Potency and selectivity indices of Myristica fragrans Houtt. mace chloroform extract against non-clinical and clinical human pathogens


 This study assessed the antimicrobial, toxicity, and phytochemical profiles of Myristica fragrans extracts. Different solvent extracts were tested for antimicrobial activity against clinical and reference microbial strains, using disc and well diffusion assays and microdilution techniques. Antioxidant potential was investigated using 2,2-diphenyl-1-picryhydrazyl (DPPH) assays. Cytotoxicity assay was conducted against human umbilical vein endothelial cells (HUVECs) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Acute toxicity was assessed in laboratory Swiss albino mice at a single dose of 2,000 mg/kg body weight for 14 days. To assess the phytochemical constituents, spectrophotometric and gas chromatography-mass spectrometry (GC-MS) methods were used. The chloroform extract revealed antimicrobial potencies against the Gram-positive bacteria and C. albicans with minimum inhibitory concentrations. In the DPPH assay, the IC50 value of the chloroform extract was determined to be 1.49 mg/mL. The phenolic and flavonoid contents were 26.64 ±  0.1 mg of gallic acid equivalents/g and 8.28  ± 0.1 mg quercetin equivalents/g, respectively. The IC50 value was determined to be 49 µg/mL against the HUVEC line. No mortality or morbidity was observed. GC-MS analysis indicated the presence of 2-cyclopenten-1-one (44.72%) as a major compound. The current results provide scientific support for the use of M. fragrans in folk medicine.


Introduction
The current attention paid to medicinal herbs is attributed to their worldwide availability and use in traditional therapies [1]. Medicinal herbs have been studied for their bioactive secondary metabolites to find alternative sources of antibiotics for pharmaceutical industries and to promote health through the consumption of products containing natural antioxidants. Natural antioxidants have the potential to scavenge free radicals and are effective in preventing diseases caused by oxidative stress [2,3], such as cancer, and neurological and cardiovascular disorders [4].
There is an urgent need for natural therapeutic products with antibacterial and antioxidant activities capable of curing microbial infection and reducing the damaging effects of free radicals [5]. Although the toxicity of most medicinal herbs has not been investigated, it is often believed that extracts derived from natural sources are safer than synthetic compounds [6][7][8]. Among these phytochemicals, phenols and flavonoids display diverse and potent biological activities [9].
Myristica fragrans Houtt., which belongs to the Myristicaceae family, is more commonly known as nutmeg [10]. The fruit, once mature, is separated into the mace (crimson-colored), which surrounds the seed [11]. Nutmeg has been used for many purposes, such as antimicrobial, psychostimulant, antioxidant, antithrombotic [12], insecticidal, or fungicidal agent [13], as well as to aid in poor digestion, insomnia, urinary incontinence [14], arthritis, and muscle spasm [11]. Some reports have highlighted its anticonvulsant, analgesic, anti-inflammatory [15][16][17], antidepressant, and anticancer activities, whereas the seed is reported to control vomiting [13] and have antiinflammatory effects [15]. The seed is also used to treat diarrhea, dysentery, vomiting, and abdominal distension, toothache, and rheumatic pain and is also used in fragrance industries [18].
However, to our knowledge, M. fragrans mace has never been extracted using a sequential solvent of increasing polarity, cytotoxicity on normal cells, and selectivity indices of the promising fraction. Finding new sources of bioactive molecules is of the highest importance, especially for drugresistant microbes.
In the present study, we aimed to further investigate the antimicrobial activity, phytochemical constituents, acute toxicity, and selectivity index (SI) of different solvent extracts of M. fragrans on noncancerous cell lines under in vitro conditions.

