Phytochemicals properties of Carica papaya Linn seeds’ essential oil and their antifungal and antibacterial activities

Carica papaya seeds have been used as traditional medicine. This study aims to determine the phytochemicals of papaya seeds essential oils their antimicrobial properties. Polyphenolic content and antioxidant properties were determined with standard methods. GC-MS was used to analyze chemical compounds. Antimicrobial properties were performed using agar diffusion method. MICs, MBCs and MFCs were established based on broth dilution methods. The results shown an average content of polyphenol compounds and antioxidant activity of 95.49 µg GAE /100 mL and 5569.06 µg AEAC/100 mL respectively. A total of 11 phytochemical compounds were identified and, Isothiocyanatomethyl (97.27%), Benzyl isocyanate (0.55%), Benzyl nitrile (0.43%)11,13-Tetradecadien-1-ol (0.28%) and Thioura, N, N'-bis (phenylmethyl) (0.26%) are major compounds. The essential oils were effective against Candida albicans , Candida spp, Escherichia coli , Salmonella spp, Shigella spp, Staphylococcus aureus and standards strains such as C. albicans 10231 S. aureus ATCC 08275, Salmonella typhimurium ATCC 14028, E. coli ATCC 25922 and E. coli ATCC 8739. Fungi MICs were from 0.16 to 0.38.10 -2 mg.mL while MFCs were between 0.31 and 0.78 10 -2 mg.mL. Bacteria MICs are from 0.09 to 6.25 while MBCs varied from 0.2 to 12.5 mg.mL. These essentials oils are possible improved traditional medicine against enterobacteria.


INTRODUCTION
Carica papaya Linn is an herbaceous and laticiferous plant cultivated throughout the tropics for food and as a cash crop.The plant belongs to the Caricaceae family and can reach up to 10 m high (Lim, 2012).The precise center of origin for this species is difficult to define as no direct archeological evidence has been found up to date (Yogiraj et al., 2014).It originated from natural hybridization (Lim, 2012).C. papaya is a tropical plant, spread to India, Oceania, Africa, and nowadays it is widely distributed throughout the tropics and warmer subtropical areas of the world (Hillocks, et al., 2002;Lim, 2012;Yogiraj et al., 2014;Yogiraj et al., 2014;Chávez-Pesqueira and Núñez-Farfán, 2017).The whole plant of C. papaya is world widely used for both food and medicine.C. papaya is low in caloric as food and rich in vitamins such as vitamin C, niacin, riboflavin, betacarotene and minerals like magnesium, calcium, phosphorus, and iron (Nwofia and Ojimelukwe, 2012).It possesses some valuable phytochemical compounds such as phytosterols, tocopherols, flavonoids, alkaloids, carotenoids, etc.These compounds have several nutritional and medical properties (Afolabi and Ofobrukweta, 2011;Barroso et al., 2016;Chai et al., 2020;Daagema et al., 2020;Silva and Jorge, 2020).As a result, C. papaya has shown anti-inflammatory, hyperglycemia, anti-hypertension, anti-dengue fever, antidiabetic, antioxidant, immunomodulatory, and antimicrobial activities, increasing scientific investigation on it.Papaya seeds are used mainly for their medical properties, and are an important source of essential oils used in medicine and other things.According to He et al. (2017), papaya seeds contain chemical compounds such as benzaldehyde, benzyl nitrile, and benzyl isothiocyanate which have some antimicrobial activities.Seeds have an effective antibacterial property against Escherichia coli, Salmonella species and Staphylococcus aureus infections.Some antihelminthic and anti-amoebic activities have also been put in evidence according to Bouanga-Kalou et al. (2011), He et al. (2017), Muhamad et al. (2017), andYi et al. (2022).The fundamental reasons for papaya seeds use as medicine are both poverty, which limit millions of people to conventional medicines, and the increase of antibiotic resistance against conventional drugs (Somda et al., 2021;Somda et al., 2023).In Burkina Faso, is already part of the plants used in traditional medicine against several diseases for children and adults (Tapsoba and Deschamps, 2006;Sawadogo et al., 2021).The study aims to identify phytochemical compounds in C. papaya seed essential oils and assesses their antifungal and antibacterial activities for improved traditional medicine purposes.

Seeds sampling and collection of strains
Papaya seeds were purchased from local markets, mainly in Ouagadougou (Burkina Faso).Based on the aspects and colors of fruits, sellers recognized different varieties of papaya that were all sampled.Papaya seeds have been dried under shade.Six papaya seed samples of 5 kg each were collected.The clinical bacteria and fungi strains were from human feces, collected at Schiphra Protestant Hospital and Charles de Gaulle Pediatric Hospital, Ouagadougou, Burkina Faso.They included Candida albicans, Candida species, E. coli, Salmonella spp., Shigella species, and S. aureus.

