GC-MS Based Metabolite Profiling, Antioxidant and Antimicrobial Properties of Different Solvent Extracts of Malaysian Plectranthus amboinicus Leaves

This study evaluates the phytochemistry, antioxidant, and antimicrobial effects of Plectranthus amboinicus leaves extracted in different solvents. The methanol extract contained the highest total phenolic (94.37 ± 1.24 mg GAE/g) and flavonoid contents (26.90 ± 1.35 mg RE/g) and exhibited the highest DPPH scavenging activity (90.13 ± 3.32%) followed by the acetone extract (80.23 ± 3.26%) at 500 μg/mL concentration. Similarly, the highest ferric ion reduction potential (849.63 ± 30.95 μM of Fe (II)/g dry weight) was exhibited by the methanol extract followed by the acetone extract (695.92 ± 25.44 μM of Fe (II)/g dry weight). The methanol extract showed greater antimicrobial activity against all the tested pathogens (Bacillus subtilis, Methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Candida albicans). However, both hexane and acetone extracts failed to inhibit E. coli. S. aureus and C. albicans were more susceptible to all the extracts. Further, GC-MS analysis confirmed the occurrence of a total 46 phytocompounds in different solvent extracts. Some of the major compounds included carvacrol (37.7%), tetracontane (16.6%), squalene (15.6%), tetrapentacontane (13.7%), and Phytol (12.9%). In conclusion, extraction solvents influenced the recovery of phytocompounds and the highest pharmacological activities of the methanol extract could be correlated to the presence of additional bioactive compounds.


Introduction
In nature, plants encompass a wide range of therapeutically valued bioactive compounds. These natural compounds are widely used in both traditional and modern therapies for improving human health with relatively less or no side effects. Globally, various medicinal plants have been well explored to discover novel drug molecules to combat the threat of everincreasing human diseases [1][2][3]. In the human body, cellular mechanisms lead to the generation of unstable compounds such as reactive oxygen species and free radicals, which can destroy living cells and may cause several clinical diseases. A range of synthetic and natural antioxidants are proved to be effective in controlling the activities of free radicals [4]. Evidence-Based Complementary and Alternative Medicine digestive, and urinary diseases [2]. Previous studies have witnessed the existence of significant variations in secondary metabolites accumulation among the same plant species grown under diverse ecological conditions [5,6]. In general, various solvents are used for extracting plant metabolites from their different parts. Moreover, the extraction of phytocompounds and yield mainly depends on the type of solvents and the method of extraction [7]. Literature survey shows less information on phytochemicals and bioactivities of P. amboinicus plants growing under tropical conditions of Malaysia [8]. With this background, we investigated the influence of different solvents to recover higher phytochemicals from a local P. amboinicus plant leaves and assessed their polyphenols and antioxidant and antimicrobial activities. Further, GC-MS analysis was carried out to explain the occurrence of bioactive nonvolatile compounds in different solvent extracts.

Plant Material Collection.
The wild growing P. amboinicus plant material was collected from the forest area near Universiti Putra Malaysia (UPM), Serdang, Selangor, Malaysia, during the month of August 2015. The plant was identified and authenticated taxonomically at the Department of Crop Science, UPM, Malaysia. The voucher specimen, PA-082015, was deposited in the department. The detached leaves from the collected plant materials were washed carefully under running tap water and dried for 1 week at room temperature in a shaded area. The dried leaves were finely powdered by using an electric blender and stored in a plastic bag container for further use at room temperature.

Preparation of Extracts.
About 25 grams of powdered leaves of P. amboinicus were kept in a beaker to which 100 mL of various organic solvents (methanol, acetone, and hexane) was added and thoroughly shaken. Later, the mixture was placed at room temperature for 48 hrs and stirred 2-3 times a day. After filtering the mixture, the filtrate was evaporated to dryness using rota-vapor. The final extracts were weighed to determine the yield (%) and the dried extracts were stored at 4 ∘ C in a refrigerator for further studies.