Plant material
M. fragrans mace was obtained from Reef al Yamen Co., an herbal shop in Al Morooj District, Riyadh, Saudi Arabia. The plant was authenticated (voucher no. KSU-BRC-136-0) at King Saud University, Riyadh, Saudi Arabia. The mace was ground using a commercial machine. About 20 g of the M. fragrans mace powder was extracted with 450 mL of different solvents (hexane, chloroform, ethyl acetate, and methanol) for 24 h in a Soxhlet extractor. The extracts were centrifuged at 10,000×g, evaporated using a rotary evaporator (Heidolph, Germany), and stored at −80°C.

Total polyphenol content
Total phenol content was assessed using the Folin-Ciocalteu (FC) reagent [19]. Two microliters of the M. fragrans chloroform (MFC) extract was added to 20 µL of the FC reagent (10%) in a 96-well plate. The solution was then pipetted up and down several times and incubated for 10 min at 25°C. Eighty microliters of sodium carbonate (7.5%) were added to the solution and mixed by pipetting up and down several times. The plate was incubated at 25°C for 2 h for color development. The absorbance at 765 nm was measured using a microplate reader (Thermo Scientific Multiskan, China). The phenolic content was calculated as mg/g gallic acid equivalent (GAE) based on the standard curve using the following equation: with an R 2 value of 0.9951.

Total flavonoid content
The total flavonoid concentration was evaluated using aluminum chloride (AlCl 3 ) colorimetric assay [19]. Two microliters of the MFC extract was mixed with 60 µL of methanol, 4 µL of AlCl 3 (10%), 4 µL of potassium acetate, and 112 µL of distilled water in a 96-well plate. The solutions were pipetted up and down several times and then incubated at room temperature for 30 min. The absorbance of the solution was measured at 420 nm. The total flavonoid content was presented as mg/g quercetin equivalent (QuE) based on the standard curve using the following equation: with an R 2 value of 0.994.

2,2-Diphenyl-1-picryhydrazyl (DPPH) radical scavenging assay
The MFC extract radical scavenging potential was assessed in vitro following the method described by Abutaha [19].  Table 1). Methanol (MeOH) was used as a negative control for the antibacterial and antifungal assays.

Culture media
Luria-Bertani agar and potato dextrose agar were employed for the disc and well diffusion assays, whereas Luria-Bertani broth was used to determine the minimum inhibitory concentration (MIC).

Disc and well diffusion assays
The test bacteria and yeast were grown on the Luria-Bertani agar and potato dextrose agar plates, respectively, for 18 h at 36 ± 1°C. Some of the isolated colonies were suspended in 0.9% saline solution, and the optical density at 600 nm was adjusted to 0.01. Fifty microliters of inoculum were swabbed onto agar plates using a sterile cotton swab. For the disc diffusion method, blank discs (6 mm diameter) were loaded with 20 μL of the MFC extract (1 mg/mL, stock solution), for a total dry weight concentration of 20 µg/disc. For the well diffusion method, a sterilized cork borer (6 mm diameter) was employed to form wells that were loaded with 20 μL of the MFC extract. Discs and wells loaded with MeOH served as a negative control. The plates were incubated for 24 h at 36 ± 1°C. The zones of inhibition resulting from treatment with the MFC extract were measured in millimeters. Tests were carried out in triplicate, and the mean values were calculated.

Broth microdilution method
The microdilution method was employed to assess the MICs of the MFC extract using 96-well plates. Briefly, 100 μL of the Luria-Bertani broth was added into each well. The MFC extract (100 μL) was then added to the first column (wells A1 to H1), and a twofold serial dilution was carried out by pipetting the contents up and down in each well of the first column and transferring 100 μL to the next well of the same row, and so on through to the 10th well. The last 100 μL from the 10th well was discarded. Rows 11 and 12 were maintained for positive control (chloramphenicol) and negative control (MeOH), respectively. Then, 100 μL of different microorganism suspensions was added and the plate was incubated overnight at 37°C. Next day, the results were read using a multi-well microplate reader. The lowest concentration of the MFC extract capable of inhibiting visible growth was considered as the MIC.