Extraction of papaya seed essentials oils
C. papaya essential oils were obtained by hydro distillation using a Clevenger-type apparatus.Papaya seeds were first dried, crushed, weighed and introduced into a pressure cooker.500 g of C. papaya powder was mixed with 5 L of distilled water and then heated up to 550°C with a heating plate, according to He et al. (2017).The essential oil was then collected in a sterile airtight container and store at 4°C.

Total polyphenolic and flavonoid content
Total polyphenols content were analyzed with gallic acid as standard according to Salla et al. (2016).Thus, 12.5 µL of Folin-Ciocalteu's reagent was added to 12.5 µL of papaya seed sample and 50 µL of distilled water.After 5 min, 125 µL of sodium carbonate (7%) was added and the absorbance was measured at 750 nm after 90 min of incubation at room temperature.Flavonoid content was performed according to Ribarova et al. (2005) method.About 25 µL of extracts and 7.5 µL of sodium nitrite (7.5%) were firstly taken.Then, 15 µL of aluminium chloride (10%), 50 µL of NaOH 1 M and 40 µL of water were added.The mixture was incubated for 5 min in the dark at room temperature, and the absorbance was measured at 520 nm.

Antioxidant properties
The DPPH radical scavenging activity was used to assess the antioxidant activity.Lamien-Meda et al. (2008) method was optimized to determine the DPPH assay.About 210 µL of DPPH (0.1 mM) was added to 40 µL of papaya seeds essential oils sample and the blank.After 90 min of incubation at room temperature, the absorbance was recorded at 517 nm for 30 min interval.The results were expressed as µg of ascorbic acid equivalent antioxidant content (AEAC) per 100 mL of papaya seeds essential oils (Lamien-Meda et al., 2008).

GC-MS analysis
Papaya seeds essential oils analysis by gas chromatographymass spectrometry (GC-MS) was carried out on Shimadzu GCMS-QP-2010 plus system coupled with TESTEK Rtx-5MS column (30 mm×0.25 mm ID × 0.25 µm film thickness (He et al., 2017).Helium was used as carrier gas with a flow rate of 1.0 mL min -1 .The column temperature was increased from 140 to280°C at 5°C min -1 and maintained for 56 min.The injection temperature was kept at 260°C.A 0.3 µL volume of essential oils was injected with 85.2 kPa as pressure.Each compound was identified based on the retention time using the National Institute of Standard and Technology (NIST) as reference database.The peak area of compound was integrated to determine their percentage.

Microbial strains and culture media
The main microorganisms responsible for gastroenteritis were the target ones.The standard strains included E. coli ATTC 8739, Salmonella typhimurium ATCC 14028, and S. aureus ATCC6538.The clinical strains from human feces were collected at Schiphra Protestant Hospital and Charles de Gaulle Pediatric Hospital and included C. albicans, Candida spp., E. coli, Salmonella spp., Shigella spp., and S. aureus.All clinical strains were previously biochemically characterized using API 20E, and human plasma only for Candida.Strains were stored at -18°C.Before being used, bacterial strains were streaked on plates containing Mueller-Hinton agar (MH) and yeasts on Sabouraud dextrose agar (SDA) and incubated for 18 up to 24 h at 37°C for bacterial and 27°C for yeasts.The operation was repeated twice to get viable and purified strains.The turbidity was then adjusted in saline solution to 0.5 according to Mac Farland standard.

The antimicrobial activity
The antifungal and antibacterial activity of papaya seeds essential oils were performed based on the inhibition diameters using agar diffusion method (KonarÃ et al., 2020).The microbial suspension was prepared in a saline solution and standardized to a turbidity of 0.5 on McFarland scale.Papaya seeds essential oils was diluted in Tween 80.A volume of 75 µL of essential oils is then used to fill wells in the plate previously performed and containing infectious microbial.Plates were incubated for 24 to 48 h at 27 and 37°C, respectively for fungi and bacterial.The evaluation of the antimicrobial activity was made by measuring zones of inhibition around wells of 4 mm diameters.Experiments were carried out in triplicate.

