Comparative Studies of Phytochemical and Antimicrobial Activity oc Carica papaya L. Extracts against Escherichia coli, Staphylococcus aureus and Candida albicans

Carica papaya extracts are known for their traditional medicinal uses. The ability of its parts to control the growth of common pathogens in the laboratory has been tested in different parts of the world using different varieties of C. papaya. This study was initiated to compare the phytochemical and antimicrobial activity of different plant parts extracts of C. papaya var. papayi GAV4 on Escherichia coli, Staphylococcus aureus and Candida albicans. C. papaya plant parts were collected from a farm in Kiboswa (Kisumu): coordinates 0.0245°S and 34.7474°E, and then were transported to Maseno University Botany Laboratory. Seeds, green leaves and bark were washed thoroughly with tap water, rinsed in sterile water and dried after which they were ground using a grinder. From each of the three plant parts, three types of extracts were prepared using water, acetone and ethanol in the concentrations 25%, 50%, 75% and 100%. The antimicrobial activity of the extracts was tested on microbes growing on agar plates by inoculation with the different concentrations using diffusion method and replicated 3 times. Extracts were isolated using Soxhlet apparatus and MIC determined by serial dilution, zone of inhibition was measured in millimeters. Means from the measurements were separated and compared at significance level P = 0.05. Phytochemicals present included alkaloids, flavonoids, tannins, phenols, saponins, glycosides, anthocyanins and terpenoids while anthraquinones were absent. Ethanol bark extract on C. albicans showed higher inhibition and there were significant differences in inhibition among the plant parts and extracts used. In concentrations used, 25% was significantly different from 50%, 75% and 100%. The results obtained in this study confirm that C. papaya has antimicrobial activity on E. coli, S. aureus and C. albicans; and has also shown high potentials for use as a potential source of antibiotics to treat diseases caused by these microorganisms. Archives of Ecotoxicology, Vol. 2, No. 3, pp. 35-42, 2020


Introduction
In traditional system of medicine, plant preparations in the forms of decoctions, concoctions, macerations, or infusions are used to treat a wide range of diseases (Tsobou et al., 2016).
Current estimates indicate that about 80 million people worldwide still depend on plants for their health needs (Dwivedi et al., 2020). Limited access to primary health care in developing countries has resulted into widespread use of herbal medicines due to the availability, accessibility, affordability and cultural acceptance across different ethnic backgrounds (Muhwana et al., 2020). There is widespread use of broadspectrum antibiotics which has led to the emergence of nosocomial infections caused by drug resistant microbes (Abubakar, 2009). Multi drug resistance and the presence of several virulence factors in the strains of many pathogens responsible for different diseases pose an increasing threat to disease treatment. There are several varieties of this plant spread throughout the world. Papaya also known as pawpaw (Carica papaya Linn) is commonly known for its nutritional and medicinal values throughout the world (Alabi et al., 2012). It is a giant herbaceous non woody plant resembling a tree from the family Caricaceae (Akujobi et al., 2010). Each part of papaya tree possesses economic value when it is grown on a commercial scale (Krishna et al., 2008; Orchue and Momoh, 2013). Even though the active compounds are normally extracted from all parts of the plant, the concentration of these compounds varies from structure to structure (Aruljothi et al., 2014). However, parts known to contain the highest concentration of the principles are preferred for therapeutic purposes and it can either be the leaves, stem, barks, roots, bulks, corms, rhizomes, woods, flowers, fruits, and the seeds (Kafaru, 1994;Emitaro et al., 2020). Various parts of the papaya plant, which include the leaves, fruit, seed, latex, and root, are known to contain bioactive compounds that contribute to reported medicinal properties (

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In Kenya there exists more than 65 varieties of C. papaya [Asundu et al., 2010], yet most C. papaya farmers around Kiboswa (Kisumu) grow the C. papaya L. var. papayi GAV4 variety. It was therefore easy to obtain C. papaya L. var. papayi GAV4 plant materials for these studies. Extracts from different varieties of the same plant may respond differently to bioassay tests conducted on microorganisms due to varying physiological and chemical characteristics where they may occur (Nirosha and , 2015). It is imperative therefore that before this study, little was known about the antimicrobial effect of bark, leaf and seed extracts of C. papaya var.papayi GAV4.The choice of C papaya var. papayi GAV4 was guided by earlier findings that showed that it is the most abundant variety in Kiboswa (Kisumu) (Asundu et al., 2010).

