Seed oils are composed of diverse triglycerides with immense potential for industrial applications, including soap production [1,2]. Oils suitable for soap-making possess specific characteristics such as high fatty acid content (oleic, palmitic, and stearic acids), high saponification and iodine values for better lathering and cleansing properties, and low unsaponifiable matter to ensure stability and prevent spoilage [3,4]. Conventional oils like olive oil, coconut oil, and palm oil are widely used due to their distinct properties; however, the exploration of underutilized seed oils offers a sustainable and economical alternative [5,6].  Traditional medical practices utilize underexploited seed oils or plant extracts for their bioactive components, which can enhance cosmetic formulations [7]. Pachira glabra, native to Brazil, is one such underutilized plant. Its seeds have an oil content of about 48% and show promise for industrial applications, including soap production, as evidenced by their reported medicinal properties [8,9]. Despite this potential, P. glabra seed oil remains largely untapped, leading to seasonal wastage of a valuable natural resource. Soap, a sodium or potassium salt of fatty acids, is primarily produced through saponification, where alkali hydrolyzes fats or oils, forming soap and glycerol [10]^. Medicated soaps incorporating bioactive agents provide effective cleansing and antimicrobial benefits, making them an attractive alternative to synthetic antiseptic soaps, especially in developing countries [11]. Ketoconazole, a synthetic imidazole antifungal agent, is widely used for treating fungal infections such as dermatophytoses, candidiasis, and malassezia-related conditions [12,13]. Fungal infections are among the most common skin ailments, requiring effective and affordable treatments [14]. The increasing demand for medicated soaps has led to innovations in soap formulations, particularly with the inclusion of natural oil bases and potent antifungal agents like ketoconazole [15,16]. However, no scientific study has explored the use of P. glabra seed oil in soap production. This research aims to formulate a ketoconazole soap using P. glabra seed oil as an oil base, evaluate the physicochemical properties of the soap (such as pH, total fatty matter, total alkali content, foam ability and drug content), and investigate the antimicrobial activities of both the oil and the soap. By utilizing this underutilized resource, the study seeks to promote sustainable agriculture, reduce resource wastage, and develop an effective antifungal product.

MATERIALS AND METHODS

Sample Collection and Identification

Pachira glabra seeds were sourced from the courtyard of Ladoke Akintola University of Technology (LAT 8.169036, LON 4.264671), Ogbomoso, Nigeria. The identification process was carried out at the Department of Pure and Applied Biology, LAUTECH. Following the method of Badejo and Ayodele (2022) with slight modifications, the fruits were first decorticated to remove the outer husk, and the seeds were removed from the pods. The seeds were oven-dried at 40 ^OC for 3 hours and 30 minutes, cooled, de-hulled, and stored in air tight container for further processing. To obtain a fine powder, the seeds were ground using a Silver Crest multifunctional electric blender (SC-1589). The powder was stored in a plastic jar and kept in a deep freezer at 4 ^OC until needed.

Extraction of oil

Extraction of oil was carried out with 250 mL Soxhlet extraction apparatus with two different solvents namely ethanol and n-hexane. Pachira glabra seed powder (200 g) was charged into the thimble, 200 mL of each solvents was used for the extraction and the whole set-up (thimble chamber, condenser and distillation flask) were carefully and effectively coupled. The whole set-up was heated by the heating mantle (WN-HM6) at 90 ^OC for 5 hours. The resulting solution containing the oil and the solvent was distilled with the aid of rotary evaporator to give the actual oil yield. The oil yield was evaluated as:

