Chemical Composition and Anti-Microbial Activity of Hog Plum (Spondias mombin L.) Peel Oil Extracted from Different Regions of Tropical Climates

The hydro-distilled essential oil of hog plum peel may be used for enhancing the flavor and taste of food products as well as for hiding the unpleasant odor of drugs. Thus, the waste peels of Spondias mombin appear to have economic importance. To find out the chemical composition and anti-microbial properties of hog plum peel oil, the samples were collected from different regions of Bangladesh for extraction and identification of volatile compounds by GC-MS, where dichloromethane was used as an extraction solvent. The required standard analytical methods were used to assay the anti-microbial properties of hog plums. In this study, pentenyl-3-thy-met-4-alpha-methyl-alpha-ethanol-oxiranen (29.04%), (3,3.1,1)-4-dimethylethyl-1,1-phenol (8.00%), cycohexanol-3 (10.85%), 4-hydroxy-penzeneethanamine (7.09%), hydroxylamine (4.63%), dibutyl phthalate (6.85%), etc., were majorly determined. Consequently, the highest content of 75.81% volatile compounds was found in the Dinajpur district, where the lowest content of 35.00% was found in the Rajshahi district. In contrast, 33 volatile compounds were identified in hog plum peels collected from the Barishal district, whereas 22 compounds were detected in the peel samples collected from the Dinajpur district. In addition, the antimicrobial activity of the oil was analyzed by the disk diffusion method, and the results revealed that the highest Ciprocin content was recorded in the hog plums of Barishal (22.0–23.0 mm), while the lowest was recorded in the Mymensingh sample (20.67–21.63 mm), which was on par with Rajshahi sample (20.70–21.50 mm). The results of the anti-fungal activities of the peel oil showed the highest zone of inhibition against the Aspergillus niger (11.63 ± 0.0003 mm) and Penicillium oxalicum (13.67 ± 1.97 mm) content of the Rajshahi and Pabna district samples, respectively.


Introduction
Hog plum (Spondias mombin L.) is a member of the Anacardiaceae family and is locally known as "Amra" in Bangladesh. Its fruit is a drupe characterized with a mixed taste of sour and sweet, and has gained increased importance in modern medicine for its possible pharmacological activities [1,2]. It grows mostly in the Indian subcontinent, e.g., in Bangladesh, India (Assam and Bombay), and Nepal. In Bangladesh, the cultivatable area of hog plum was about 1247 acres, and the corresponding total production was 36,068 metric tons (MT) in 2016-2017 [3,4]. Hog plum is mainly consumed as fresh fruit and has other uses such as making jam, jelly, squash, and marmalades, on a small scale and on a commercial scale. After the consumption and manufacturing of the above products, the peels are discarded as waste that holds almost 20% of the fresh fruit [5,6]. These wastages are considered a problem for the food processing industry and pollution monitoring organizations [7]. Nevertheless, these waste portions could be used as a potential source of valuable by-products like essential oils (EOs) which could be extracted from flowers, leaves, stems, roots, seeds, barks, resins, or fruit rinds [6].
Essential oils from citrus fruits are a group of natural flavors and fragrances which are popularly used in the food and pharmaceutical industries, daily chemical products, and in the health care field [8][9][10][11][12]. The products of medicinal plants like EOs and their antimicrobial properties have been empirically recognized for centuries, but recently the antimicrobial properties have been studied and confirmed scientifically [13][14][15]. These EOs effectively control the growth of different microorganisms like fungus, yeasts, bacteria, etc., which has been reported in several studies [16][17][18][19][20][21]. The bark extract of hog plums exhibits a valuable antibacterial activity, while the aqueous extract of the bark has shown a moderate antibacterial activity against Escherichia coli, Salmonella typhimurium, and Vibrio cholerae [22,23].
Many studies have been carried out regarding the fruit, bark, and leaf extracts of hog plums [24][25][26][27][28], but there has been little research work on hog plum peels, which may contain various bioactive volatile and antimicrobial compounds. The extraction and identification of bioactive volatile compounds (VCs) in hog plum peels and their preservation are a potential source of medicine as well as nutrients for functional foods and feed industries. Therefore, the present study was conducted to discover the chemical composition and anti-microbial properties of hog plum peel oil collected from different regions of Bangladesh.

