Phytochemical Analysis, Antioxidant and Antibacterial Activity Determination of Ethanolic Extract of Carica Papaya Seeds

In the present study, the Carica Papaya Seeds (CPS) were extracted by using 95% ethanol with Soxhlet extraction method. After the plant extract was subjected for the sterility test and results showed no growth of bacteria, the plant extract was examined for the phytochemical screening. The antibacterial test was carried out with agar well diffusion via examination of nutrient and tryptic soy agar. MIC test is used to determine whether the bacteria can be inhibited by plant extract concentrations that are achievable and safe in the blood stream, it was conducted by culturing microorganism in liquid media. The antioxidant activity and total phenolic content were carried out to identify the IC50 value and the total amount of phenolic content of CPS. For the phytochemical screening test, the results showed the presence of alkaloids, anthraquinone, flavonoids, and carbohydrate. The DPPH radical scavenging assay and total phenolic test were shown positive for the antioxidant activity of the CPS. As a basis, Folin-Ciocalteu reagent was used to measure the phenolic content of different concentrations of plant extract. In this test, the Gallic acid was used as a standard and the total phenolic content was calculated by using the formula C = (A/B) x dilution factor. In the present study, the total phenolic content found was 6.420 mg GAE/g and 6.097 mg GAE/g for the concentrations of 50 and 200μg/ml respectively. According to the results obtained, the IC50 was calculated for the BHT as well as CPS extract, the value was 84.04 μg/ml and 182.82 μg/ml, respectively. Besides that, the percentage scavenging value obtained for the ethanolic C. papaya seed was inconsistent and not precise, therefore the results cannot be considered or equivalent to the antioxidant activity exerted by the CPS as the results are not reliable. However, it can be concluded that the CPS are exhibiting antioxidant effect as it shows positive results for the Total Phenolic Content (TPC) as well as DPPH scavenging method. Received: February 02, 2021


Introduction
Plants, the greatest invention of Almighty God are the 'lungs' of our lovely planet. Plants are just like the 'treasure box' hidden with a lot of active components that are essential for the process of developing new medicine. Medicinal plants not only considered as a readily available and affordable source, but they are also able to synthesize diverse active compounds which are effective in controlling and treating many diseases. These active compounds are known as secondary metabolites, such as phenols, tannins, Recently, the CPS has been studied further for its uses and identified the presence of any medicinal values and nutrients.
Unlike the 'old-fashioned' way it is dealt with the papaya seeds, nowadays, the seeds are used for many purposes, for example the seeds are processed and grounded to substitute black pepper. Even though the papaya seeds are unpalatable due to its peppery and spicy taste, it is however, having potent activity compared to the flesh of CP [2]. Other than consumption purposes, the papaya seeds can be used as an antibacterial agent as it is effective against many microorganisms, such as E. coli, S. typhi and S. aureus. Some studies showed significant anti-helminthic and anti-amoebic properties.
The Taxonomical classification of CP is shown in Table 2

Sample Preparation / Extraction Process
Generally, sun drying is the most acquired method to dry the plant sample because it is cost-saving. A minimum temperature of 30 ˚C or higher is required and several days will be taken to eliminate the excessive moisture content of the sample [3]. After the drying process, the dried plant sample must be preserved in a dry container with low humidity conditions to prevent the absorption of the moisture by the dried sample. Next, the dried sample is needed to reduce in size as the bigger surface area can enhance the contact point between the sample and the solvent utilised, thus facilitate the extraction process. The dried sample can be ground into either powder or pieces form by using a grinding machine or mortar and pestle. Next, the extraction process can be started by placing the dried sample and solvent together in a vessel or extraction instrument. The extraction of CPS were carried out with ethanolic Soxhlet extraction, the evaporation and total dryness over hot water bath.
Phytochemical Screening Unlike pharmaceutical chemicals, phytochemicals are safe and dependable, compared to costly synthetic drugs which are invariably associated with adverse effects, therefore it is considered as "man-friendly medicine". Some phytochemicals are known to possess medicinal and physiological activities which are alkaloids, saponins, flavonoids, tannins, glycosides anthraquinones and terpenoids [4]. 2%

