2.1. Plant Material

P. pellucida plants were collected and identified from a population growing at the Institute of Biological Science’s garden at the University of Malaya (UM) as well as from various areas around the UM. The plants were then maintained at the Institute of Biological Sciences garden at the Faculty of Science, UM, Kuala Lumpur, Malaysia. The voucher specimens were deposited in the UM Herbarium (KLU), Kuala Lumpur, Malaysia, for identification. The intact plants without the roots were collected in the year 2017 from the ISB garden at the UM, and the in vitro regenerated plantlets of P. pellucida grown on MS medium were used as a comparison.

2.2. In vitro Regeneration of P. pellucida

Above ground, vegetative shoots of P. pellucida were collected from the ISB garden, UM. The plants were first washed under running tap water for 30 min. Then, the plants were soaked and washed with three drops of teepol with sterile distilled water for 2 min and rinsed thoroughly 5 times with sterile distilled water. Next, the plants’ surfaces were sterilized with 50% sterile sodium hypochlorite for 2 min, followed by a 50% disinfectant solution (Dettol, Reckitt Benckiser) for 10 min. This was followed by rinsing the plant 5 times with sterile distilled water. Finally, the P. pellucida plants were treated with an antifungal, 0.01% (w/v) carbendazim diluted in 70% (v/v) ethanol for 30 s and rinsed 5 times with sterile distilled water. Then, the sterilized internodes were tapped dry using sterile tissue paper and intersected small pieces (10 mm) were cultured onto the MS basal medium [25] comprised of 30 g/L sucrose (System), 4.4 g/L MS powder (Duchefa Biochemie), 3.3 g/L gelrite (Duchefa Biochemie), and 0.01% carbendazim, adjusted to pH 5.7 before being autoclaved at 121°C for 20 min. Cultures were maintained at 25 ± 2°C, 16 h/8 h (light/ dark) photoperiod with 25 mmol m-2s-1 of light intensity.

2.3. Extraction of Plant Material

The harvested in vivo or in vitro generated plants were separately washed clean from soil or media. The clean, intact plants were freeze-dried, after which the dried samples were ground to a uniform powder and sorted out according to size using 250 mm sieves. In brief, dry powdered plant material (10 g) was weighed out and filled into a cellulose extraction thimble (Whatman), then inserted into a Soxhlet apparatus and extracted with 300 mL ethanol (R&M Chemicals) for 48 h or methanol (R&M Chemicals). Separately, all the extracts were then concentrated using a rotary evaporator under reduced pressure at 20°C, relatively low temperature and kept in an airtight container and preserved at −20°C until further use.

2.4. Analysis of Plant Extracts Chemical Composition

The phytochemical components of the P. pellucida extractions were identified using Agilent GC-MS. GC-MS was carried out on a 7890A GC/MS Agilent 5975, Shimadzu QP2010 ULTRA GCMS equipped with RTX-5 column. Sample injected: 2 ml. The oven column temperature was pre-set at 60°C, with an initial hold time of 2 min, to 280°C at the rate of 5°C/min with a final hold time of 9 min. The injector temperature was set to 240°C. The total GC running time was 47 min. Interpretation of GC-MS was conducted, using the National Institute Standard and Technology Version – Year 2011 library. The name, retention time, molecular weight, and molecular structure of the compounds of the test substances were ascertained using the database.

2.5. Antimicrobial Activity

All the isolated bacteria were provided by the Microbiology Laboratory at the Institute of Biological Sciences, Faculty of Science, UM. The microorganisms used in this study are as follows: Salmonella typhi, Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus. All the bacteria were cultured in Nutrient broth (Difco) and incubated for 24 h at 37°C. The disk diffusion method by Bauer et al. [26] was used with slight modification. A crude extract of in vivo and in vitro grown plants was diluted in distilled water to a concentration of 10 µg/mL. 10 µg/mL of chloramphenicol (Duchefa Biochemie) was also prepared as a positive control for bactericidal activity. Sterile blank disks sized 6 mm in diameter (Oxoid) were impregnated with 20 mL of each diluted extract and chloramphenicol. The impregnated disks were allowed to fully dry in the laminar flow before the application on the bacterial lawn. The 10⁻⁹ serially diluted bacteria were aseptically swabbed on the surface of sterile 4% nutrient agar (Difco) prepared plates using sterile cotton swabs. Using sterile forceps, aseptically, the prepared antibiotic disks were placed over the bacteria seeded agar plates. The disks were separated sufficiently from each other to avoid overlapping of inhibition zones. The plates were then incubated at 37°C for 24 h. Finally, antibacterial activity was expressed as the mean zone inhibition diameter (mm) produced by the extract.

