{'fraction_non_alphanumeric': 0.029381121066888935, 'fraction_numerical': 0.017573105508091963, 'mean_word_length': 5.179399141630901, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Antibacterial studies on fruit-skin and leaf extracts of Annona muricata in Ota, Nigeria

The current study examined the phytochemical constituents and antibacterial activity of leaf and fruit-skin extracts of Annona muricata Linn. The extracts were screened qualitatively for their phytochemical constituents following established protocols; disc diffusion method was employed for the antibacterial study on Klebsiella pneumoniae, Staphylococcus aureus and Pseudomonas aeruginosa, after which their minimum inhibitory concentrations were determined via double dilution method. Furthermore, selected extracts were screened against 50 multidrug-resistant clinical isolates from immunocompomised patients who are already down with secondary acute respiratory infections. The phytochemical screening of the extracts collectively revealed the presence of tannins, saponins, terpenoids, steroids, phenols, flavonoids, coumarin, alkaloids, antocyanins and betacyanins. Only aqueous leaf (ALA) and fruit-skin ethanol (ESA) extracts showed remarkable activity with 15 – 17 mm inhibition diameter (DIH) against the test organisms. The minimum inhibitory concentrations (MIC) for the ALA and ESA were 25 and 6.25 mgmL-1 respectively. The two extracts further showed activity (6-18 mm DIH) against 80% of the multidrug resistant isolates tested. It can therefore be inferred from this study that Annona muricata possibly contains potent bioactive antibacterials that can be developed into nature-friendly drugs to counter the effects of pathogenic bacteria, thereby preserving lives as well as sustaining a viable environment.


Introduction
Provided the indispensable activity of a class of supramolecular complexes called viruses were not in existence, bacteria would have overrun the entire planet [1][2][3]! Bacteria are a set of highly prolific microscopic organisms that occupy any and everywhere called space. They fill up both habitable and non-habitable zones of the planet Earth. It is quite alarming to note that the total bacterial soil carbon in existence worldwide today, is estimated at the entire weight of the United Kingdom [4][5][6][7]. Be that as it may, bacteria can be as much beneficial as they are detrimental [8][9][10]. In the past few decades, Scientists have harnessed the use of bacteria through diverse technologies to better the lot of mankind, other living systems as well as the environment. In a bid to further ameliorate the impact of deleterious bacteria, natural herbs with ancient folklore claims are also being investigated worldwide to establish their ethnopharmacological effect [11][12]. Anyanwu and Okoye [13]  Deploying state-of-the-art technologies into exploring these medicinal plants are most likely the way out of the currently emerging threats of antimicrobial resistance that is rendering modern medicine ineffective in so many ways. One of such herbs that has been designated as a crop for the future to combat bacterial infections amongst other disease conditions is Annona muricata Linn. Annona muricata also commonly called soursop is an evergreen low-branching, slender and bushy tree towering between 5 and 9 metres and 0.15-0.83 metres wide. The soursop tree is extravagantly distributed in the tropics largely because of the favourable climatic conditions such as annual rainfall above 1500 mm, altitudes below 1200 m above sea level, 60 to 80% relative humidity between and temperatures between 25 and 28 °C. A remarkable feature of the tree that makes it of utmost economic importance is its high and continuous productive profile [14][15][16][17]. For many decades now, the antibacterial activity of A. muricata have been explored on several gram-negative and gram-positive organism with interesting results [18]. A review report showed that A. muricata extract demonstrated antibacterial activity comparable to streptomycin which is a potent standard antibiotic [19]. One very key observation in the antibacterial activity studies of A. muricata is that extract activity is largely dependent on extraction solvent; denoting the fact that extracts prepared with organic solvents for instance, showed higher activity over aqueous extracts. Viera et al. [20] highlighted and confirmed this phenomenon, where ethanolic and methanolic A. muricata extracts were active against Staphylococus aureus, whereas no such activity was observed in the aqueous extract of the fruit skin. As a result, the polarity of the organic solvent used in extraction further significantly influenced antibacterial activity. Amidst several studies, the current examination of the fruit-skin and leaf extracts of this unique crop is hereby presented.

