Comparative study of Biological activity and chemical composition of Methanolic and Ethanolic plant extracts of Persea americana leaves in-vitro

The aim of this study was to evaluate and compare biological activity, chemical composition, antioxidant and antimicrobial activity of Persea Americana mill refluxed in various solvents, ethanol and methanol respectively. Persea Americana is a plant that is quite new to the turkish medicine and found its way to Turkey as it is little over 10 years old in use. It has been used to treat certain skin problems anddiahrea. The plant extraction was done with various solvents and comparison was done between the extracts produced by ethanol solvent and that of methanol solvent. Chemical constituents of the leaf extract were analyzed using techniques of gas chromatography/mass spectrometry (GC/MS). The methods employed for in vitro antimicrobial screening were the broth microdilution assay and Kirby bauer assay. The technique 2,2-diphenyl-1-picrylhydrazyl (DPPH) free-radical scavenging was applied for antioxidant activity. The ethanol aqueous extract (EAE) IC50 value of 2,4 mg / mL and methanol aqueous extract (MAE) IC50 value was 2,51 mg/ml respectively. According to literature the lesser the IC50 value the more potent the molecule is in the DPPH assay, therefore the results of the IC50 for could be viewded as a powerful antioxidant thus it is highly potent. Scavenging activity for the MAE and EAE were analyzed. Scavenging/inhibition activity increases as concentration increases. The antibacterial activities of the various solvent extract were screened and compared against to Bacillus cereus, Escherichia coli, Staphylococcus Aureus and Salmonella typhrium. The aqueous extracts showed a favourable amount of microbial activity, especially on S. Aureus and E. coli. Persea Americana in both extract conditions showed strong antioxidant ability, strong antimicrobial activity and gave an impressive amount of chemical components which reveal the compounds responsible for its biological activities.


Introduction
Persea americana which is called avocado; is a fruit originalily grown in tropical and subtropical regions that is greatly known around the world. The avocado has gained large stride in worldwide popularity. It has been exposed to intense marketing and an extensive distribution, largely promoted by its health benefits (Araújo et al., 2018). Persea americana is cultured in the southern coastal region of Turkey due to its commercial importance. The leaves are widely used for pass kidney stone and against the urinary tract infections as therapeutic among the people in Turkey (Kendir & Köroğlu, 2018).Currently Persea Americana is gradually finding its relevance in turkish medicine since its arrival a little over 10 years ago. Avocado belongs to the plant kingdom, and of the Lauracea family under the order Laurales, and genus Persea, and specie P. Americana (Zafar & Sidhu, 2011). The whole avocado fruits are rich in lots of biocompounds (pulp, seed and peel) and has enormous adavantages, amongst which include antioxidant and anticancer activities as well as skin exfoliation uses (Araujo et al.,2018). Research has shown that avocado seeds whether toasted or dried can be used to treat rashes, diahrea amongst other common household diseases (Jiménez-Arellanes et al., 2013). Ancient and cultural folklore in mexico explain that avocado seeds whether toasted/dried can be used to treat rashes, diahrea amongst other common household diseases (Jiménez-Arellanes et al., 2013). The antioxidant activity of avocado fruit as a whole is a phenomena to reckon with in the ongoing cancer research around the globe as there is the existence of radicals such as superoxides and hydroxyl which is a large scale problem to human health. (Antasionas et al., 2017). Antioxidants are substances that are capable of adequately reducing the effects of the free radicals and oxidative reactions of this macromolecules (Antasionas et al., 2017). Inorganic sources of antioxidants amidst their commendable performance also pose disadvantages that are gradually outweighing their advantage. Research therefore is gradually moving towards greener and natural sources from living things especially plants. Plants therefore have established a widely and far more commendable stride as they feature for therapeutic agents for treatment of diseases. Avocado pears are rich source of soluble phenolics, flavonoids and also retain some therapeutic activity against certain strains of bacteria (Ogundare & Oladejo, 2014). The complement system is an significant buffer of the frequently experienced inflammatory process, so an active study of the biological activity of molecules that can add to this scheme, and more specifically, prevent the activation of this process, is of excellent significance to different research advancement. Compounds showing both complement-inhibiting property and antioxidant activity could be considered prospective therapeutic agents in this note (Yamasaki, 2018).Previous studies (Ogundare & Oladejo, 2014); (Osuntokun et al., 2017) have extensively described the antimicrobial activity of Persea Americana against Salmonella typhi, Escherichia coli, klebsiella pneumonia amongst others. The aim of this study was to identify the chemical compounds within the P. Americana (avocado) leaves from Yeşilırmak Region and to extract with various solvents, and after which to monitor the effect of the solvent on the in-vitro biologic activity of Persea americana relative to its antioxidant ability and antimicrobial activity.