Antibacterial Assay
According to the report shown, the skin, pulp and seeds of Carica papaya contain numerous types of phytochemicals which may be one of the sources to synthesize antimicrobial drugs. This is one of the aims of this study which is to identify the antibacterial potential of ethanolic CPS [7]. The antimicrobial assay was carried out by using gram-positive and gram-negative bacteria, such which media is suitable for that particular bacteria followed by the cultivation of the bacteria. The two main types of bacterial growth media are broth and agar [8]. For some bacteria such as S.
aureus, E. coli and S. typhi, they are suitable to be cultured in the nutrient agar and nutrient broth, whereas for both P. aeruginosa and B. subtilis, they are usually cultivated in the tryptic soy broth and tryptic soy agar. The broth will be initially sterilized and then inoculated with the bacteria of interest, followed by incubation for 24 hours to encourage bacterial growth. After the incubation step, the broth will be incorporated into the agar in order to carry out the antibacterial assay [9].
Commonly, the method that employed the antibiotic susceptibility test is the agar diffusion method. It involves the application of different concentrations of antibiotics and added into the wells, made with the cork borer, of the agar plates seeded with the bacterial strain [10]. The slow diffusion of the antibiotic into the agarose medium leads to the inhibition of the bacterial growth around the wells, in which there will be the formation of a clear inhibition zone that can be observed clearly. The diameter of the zone of inhibition will increase as the concentration of the antibiotic and the sample increase [11]. As an observation, the diameter of the zone of inhibition will be measured by using calliper and the values will be recorded as results. After the identification of the concentration effective in inhibiting the bacterial growth in the agar diffusion assay, it is then carried on with the minimum inhibitory concentration test.
It is important to determine the lowest concentration of an antimicrobial agent that inhibits the growth of microorganisms. As an effective antimicrobial agent, the MIC scores should be lower as it indicates that less concentration is required to prevent the growth of microbes. After 24 hours of incubation, if the mixture of the mi-croorganism and antimicrobial agent turns cloudy, this indicates the microorganism had maximum growth and the test agent does not pose antimicrobial activity at that particular dilution [12]. However, the viable microorganism in low-level that remains in the test tube after incubation cannot be differentiated from the microbes that have been killed by the antimicrobial agent. Due to that, MIC assay is not used as an indicator of bactericidal activity. The Minimum Bactericidal Concentration (MBC) is compulsory to the MIC.
The MBC of an antibacterial can be determined by sub-culturing the last clear MIC tube onto the growth medium and examining for bacterial growth. The plate that does not show any bacterial growth will be the minimum bactericidal concentration [13].  Shimadzu UV-Visible Spectrophotometer.

Bacterial Strains
The cultures were used for the antibacterial assay. These cultures were obtained from microbial culture bank of faculty of pharmacy (FOP), Aimst University.

Antioxidant Assay
The CPS were cleanly washed and air-dried at room temperature for 1 week to prevent the seeds from undergoing deterioration [14]. The dried seeds were passed through a sieve 1.40 mm to remove the unwanted impurities and then pulverized into powder form using the mortar and pestle. Figure 2a&2b indicated fresh and dried seeds respectively. The extraction of the seeds was carried out using ethanol as an extracting solvent. The Soxhlet extraction method was employed to extract the chemical constituents in the C.
papaya seeds [15]. 19.38 gm of the CPS powder was inserted into a thimble 300 ml of ethanol was measured accurately and added into the round bottom flask. The vapour formed during the extraction process were allowed to condense and accumulate back in the Soxhlet chamber. The running of tap water has remained until the whole apparatus cools down. The C. papaya seed extract contained in the round bottom flask was poured into a cleaned and dried conical flask (1000 ml). The conical flask was covered with cotton wool and aluminium foil to prevent evaporation [16].

Figure 2:
(a) Fresh C. papaya seeds The ethanolic extract of CPS was evaporated by using the rotary evaporator. The temperature of the rotary evaporator was set at 80 ˚C. The process was continued until no notable changes were observed. The pure CPS extract was poured into a clean beaker. The collected pure extract was introduced into a china dish and placed on the hot water bath for further evaporation [17]. After a few hours, a concentrated extract was formed and the china dish was removed from the hot water bath. The extract was filtered by using the Buchner funnel topped with filter paper. A semi-solid residue of CPS extract was obtained and it was put in the china dish. The china dish was covered with double-folded aluminium foil [18]. The volume was then made up to 10ml with 95% ethanol. Another 6 clean tubes were prepared [19]. 0.2ml of the standard solution were taken and transferred into the test tube prepared. 4 ml of 2.5% sodium carbonate and 0.2ml of Folin-Ciocalteu reagent was added to each of the test tubes. The test tubes were allowed to sit for 2 hours. The absorbance of the standard solution was examined by 750nm UV spectrophotometry and was recorded [20]. The mixtures were shaken and labelled accordingly. The test tubes were allowed to keep in the dark for 30 minutes. The absorbance was measured at 518nm using a UV spectrophotometer.

Preparation of Sample:
The steps in preparation of DPPH solution were repeated in the preparation of the sample, in which the BHT was changed to the plant extract [23]. The absorbance was measured at 518nm using a UV spectrophotometer.

Preparation of Blank :
The blank solution was prepared by mixing 3ml of 0.1mM DPPH reagent and 2.5ml of methanol, the solution was allowed to stand in the dark for 30 minutes. The absorbance was measured at 518nm using a UV spectrophotometer [24]. The values of absorbance of standard and sample obtained were then utilised in plotting the graph of UV absorbance against concentration of the sample and standard.