Synergistic testing
The fractional inhibitory concentration (FIC) index was employed to calculate the synergistic potential between the MFC extract and chloramphenicol against resistance to Staphylococcus epidermidis and MRSA. Antimicrobial activity was measured using the checkerboard assay [20] with chloramphenicol in combination with the MFC extract.
FIC index values <0.5 suggested synergy, those between 0.5 and 0.75 suggested partial synergy, those between 0.76 and 1 suggested an additive effect, and those >2 suggested antagonism.

In vitro cytotoxicity
Compounds were assayed at concentrations of 1,111-0.006 μg/mL against a human umbilical vein endothelial cell (HUVEC) line (ATCC CRL-1730™) to assess potential toxicity in vitro. Cells were grown in Dulbecco's modified Eagle's medium (UFC Biotech, Saudi Arabia) with 10% fetal bovine serum (Gibco, UK) in 5% CO 2 at 37°C. Controls were treated with methanol alone (0.01%). The cells were incubated with the MFC extract in a 96-well plate for 48 h, and then the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent was added (MTT, Invitrogen). Absorbance was read at 540 nm using a multi-well microplate reader (Thermo Scientific Multiskan, China). The number of viable cells were expressed as a percentage of the methanol control.

Selectivity index
SI values of the extract were calculated by dividing the IC 50 (μg/mL) by the MIC (μg/mL). SI values <1 imply that the extracts are safe for use in mammalian cells and toxic to bacteria.

In vivo study
This study was carried out using male Swiss albino mice weighing 28 ± 2 g. The mice were maintained in polyacrylic cages under standard environmental conditions and provided with free access to water and a standard diet. For the acute toxicity test, ten mice were distributed into two groups, each consisting of five mice. Group I (controls) received only phosphate-buffered saline (PBS).
Group II received a single oral dose of 2,000 mg/kg body weight (BW) extract. All procedures were performed according to the guidelines of the National Institutes of Health. The protocol was approved by the Ethics Committee of King Saud University, Saudi Arabia. All procedures were performed with the approval of the Animal Ethics Committee of the King Saud University Zoology Department.

Hemolytic activity (HC)
The HC of the MFC extract was assessed on mouse and human erythrocytes following the modified method of a previous study [21]. A 3% (v/v) suspension of erythrocytes was incubated with various concentrations of the MFC extract in a 96-well plate for 30 min at 37°C. The plates were centrifuged at 3,000 rpm, and the supernatant was aspirated and used to calculate the released hemoglobin at 540 nm. The percentage of hemolysis was expressed compared to the positive control (0.1% Triton X-100); PBS was used as a negative control. Experiments were carried out in triplicate. The hemolysis percentage was calculated using the following equation:

= ×
Hemolysis percentage Absorbance of sample Absorbance of positive control 100.
2.11 Gas chromatography-mass spectrometry (GC-MS) analysis The MFC extract was analyzed via GC-MS (Agilent Technologies, USA) using helium as the carrier gas. The flow rate was 1 mL/min, and the program was as follows: Maintaining at 50°C for 10 min and then heating from 50 to 220°C at a rate of 4°C/min, hold at 220°C for 10 min and then increase to 240°C at a rate of 1°C/min, followed by a final hold at 240°C for 10 min. Identification of compounds was carried out by comparing their mass spectra to the available literature (NIST11).

Total phenol and flavonoid contents
Total phenolic and flavonoid contents were assessed using gallic acid and quercetin standards, respectively. The MFC extract contained a considerable amount of phenols (26.64 ± 0.1 mg GAE/g extract) and flavonoids (8.28 ± 0.1 mg QuE/g extract).

DPPH radical scavenging activity
The scavenging activity of the MFC extract is presented in Figure 1. The MFC extract showed concentration-dependent scavenging activity with an IC 50 value of 1.49 mg/mL. Although the extract showed lower IC 50 value than gallic acid (IC 50 = 81.2 μg/mL), we revealed that the extract does contain compounds that can donate protons to the free radicals.