Chemicals and reagents
Mateials used in the experiments encompassing reagents and chemicals, were anaytical grade and obtained from Merck (Darmstadt, Germany).

Plant sample collection
Avocado (Persea Americana) was collected in February 2019 from the region of Yeşilırmak of Northern Cyprus (35° 08' 40" N;32° 43' 21" E). Avacado leaves were washed with deionized water and dehydrated at room temperature for about 10 days and weighted. Theprocess of drying the Persea americana leaves was carried out until they were kept at a steady weight and free of moisture. Then Persea americana leaves were grinded to fine powder using a simple laboratory mill. Botanical identification was done in the Faculty of Agricultural Sciences and Technologies by Prof. Dr. İbrahim Baktır (Dean) from Cyprus International University. Persea americana methanol and ethanol extract was prepared according to (Ribeiro et al., 2013). Dried P. Americana leaves were grinded by a miller at the laboratory. To 2 aliquot of 20g ground leaves of P. Americana, 200 ml of 95% ethanol and methanol HPLC grade was added and allowed to shake for 48 h using an overhead shaker. After 48hrs the methanol aqueous extract (MAE) and the ethanol aqueous extract (EAE) were strained with the use of a Whatman filter paper. The extract was collected by the use of rotary evaporator at 40rpm at a temperature of 50°C for 1hr. Dry mass of extracts were stored for further use.

Determination of total phenolic content (TPC)
The TPC values were established by applying the Folin-Ciocalteu colorimetric assay described previously by Büyüktuncel et al, 2014. The preparation of calibration curve was done by obtaining 0.1 mL volume of various w/v concentrations of 50, 100, 150, 200, 250 and 300 mg/L gallic acid solutions.0.1 mL 20-times dilution samples of P. Americana (diluted with 13% (w, v) ethanol and methanol) were transfered into a glass test tube and then 2 mL of 2% disodium carbonate was added. After incubation for 2 min, 0.1 mL of Folin-Ciocalteu's reagent was added. Sequel to this, at a 760 nm wavelength the absorbance was detected by using a UV Visible Spectrophotometer (Shimadzu UV-2450). The amount of phenols was shown as mg of gallic acid equivalent per gram of extract (mg GAE/g). Spectrophotometric determination of the TPC was carried out with Folin-Ciocalteu method as adapted for P. Americana leaves extract analysis, standardised with gallic acid. The method works by the principle of reduction of a phosphotungstenphosphomolybdate complex by phenols present to blue colored products.

Determination of total flavonoid content (TFC)
The TFC data were analysed with the aluminum chloride colorimetric method as discussed by Marinova et al., (2005). Absorbance measured at a wavelength of 510 nm. The total amount of flavanoids was quantified in mg (weight) of quercetin equivalents per gram of extract (mg QE/g). All tests were performed in three sets.

