*3.1. 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.


### *3.2. Chemical Composition of Volatile Oil*

There were 95 chemical compounds in the collected samples of hog plum peels (Table 2), and among them, the esters, alcohols, ketones, aldehydes, acids, hydrocarbons, phenols, and others comprised 26 (27.37%), 23 (24.21%), 16 (16.84%), 9 (9.47%), 6 (6.32%), 4 (4.21%), 5 (5.26%), and 6 (6.32%) of these compounds, respectively. Among the total (95) volatile compounds (VCs) of the five light yellowish oil samples, the Barishal sample oil was found to be rich in pentenyl-3-thy-met-4-alpha-methyl-alpha-ethanol-oxiranen (29.04%), and 5-heptone-methyl-one, 6-2 ester (3.24%); the Dinajpur sample oil contained pentenyl-3-thy-met-4-alpha-methyl-alpha-ethanol-xiranen (21.57%), cycohexanol-3 (8.59), picolylamine (10.49%), (3,3.1,1)-4-dimethylethyl-1,1-phenol (8.00%), 4-hydroxypenzeneethanamine (7.09%), and dibutyl phthalate (4.32%); the Mymensingh sample oil contained pentenyl-3-thy-met-4-alpha-methyl-alpha-ethanol-oxiranen (28.52%), cycohexanol-3 (10.85%), dibutyl phthalate (6.85%), and cyclotetrasiloxane (1.94%); the Rajshahi sample oil contained pentenyl-3-thy-met-4-alpha-methyl-alpha-ethanol-oxiranen (9.27%), cycohexanol-3 (5.80%), borneol (2.35%), and hydroxylamine(4.63%); and the Pabna sample oil was found to be rich in pentenyl-3-thy-met-4-alpha-methyl-alpha-ethanoloxiranen (11.45%), cycohexanol-3 (25.00%), borneol (4.96%), dibutyl phthalate (3.03%), and 3-cyclohexen-1-ol (3.57%). Furthermore, pentenyl-3-thy-met-4-alpha-methyl-alphaethanol-oxiranen was detected in all the samples, but cycohexanol-3 was present only in the Mymensingh, Dinajpur, Rajshahi, and Pabna samples, and was absent in the Barishal samples. (Methylyethyl-1)-4-methyl-oxabicyclo, 1-7 was present in the Dinajpur, Barishal, and Pabna samples but was absent in the Rajshahi and Mymensingh samples. Cycohexanol-3 was present in the Mymensingh, Rajshahi, Dinajpur, and Pabna samples but was absent in the Barishal sample. Dibutyl phthalate was present in the Dinajpur,

Pabna, and Mymensingh samples, but was absent in the Barishal and Rajshahi samples, and so on.


**Table 2.** Chemical compounds obtained from hog plum samples by GC-MS (Retention time: 5.52–22.34 min).



**Table 2.** *Cont.*

Significant spatial variations were observed in the number of VCs detected in the hog plum peel samples (Figures 1–3). The number of VCs varied among hog plum peel samples (Figure 1 and Table 2). The highest number of VCs (75.81%) was recorded in samples collected from Dinajpur followed by those collected from Pabna (65.30%), Mymensingh (57.01%), and Barishal (47.79%), while the lowest number of VCs (35.00%) was observed in the samples collected from Rajshahi. Figure 2 represents the number of VCs, which were identified by GC-MS analysis of *S. mombin* from the five districts of Bangladesh. The numerical values of the chemical compounds of hog plum peel essential oil from Dinajpur, Mymensingh, Barishal, Rajshahi, and Pabna were 22, 23, 33, 24, and 30, respectively. It was reported earlier that the number of VCs present in the essential oils of hog plum peels is comparatively higher than that of mango peels [34]. This is in agreemen<sup>t</sup> with the earlier literature studies, which showed a considerable variation in the chemical composition of peel EOs concerning varieties and drying conditions [36]. The investigational components of the oil were higher in number than those of earlier literature which was held on the fresh fruits, leaves, and barks of hog plum cultivated in the tropical conditions of Brazil [37]. Several studies also considered the volatile oil of mango fruits, leaves, and peels with results comparable to those of hog plum peel oil. Each of the volatile oils exhibited different chemical constituents that may be attributed to several factors such as ecological and climatic conditions, plant age, and genotypic characteristics [38–43]. The

esters showed the highest value (27.37%), and hydrocarbons showed the lowest value (4.21%) among the volatile substances in this study. Phthalic acid, isobutyl nonyl ester, and 2-phenylheptyl ester-acetyl-phthalic acid were detected in lower quantities. These substances, produced biosynthetically from unsaturated fatty acids, are precursors for the straight-chain esters [44]. Dichloromethane was used because generally, it appears as the best solvent for extraction of a wide class of flavors. The GC-MS analysis revealed that the hog plum peel oil contained different kinds of chemical compounds and/or EOs (Table 2) which have numerous antimicrobial properties that efficiently regulate the growth of different microorganisms such as fungus, bacteria, and yeasts, as envisaged earlier in several studies [21,22]. It has been proven that these compounds are potentially useful additives for food preservation against mycotoxigenic fungi and bacteria. A similar conclusion was suggested from a recent study, which ascertained that these additives prolong the shelf life and improve the quality of stored food products [45].

**Figure 1.** Yield of volatile compounds from hog plum peel samples collected from different districts of Bangladesh.

**Figure 2.** Number of volatile compounds identified from hog plum peel samples collected from different districts of Bangladesh.

**Figure 3.** GC-MS chromatogram of *S. mombin* peel oil collected from (**a**) Dinajpur, (**b**) Barishal, (**c**) Mymensingh, (**d**) Rajshahi, and (**e**) Pabna districts.

### *3.3. 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].

### *3.4. 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 [50–53]. These monoterpene hydrocarbons inhibit both bacteria and fungi via interference with spore germination and mycelia growth [54–56]. 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].



**Table 3.** Antibacterial activity of hog plum (*Spondias mombin* L.) peel oil extracted from different regions of Bangladesh.

**Table 4.** Antifungal activity of hog plum (*Spondias mombin* L.) peel oil extracted from different regions of Bangladesh.


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