Determination of Total Phenolic Content in P. amboinicus
Extracts. Using the FC (Folin-Ciocalteu) colorimetric assay method, total phenolic content occurring in different solvent extracts of P. amboinicus leaves was evaluated [4]. Briefly, dried plant extract (0.1 g) was dissolved in 1 mL of distilled water. About 0.1 mL of this solution was later added to a solution containing 20% sodium carbonate solution (1 mL) and 50% FC reagent (0.5 mL). This solution was mixed thoroughly and allowed to stand by at room temperature for 20 min to facilitate the reaction to occur. Afterwards, the absorbance of the solution was recorded at a wavelength of 730 nm against the blank (water). Likewise, a standard calibration curve was generated by using gallic acid (standard) at different concentrations. The gallic acid curve was used to convert the absorbance of different plant extracts into total phenolic content and expressed in mg GAE (gallic acid equivalents) per gram of dried solvent extract.

Determination of Total Flavonoid Content in P. amboinicus
Extracts. The presence of total flavonoids content in various solvent extracts of P. amboinicus leaves was determined by using the colorimeter assay method as described by Swamy et al. [4]. In short, about 0.5 mL of the solvent extract (1 mg/mL) was suspended in 2 mL of distilled water and then added with 150 L of 5% NaNO 2 solution and incubated at room temperature for 5 min. Thereafter, the solution was added to 2 mL of NaOH (4%) and 600 L of AlCl 3 (10%) and made up to 5 mL using distilled water. The solution was mixed thoroughly and incubated for 15 min at room temperature. The absorbance of the solution was documented at a wavelength of 510 nm against the blank (water). In a similar way, a standard calibration curve was prepared by using the standard, rutin, at different concentrations. By using the standard curve of rutin, total flavonoid content was determined and expressed in mg of RE (rutin equivalents) per gram of dried solvent extract. properties of different solvent extracts of P. amboinicus leaves were evaluated by using disc diffusion method as described by Kumara et al. [10] with little modifications. In short, 10 mg of each solvent extract was dissolved in 1 mL of dimethyl sulfoxide (DMSO) and about 10, 20, and 30 L of this solution were impregnated on 6 mm sized sterilized filter paper discs. Later, the air-dried discs were placed on individual MHA or PDA plates and previously preinoculated uniformly with a known bacterial or fungal strain, respectively. The discs saturated with DMSO (20 L) served as a negative control for all microbes while streptomycin (100 mg/mL) and nystatin (100 mg/mL) served as a positive control for bacterial and fungal species, respectively. All microbes were incubated in an incubator at 37 ∘ C for 24 hrs to observe the zone of growth inhibition (mm) around each disc. For each microbial strain, the experiment was repeated 3 times.

GC-MS Analysis.
Each solvent extract was subjected to GC-MS analysis using the model instrument, GCMS-QP2010 Ultra (Shimadzu Co., Japan) attached with a capillary column DB-1 (0.25 m film × 0.25 mm I. d. × 30 m length). Analysis was performed by injecting 1 L of the sample with a split ratio of 20 : 1. Helium gas (99.9%) was used as the carrier gas at a flow rate of 1 mL/min. The analysis was performed in the EI (electron impact) mode with 70 eV of ionization energy. The injector temperature was maintained at 250 ∘ C (constant). The column oven temperature was set at 50 ∘ C (held for 3 min), raised at 10 ∘ C per min to 280 ∘ C (held for 3 min), and finally held at 300 ∘ C for 10 min. The compounds were identified after comparing the spectral configurations obtained with that of available mass spectral database (NIST and WILEY libraries).

Statistical Analysis.
In each experiment, the data recorded was from 3 replications ( = 3) and all the results are represented as mean ± SD. One-way analysis of variance (ANOVA) was carried out to compare the data. Further, to determine the statistically significant differences, Tukey's test was performed at < 0.05 level using GraphPad Prism (version 5.0) statistical software.