Gas chromatography-mass spectrometry (GC-MS)
The different solvent extracts were analyzed by gas chromatography-mass spectrometry (GC-MS) according to a certain conditions stated by (Ukwubile et al., 2019). The GC or GC-MS conditions were optimized based on the property of the various solvent extract. A 30 m×0.25 mm, (thickness of film) 0.25 μm, HP-5 fused silica capillary column was utilized. The carrier gas, Helium (purity 99.999%), had a flow rate of 0.9 mL/min. The oven temperature of the column was programmed from 50 •C (hold 1 min) to 240 •C (hold 10 min) at a 5 •C/min rate. EAE and MAE was dispersed in methanol and ethanol respectively introduced in an Agilent 7890A GC system joined with an MS (Agilent technologies) by author injection at the environmental laboratory, Cyprus international university. The operating conditions of the GC-MS used in the study were as follows: temperature of oven was set at 50°C for 2 min then 100°C with the rate of 10°C/ min and steadily increased to 200°C and held at a constant temperature for a period of 10 min. The quantity of the sample was 2 μL and the carrier gas (helium) was 1 mL / min. 70 eV was used to ionize the sample parts. The methanol and ethanol P. Americana extracts were analyzed for a time period of 24.50 min.
The ethanol extract was analysed by the following GC conditions stated by (Abdel-Naime et al., 2019). l μL of the sample was injected and the extract analysis was done as described by a flame ionization detector programmed temperature method. Initial temperature was set as 70 °C, and rate of increase was arranged as 5 °C/min to 220 °C. The total time using technique was 60 min and the split ratio was 1:50. Carrier gas; Helium with a flow rate of 0.8 mL/min. To identify the compounds, a comparison of their mass spectra ws done against the database the (NIST library) and Wiley library 9.

Antioxidant activity by DPPH scavenging assay
The antioxidant activity was deterrmined by using 1, 1-diphenyl-2-picrylhydrazyl (DPPH) as a scavenging radical on ethanolic and methanolic extracts of P. Americana respectively with a few modifications. DPPH ethanolic and methanolic solution (1mM) were prepared respectively. 3 mL of solution separately added to 100mg/ml, 75mg/ml, 50mg/ml, 25mg/ml and 12.5mg/ml respectively and kept in the dark. This was done for both the EAE and MAE. Absorbance values of the DPPH ethanolic and methanolic solutions at different periods were established at 517 nm by a spectrophotometer (Shimadzu UV-2450).
The follow equation was utilised to determine the %DPPH Scavenging activities of examined samples. %DPPH = [1-(As/Ac)] x 100 Where: As (tested sample) and Ab (blank sample) is the absorbance of the the sample with no reagent within. To the labelled microliter plate containing nutrient Broth (NB), EAE and MAE at different volumes of the stock solution was added to the well plates ranging from 100ul, 50ul, 25ul, 12.5ul, 6,25ul respectively. Sterilized culture medium including tested samples DMSO was adopted as negative controls. The well concentrations where no turbidity was noted in the wells after a 24hour were stated to be the MIC of the ethanolic and methanolic extracts. The potency of bacteria in the culture of Mueller Hinton Broth (MHB) was further established by transferring a volume of the microbial suspension to a petri dish containing Muller Hinton Agar (MHA), following which incubation at a stable temperature of 37 ° C. The MBC were determined as the least concentration of ethanolic and methanolic P. Americana extract required to totally restrict the growth of the microorganisms after 24 h. All analysis was carried out twice on two separated tests.

Disk diffusion (Kirby Bauer) assay
The protocols in this study was according to (Umar et al., 2019) with slight modifications. Briefly, each sterile Petri dish with a diameter of 9 cm was prepared with 20 mL of Mueller-Hinton medium. Standard quantity of a bacterial suspension (108 CFU / mL) was dispersed on the plates after solidification. A sterile paper filter disk (6 mm) with 20 μL of P. americana was put on the plate surface after 5 min.
To accelerate extact diffusion into the agar, the plates were incubated at 4 °C for 1 h and then incubated at 37 °C, for 24 to 48 h. Bactericidal areas (mm) diameters were evaluated, including control disks' diameter. Gentamycin (30 μg / disk) was used as positive controls for gram-positive fungi and chloroamphenicol (30 μg / disk). All experiments were carried out in duplicates.