Determination of MICs, MBCs and MFCs
MICs, MBCs and (MFCs of papaya seeds essential oils were recorded according to broth dilution method.MICs were determined according to the CLSI M27-A3 guidelines (Clinical and Laboratory Standards Institute, 2008).The tests were performed in sterile 96-microwell plates, into which 100 μl of the RPMI-1640 medium (pH 7•0) was added for each well.Before inoculation, 100 μl of the essential oil solution (with <0•01% Tween 80) was added to the first well and serially diluted from the first well by taking 100 μl into the next.This twofold dilution was continued until the 10th column of the plate was reached.The wells in the 11th column of the plate were reserved for the negative control (without inoculation) and the wells in the 12th column were reserved for the positive control (with FLZ).The final concentrations of essential oils and FLZ in the wells were in the ranges of 256 to 0•5 μg ml −1 and 64 to 0•125 μg ml −1 , respectively.The microbial colonies were suspended in the RPMI 1640 medium and the concentration was adjusted to 1 to 5 × 10 3 CFU/ml.The solution (100 μl) was added to each well except those in the 11th column, which was supplemented with 100 μl of the RPMI 1640 medium.The 96-microwell plates were incubated at 35°C for 48 h.Finally, the optical density values were measured at 630 nm by enzyme-linked immunosorbent assay (ELISA, Biotek Synergy HTX, Vermont, MA, USA).The MICs were determined as the lowest concentration of the test substances that caused 50% inhibition.After the 96-well plates were incubated at 35°C for another 24 h, 20 μl of solution from each test well was streaked onto an SDA plate or on Mueller-Hinton agar (MH), followed by incubation at 35°C for 48 h.The MFCs and MBCs were determined as the minimum drug.

Statistical analysis
The data collection was performed with Excel version 2010.Analysis of variance and average were done using XLSTAT 2014.5.03.

Total polyphenolic and flavonoids content and antioxidant activity
Polyphenols, flavonoids content and antioxidant activity are presented in Table 1.Flavonoid compounds were not found in papaya essential oils.The average content of polyphenol compounds was about 95.49 µg GAE/100 mL and the average antioxidant capacity based on the DPPH radical scavenging power was 5569.06 µg AEAC/100 mL.

Antifungal activity
C. papaya essential oils have presented inhibition activity against both clinical and referenced Candida strains, shown in Table 3.The inhibition zone diameter ranged between 16.5 and 60 mm.The widest zone of inhibition was against Candida spp.438 (60 mm) and the smallest one against Candida spp.1166 (16.5 mm).The standard reference strains (C.albicans 10231) presented 50.5 mm as inhibition zone diameter.

Antibacterial activity
The essential oil of papaya seeds showed an antibacterial activity against all clinical isolated strains and standard strains.The inhibition diameters ranged between 10 and 20 mm for the clinical strains and from 12 to 18 mm for the standard strains.Table 4 presents the inhibition zone diameters of all strains.
The antibacterial activity of standard antibiotic against both clinical and standard strains is presented in Table 5.
In average, papaya seeds essential oils antibacterial properties were less effective than standard antibiotic.

Minimum fungicidal and bactericidal inhibitory concentrations
The minimum inhibitory concentration against fungi ranged between 0.09 and 0.38 10-2 mg.mL -1 and the minimum fungicidal concentration ranged between 0.31 and 0.78 10-2.mg.mL -1 .The smallest inhibitory and fungicidal concentrations were found against Candida  spp.27 (Table 3).Concerning the antibacterial activity, MICs ranged between 0.09 and 6.25 10-2 mg.mL -1 and the MBCs ranged between 0.2 and 12.5 10-2 mg.mL -1 .The smallest minimum inhibitory and bactericidal concentrations were against E. coli 1146 and the highest minimum inhibitory and bactericidal concentrations tested were against S. aureus ATCC 08275 and S. Typhimurium ATCC 14028 (Table 4).