Results
The dry weight and final yield of the leaf extract were significantly affected by different solvents used for the extraction Values in the column followed by a different letter superscript are significantly different ( < 0.05). ( Table 1). The methanol solvent recorded the highest weight of the extract (131.6 ± 2.6 mg) and its yield (1.3%). The extractable elements recovered from different solvents are in the following order, that is, methanol > hexane > acetone. Further, solvent extracts had a significant difference ( < 0.05) in their total phenolic and total flavonoid content ( Table 2). In the methanol leaf extract, the highest total phenolic content (94.37 ± 1.24 mg GAE/g) and flavonoid content (26.9±1.3 mg RE/g) were recorded. In hexane extract, total phenolic content was found to be 75.39±1.07 mg GAE/g. However, total flavonoid content (16.46 ± 0.9 mg RE/g) was relatively lesser when compared to the acetone extract (21.27 ± 0.8 mg RE/g). DPPH free radical scavenging property was observed to be dependent on the concentration for all the tested solvent extracts. The methanol extract exhibited the highest DPPH scavenging activity (90.13 ± 3.32%) followed by acetone extract (80.23 ± 3.26%) and hexane extract (73.52 ± 3.18%) at 500 g/mL concentration ( Figure 1).
However, DPPH scavenging activity of all extracts was inferior to the standard (ascorbic acid). Similar pattern of antioxidant activity was evidenced from FRAP assay method where the highest ferric ion reduction potential was exhibited by the methanol extract (849.63 ± 30.95 M of Fe (II)/g dry weight) followed by acetone extract with 695.92±25.44 M of Fe (II)/g dry weight ( Figure 2). The lowest FRAP activity was observed in hexane extract (376.98±15.42 M of Fe (II)/g dry weight). However, the superior activity was evidenced in the standard, -tocopherol.
The microbial inhibitory potential of different solvent extracts of P. amboinicus is depicted in Table 3. The results clearly showed the existence of a varied and selective antimicrobial activity of different solvent extracts against each microbial species tested. Further, increased concentration 4 Evidence-Based Complementary and Alternative Medicine   belong to different chemical classes and most of them are reported to exhibit important biological activities. Figure 3 shows the distinct chromatogram of P. amboinicus leaves extracted in different solvents. The identified compounds with their peak number, retention time (RT), and peak area (%) are presented in Table 4. Out of 39 peaks observed in the chromatogram of methanol extract, 19 compounds were identified with 6 major peaks. The major compounds included tetracontane (16.6%), tetrapentacontane (11.3%), pentacosane (7.8%), and n-hexadecanoic acid (4.9%).  Evidence-Based Complementary and Alternative Medicine