Sterility test of Ethanolic CPS Extract
Before the initiation of phytochemical screening, antibacterial and antioxidant assay of C. papaya seed extract, the extract was streaked on nutrient agar and was incubated for 24 hours at the temperature of 37 ˚C. Due to the movement control order in Malaysia, the plant extract was kept in the fridge for about 3 months, therefore the growth promotion test is able to determine whether microbes were present in the plant extract. This enables us to ensure positive results for all tests, especially antibacterial assay. There was no bacterial growth observed after 24 hours of incubation.

Serial Dilution of Plant Extract
Before the dilution of plant extract, the 0.

Agar Well Diffusion
Before the initiation of the agar well diffusion test, the biosafety cabinet was sterilized with 70% alcohol and the UV light was switched on for 10 minutes. Apart from that, the plant extract

Minimum Inhibitory Concentration (MIC)
5 loops of the bacterial strains were cultured in each sterile nutrient broth and then incubated at 37 ͦ C for 24 hours. Afterward centrifuged at 5000 rpm for 10 minutes.

Preparation of Standard McFarland Bacterial Culture:
The nutrient broth as well as tryptic soy broth were prepared freshly.
These broths were sterilized for 2 hours at the temperature of 121˚C and were allowed to cool down. In the biosafety cabinet, 5 loopful of Bacillus subtilis bacteria colonies were inoculated into the tryptic soy broth. This step was repeated for the other three bacteria which were inoculated in the nutrient broth. These broths were incubated for 24 hours at the temperature of 37 ˚C in an incubator. Then, these broths that contained bacteria strains were centrifuged at 5000rpm for 10 minutes. The supernatant was discarded and the cell pellet formed was collected and re-suspended in the sterile nutrient broth and tryptic soy broth. By using the UV spectrophotometer, the seeded broths were standardized to the value 0.5 of the absorbance according to McFarland standard at the wavelength of 517nm.

Determination of Total Phenolic Content (TPC):
In this research, the antioxidant potential of the C. papaya seeds was determined by using DPPH free radicals scavenging method and BHT was used as a standard. The DPPH is a stable free-radicals which has a colour of purple, as the antioxidants react with the DPPH, the DPPH will be converted into non-radical DPPH-H form. Besides that, the colour of the DPPH will be decolourised from purple to light yellow colour. The degree of decolourisation indicates the potency of the plant extracts in scavenging the free radicals.  Table 6. The concentration, absorbance and the corresponding percentage of scavenging of ethanolic CPS extract is placed in Table 7 while Graph of percentage scavenging against the standard concentration is drawn in Figure 5. The Figure 6 is indicating percentage scavenging against the sample concentration.

DPPH Free Radical Scavenging Assay
Antimicrobial Assay: Tables 8-15 Table 9: Zone of Inhibition of Escherichia coli in comparison to Ciprofloxacin.

Set 1 Set 2 Set 3 Average
Plant Extract

Set 1 Set 2 Set 3 Average
Plant Extract

Set 1 Set 2 Set 3 Average
Plant Extract

Set 1 Set 2 Set 3 Average
Plant Extract   Table 15.

Set 1 Set 2 Set 3 Average
Plant Extract

Discussion
One of the objectives of this research was to determine the phytochemical properties of Carica papaya seeds. The Carica papaya seeds were extracted with 95% ethanol and the plant extract was undergone phytochemical screening. The presence of phytochemicals is a marker that the plant can be an essential source of precursors in the formation of newer synthetic drugs.
In the present study, the results obtained for the phytochemical screening of C. papaya seed ethanolic extract was analysed and it was found out that alkaloids, anthraquinone, carbohydrate and flavonoids were present in the sample. Moreover, the extractive value estimation should be carried out to determine the amount of the active constituents in a given amount of plant material and identify which extraction solvent is more suitable for the given plant sample. By using this method, phytochemicals present in the plant sample can be extracted maximally. Phenolic compounds are very essential plant constituents which responsible for the antioxidant activity due to its redox properties. As a basis, Folin-Ciocalteu reagent was used to measure the phenolic content of different concentrations of plant extract. In this test, the Gallic acid was used as a standard and the total phenolic content was calculated by using the formula C = (A/B) x dilution factor. The results were derived from the calibration curve of Gallic acid, it was expressed as Gallic acid equivalents (GAE) per gram of samples. In the present study, the total phenolic content found was 6.420 mg GAE/g and 6.097 mg GAE/g for the concentrations of 50 and 200µg/ml respectively.
In this research work, the antioxidant potential of the C. papaya seeds was determined by using DPPH free radicals scavenging method and BHT was used as a standard. The DPPH is a stable freeradicals which has a colour of purple, as the antioxidants react with the DPPH, the DPPH will be converted into non-radical DPPH-H form. Besides that, the colour of the DPPH will be decolourised papaya seeds is a natural source that worth for further explore as it is inexpensive, natural, harmless and easy to obtain.

Interests of Conflict
The authors declare that there is no conflict of interests in the present publication.