Study area
After identification of the plant (Figure 1) was conducted using a taxonomic key (Cowan, 1999), the materials used in this study were collected from a farm located at Kiboswa (Kisumu)within the geographical coordinates 0.0245°S and 34.7474°E. All plant parts including; fruits, leaves and bark were then transported to the botany laboratory at Maseno University (Maseno Kenya) located within the geographical coordinates 0.0°0S and 34.36°E where analysis was conducted.

Preparation of seed extract
Seeds were obtained when fruits earlier acquired from Kiboswa (Kisumu County), and taken to Maseno University botany laboratory were washed with clean tap water, rinsed in sterile distilled water and cut open using a kitchen knife, then left to dry for 30 days at 25 0 C at the Botany Laboratory, Maseno University (Jyotsna et al., 2014). The seeds were then powdered using a grinder to produce a fine powder ( Figure 2).

Preparation of leaf extract
Disease free green leaves of C. papaya var. papayi GAV4 earlier collected from Kiboswa were washed in tap water, rinsed in sterile distilled water, dried at 25°C for 20 days before being grounded into a green powder using a grinder to produce a fine powder (Jyotsna et

Preparation of bark extract
Diseases free pawpaw bark were cut from the tree using a sharp kitchen knife, washed in tap water, rinsed in sterile distilled water then dried at 25°C for 30 days before being ground to produce a brown powder using a grinder (Jyotsna et   2.5 Ethanol extraction of seed, leaf and bark extracts One hundred grams of powdered dried seeds, leaf and bark were weighed using a weighing machine and powder transferred into 500 ml glass conical flasks as earlier described by Okunola et al. (2012). Ninety five percent of 500 ml ethanol was measured and poured onto the conical flask containing the seed, leaf and bark powder and stirred to produce mixtures that were allowed to stand for 24 hours before decantation and filtration through a Whatman filter paper No 1. (Okunola et al., 2012). The resulting filtrates were concentrated in a rotary evaporator at 79 0 C resulting in concentrates that were stored in the refrigerator at 4 0 C until required for use.

Water extraction of seed, leaf and bark extracts
One hundred grams of powdered dried seeds, leaf and bark extracts were transferred into 500 ml glass conical flask into which two hundred millimeters distilled water was poured then stirred to produce mixtures that were allowed to stand for 24 hours before decantation to produce filtrates using a Whatman filter paper No 1.

Acetone extraction of seed, leaf and bark extracts
One hundred grams of powdered dried leaves, seed and bark extracts were transferred into 500mls glass conical flask into which five millimeters of 95% acetone was poured then stirred to obtain mixtures that were allowed to stand for 24 hours before decantation and filtration through Whatman filter paper No 1. resulting in filtrates that were concentrated with a rotary evaporator at 45 0 C to produce concentrates that were later stored in the refrigerator at 4 0 C until required for use.

Test microorganisms
The test organisms that were used in this study were pure strains of human pathogenic organisms of clinical origin obtained from Centre for Disease Control (KEMRI/CDC) located in Kisian (Kisumu) and maintained on Mueller Hinton Agar (Oxoid, UK) medium as stock cultures in the laboratory refrigerator set at 4 0 C.The isolates included pure strains of gram negative bacteria E. coli (ATCC 25922) gram positive bacteria S. aureus (ATCC 25923) and an imperfect yeast C. albicans (ATCC 1405). The C. albicans strain was grown in Nutrient agar (NA) media, E. coli grown in Nutrient agar (NA) media while the S. aureus was grown in Potato Dextrose agar (PDA).