2.6. Anticancer Activity

To test the anticancer properties, both the methanol-extracted and ethanol-extracted compounds from in vitro and in vivo conditions were prepared in seven different concentrations, serially diluted with a dilution factor of two. As such, the final concentrations of each condition were as follows: 30 µg/mL, 15 µg/mL, 7.5 µg/mL, 3.75 µg/mL, 1.875 µg/mL, 0.9375 µg/mL, and 0.469 µg/mL.

The cell culture works were performed as described previously with a slight modification [27]. The anticancer properties of extracted compounds were tested on two different types of commercially available cancer cell lines, namely adenocarcinomic human alveolar basal epithelial cells (A549, ATCC, USA) and human breast adenocarcinoma cell lines (MCF-7, ATCC, USA). They were seeded at 1000 cells/100 uL/well in a 96 well plate format (Corning, USA). The anticancer effects of the aforementioned compounds were evaluated by utilizing a viability assay using 10% Alamar Blue reagent (Thermo Fisher Scientific, MA, USA). Cell viability was analyzed through absorbance at 450 nm with a reference wavelength set at 590 nm by the Tecan microplate reader (Infinite 200 PRO Tecan, Life Sciences, Switzerland). From the viability profile, the respective half-maximal inhibitory concentration (IC₅₀) was calculated from the general equation y =mx + c to indicate the toxicity level of each compound. All assays were performed in triplicates to ensure consistency.

2.7. Statistical Analysis

For the antimicrobial study, data were collected and statistically analyzed using Duncan’s Multiple Range Test. It should be noted that the mean with different letters in the same column indicates a significant difference with P < 0.05. For the anticancer study, data were collected and analyzed using two-way ANOVA with Tukey’s post-hoc.

3.1. Analysis of Plant Extracts Chemical Composition

In the recent study, GC-MS analysis had identified the presence of phytochemical compounds in both ethanol and methanol extracts of in vivo and in vitro grown P. pellucida. The ethanol extract of the in vitro grown P. pellucida yielded 24 constituents [Table 1], while the ethanol extract of the in vivo grown P. pellucida only yielded 23 constituents [Table 2]. For the methanol extract of in vitro grown, P. pellucida gave rise to 13 constituents from the chromatogram of the extract [Table 3], while the methanol extract of in vivo grown P. pellucida yielded a total of 15 constituents [Table 4]. At present, no studies are reporting the antibacterial activity using extracts from tissue cultured (in vitro) P. pellucida plants. Our study is the first ever to report the constituents of phytochemical compounds from tissue cultured (in vitro) P. pellucida plants. Since methanol and ethanol are efficient in traversing cell walls and seed pods which have more unipolar character and would cause cells to release their chemical compounds, thus they are chosen for extraction. The number of chemical compounds extracted varied depending on the usage of different materials and solvents. The chemical constituents obtained showed that solvent polarity influenced the outcome of the extraction process as the solubility of components depends on the number, type, and linkage position of the compound [28,29].

Table 1: GC-MS of ethanol extract analysis revealed the presence of phytochemical components in in vitro grown Peperomia pellucida.

Table 2: GC-MS of ethanol extract analysis revealed the presence of phytochemical components in in vivo grown Peperomia pellucida.

Table 3: GC-MS of methanol extract analysis revealed the presence of phytochemical components in in vitro grown Peperomia pellucida.

Table 4: GC-MS of methanol extract analysis revealed the presence of phytochemical components in in vivo grown Peperomia pellucida.

The ethanol extracts from in vivo and in vitro grown plants showed a greater number of chemical compounds as compared to methanol extracts and these identified compounds possessed biological properties. This shows that P. pellucida contains more non-polar compounds that are more readily extracted with ethanol compared to methanol. A comparison was done between the extracts and it was found that certain compounds that are identified in ethanol and methanol extracts of in vitro and in vivo grown plants are nearly similar. For instance, in this study, apiol was found in all the extracts. However, the apiol content was found to be higher in ethanol extracts of in vitro and in vivo grown plants compared to methanol extracts. Previous studies have reported the presence of apiol in the ethanolic extracts [29] and in the essential oil of in vivo grown plants [9,18,30,31]. Apiol was reported to have antifungal activities against Trichophyton mentagrophytes [21,32] and interestingly, it is also used as birth control as well as to treat menstrual disorder [33].

Apart from apiol, the present study identified the presence of phenolic compounds in the ethanol extracts of in vitro grown plants and methanol extracts of in vivo grown plants, as reported previously [34]. The current study reported that the detection of cyclohexane, 1-ethenyl-1-methyl-2,4-bis(1-methylethenyl)-, and caryophyllene was in the methanol extracts of in vitro and in vivo grown P. pellucida. Narayanamoorthi et al. [35] had reported the presence of cyclohexane, 1-ethenyl-1-methyl-2,4-bis(1-methylethenyl)-, and caryophyllene in the ethanol extract of an in vivo grown P. pellucida whole plant. These two compounds have shown to be effective as an anticancer, analgesic, antibacterial, anti-inflammatory, sedative, and also as a fungicide [22,35].