Collection and identification of plant samples
Unripen mature fruits of Annona muricata were bought from local fruit markets in Lusada and Agbara, Ogun state, Nigeria; and the leaves were collected from trees on farmlands also in Ota, Ogun state, Nigeria. Fresh samples of the healthy fruits and leaves were deposited in the Forestry herbarium of the Forestry Research Institute of Nigeria (FRIN), Ibadan, Oyo State, Nigeria. The plant samples were identified and authenticated with Voucher referencing Number FHI. 110177.

Test Organisms
Clinical isolates of Klebsiella pneumoniae, Staphylococcus aureus and Pseudomonas aeruginosa coupled with those listed in Table 4 which were isolated from HIV/AIDS patients already down with secondary lower respiratory infections.

Culture media and plates
Mueller-Hinton agar (HiMedia Laboratories Ltd, Mumbai, India -ISO 13485-2003) and MacConkey agar (without salt: Rapid Labs), petri dishes and universal bottles were all purchased from standard scientific stores.

Methods Preparation and extraction of plant samples
The plant samples of A. muricata were washed properly in clean running water. The fruits were peeled manually with knife, a portion of the peels (fruit skin) were blended and frozen while another portion was frozen directly for subsequent extractions. Also, a portion of the leaves were shade-dried and blended, while the remaining portion was frozen for respective extraction. Both fruit skin and leaves of A. muricata were extracted with five different categories of solvents following stipulated protocols itemized as follows. The extracts were concentrated using a rotary evaporator and the percentage (%) yield of the dry residue calculated as described by [21].

Aqueous extraction
Weighed amount of powdered leaves of A. muricata were soaked in distilled water (1:10) for 12 hours and sieved with muslin cloth to remove fiber particles; then filtered with a vacuum membrane filter device as well as filter paper into conical flasks. The filtrate was concentrated in a rotary evaporator and dried to powder in a water bath. The powder was weighed and percentage yield calculated. The aqueous leaf extract is labeled as subsequently referred to as ALA. The blended and frozen fruit skin were thawed and extracted with distilled water (1:5) for 12 hours, sieved and filtered. The filtrate was frozen and dried in a freeze dryer; it is labelled ASA [22] Sodium chloride extraction A specified volume of 1% sodium chloride was poured into weighed powdered leaves of A. muricata in a glass jar and left for 12 hours. The mixture was sieved, filtered and concentrated in a rotary evaporator and subsequently freeze-dried. The resulting powder was weighed and percentage yield calculated. Sodium chloride leaf extract is labelled as SLA. Frozen peels of A. muricata are weighed and blended into fine extractible particles; this was then soaked in a known volume of 1% sodium chloride for 12 hours. The mixture was also sieved, filtered and concentrated in a rotary evaporator and freeze-dried. Sodium chloride fruit skin extract is labelled as SSA [23][24].

Methyl-tert-butyl ether (MTBE) extraction
Fresh leaves and fruit skin of A. muricata were extracted with Methyl-tert-butyl ether (MTBE) amongst other solvents (MTBE/methanol/water: 10/3/2.5; volume by volume) for an accurate lipidome profiling. Methanol (970 mL) was added to 900g of blended fresh leaves of A. muricata in a cylindrical glass jar with lid; the content was shaken together to mix well. This was followed by adding 3.2 L of MTBE and incubating the mixture at room temperature for 55 minutes. After the incubation phase separation was induced with the addition of 810 mL of distilled water. Following 10 minutes of incubation at room temperature, the mixture was sieved with 8-fold muslin cloth and the resultan filtrate was poured into a separating funnel. The upper organic phase was collected, concentrated in a rotary evaporator and dried in a weighed beaker on a heat block set at 55 o C. Extracted lipids were kept in the refrigerator. Methyl-tert-butyl ether leaf and fruit skin extracts of A. muricata were labelled as MLA and MSA respectively [25].

Ethanol extraction
Powdered leaves of A. muricata were extracted with 95 % ethanol using soxhlet apparatus. Likewise the blended fresh peels of A. muricata were soaked in 95 % ethanol overnight (8-12 hours), sieved with an 8-fold muslin cloth to take out fiber particle and then filtered a vacuum membrane filter. Both the extract from the leaves and the filtrate from the fruit skin were concentrated in a rotary evaporator and dried in a weighed beaker on a heat block at 65 o C. The ethanolic leaf and fruit skin extracts of A. muricata were labelled as ELA and ESA respectively [26].