DISCUSSION
C. papaya seeds essential oils showed an important polyphenols content (95.49±41.82µg GAE/100 mL) with a high antioxidant activity (5569.06±454.59µg AEAC/100 mL) and a diversity of phytochemical compounds, mainly Methyl Isothiocyanate, Benzene, Glycerin, 1,2-Dicycopropylacetylene, 14-Octadecenoic acid, Methyl ester, Benzyl isocyanate, Urea, and Benzyl nitrile (Table 2).The antimicrobial inhibition diameter was around 20 mm for bacteria and up to 60 mm for fungi.The tested essential oils were effective against both clinical strains and standard ones.The clinical strains were E. coli, Salmonella spp., Shigella spp.and references ones were S. aureus ATCC 08275, Salmonella Typhi ATCC 14028, E. coli ATCC 25922, and E. coli ATCC 8739.Polyphenols are secondary metabolites compounds widely distributed in plants, where they perform various functions related to growth, reproduction and protection against pathogens, predators, diseases, and UV radiation (Aguilar-Veloz et al., 2020).The polyphenol content as well as the other phytochemicals have some antimicrobial activities (Singh and Ali, 2011;Yanty et al., 2014;Igwe, 2015;Salla et al., 2016;He et al., 2017;Zhang and Chen, 2017;Tan, 2019;Othman et al., 2019;Manso et al., 2021).The antimicrobial activity of papaya seeds essential oils is then tributed to the phytochemicals compound.The content in polyphenols in papaya seeds essential oils in the present study is less important as compared to other plants and this high value is correlated with the antioxidant activity.Previous researches showed a value of polyphenols content, ranging between 4.83 and 22.59 mg GAE/g DW (Afolabi and Ofobrukweta, 2011;Salla et al., 2016;Muhamad et al., 2017;Muntholib et al., 2020) but also some flavonoids content of about 2.68 mg GAE/g DW in papaya seeds extracts (Salla et al., 2016).The appreciable value of antioxidant activity is of nutritional interest, added to their antimicrobial activity.Antioxidant activities are chemicals properties of the phytochemical compounds.These compounds reduce the risk of coronary heart diseases by preventing the oxidation of low-density lipoprotein (LDL) cholesterol, reducing the synthesis or absorption of cholesterol.They also appear to neutralize free radicals, inhibit enzymes that activate carcinogens, and activate enzymes that detoxify carcinogens (Saxena et al., 2013).The antioxidant activity of papaya seeds essential oils found here is higher than those of Dadoosh (2021) and Kothari and Seshadri (2010).The diversity in the chemical compounds is also more than He et al. (2017) who found only three compounds (Benzaldehyde, Benzyl nitrile, and Benzyl isothiocyanate).
Papaya seeds essential oils in this study have been found to be effective against both clinical and references microbial trains (Tables 3 and 4).Shigella spp.1047 and Shigella spp.1183 which were resistant to commercial antibiotic (Table 5) were sensitive to papaya seeds essential oil at different concentrations (Tables 3 and 4).The inhibition activity was more important for fungi (16.5 to 60 mm) than bacteria (10 to 20 mm).Antimicrobial activities of papaya seeds essential oils was also previously proved (Tapsoba and Deschamps, 2006;Singh and Ali, 2011;Muhamad et al., 2017;Zhang and Chen, 2017;Seshamamba et al., 2018;Heena and Sunil, 2019;Chai et al., 2020;Dotto and Abihudi, 2021;Li et al., 2021;Yi et al., 2022).The inhibition diameter for all Candida strains is higher than those of all commercial antibiotics and also higher than those found by He et al. (2017) which was less than 34 mm.But, on average, the inhibition diameter was higher with the commercial antibiotic than papaya seeds essential oils for bacteria strains.The MICs for fungi (0.09 to 0.38) and MFC (0.20 to 0.78) were also low than MICs (0.09 to 6.25) for bacteria and MBCs (0.2 to 12.5).He et al. (2017) also showed that the antifungal activities against Candida strains include C. albicans, Candida glabrata, Candida krusei, Candida parapsilosis and Candida tropicalis.Among fungi strains, Candida spp.438 (60 mm of inhibition diameter) was the more sensitive to papaya seeds essential oils while Candida spp.1166 (16.5 mm) was the less sensitive.And, among bacteria, Salmonella 410 was the less sensitive clinical strains.Salmonella Typhi ATCC 14028 was the less sensitive standard strain, while Salmonella 03 and E. coli ATCC 25922 were the more sensitive clinical and standard strains, respectively.The two clinical strains (Shigella spp.1183 and Shigella spp.1047) which were resistant to commercial antibiotic were sensitive to papaya seeds essential oil.But, MICs and MBCs of C. papaya essential oils microbial activity against pathogens are more weak than those of some other plants' essentials oils (Kim et al., 1995;Bosnić et al., 2006;Fu et al., 2007;Thosar et al., 2013).

Conclusion
This study showed an interesting chemical compounds and antioxidant activities with an effective antimicrobial property of C. papaya essential oils against both clinical and referenced fungi and bacteria.With these properties and antimicrobial activity, papaya seeds essential oil extracts are potential candidates in the improvement of traditional medicine against enterobacteria, but there is a need to specifically focus on their antimicrobial activity against microbial strains which are resistant to commercial antibiotic and complete the study with clinical test and toxicological activities.

Table 1 .
Phytochemical content and antioxidant capacity.

Table 2 .
GC-MS phytochemical composition of papaya seeds essential oil

Table 3 .
Antifungal activity of papaya seeds essential oils.

Table 4 .
Antibacterial activity of papaya seeds essential oil.