Phytochemical compounds extraction and screening
Twenty-five grams of dried leaves, seed and bark powder were extracted in Soxhlet apparatus by using 25 ml of solvent having polarity of ethanol for 48hrs and then concentrated by evaporation. These prepared extracts were used for phytochemical screening for alkaloids, flavonoids, tannin, phenols, saponin, terpenoids, anthraquinones, Cardiac glycosides and anthocyanins as earlier described by

Determination of alkaloids
Two grams of the extract were extracted by warming it for 2 minutes with 20ml of 1% H2SO4 acid in a 50ml conical flask on a water bath, with intermittent shaking. It was then centrifuged and the supernatant was pipetted off into a small conical flask. One drop of Meyer's reagent was added to 0.1ml supernatant in a semi micro tube. A cream precipitate indicated the presence of alkaloids.

Determination of flavonoids
5ml of dilute aqueous ammonia solution were added to a portion of the plant extract followed by addition of concentrated sulphuric acid. Positive test was indicated by yellow colouration which disappeared on standing.

Determination of tannins
About 0.5 g of the dried powdered samples was boiled in 20ml of water in a test tube and then filtered through Whatman No. 42 filter paper. A few drops of 0.1% ferric chloride was added. A brownish green or a blue-black coloration indicated the presence of tannins.

Determination of phenols
Ferric chloride test was carried out where the extract was diluted to 5ml with distilled water. To this, a few drops of neutral 5% Ferric chloride solution was added. A dark green or a blueblack color indicated the presence of phenolic compounds.

Determination of saponins
About 2 g of the powdered sample was boiled in 20ml of distilled water in a water bath and filtered. Ten milliliters of the filtrate were mixed with 5ml of distilled water and shaken vigorously to form a stable persistent froth. The froth was mixed with 3 drops of olive oil and shaken vigorously, and then was observed for the formation of emulsion.

Test for anthraquinones
Powdered plant material was boiled with 10% HCl for a few minutes, then filtered and allowed to cool. This was then partitioned against equal volume of chloroform. Formation of rose-pink color upon addition of 10% aqueous ammonium solution, indicated the presence of anthraquinones.

Test for Cardiac glycosides
Five ml of extract was treated with 2ml of glacial acetic acid containing a drop of FeCl3 solution. This was then underplayed with 1ml conc. H2SO4. A brown ring of the interface indicated a deoxy-sugar characteristic of cardenolides.

Test for Anthocyanins
2ml of 2M sodium hydroxide (NaOH) solution was added to few extracts in a test tube. The formation of blue-green colour compound confirmed the presence of anthocyanins.
2.9.9 Test for terpenoids 5ml of the extract was mixed with 2ml of chloroform followed by addition of 3ml of concentrated sulphuric acid to form a layer. Positive test was indicated by formation of a red colouration at the interface.

Antimicrobial susceptibility test for bacteria
The disc diffusion method on Mueller Hinton agar (Yahaya et al., 2017) was used to determine the antibacterial activity of the plant extracts. An overnight culture of the bacterium was diluted to 10 5 cells/ml using a spectrophotometer at a wavelength of 625 nm. One milliliter of the bacterial suspension was introduced into sterile petri dishes and 20 ml of Mueller -Hinton agar at 40 0 C poured into the inoculated dishes before the plates were allowed to cool and solidify. A sterile filter circular discs, 8mm in diameter each were cut from Whatman No.1 filter paper using a paper punch and each dipped in a known concentration of 25, 50, 75 and 100% of the extracts for about 2 minutes, then gently transferred to the centre of the inoculated agar media. Petri dishes inoculated with bacteria and fungi were kept for incubation for 24 hrs at 37 0 C and 25 0 C respectively. The diameter of inhibition zone was measured using 12.5 cm Vernier calipers. This was carried out in triplicates in a completely randomized design.

Determination of Minimum Inhibitory Concentration (MIC)
The MIC of the extracts was determined by using Muller-Hinton broth dilution (Anibijuwon and Udeze, 2009) made and sterilized using an autoclave. Serial dilutions of the extract in liquid medium were prepared and 1.0 ml of the prepared broth dispensed into the test tubes labeled from 1 to 4 using sterile syringe and needle. A stock solution containing 100mg/ml of the extract was prepared. 1.0 ml of the solution was dispensed into the tube 1. Subsequently, from tube 1 solution was serially transferred until 4.0-1.0 ml of the solution was discarded from it. An overnight culture of each of the test isolates was prepared in sterile nutrient broth. 1 ml inoculum was transferred into each tube from tube 1 to tube 4. The final concentrations of the extract in each of the test tubes after dilution i.e. 100, 50, 25 and 12.5 mg/ml was incubated at 37°C for 24 hrs and examined for emergent growth. To measure the MIC values, various concentrations of the stock, 25, 50, 75 and 100 mg/ml were assayed against the test bacteria where the minimum inhibitory concentration was defined as the lowest concentration able to completely inhibit any visible microorganism growth after overnight incubation with media (Prescort, 1999 ; Yahaya et al., 2017).