Discussion
Recent years have witnessed the importance of phytochemicals due to their remarkable health benefits. Plant metabolites are extracted by using various ways, such as maceration, decoction, soxhlet extraction, microwave-assisted extraction, supercritical fluid extraction, and ultrasound-assisted extraction method. As maceration technique is a simple and the easiest method, it is widely used at the preliminary research level [11]. The efficiency of extraction depends on several factors, including the nature of phytochemical constituents, the method of extraction, particle size of the sample, extraction time, temperature, pH, solute to solvent ratio, and the solvent polarity [12]. The appropriate use of solvent system is crucial in order to recover higher extract yield, polyphenols, and some other bioactive compounds from a sample [13]. Plant derived polyphenols including flavonoids possess several biological properties and thus necessitate evaluating their presence in different plant parts extracted in different organic solvents [4,14,15]. This study revealed the existence of a significant difference in the extract yield obtained by different solvents. Further, the highest extract yields and total phenolic content obtained in the methanolic leaf extract of P. amboinicus could be attributed to its high polarity [13]. Another possible reason might be due to the establishment of complexes by phenolic constituents with other biomolecules such as proteins, carbohydrates, terpenes, and inorganic compounds that can be more easily recovered from methanol in comparison to other solvents [16]. Similarly, the use of methanol as the best solvent for extraction is well documented in the literature [4,13,17]. On the contrary, methanol, acetone, and water are reported to be ineffective for extracting polyphenols from the seeds of grapes [18]. In the present study, the extractive yield and total phenolic content were better evidenced in the nonpolar hexane extract when compared to the polar solvent, acetone. In contrast, the hexane extract yielded much lesser total flavonoid content compared to the acetone extract. This difference in the extract yields could be attributed to the occurrence of a wide range of diverse phytochemical components in P. amboinicus leaves and differences in the solvent polarity. Furthermore, it is reported that the level of solubility of a phenolic compound decides the recovery percentage of a particular solvent type [4,13]. In contrast, according to the report by Bhatt and Negi, [19], the acetone extract contained significantly higher total phenolics compared to the methanol or hexane extracts, which was endorsed for the difference in the geographic location and other environmental factors [20]. Likewise, earlier study has identified the presence of polyphenols such as caffeic acid, coumaric acid, rutin, quercetin, and gallic acid in the acetone leaf extract of P. amboinicus [21]. As there is a dearth of scientific details on phytochemistry of Malaysian P. amboinicus, this study will be certainly appreciated. In general, this study suggests the use of methanol and hexane solvents for higher recovery of extractable compound from P. amboinicus leaves. Because of the lower extractive yield obtained in this study, it is recommended to use other better extraction methods in P. amboinicus. Nevertheless, it is reported that the biological properties depend not only on the total extract yield but also on its phytochemical composition [22]. In the human body, cell damage may induce the generation of increased levels of free radicals. Various disorders including myocardial infarction, cancer, atherosclerosis, and neurogenerative disorders are mainly correlated to these free radicals [4]. Antioxidants are chemical compounds and have the ability to prevent various oxidative stress related cell damages mediated by free radicals. Many antioxidant molecules are recorded in several medicinal plants and thus will be beneficial in the treatment of several human diseases [15]. Most frequently, the DPPH and FRAP assay methods are preferred for determining antioxidant activity [9]. In this study, radical scavenging activity was observed to be concentration dependent and corroborates with the previous reports on several other plant species [4,9,23]. The superior radical scavenging potential of plant solvent extracts may be interrelated to the presence of various antioxidants such as polyphenolic compounds [14,23]. Even though earlier reports have stated that P. amboinicus leaf extracts possess antioxidant properties [2], none of them have reported the systematic study comparing the effect of different solvents on the antioxidant potential of P. amboinicus species growing under tropical conditions of Malaysia. The methanol extract was found to exhibit the highest scavenging activity when compared to other solvent extracts. This could be attributed to the fact that the methanol extract possesses higher antioxidant molecules such as phenolic compounds as evidenced by our phytochemical analysis ( Table 2). In contrast, Bhatt and Negi [19] reported that acetone extract of P. amboinicus possessed superior antioxidant activity when compared to hexane, ethyl acetate, or methanol extracts. According to Rai et al. [24], the effective antioxidant activity of P. amboinicus leaves was observed when extracted with the solvent, ethanol. This difference could be could be due to differences in the geographical growth conditions as stated previously by Swamy et al. [4]. As stated by the earlier reports, the free radical scavenging activity of different solvent extracts of plants mainly depends on the existence of different bioactive chemical constituents [1,5]. Further, more polar solvents can often extract antioxidant compounds in higher quantities. Overall, high-polarity solvent (methanol) was very effective in extracting more antioxidant compounds when compared to an intermediate polar solvent, acetone, and nonpolar solvent, hexane.