Statistical analysis
All experiments were carried out parallely duplicated with three replicates. Graphs and peaks were plotted using the Origin Pro.

Results and Discussion
This study conducted to identify the chemical compounds within the P. Americana (avocado) leaves and to extract with various solvents, and after which to monitor the effect of the solvent on the in-vitro biologic activity of P. Americana relative to its antioxidant ability, physicochemical compositionand antimicrobial activity. Total phenols and flavannoid are presented in Table 1. **Total amount of phenols was expressed as Gallic acid equivalents (mg GAE/g of extract).

Total phenols and total flavannoid content
***Total number of flavonoids was expressed as Quercetin equivalents (mg QE/g of extract).
The extraction method of Persea americana (avocado) leaves using 95% HPLC grade ethanol and methanol as a universal solvent for refluxing is quite capable of dissolving polar compounds, so that multiple polar and non -polar compounds, such as certain bioactive elements contained in avocado leaves, can then be attracted to the solvent, although study has shown that ethanol is less effective in screening antioxidants (Rahman et al., 2018). Methanol in comparison to ethanol has proven to be a better solvent for extraction of bioactive and antioxidant but the use of ethanol is safer compared to methanol (Rahman et al., 2018). The Table 1 shows the total phenolic content of Persea Americana extracted by both 95% methanol and ethanol respectively. It wasobserved that for the total phenolics in the methanol extract appeared quite higher than that of ethanol extract.
P. Ameriana's total phenolic content and avocado's antioxidant potential were influe nced by the type of avocado and solvent extraction. By donating hydrogen atoms or by transferring single electrons, flavonoids can immobilise free radicals. Flavanoids are abundant in fruits and vegetables, but they form an significant cereal and pulse gro up. (Saharan et al., 2017). The flavanoid content for the methanol extract of P. Americana was 71,49 mg QE/gof extract and that of ethanol extract was which can be compared to that of Rahaman et al., 2017 where values of flavanoid in avocado leaves showed to be strongly positive which encourages the result of a high amount og of flavanoid observed in this study.

GC-MS analysis of bioactive compounds
The Table 2 and Figure 1. showed occurrence of the various bioactive compound as observed by different organic solvents. Some compounds were in increased occeurence depending on the solvent of choice.