Phytochemical screening
Information on the phytochemical constituents of plant materials are generally required for the discovery of novel drugs. The phytochemical screening of C. papaya plant materials carried out revealed the presence of alkaloids, flavonoids, tannins, terpenoids, anthraquinones, phenolic compounds and saponins in the extracts of leaf, seed and bark (Table 1), while anthraquinones were not detected in the seed and bark extracts.  Table 2). There was no visible inhibition exhibited by C. albicans on the acetone extracts, yet when comparison was made for the 3 extracts, there were significant differences among the extracts ( Table 2).   The effect of C. papaya water bark extract on growth of the 3 microorganisms ( Figure 5 & 6) indicate that even the water extract has visible inhibitory effects against the growth of E. coli, C. albicans and S. aureus in the Petri dishes. Yet the effect of C. papaya seed extract on growth of E. coli, C. albicans and S. aureus when measured indicated that the highest zone of inhibition was demonstrated against E. coli at 8.13 mm by ethanol extract of dried seeds ( Table 3). The lowest zone of inhibition was demonstrated against E. coli at a measurement of 5.09 mm by the acetone extracts of dried seed (Table 3). There was no inhibition at all for the acetone extract of C. albicans and S. aureus. There appeared to be no significant differences among the extracts.  Table 3). The lowest zone of inhibition was demonstrated against E. coli with a measurement of 2.97 mm by the water extract of dried seeds. There was no inhibition for the acetone extracts on C. albicans and S. aureus (Table 4). The results obtained indicated that there were significant differences among the extracts (Table 4). The disc diffusion method on Mueller Hinton agar was used to determine the antimicrobial activity of C. papayavar. papaya GAV4 leaf, seed and bark extracts with different concentrations ( Table 5). As shown in table 5, increase in the concentration has different effects on the microorganism, plant part and extract used. It is shown that ethanol, acetone and water leaf extracts did not inhibit the growth of C. albicans and acetone leaf extracts did not inhibit the growth of S. aureus. At 25% concentration, there was no inhibition of growth of E. coli by ethanol, water and acetone leaf extracts and water seed extracts. Table 6 shows Minimum Inhibitory Concentration (MIC) of various extracts of leaf, seed and bark extracts of C. papaya var. papayi GAV4 on the microorganisms, the minimum inhibitory concentration was defined as the lowest concentration able to inhibit any visible bacterial or fungal growth, the MIC varies with the microorganism, plant part and extract used.  (Musyimi et al., 2007). For example, alkaloids isolated from plants have been found to have antimicrobial properties (Sikandar et al., 2013), and are one of the most efficient therapeutants that were isolated from the plant extracts during these studies. Flavonoids represent the common and widely distributed group of plant phenolics; their biological functions include protection against allergies, inflammations, platelets aggregation microbes, ulcer, viruses and tumors (Okwu and Okwu. 2004). The presence of tannins in the C. papaya can support its strong use for healing of wounds, ulcers, hemorrhoids, frost-bites and burns in herbal medicine (Igboko, 1983). Tannins have astringent properties which hasten the healing of wounds and inflamed mucous membrane (Igboko, 1983;Maduinyi, 1983). The presence of phenolic compounds in the extracts of C. papaya shows that the extracts may have antimicrobial potential, because phenols and phenolic compounds have been extensively used in disinfections and remains the standard with which other bactericides are compared (Oakenful, 1981).The presence of saponins supports the fact that C. papaya extracts may have cytotoxic effects (Okwu and Okwu, 2004; Okigbo et al., 2009). Saponins exhibit broad range of pharmacological actions, such as ability to heal wounds and inflamed mucous membranes. Therefore, in view of the occurrence of phytochemicals in the extracts it is more appropriate to state that the antimicrobial activity of the C. papaya extracts may be attributed to the presence of the bioactive compounds. The confirmed presence of bioactive substances in these extracts is very important, such substances has been reported to confer resistance to plants against bacteria, fungi and other microorganisms, this