Antimicrobial agents (synthetic or plant based) effectively kill a wide range of infectious microbes. However, their improper and overuse has permitted microbes to develop multidrug resistance ability which is a major threat and a bigger challenge to the medical world [25]. Hence, there is a need to investigate novel molecules to overcome antimicrobial resistance. Further, present research emphasizes on the development of plant based drugs due to the fact that synthetic drugs cause several side effects in humans. In this regard, we evaluated the antimicrobial properties of P. amboinicus leaf extracts and the results were very conclusive. All the tested 8 Evidence-Based Complementary and Alternative Medicine microorganisms were effectively inhibited by all solvent extracts with the exception of a gram-negative bacteria E. coli, which was inhibited only by the methanolic leaf extract. MRSA strain exhibited less susceptibility to all solvent extracts when compared to other gram-positive strains. B. subtilis was highly susceptible to the methanol extract. Interestingly, methicillin-resistant S. aureus and C. albicans were highly susceptible to all the extracts. Similarly, Gurgel et al. [26] reported the efficacy of the hydroalcoholic leaf extracts of P. amboinicus from Brazil against MRSA strains while Manjamalai et al. [27] report the anticandidal activity of the methanolic leaf extract of Indian P. amboinicus. To support this, researchers have stated that mostly gram-negative bacteria exhibit more resistant properties against a wide range of antibiotics or chemical drugs when compared to grampositive bacteria [19,28]. This difference in the susceptibility of a microbe to plant extracts is also linked to differences in their cell wall and outer membrane structures [29]. MRSA cause several human health problems including skin infections, sepsis, pneumonia and other infections, and C. albicans is a prime causative agent of candidiasis. Thus, P. amboinicus leaf extracts may be beneficial in treating these diseases causing microbes. Microbial inhibition was found to vary with the type of solvent extracts evaluated. Likewise, previous researchers also have testified the wide-ranging antimicrobial activity of this plant [2,19]. However, these results contradict with the findings by Bhatt and Negi [19], where the superior antimicrobial activity was shown by the acetone extract, while Jiyauddin et al. [8] report the highest antimicrobial activity in the ethanolic leaf extract. This discrepancy could be due to differences in the extraction methods and microbes tested. The highest antimicrobial activity of methanol extract against all the examined microorganisms could be due to more soluble bioactive compounds extracted in the methanol solvent. These bioactive compounds, perhaps, may inhibit microbial growth effectively by binding to their cell surface. In support of this, previous studies have stated that the antimicrobial action of plant extract is presumably related to the collective effect of phenolic compounds adsorption onto the cell membrane leading to its disruption and cell leakages and the generation of hydroperoxides by phenolic compounds [2,30]. These results support the reports emphasizing on the curative property of P. amboinicus plant extracts and its essential oil [2]. However, most of the research studies have evaluated antimicrobial activities of essential oil of P. amboinicus and limited importance is given on different plant extracts, especially, in those plants growing under Malaysian conditions [2,8,20,26,27,31]. Furthermore, this study establishes the importance of the solvents in recovering higher extractable compounds with potent antimicrobial action against human pathogens for the first time.
Various biological activities of P. amboinicus leaf extract can be described by investigating the chemical composition of each extract using GC-MS analysis. The literature survey reveals the occurrence of about 30 nonvolatiles from methanol, ethyl acetate, water, and chloroform extracts of P. amboinicus leaves, stems, and roots around the world [2,19,32]. However, to the best of our knowledge, there is no report of GC-MS based metabolite profiling to detect the presence of various bioactive compounds in hexane and acetone extracts of P. amboinicus leaves. In addition, compounds in different solvent extracts of Malaysian P. amboinicus plants are yet to be studied. Accordingly, GC-MS analysis was performed in the preset study and identified a total of 46 compounds from different solvent extracts. The highest number of compounds (19) was evidenced in the methanolic leaf extract followed by a hexane extract (16) and acetone extract (11). Most of these compounds are known to exhibit various pharmacological activities. In this study, the higher antioxidant and antimicrobial activities of the methanol extract could be correlated to the occurrence of more number of bioactive compounds including n-hexadecanoic acid, Phytol, and squalane. Similarly, the occurrence of these compounds is also evidenced in many medicinal plants leaves extracted with methanol [5,33,34]. Phytol is an important diterpene that possesses antimicrobial, antioxidant, and anticancer activities [5,35,36]. Hexadecanoic acid is known to exhibit strong antimicrobial and anti-inflammatory activity [5,37]. Squalene is a triterpene that act as natural antioxidants and possess various pharmacological importance [38,39]. Neophytadiene is a good analgesic, antipyretic, anti-inflammatory, antimicrobial, and antioxidant compound [33]. Similarly, the compounds and -Amyrin present in hexane and methanol extracts are known to possess antidiabetic, anti-inflammatory, antiarthritic, and anticancer activities [34]. The presence of compounds such as carvacrol, caryophyllene oxide, and stigmasterol in hexane extract is also reported with various biological activities [2,33]. Similarly, Kunle et al. [39] also showed the occurrence of carvacrol in the hexane extract of Lippia multiflora leaves and showed potent antimicrobial activity [39]. Carvacrol, an isoprenyl phenol is reported as one of the strongest antimicrobial agents [40]. Moreover, it has shown to inhibit the biofilms formation by Chromobacterium violaceum, Typhimurium DT104, Salmonella enterica, and Staphylococcus aureus [41]. The compound, caryophyllene oxide, exhibits a wide range of antimicrobial properties [42]. Likewise, a steroid compound was also isolated from the hexane extract of Hydnophytum formicarum [43]. In the same study, stigmasterol was shown to possess better antimicrobial and antioxidative properties. This could be one of the reasons for the higher antimicrobial activity exhibited by hexane extract compared to acetone extract as stated earlier.
However, some of the other major compounds, including tetracontane, pentacosane, tetratriacontane, tetrapentacontane, and hentriacontane, are yet to be described in detail. Nevertheless, more research efforts are required to isolate, characterize, and evaluate these compounds from P. amboinicus leaves to validate their various pharmacological importance.

Conclusion
In this study, the presence of various soluble bioactive compounds in P. amboinicus leaf extracts greatly contributed to antioxidant and antimicrobial activities. Phytochemical composition and yield of the extract varied depending on the solvent types used for the extraction. In the methanolic extract, more soluble phytocompounds were noticed. The Evidence-Based Complementary and Alternative Medicine 9 results clearly support the use of P. amboinicus in traditional medicinal practices to treat various diseases. Thus, this plant can serve as a new natural source for obtaining many therapeutically valued metabolites against various diseases.

Conflicts of Interest
The authors declare that they have no conflicts of interest.