Sample Collection
The samples of Spondias mombin were collected in the early summer season from the local market of the Dinajpur (Latitude: 25.

Preparation of Hog Plum Peel Powder
The samples were washed and rinsed with deionized water and subsequently peeled with a knife carefully. The peels of each of the five samples were weighted and spread in five different trays for drying in a cabinet dryer (Model FMA-275) at 60 • C for three days until they were completely dehydrated. Then the samples were taken out from the drier and put into desiccators for a few minutes to adjust to the ambient temperature. Then, the dried samples were blended by an electric blade blender (Vitamix 5200 Series) to make the powdery form and to prepare the peel samples for hydro-distillation.

Extraction of Essential Oil
Five samples of hog plum peels were collected in mid-June 2018 from the five districts. Each sample of peel powder, weighing 20 g, was suspended in 300 mL of deionized water and subjected to steam distillation using a Clevenger-type apparatus for 4 h. Then the sample oils were collected, dried over anhydrous sodium sulfate (Na 2 SO 4 ), filtered, and stored in sealed vials under refrigeration at 4 • C until analysis [29]. The yield (%) of oil was calculated by the following formula: Yield (%) = (Weight of oil)/(Weight of the fresh sample) × 100

Gas Chromatography and Mass Spectrometry (GC-MS) Analysis
GC-MS was conducted with a Varian Saturn 2200 equipped with an ion trap detector (ITD) for the identification of different components of essential oil. To obtain better results, dichloromethane was used as solvent. The sample of 2.0 µL was injected on a DB-5 MS (30 m, 0.25 mm ID, 0.25 µm film thickness) column. Helium was used as a carrier gas with a flow rate of 1 mL/min and a split ratio of 1:5. The temperature in the oven-dryer was set at 50 • C for 1 min, followed by a temperature gradient of 2.5 • C/min to 280 • C/min for 40 min. The injector and transfer line temperatures were set to 250 • C and 280 • C, respectively. Various components were identified by their retention time (5.52-22.34 min) and peak enhancement with standard samples in gas chromatographic mode and a National Institute of Standards and Technology (NIST 20) library search from the derived mass fragmentation pattern of various components of the essential oil [30].

Determination of Antimicrobial Activity
The antimicrobial activity of the tested essential oil was monitored using the disc diffusion method [31] against different food-borne pathogens including bacteria (Escherichia coli, Salmonella spp., and Staphylococcus aureus), and two selected fungi (A. niger and P. oxalicum). The antibacterial and antifungal screening was performed briefly using ciprofloxacin (10 µg/disc) and fluconazole (10 µg/disc) as a positive control and sterile water as a negative control. Standard culture media of each type of bacteria and fungi were employed on NA and SDA plates (100 mL each), where 5 µL (1000 ppm) of the essential oil was used for each test sample. During the investigation, the incubation temperature was maintained for both fungi (25 • C) and bacteria (37 • C). The zones of inhibition thus developed against the tested microorganisms were measured after a period of 48 and 96 h. All experiments were conducted in triplicate. The results of the antimicrobial activity of the peel oil against the different microorganisms were expressed as resistant, intermediate, and sensitive.

Statistical Analysis
Antibacterial and antifungal experiments were performed in triplicate and the analyzed data were presented as mean ± SE. The obtained data were subjected to one-way ANOVA using MS Office 2007 (significance: p ≤ 0.05; coefficient interval (CV): >95%).