Figure 2. Chromatogram showing the P. Americana extracted by Ethanol
It was gathered by observation that the qualitative extraction of P. Americana using methanol and ethanol showed the presence of varying chemical components such as Globulol which is one of the compounds responsible for antimicrobial activity according to (Manliang et al., 2007;Manliang 2008). Nerolidol also known as peruviol was present in the ethanolic extract of P. Americana; Nerolidol has a floral odor, a type of alcohol and exists as a vital bioactive component in some plants including P. Americana and possess strong anticancer and antitumor ability (Chan et al., 2016). In another study, they found some of the chemical compounds like palmitic acid, isochiapin B which were part of the components observed in the profiling carried out. Besides these aforementioned most of the represented components are not yet reported or accessible (Senthilkumar et al., 2012).  In human's research has shown that oxidative stress results as a result of increased oxygen radicals which could affect the function of some bio-molecules (Nathaniel et al., 2016). In the In vitro antioxidant assay, ascorbic acid was utilized as a standard control. Antioxidants yields from food or natural sources, has a strongly correlation with various extraction parameters like solvent concentration and physiochemical characteristics (Rodríguez-Carpena et al., 2011). Ascorbic acid was weighed anddissolved in both methanol and ethanol respectively. These methods were done to determine the antioxidant potential of P. Americana ethanolic and methanolic extracts and the interactions with free radicals. In vitro antioxidant assays have the potential to give a certain level of information that could contribute and give insight as to what happens in in vivo assay. Ascorbic acid has very high standard antioxidant activity, the percentage DPPH scavenging activity appeared to increase as concentration increased for both the standard and the other solvent extracts of consideration. The absorbance appeared to reduce as concentration increased and it correlated with what (Rahman et al., 2018) reported in a similar research. The varying of of IC50 value of P. Americana extract in ethanol and methanol is worthy of note. Compared to the IC50 values of Ascorbic acid which was 5mg/ml, methanolic extract had a lower IC50 value is 3.2mg/ml which according to previousliterature is a good one given that lower IC50 values indicate great antioxidant activity (Rahman et al., 2018). Ethanolic plant extract appeared to have the lowest IC50n value amongst the three of them 2.5 mg/ml which was lower that both the methanolic extract and the standard. Methanolic extract 91.7 (Salim, 2013) This research recorded a highest antioxidant activity of 74.8 % for Persea Americana ethanolic extract and for P. Americana methanolic extract 91.8%. It's safe to say that increasing percentage of scavenging activity will mean a reducing IC50 value. In comparison to other authors, it is worthy of note that the P. Americana ethanolic extract considered within this study had a higher antioxidant activity than other studies expressed in the Table 5. Similar to most studies, it has been observed that ethanolic extracts of P. Americana leaves have relatively low antioxidant activities in comparison to the counterpart methanolic extracts of the same plants. Further observation and comparison of the methanolic extract showed that across the authors evaluated, methanol extracts of P. Americana leaves appear to have higher antioxidant activities. The antioxidant activity recorded for this study appeared to be higher than the studied authors. This variances might be as a result of many factors like human and equipment errors and maturity of the leaves in question. Some research have reported that maturity of the P. Americana leaves used for the extract could affect the result of the antioxidant activities thus younger leaves are more likely to have higher antioxidant activity than mature leaves ( Kingne et al., 2018). The minimum inhibitory concentration is the weight per unit volume of a drug capable of inhibiting the growth of microorganisms. The MIC for this research was determined as the lowest concentration that didn't have color change after inoculating the microorganism with the similar concentration as the Mc farland standard. In this research, the various wells were plated out for better observation. Keen observation of the interaction between the microrganisms and the plant extracts helped to assess the viability and antimicrobial activity of P. Americana (Chakraborty, M., & Mitra, 2008). E. coli and S. aureus had a minimum inhibitory concentration (50mg/ml) as the ethanolic extract was able to inhibit its growth at the said concentration, while E. coli and S. aureus MIC for methanolic extract was 25mg/ml. This explains that for the E. Coli and S. aureus, the methanolic extract has a lower MIC and thus was more effective than the ethanolic extract. The S. typhirium and B. cereus had very low MBC an MIC because the plant exract had the ability to completely inhibit the proliferation of the said bacteria. In the metanolic extract of P. Americana, on an average a lower MIC was observed for all the microorganisms. A few factors may have been responsible for the different MIC of P. Americana in various solvents like polarity which might be as a result of the solvent of extraction and or maybe thepolarity of the solvent. For E. Coli and S. aureus the MIC was 50mg/ml with the ethanolic exract while that of the methanolic extract was 25mg/ml. The P. americana ethanolic and mehanolic extract was more effective on S. typhirium and B. cereus repectively. The efficacy of various extracts can be described by the polarity of the solvents which might affect how certain compounds within the samples dissolve or interact with the solvent (Cardoso et al., 2016).

Conclusions and Recommendations
The research intends to compare activities of Persea Americana in two different solvents with respect to the antioxidant activity, chemical composition and antimicrobial analysis. From the antioxidant test, we observe different % scavenging activity and IC50 between the solvent extracts; for the microbial test some microorganisms were more sensitive to one of the extracts than the other; for the chemical composition certain compounds were more visible with significant amount in methanol that in ethanol and vise versa. In other words it is safe to say that the solvent of choice for extraction plays a role in the bioactivity of a given plant material as different solvents interact differently with certain components of this biological material. As a result, Persea Americana in both extract conditions showed strong antioxidant ability, strong antimicrobial activity and gave an impressive amount of chemical components which reveal the compounds responsible for its biological activities.

Acknowledgement
Due recognition goes to Cyprus International University Biotechnology Research Laboratory directors who contributed their quota to make this sresearch a grand success.