(1) Where W₀ = Weight of empty flask

W₁ = Weight of flask after distillation

W₂ = Weight of P. glabra sample

Determination of Total phenolic Content

With some modifications, the spectroscopic method previously described by Edewor et al (2021) was used to determined the concentration of phenolic in the seeds oil. Folin-Ciocalteu assay method was used for the determination of total phenolic content. Into a 25 mL volumetric flask 1 mL of P. glabra seed oil and 9 mL of distilled water were added, the mixture was shaken after which 1 mL of Folin-Ciocalteu phenol reagent was added. After 5 minutes, 7% sodium carbonate solution (10 mL) was added and the mixture was made up to 25 mL volume. Standard solution of gallic acid (20, 40, 60, and 80 µg / mL) were prepared and treated following same procedure as sample. Both the sample and standard solution were incubated for 90 minutes at room temperature, then, the absorbance of the sample and standard solutions was measured using a UV/Visible spectrophotometer (ASUV-6300 PC, USA) against the reagent blank at 550 nm. The amount of total phenol content was reported as milligrams of gallic acid equivalents (mg GAE/g) per gram of sample.

Determination of Total Flavonoid Content

With some modifications, the aluminum chloride colorimetric assay previously reported by Edewor et al, (2021) was used to measure the total flavonoid content. P. glabra  seed oil (1 mL) was put in a 25 mL volumetric flask and 4 mL of distilled water was added, after five minutes, 0.3 mL of 10 % aluminum chloride was mixed with 0.30 mL of 5 % sodium nitrite in the flask. One molar sodium hydroxide (2 mL) was added after 5 minutes, and it was diluted to 10 mL with distilled water. Standard solution of quercetin (20, 40, 60, and 80 µg / mL) were prepared and treated following same procedure as sample. Using a UV/Visible spectrophotometer (ASUV-6300 PC, USA), the absorbance of the sample and standard solutions were measured at 510 nm in relation to the reagent blank. The total flavonoid content was expressed as mg amount of quercetin extract per gram of sample (mg QE/g).

Determination of saponification value

Extracted oil (2 g) was mixed with 25 mL of methanolic KOH in 250 mL conical flask and heated in a water bath for 30 minutes with frequent refluxing, 1 mL of phenolphthalein was added and the content (as hot) was titrated against 0.5 M HCl until the pink colour disappeared. The discolouration indicates the end point.¹⁸ Saponification value was calculated using the equation (2).

(2)where B = blank titre value (mL)

S = sample titre value (mL)

M = Molarity of KOH

Soap formulation (saponification of oil)

A basic saponification reaction is used in the soap formulation, in which KOH and Pachira glabra seed oil react to produce soap. Following the method of Pandey et al. (2023) with modification, 40g of seed oil was placed in a beaker and heated to 55 ^OC in a water bath. Concurrently, in a separate beaker, 7.32 g of KOH was dissolved in deionized water. The KOH solution and oil were mixed and constantly stirred for about half an hour, or until the oil completely turns into a uniform solution. After the mixture was cooled, 350 mL of saturated sodium chloride solution was added to precipitate the soap. After separation, the precipitate was transferred into a clean beaker.

Formulation of 2 % w/w ketoconazole soap

For the production of 25 g medicated soap, 24.5 g of the soap that was made and 0.5 g of ketoconazole tablet (King sales agency, India) that was purchased from a pharmacy store was combined and heated gently for 20 minutes while being constantly stirred. The medicated soap was filled into the proper mold and allowed to solidify for duration of 24 hours [13].

Physicochemical characteristics of ketoconazole soap produced

pH measurement

The pH of the soap produced was measured using a pH meter. A 1 % (w/v) soap solution was prepared by dissolving 1 g of soap in 100 mL of distilled water. The pH meter electrode was then immersed in the solution and the reading was recorded [1].

Foam forming ability and retention test

Soap sample (0.5 g) was mixed with 10 mL of distilled water in a 100 mL measuring cylinder. After vigorous shaking for 2 minutes to produce foam, the flask was left undisturbed for 10 minutes and the foam height was measured with ruler (cm) and the time taken for the foam to disappear was recorded as the foam retention time [13].