Isopropanol extraction
Frozen leaves and fruit skin of A. muricata were blended with a mixture of isopropanol/hexane/water in the ratio 55:20:25 (volume by volume) and incubated at 55.5 °C for 15 min with occasional shaking. After incubation, the mixture was sieved with an 8-fold muslin cloth and transferred to a separating funnel. The upper organic phase was collected with a micropipette into a weighed beaker and dried on a heat block set at 55 o C. isopropanol/hexane/water leaf and fruit skin extracts of A. muricata were labelled as ILA and ISA respectively [27].

Phytochemical screening
Selected qualitative phytochemical screening tests were carried out according to methods previously described on the extracts for the detection of the following compounds: alkaloids, anthocyanins & betacyanins, coumarins, flavonoids, glycosides, phenols, quinones, saponins, steroids, tannins, terpenoids and triterpenoids. Prior to the screening, the extracts were dissolved in their respective solvent [28][29][30] .

Antibacterial studies In vitro screening for antibacterial activity
The agar well diffusion method of sensitivity testing were used to determine the growth inhibition of bacteria by the plant extracts [31][32][33].

Determination of minimum inhibitory concentrations
Minimum inhibitory concentrations (MIC) of the extracts were determined by agar well dilution method as described by Russell and Furr (1972). Different dilutions of the extracts were prepared to give final concentration in the range of 100.0, 50.0, 25.0, 12.5, 6.25, 3.12 and 1.56 mg mL -1 . Two milliliter (2 mLs) of each dilution was mixed with 18 mL of Mueller Hinton agar (HiMedia Laboratories Ltd, Mumbai, India -ISO 13485-2003), poured into petri-dishes and allowed to set. The agar was streaked with an overnight broth culture of the bacterial isolates and incubated overnight. The plates were then examined for the presence or absence of growth. The minimum concentration that completely inhibited macroscopic growth was regarded as the minimum inhibittory concentration of the respective extracts [18].   ALA2

Minimum inhibitory concentration (MIC) of aqueous leaf and ethanol skin extracts of Annona muricata against Staphylococcus aureus
The minimum inhibitory concentration (MIC) of aqueous leaf (ALA) and ethanol fruit-skin (ESA) extract of Annona muricata against Staphylococcus aureus are 25 mgmL -1 and 6.25 mgmL -1 respectively as shown in table 3.

Antibacterial activity of aqueous and ethanol leaf extracts of Annona muricata against multidrug resistant (MDR) organisms isolated from immunocompromised (HIV/AIDS) patients with lower respiratory infections.
The two active extracts of Annona muricata (ALA and ESA) further screened against multidrugresistant organisms isolated from immunocompromised patients co-infected with acute respiratory infections demonstrated antibacterial activity; and most of the organisms were sensitive to the extracts with zones of inhibition ranging from 7-18 mm (Table 4.6). Furthermore, a remarkable phenomenon was observed in the activity of ESA apart from showing a zone of inhibition such that the organisms appeared morphologically altered. This alteration occurs immediately after the zones of inhibition forming a purported zone of reversion which can be as wide as 28 mm in diameter.

8.
Klebsiella pneumoniae pneumoniae 0.7 16 [35] in inhibition zone and that of Martínez et al. [38] with Meléndez and Capriles [39]; who reported Staphylococcus aureus as one the most susceptible bacteria to plant extracts. The non-significant difference in antibacterial activities observed between the aqueous leaf and fruit-skin ethanol extracts of A. muricata implies that the antibacterial activities of both extracts against selected organisms are closely related; although several studies have proved that the extent of antibacterial activity for Annona muricata is solventdependent; affirming that ethanolic and methanolic extracts showed higher activity compared to aqueous extract which often times show low or no antibacterial activity [40]. The antibacterial activity demonstrated by ALA and ESA against 80% of the multidrug-resistant organisms isolated from immunocompromised patients co-infected with acute respiratory infections, with zones of inhibition ranging from 7-18 mm agree well with the findings of [40].

Conclusion
Extracts from the fruit-skin and leaves of Annona muricata are composed of diverse phytochemicals that possess both antibacterial and possible antimultidrug-resistant bioactive principles that may serve as potential antidote against deadly bacterial infections. There is need for more research on the fruitskin extracts of Annona muricata Linn., as future findings from this may further enhance life quality via ameliorating targeted infections. Bioactive-directed fractionation of the fruit-skin extract to isolate pure bioactive compounds is also highly recommended.