphorbia grantii

Photochemical constituents of crude extracts from E. grantii

Phytochemical analysis of crude extracts from Euphorbia grantii revealed the presence of cardiac glycosides, saponins, tannins, anthroquinones, alkaloids, flavonoids and phenolics (Table 2).

Table 2: Photochemical constituents of Euphorbia grantii

+; Presence, -; absence

Antibacterial activity of crude extracts from E. grantii

The zones of inhibition of the test pathogens by the crude extracts from E. grantii varied significantly (F= 5.573 P=0.008197). The zones of inhibition varied from 13.2±0.3mm to 23.1±0.2mm in hexane extracts, methanol (18.2±0.1-25.3±0.3mm), distilled water (11.3±0.2-21.2±0.2mm) and in amoxicillin (22.1±0.3-26.2±0.1mm) (Table 3).

Table 3: Zones of inhibition (mm) of the test pathogens by crude extracts from Euphorbia grantii

Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the crude extracts

The MIC varied significantly between hexane, methanol and distilled water crude extracts (F=6.6400 P=0.005). However, the MIC and MBC were not significantly different among the crude extracts (P=0.067). MIC in hexane extracts varied from 110±2 to 125±1 mg/mL, methanol (100±1-150±3 mg/mL), distilled water (110±1-150±3 mg/mL) and amoxicillin (15±1-25±3 mg/mL) (Table 4). The MBC ranged from 110±1 to 125±2 mg/mL in hexane, methanol (125±1-150±2 mg/mL), distilled water (110±1-150±2 mg/mL) and amoxicillin (15±2-25±3 mg/mL).

Table 4: Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of crude extracts of Euphorbia grantii

S. aureus; Staphylococcus aureus, S.  epidermidis; Streptococcus epidermidis, S. dysentriae; Shigella dysentriae, S. typhi; Salmonella typhi and K. pneumonia; Klebsiella pneumonia.

Cytotoxicity of crude extracts from E. grantii

In testing for cytotoxicity, the concentration of the crude extracts was increased from 1 to 31 µg/mL (Table 5). The percentage mortality of the brine shrimps ranged from 31-100 in methanol extracts, hexane (25-100) and distilled water (15-100). The LC50 (µg/mL) varied from 14.20 in methanol to 18.30 in distilled water.

Table 5: LC50 (µg/mL) values of crude extracts on brine shrimp lethality bioassay.

Discussion

The percentage yield of crude extract from E. grantii in this study was high in methanol compared to hexane and distilled water. Comparatively, previous studies obtained higher yields. El-Mahmood (2009) reported a yield of 3.9% for water extract, 1.3% for hexane extract and 1.8% for methanol. Besides Owolabi et al. (2007) reported 10.4% crude extracts using water as a solvent and ethanolic extracts (3.78%). Further, Ogbolie et al. (2007) reported 9.1% yield from water extracts of E. grantii. Polarity of the solvent and age of the plant has been shown to affect yield of crude extracts in plants (Sharma, 2024).

The crude extracts obtained from the current study contained cardiac glycosides, saponins, tannins, anthroquinones. Alkaloids and phenolics. These compounds have been shown to have antimicrobial activity (Patidar & Khan, 2024). Previous studies have linked the presence of these bioactive compounds to the antimicrobial properties of crude plant extracts (Ghosh et al., 2019; Ghosh et al., 2020; Ogbole et al., 2021). According to Ojedirah et al. (2024) alkaloids are used as analgesics, antimalarials, and stimulants. The presence of glycosides moieties like saponins, anthraquinones, cardiac glycosides and flavornoids have anticancer properties in addition to having antibiotic properties (Cayona & Creencia, 2022). In addition, herbs that produce tannins have been used in in treating intestinal disorders such as diarrhea and dysentery in traditional medicine due to their antibiotic properties (Mohammad et al., 2017). In addition, tannins are used in traditional medicine in treating wounds and arresting bleeding (Magozwi et al., 2021). Besides these bioactive compounds produced as secondary metabolites as the plant grows, protect the plant against infections and predation by animals (Srivastava & Soni, 2019).

The selected test pathogens were susceptible to crude extracts from E. grantii at varying degrees. Methanol extracts presented the highest zones of inhibition followed by hexane extracts and the least zones of inhibition were presented by distilled water. This contradicted the finding obtained by Cano et al. (2024) where water extracts presented the biggest zones of inhibition. According to Elisha et al. (2023), the susceptibility of bacteria to plant extracts, varies depending on strains and species. In addition, the zones of inhibition in Gram positive pathogenic microorganisms were bigger than in Gram negative pathogens. This concurred with a study carried out by Iskandar et al. (2022). Ojedirah et al. (2024) explained that Gram negative bacteria are resistant to most antimicrobial agents.  Gram negative pathogens have lipopolysaccharide in their phospholipids membrane which confers to them impermeability to antimicrobial agents (Gigante et al., 2023). Amoxicillin served as a positive control in this study. At low concentrations, it gave larger zones of inhibition than the crude extracts. This could be attributed to the pure form in which it was used (Mekam et al., 2019).

The MIC and MBC values for methanol were lower than in hexane and distilled water. In addition, The MIC and MBC values for amoxicillin were lower than those from crude extracts. However, the values varied among the test pathogens with Gram positive pathogens having lower MICs and MICs than Gram negative test pathogens. These findings agreed with those of a previous study by Igwe et al. (2021). The differences in MIC and MBC among test pathogens could be attributed to variation in the structure of the cell wall (Ballentes & Pradera, 2019). Besides, Maria et al. (2021) maintained that microorganisms varied widely in their degree of susceptibility to antibiotics. Panzu et al. (2020) observed that crude extracts with low activity against a particular organism gave high MIC and MBC values, while a highly reactive extracts gave low MIC and MBC values. In the current study, the MIC and MBC values obtained had very small differences. This indicated that the crude extracts were bactericidal and not bacteriostatic (Taib et al., 2022).

The lethality of the crude extracts of E. grantii increased with concentration suggesting the presence toxic compounds. This correlated well with previous reports regarding cytotoxicity of E. grantii and other species in the genus (Behravan et al., 2010). Brine shrimp lethality test is correlated to various biological activities such as anticancer, larvicidal and antimicrobial activities (Runyoro et al., 2017). 

Conclusion

E. grantii plant samples contained bioactive metabolites. The extracts inhibited growth of the test pathogenic microorganisms. However, the metabolites inhibited growth of Gram positive more than the Gram-negative pathogens. The crude extracts were toxic to brine shrimps.

Recommendations

The crude extracts from E. grantii need to be tested against other pathogens. Structure elucidation of the crude extracts need to be carried out. In addition, there is need to carry our mass production of the crude extracts.

Acknowledgments

Many thanks to Kirinyaga University for giving us the laboratory space for carrying out this work.

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