Determination of total fatty matter (TFM) in soap produced

The TFM was determine by the petroleum spirit extraction method earlier stated by Olabanji et al (2016) with modifications. Soap sample (10 g) was dissolved in 100 mL of warm water (80 ^OC) and transferred to a separating funnel. Methyl orange indicator (3 drops) was added, followed by 10 mL of 50 % H₂SO₄ until the color changed. After adding 1 mL of petroleum spirit and vigorously shaken, the mixture was allowed to settled, and the liberated fatty acids formed a clear layer on top. Saturated NaCl solution (50 mL) was added to the mixture and washed with 25 mL of n-hexane twice, followed by 25 mL distilled water twice in the separating funnel and the water layer was discarded. The remaining mixture was transferred into the pre-weigh beaker and dry to constant weight in an oven at 60 ^OC. The percentage TFM was determined from the weight of the fat obtained and the soap using the (Equation 3)

(3)Where W₀ = Weight of empty beaker

W₁ = Weight of beaker + fatty matter

W₂ = Weight of ketoconazole soap

Determination of total alkali in soap produced

The determination of total alkali was carried out using the method described by Arasaretnam and Venujah (2019).  Soap sample (5.0 g) was dissolved in 100 mL distilled water and heated for 20 - 30 minutes. Concentrated sulfuric acid was added to separate the fatty acid layer. The mixture was extracted with chloroform and transferred to a separating funnel. After shaken thoroughly, the chloroform layer was separated and the aqueous solution was measured. A 10mL aliquot of the aqueous solution was titrated with standard KOH, using methyl orange as an indicator, to determine the alkali content of the soap.

Determination of drug content

The determination of drug content was carried out following the method of Pandey et al (2023) with modifications. Ketoconazole soap produced (2.5 g) was dissolved in the 100 mL volumetric flask using 50 mL methanol, and the sample solution was sonicated for 30 minutes to ensure complete solubilization of soap, followed by filtration. Similarly, standard solution of ketoconazole was prepared by dissolving 50mg of the ketoconazole drug in 100 mL of methanol in a volumetric flask. With proper dilution, 25 ppm and 12.5 ppm concentration of the standard were prepared. Thus, the absorbance of both sample and ketoconazole solution were measured at 240 nm using a UV-visible spectrophotometer (ASUV-6300 PC, USA) and the calibration curve was drawn using the absorbance obtained and the concentration of ketoconazole soap was obtained. The percentage drug content was obtained using the Equation 4 and 5.

     (4)

(5)

Antimicrobial activity

Antimicrobial activity comprises Minimum Inhibitory Concentration (MIC) and Minimum Microbicidal Concentration (MMC) of oil extracted from P. glabra seed using n-hexane and ethanol and the ketoconazole soap produced were determined by the broth micro dilution method against some microbial organisms; Gram positive (Staphylococcus aureus, Bacillus subtilis), Gram negative (Escherichia coli, Klebsiella pneumonia) and fungi (Trichophyton rubrum, Candida albicans) following the method of Oulkheir et al. (2017).

Determination of Minimum Inhibitory Concentration (MIC)

The Minimum Inhibitory Concentration (MIC) was determined using the broth micro dilution method in 96-well plates. Samples were initially dissolved in double-strength Tryptone soya Broth to create a 50% solution, which was serially diluted to achieve concentrations of 25 %, 12.5 %, 6.25 %, 3.125 %, 1.5625 %, 0.78125 %, and 0.3960 %. Ciprofloxacin (10 ?g/mL) and ketoconazole (1 %) served as reference drugs for antibacterial and antifungal assays, respectively. These reference drugs were serially diluted to achieve concentrations of 10 ?g/mL, 5 ?g/mL, 2.5 ?g/mL, 1.25 ?g/mL, 0.625 ?g/mL and 0.1325 ?g/mL for ciprofloxacin and and 1 %, 0.5 %, 0.25 %, 0.125 %, 0.0625 %, and 0.031255 % for ketoconazole. Microplate wells were inoculated with 10 ?L of microorganisms and incubated at 37 ^OC (bacteria) and 25 ^OC (fungi) for 24 and 48 hours, respectively. The lowest concentration showing no growth or turbidity was considered the Minimum Inhibitory Concentration (MIC) [21].