Yield
The yield of essential oil by the hydro-distillation method was satisfactory from the Spondias mombin peel samples collected from the five selected districts of Bangladesh (Table 1). However, the Barishal sample showed the highest production of EOs (75%), while the Pabna sample showed the lowest value (47%). Based on the yield of essential oil, the samples can be ranked as Barishal > Dinajpur > Mymensingh > Rajshahi > Pabna. Natural plant-derived non-phytotoxic substances such as EOs may increase the shelflife of processed food products by destroying the cell wall of bacteria and fungi. Thus, researchers have devoted their interests to producing natural medicinal and value-added food products from plant-based extracts. The experimental volatile oils of hog plum peels exhibited strong flavor alike to that of the fresh raw samples. Table 1 shows a higher yield of oil from the peels than that obtained from the fruits, leaves, and barks of hog plums [32]. On the other hand, Mangifera indica is taxonomically close to S. mombin which also had similar results [33,34]. The yield was comparable to that reported in previous studies. Oven-dried citrus peels exhibited higher oil yield followed by the ambient-dried and fresh samples. Soumaya et al. [35] also reported that the yield of volatile compounds varied during ripening and reached the maximum values during the middle stage of maturity (second stage) for citrus fruits, while the highest lemon yield was determined at the beginning of fruit maturation and decreased thereafter. Table 1. Regional samples, oil mass, and its product percentages.

Anti-Bacterial Activities of Essential Oil
In Table 3, antibacterial activity of peel oil was determined against three bacterial pathogens, namely, Escherichia coli, Salmonella spp., and Staphylococcus aureus. The disk diffusion method was used to determine the antimicrobial activity by measuring the zone of inhibition. All peel oils exhibited a moderate clearance zone of inhibition against Escherichia coli, Salmonella spp., and Staphylococcus aureus. Among those, the Barishal sample oil showed the highest antibacterial sensitivity. From the experimental data, the Barishal sample oil showed inhibition diameters of 14 ± 0.17 mm, 13 ± 0.29 mm, and 12 ± 0.17 mm for Staphylococcus aureus, Escherichia coli, and Salmonella spp., respectively. However, the standard Ciprocin exhibited anti-bacterial activity ranging from 22 to 23 mm. The obtained oil samples showed significant or moderate antimicrobial and antifungal activity against clinically isolated pathogenic microbial strains in comparison to standard Ciprocin and fluconazole; hence, it might be considered essential to their potential for maintaining hygienic, healthy conditions. These results are similar to those of Chacko and

Anti-Bacterial Activities of Essential Oil
In Table 3, antibacterial activity of peel oil was determined against three bacterial pathogens, namely, Escherichia coli, Salmonella spp., and Staphylococcus aureus. The disk diffusion method was used to determine the antimicrobial activity by measuring the zone of inhibition. All peel oils exhibited a moderate clearance zone of inhibition against Escherichia coli, Salmonella spp., and Staphylococcus aureus. Among those, the Barishal sample oil showed the highest antibacterial sensitivity. From the experimental data, the Barishal sample oil showed inhibition diameters of 14 ± 0.17 mm, 13 ± 0.29 mm, and 12 ± 0.17 mm for Staphylococcus aureus, Escherichia coli, and Salmonella spp., respectively. However, the standard Ciprocin exhibited anti-bacterial activity ranging from 22 to 23 mm. The obtained oil samples showed significant or moderate antimicrobial and antifungal activity against clinically isolated pathogenic microbial strains in comparison to standard Ciprocin and fluconazole; hence, it might be considered essential to their potential for maintaining hygienic, healthy conditions. These results are similar to those of Chacko and Estherlydia [7]. Ciprocin (Ciprofloxacin) is a fluoroquinolone antibiotic used to prevent bacterial infections such as bone and joint infections, intra-abdominal infections, certain types of infectious diarrhea, respiratory tract infections, skin infections, typhoid fever, urinary tract infections, etc. Results from the third disk diffusion test of the peel oil, represented in Table 3, show that the gram-positive S. aureus bacteria was more sensitive to the tested extracts than the other two gram-negative bacteria. Most studies have investigated the action of EOs against food spoilage microorganisms and food-borne pathogens and showed that these oils are slightly more active against the gram-positive than the gram-negative bacteria [46]. They studied the resistance against bacteria and represented the inhibition zones against the ciprofloxacin and erythromycin. These results are comparable to those of our research. Kalemba et al. [47] classified the antimicrobial activity of EOs into three levels: weak activity (inhibition zone ≤ 12 mm), moderate activity (12 mm ≤ inhibition zone ≤ 20 mm), and strong activity (inhibition zone ≥ 20 mm), while our samples showed moderate to strong antimicrobial activity (Table 3). Moreover, several researchers have studied the antifungal activities of important Spondias species [48].