Furthermore, detected was the presence of 9-octadecenoic acid in the ethanol extract of in vivo grown P. pellucida. Omotoso et al. [36] had reported that 9-octadecenoic acid (z) contains anticancer properties, acts as a lubricant, as an anti-inflammatory agent, able to inhibit hemolytic 5-a reductase, acts as a diuretic agent, able to stimulate immune system, antiandrogenic, antifungal, antibacterial, and lipoxygenase inhibitor activities. Decane is normally used in chemical tests as a function of concentration and temperature [37]. In the present study, the detection of phytol is positive in both the ethanol extract of in vitro grown plants and methanol extract of in vivo grown P. pellucida. Narayanamoorthi et al. [29] reported the presence of phytol in the ethanolic extract of the in vivo grown plant, while Wei et al. [38] reported phytol in the methanol extract of an in vivo grown plant. Whereas, Okoh et al. [39] reported the presence of phytol in essential oil. Previous research has shown that phytol is known to have antimicrobial, anticancer, diuretic, and anti-inflammatory activities [22].

In a previous study, Wei et al. [38] had reported the presence of phytol, 2- naphthalenol, decahydro-, hexadecanoic acid, 9,12-octadecadienoic acid, and methyl ester from methanol extraction of in vivo grown P. pellucida. Verma et al. [9] has reported the detection of a total of 50 compounds from the essential oil of P. pellucida. Among the 50 compounds, apiol was also present in the essential oil. A separate study reported the presence of dillapiole in P. pellucida extract and tested on Wistar rats for gastroprotection. The findings have concluded that dillapiole is the most active gastroprotective agent of P. pellucida [8].

3.2. Antibacterial Activity

P. pellucida has known to be a medicinal plant and is widely used for skin, eye, throat, and gout as well as for antibacterial activity. In the present study, ethanolic and methanolic extracts of in vivo and in vitro grown plants were found to be effective against the following bacteria: S. typhi, P. aeruginosa, E. coli, and S. aureus [Table 5]. Tests of the extracts against several organisms have displayed comparable results that of the standard antibiotics, chloramphenicol. The GC-MS chemical analysis result of P. pellucida shown it to contain bioactive compounds and has been reported to exhibit antibacterial activities [22,35,38]. Based on Table 5 in the current study, the methanol extract from an in vitro plant showed the highest inhibition zones (12.5 mm) against S. aureus with a lower concentration compared to the in vivo grown plant extracts from other studies. The ethanol extracts from an in vivo grown plant (6.75 mm) and in vitro grown plant (10.75 mm) showed a contrasting result with the methanol extract from an in vitro grown plant, which was less effective against S. aureus. This result is also highly comparable to previous studies [Table 5]. S. aureus is a major human pathogen that affects the skin and soft tissues, pleuropulmonary, bloodstream respiratory tract, and can also contaminate medical devices [40,41]. In treating multiple resistant S. aureus (MRSA), there are proposals for the usage of the methanol extract of the in vitro grown plant. The methanol extract of the in vitro grown P. pellucida can be a promising potential inhibitor against MRSA. GC-MS analysis of the methanol extract from the in vitro grown plant has detected the presence of previously described antimicrobial agents such as Cyclohexane, 1-ethenyl-1-methyl-2,4-bis(1-methylethenyl)-, and Caryophyllene. However, in the current study, the ethanol extract from the in vitro plant showed the highest inhibition zones (12 mm) against S. typhi and P. aeruginosa compared to methanol extracts from in vivo and in vitro grown plants or the ethanol extract from in vivo grown plants. In the current study, the ethanol extract from the in vivo grown plant showed greater activity (11.5 mm) against E. coli compared to the ethanol extract from an in vitro grown plant (8.00 mm). A study conducted by Edewor-Kuponiyi [42] showed that the crude methanol extract from the in vivo grown leaves at 1 mg/mL concentration showed a higher inhibition zone compared to the current study. Edewor-Kuponiyi used dimethyl sulfoxide (DMSO) to dissolve the extracts. A study reported DMSO could inhibit the growth of bacteria [43]. This implies that the presence of DMSO can enhance the effect of the inhibition zone. At present, no studies are reporting the antibacterial activity using extracts from tissue cultured (in vitro) P. pellucida plants. The results obtained in this study on the antimicrobial efficacy of dried crude extracts of in vitro regenerated P. pellucida showed the highest inhibitory activity using ethanol and methanol as the medium of extraction. The plant extracts showed bacteria inhibition. This could be due to the presence of various active compounds such as polyphenols and flavonoids from the plant extracts that could be the antimicrobial components [17]. The topical application of the ethanolic and methanolic extracts of in vivo and in vitro grown plants is possible as both extracts displayed as antibacterial agents. All parts of P. pellucida have potent antibacterial activity and it has also been reported that crude methanol extracts of P. pellucida have a broad spectrum of antimicrobial activity [23]. In vivo and in vitro grown plants produce various compounds which show different bioactivity prospectives [44].