Minimum Microbicidal Concentration (MMC)

To determine the MBC/MFC, 10?L of p-INT  (piodonnitrotetrazolium violet) solution was added to the MIC test plates. After 30 minutes of incubation at 37 ^OC, wells with color change from yellow to pinkish-red indicated microbial growth, and the lowest concentration with no growth was considered the MBC/MFC (MMC) [20].

RESULTS AND DISCUSSION

Some characterization of P. glabra seed oil were reported in Table 1. The percentage oil yield from P. glabra seed using ethanol and n-hexane were 12.1% and 47.8 % respectively  but the later compared favourably with 47.1 % reported for the same sample using n-hexane as extraction solvent, groundnut (31.7 - 57 %), neem oil (38.8 %), and poppy seed oil (30 - 44 %) [22,23,24,25]. The high level of oil yield will enhance economic importance as raw material for commercial industrial production. Thus, the n-hexane extract was considered for the production of ketoconazole soap. The total phenolic content  4.61 and 4.69 mg/g were obtained for oil extracted from P. glabra using ethanol and n-hexane while the total flavonoids obtained were 0.09 and 0.11 mg/g respectively. Meanwhile, phenolics and flavonoids are bioactive compounds commonly found in plant extracts, including oils, which have several skin benefits including skin soothe, inflammation reduction, and could promote wound healing.

The saponification value of P. glabra seed oil obtained using ethanol and n-hexane were 188 and 178 mgKOH/g respectively, which compared favourably with ranges  161.09 - 183.03 mgKOH/g reported for the same sample [22], and some other seed oil such as sunflower oil (184.50 mgKOH/g), bran oil (184.50 mgKOH/g), palm oil (192.79 mgKOH/g), and mustard oil (193.36 mgKOH/g) but lower compared with Citrus sinensis seed oil (222.58 mg KOH / g) and Palm kernel oil (280.5 mg KOH / g) [19,26]. Meanwhile, oil ranging from 180 - 300 mg KOH/g are considered to have high saponification value, which indicated that the oil will be useful in the production of soap and other cosmetic products

Table 1: Characterisation of P. glabra seed oil extracts

Table 2: Physicochemical characteristics of the soap produced from P.glabra seed oil

Figure 1: Picture of 2% w/w ketoconazole soap produced from P. glabra seed oil
       
           
       