Anti-Fungal Activity of Essential Oil
As shown in Table 4, the antifungal activity of the peel oil samples was determined against two fungal pathogens, namely, A. niger and P. oxalicum. The disk diffusion method was used to determine the bioactivity by measuring the zone of inhibition. From our research data, peel oil from the Pabna sample produced satisfactory inhibition against A. niger and P. oxalicum. However, the highest inhibition zone of P. oxalicum (13.67 ± 1.97 mm) was observed against the Pabna sample oil, whereas A. niger showed the best inhibition against the Rajshahi sample oil (11.63 ± 0.0003 mm). The Barishal sample oil showed the secondhighest zone of inhibition against P. oxalicum (13.17 ± 0.17 mm) followed by A. niger (11.33 ± 0.17 mm). On the other hand, standard fluconazole exhibits anti-bacterial activity ranges from 19 to 20 mm. The antifungal results of all the essential oil samples in our study were similar to the results of previous research [49]. The monoterpenes of the volatile oils were mainly terpene hydrocarbons that have antimicrobial activity [50][51][52][53]. These monoterpene hydrocarbons inhibit both bacteria and fungi via interference with spore germination and mycelia growth [54][55][56]. Most of the terpenoids and their derivatives found in this study are important natural medicinal chemical constituents with wide biological activities [33]. The phenolic compounds can donate a hydrogen atom to the free radicals, thus breaking the propagation of chain reactions during the lipid oxidation process [57,58]. The oil can also inhibit the activity of protective enzymes and sequentially inhibit one or more biochemical pathways [11]. Lu et al. [59] explained the effect of oxygen availability on the antimicrobial efficacy of the oil on Staphylococcus aureus and Salmonella enteritidis. Microaerobic or anaerobic conditions were greatly enhanced when these organisms were incubated. The antimicrobial components of the essential oil cross the cell membrane, interact with the enzymes and proteins of the membrane, and hence produce a flux of protons towards the cell exterior, which induces the changes in the cells and ultimately, promotes their death. Due to the presence of monoterpenes in EOs, they may be used extensively as natural preservatives in many food products, soaps, soft drinks, cosmetics, and perfumes for their lemon-like flavor and odor [48].

Conclusions
This study showed that the peel of S. mombin appears to be unique in terms of its volatile composition. The results indicate that the essential oils of different regions showed varying anti-microbial activities against the various food-borne pathogenic bacteria and fungi tested. The highest zone of inhibition was shown against Staphylococcus aureus and Penicillium oxalicum. On the other hand, the highest number of VCs was identified in peel samples from the Barishal district, whereas the lowest number was in the Dinajpur district samples. On the contrary, the highest number of VCs was found in the Dinajpur district samples, whereas the lowest number was in the Rajshahi district samples. However, the variation in the number of VCs of the samples was very small. Thus, S. mombin could become an alternative to synthetic bactericides and fungicides for use in agro-industries. This study provides data for the development of a more economical, useful, and ecofriendly bio-alternative to existing usages of hog plum peels. However, further studies on the safety and toxicity of these oils and their possible in vivo bioactivity should be carried out before use.