Table 5: Antibacterial activities of ethanolic and methanolic extracts of in vivo and in vitro Peperomia pellucida.

Biotechnological methods through plant tissue culture provide a promising bio-production platform for desired natural products. Cultures developed from shoots or roots revealed an undifferentiated metabolic characteristic compared to their parent plant. Tissue culture offers an alternative potential for the fabrication of high-value natural products in the plant [6]. It is important to note that the antimicrobial activities differ in in vivo and in vitro grown plant extracts, probably due to the inherent characteristics of the fully grown plants and the maturity of their chemically active constituents.

3.3. Anticancer Activity

Apart from the aforementioned characteristics of P. pellucida, the anticancer properties of the said extracts were tested on two commercially available cancer cell lines, namely; adenocarcinomic human alveolar basal epithelial cells (A549) and human breast adenocarcinoma cell lines (MCF-7). As shown in Figure 1, with increasing concentration of both ethanol extracts of in vitro and in vivo grown plants shows a general decrement of the viability of the cells. For instance, at 30 mg/mL, the viability of A549 cells has reduced from 100% to 45.3%. The IC₅₀ of this extract was found to be 13.02 mg/mL based on the equation y = −7.335ln(x) + 68.828 [Figure 1a]. Similarly, the viability of A549 cells was also checked upon exposure to both methanol extracts of in vitro and in vivo grown plants [Figure 1c and d]. In particular, the viability of A549 cells at 30 mg/mL for both methanol extracts of in vitro and in vivo grown plants were 35.8% and 33.3%, respectively, hereby indicating the potency of this extract, especially upon methanol extraction. The IC₅₀ for the extracts was found to be 8.11 mg/mL and 6.76 mg/mL, respectively.

Figure 1: (a-d) Viability assay of A549 cells response [Click here to view]

In addition to assessing the viability of A549, a similar assay was also conducted on another robust cancer cell line, MCF-7 [Figure 2]. Upon exposure to the ethanol extract from an in vitro grown plant, the viability trend showed a typical decrement with increasing concentration. The observation for the exposure of the 30 g/ml extract shows the viability to be at 38.6% compared to 45.3% in its A459 counterpart with IC₅₀ of 22.88 g/mL [Figure 2a]. Finally, the viability of MCF7 upon exposure to the methanol extracts from an in vivo grown plant also displayed an augmented survival pattern as compared to its A549 counterpart. For instance, the exposure to 30 mg/mL of the extract revealed a viability percentage of 49.94%, as compared to 33.3% in A549 cells with IC₅₀ 52.56 mg/mL [Figure 2c]. A similar study by Wei et al. [38] also confirmed the anticancer findings, thus strengthening the possibility that P. pellucida extract can be commercially harnessed for anticancer treatment. Although the study reported a much lower IC₅₀ than our findings, it is believed that this variation could be due to the number of plants used. In this study, two different types of cancer cells, namely adenocarcinomic human alveolar basal epithelial cells (A549) and human breast adenocarcinoma cell lines (MCF-7), were chosen based on the frequent occurrence of such cancers in human [45]. In general, an increase in extract concentration resulted in the poor survival of these cancer cells over time. However, the total effect varied among the type of cancers as well as the origin of the extract being used. For instance, in A549 cells, extracts from methanol resulted in a lower survival rate upon exposure at 30 mg/mL for 24 h. This shows that the presence of more active compounds in the methanolic extracts had displayed higher cytotoxicity on A549 cells as compared to those from ethanol extraction. This was also supported with IC₅₀ values found alongside. Contrastingly, in MCF7 cells, compounds through ethanol extraction presented higher cytotoxicity coupled with lower IC₅₀ values, as compared to those of methanol extraction.

Figure 2: (a-d) Viability assay of MCF7 cells response [Click here to view]

Although this study did not address the possible mechanism behind the cytotoxicity effect of these compounds, it is speculated that key cellular pathways such as TGF-b/Smad and PI3K/Akt/mTOR signaling pathways could be one of which the active compounds exert their effects [46]. Further studies could clarify therapeutic potentials and toxicity issues should these compounds move toward clinical and therapeutic usages. Anticancer activities differ in in vivo and in vitro grown plant extracts, probably due to the inherent characteristics of the fully grown plants and the maturity of its chemically active constituents.