therefore may explain the reasons for the demonstrated antibacterial activity by the plant extracts used in this study. Antimicrobial properties infer that any of these properties: i.e. anti-bacterial (antibiotics), anti-fungal (antimycotic), anti-cancerous (anti-oncogenic) or anti-viral is inherent (Baskaran et al., 2012). The present study has clearly shown that the different parts of C. papaya possess antimicrobial potential against S. aureus, E. coli and C. albicans. In line with the present findings, several other studies have reported other varieties of C. papaya leaves (Kafaru, 1994 ; Rahman et al., 2011) have antimicrobial potentials. Additionally, the reports of other workers (Yahaya et  al., 2017) have also shown that other varieties of C. papaya leaves and stem barks have significant antibacterial activity in extracts from different tree parts. Other workers concur with our findings that C. papaya had significant antibacterial activity (Nirosha and Mangalanayaki, 2013; Douhari et al., 2007). The gram-negative bacteria display some particularities that inhibit antibiotics penetration, as the lipopolysaccharide layer that determines the permeability and susceptibility to antibiotics. In the antimicrobial test for bacteria, it was observed that the potency of the activity of C. papaya depends on the extraction solvent used; organic extracts such as ethanol were more effective than C. papaya in aqueous extracts may be as a result of the better solubility of the active components in organic solvents. The ethanol extracts clearly demonstrated a higher activity than the acetone and water extracts, the better efficacy of the ethanol extract against the acetone and extract may be because different solvents have different polarities, hence different degrees of solubility of the various phytoconstituents (Rahman et al., 2011).Based on the limited spectrum of activity of the other extracts compared with the ethanol extracts, it suggests that the active component is more soluble in ethanol than in the other solvents. This is in agreement with The results obtained during this study have clearly shown that the MIC values varied from 0.025-0.1mg/ml for the three extracts. Lowest MIC value 0.025mg/ml was recorded against E. coli and S. aureus where against C. albicans the lowest MIC observed was 0.05mg/ml. these results indicates significant antimicrobial potential of extracts. High minimum inhibitory concentration observed for Candida albicans, high MIC may be an indication of low efficacy or that the organism has higher potential for developing resistance to the bioactive compounds in the plant, which is said to be related to a thick layer in their outer membrane which prevents the entry of inhibition substances (Chima et al., 2016). High MIC may also mean that a higher concentration of the extract is required to inhibit the organism's growth. The low MIC value observed for E. coli and S. aureus is a good indication of high efficacy against these microorganisms. This also means that lower concentration of the extract is required to inhibit the organism's growth. On the other hand, disparity in Minimum Inhibitory Concentration may be due to variable sensitivity to the chemical substances related to different resistant levels among strains.

Conclusions
Carica papaya var. papayi GAV4 leaves, seeds and bark contain alkaloids, saponins, tannins, glycosides, phenols, terpenoids, anthocyanins and flavonoids. Anthraquinones were absent in seeds and bark. This plant extracts showed antibacterial and antifungal activities against S. aureus, E. coli and C. albicans, thus an indication that the plant can be a potential source for production of drugs with a broad spectrum of activity. Additionally, the Minimum inhibitory concentration for S. aureus and E. coli was 0.025mg/ml while that of C. albicans was 0.05mg/ml. Further pharmacological evaluations, toxicological studies and possible isolation of the therapeutic antibacterial substances from plants are some of the future challenges that will be faced by workers studying new substances with antimicrobial properties. C. papaya var. papaya GAV4 may be recommended as a useful source to prepare natural bioactive products from which we can develop new antimicrobial drugs which will be costeffective. We suggest that in the search for new pharmaceuticals substances, screening of various natural organic compounds and the identification of active agents must be considered as a fruitful approach.