Figure 2: Foam ability of ketoconazole soap produced from P. glabra seed oil

According to the Bureau of Indian Standards (BIS), soaps with TFM greater than 76 % are classified as Grade I, while those with TFM above 60 % are Grade II, and soaps with TFM above 50 % are classified as Grade III. Although the TFM of the soap from P. glabra seed oil falls below the Grade III threshold, it is still within an acceptable range based on industry standards. Furthermore, a higher TFM typically correlates with better cleansing, moisturising properties, and more lather, as highlighted by Abba et al. (2021). The total alkali content of the soap produced in this study was found to be 3.06 %, which is lower than the 4.65 % reported for soap made from Citrus sinensis seed oil. However, it is consistent with the alkali content found in soaps produced with other seed oils, such as coconut oil, olive oil, and palm oil, which fall within the 2.7 – 4.48 % range [19,20]. The value of total alkali content obtained was higher than the 2.03 – 2.04 % observed in commercial soaps like Septol, “Dudu Osun”, Farha, and Giv [4], but it still falls within the acceptable limits of ? 5 % as specified by the Bureau of Indian Standards (BIS). Alkali content is an important parameter that influences the abrasiveness and overall harshness of soap on the skin. A moderate alkali content of the ketoconazole soap produced will enhance an effective cleansing action without causing excessive irritation. The drug content of ketoconazole in the soap produced was measured to be 80.7 %. The ketoconazole content of the soap was lower than the 99.37 % reported for Prinsepia utilis seed oil-based ketoconazole soap [13]. The ketoconazole content does not fall within the 95 – 105 % range specified by the US Pharmacopoeia (2020), but it still demonstrates a reasonable concentration of the active ingredient. The results of the antimicrobial activities of the oil extracted from P. glabra seed using n-hexane and ethanol, as well as the ketoconazole soap produced using P. glabra seed oil were reported in Table 3, which revealed varying degrees of effectiveness against the tested microorganisms. Both the n-hexane and ethanolic extracts (oil) exhibited similar growth inhibitory effects against Staphylococcus aureus and Escherichia coli, with comparable values for the Minimum Inhibitory Concentration (MIC) and Minimum Microbicidal Concentration (MMC). Notably, the n-hexane extract demonstrated a maximum inhibitory effect against Trichophyton rubrum, Candida albicans, and Escherichia coli, with MIC and MMC values that surpassed even the positive controls (ciprofloxacin and ketoconazole). This suggested that the n-hexane extract possesses significant antimicrobial potential. On the other hand, the ethanolic extract showed lower activity against Bacillus subtilis, Klebsiella pneumoniae, Candida aureus, and Trichophyton rubrum compared to the positive controls, indicating a relatively weaker antimicrobial effect. Both oils, however, were particularly effective against Escherichia coli, with MIC values higher than the positive control (ciprofloxacin), indicating stronger inhibition than ciprofloxacin. This highlights the robust antimicrobial action of the P. glabra seed oil extracts, particularly against E. coli. Regarding the ketoconazole soap produced using P. glabra seed oil, it exhibited strong fungicidal and growth inhibitory effects against Candida albicans and Trichophyton rubrum, with MIC and MMC values higher than those of the positive control. However, the ketoconazole soap displayed similar MIC and MMC values to the control against Escherichia coli and Klebsiella pneumoniae. In contrast, the positive control was found to be more effective against Staphylococcus aureus and Bacillus subtilis when compared to the ketoconazole soap. The high effectiveness of the n-hexane extract can be attributed to the presence of bioactive compounds such as phenols and flavonoids, which are known for their antimicrobial properties. Additionally, the fatty acid composition of the oil plays a significant role in its antimicrobial efficacy, particularly fatty acids with 18 carbon atoms, such as linolenic, linoleic, and oleic acids [29]. These fatty acids are more abundant in the n-hexane extract, which could explain the superior antimicrobial activity observed. In contrast, the ethanolic extract contains lower levels of these key fatty acids, which may account for its reduced antimicrobial effectiveness. The MIC is defined as the minimum concentration of the tested plant oil that inhibits visible growth of microorganisms (bacteria, fungi, or yeast) after a specified incubation period [30,31]. On the other hand, the MMC refers to the minimum concentration of the plant oil required to kill 99.9% of the microorganisms after a specified incubation period [32]. These parameters provide insight into the potency and effectiveness of the antimicrobial agents in the oils and soaps tested.

CONCLUSION

Pachira glabra oil can serve as pharmaceutical ingredient, as it showed broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria, as well as fungi. Its potential as an antimicrobial agent makes it a valuable ingredient in treating various infectious diseases, particularly those caused by Trichophyton rubrum. Furthermore, development of ketoconazole soap using P. glabra seed oil has highlighted its potential use in soap formulations targeting susceptible microorganisms. However, cultivation and commercialization of this plant seed could find utility from non-conventional seed oil, thereby, prevent wastage of natural resources, promote sustainable agriculture and clean environment thus, promoting good health and wellbeing.

Conflict of interest

The authors declare no conflict of interest

Author’s declaration

The authors hereby declare that the work presented in this article are original and has not been publish elsewhere

Table 3: Antimicrobial activities of P. glabra seed oil and its soap (MIC and MMC mg/mL)

KEY: S.A - Staphylococcus aureus        B.S-Bacillus subtilis                 E.C - Escherichia coli  

Kleb - Klebsiella pneumonia                  T.R - Trichophyton rubrum        C.A - Candida albicans

MIC - Minimum Inhibitory concentration            MMC - Minimum microbicidal concentration   

Ket - Ketoconazole                               Cipro - Ciprofloxacin                  NA - Not applicable