In vitro susceptibility and synergistic studies
In in vitro susceptibility assays, none of the tested extracts (hexane, ethyl acetate, and methanol) showed antimicrobial activity against tested organisms except MFC extract. Figure 2 revealed that the MFC extract had antimicrobial effects against Gram-positive bacteria and C. albicans ( Table 1) as evaluated by the disc and well diffusion methods, as well as by MIC using the microdilution assay. The inhibition zones measured against the Gram-positive bacteria and C. albicans ranged from 9 to 15 mm at 20 μg extract treatment per disc/well. The MIC results confirmed the results obtained using the disc and well diffusion methods. The MFC extract showed antibacterial activity against Gram-positive strains, with values ranging from 1.56 to 3.12 μg/mL. However, the MIC of the MFC extract against C. albicans was 25 μg/mL. Only resistant bacteria were selected, namely, MRSA and S. epidermidis. The MFC extract resulted in an FIC index value of 1.49 and 1.24 against MRSA and S. epidermidis, respectively, which demonstrated no effect in combination with chloramphenicol ( Table 2).

Cytotoxicity on HUVECs
There was a concentration-dependent decrease in cell survival with almost complete inhibition of proliferation at 1,111 µg/mL; the IC 50 values of the MFC extract are presented in Figure 3. To assess the effect of the extract  on HUVECs, the morphologies were observed with inverted light and fluorescent microscopes after staining with Hoechst. As shown in Figure 3, after treatment with 123.4 µg/mL of MFC for 48 h, the morphology of the cells was altered, and the density of cells was reduced. Cells were found floating, as well as rounded, fragmented, and shrunken. The treated cells showed a decrease in cell count with an increase in the concentration of the extract.

Acute toxicity study
The oral toxicity of the MFC extract was assessed as per OECD guideline 423 [22], where the highest test dose of 2,000 mg/kg BW was administered. No mortality was observed, and toxicity symptoms of the extract-treated mice and control groups were observed first for 6 h and then for 14 days. Treated mice showed no sedation, lethargy, drowsiness, or changes in breathing. Therefore, the extract seems to be safe at a dose of 2,000 mg/kg, and the LD 50 was higher than 2,000 mg/kg.

Hemolytic activity
The HC assay is valuable to assess whether the cytotoxicity is attributed to direct cell membrane damage. Figure 4 shows the toxicity results for mouse and human erythrocytes treated with the chloroform extract. A significant (p ≤ 0.05) variation was detected among the treatments; the positive control (Triton) displayed 100% lysis, whereas PBS displayed no RBC lytic activity. The chloroform extract showed low HC (under 10%) below 3.1 μg/mL. Then, from 6.2 to 50 μg/mL, the HC increased considerably, demonstrating the toxicity of the MFC extract toward mouse erythrocytes. In contrast, the chloroform extract showed HC 50 equal to 220 µg/mL demonstrating lower toxicity of the MFC extract toward human erythrocytes.

Discussion
Many antibiotics have lost their efficiency because of the development of bacterial resistance and the expression of resistance genes [23]. Antibiotics can also be responsible for allergic reactions, immune suppression, and hypersensitivity [24]. Therefore, it is necessary to identify  promising antimicrobial agents for the treatment of infectious diseases.
To survive, plants have developed defense mechanisms against microbes, herbivores, and other plants competing for nutrients, space, and light [25] by producing secondary metabolites that also function in physiology and structural maintenance [25]. As presented in Table 1, the antimicrobial potential of the MFC extract was investigated against Gram-negative and Gram-positive bacteria. In this investigation, the inhibitory effect of the MFC extract was higher than that determined by Shafiei et al. [26] who found lower antibacterial activity of the ethyl acetate and ethanol extracts of mace with MICs ranging from 20 to 40 mg/mL against different oral pathogens. In another study, the MIC values of nutmeg essential oil showed inhibitory effect against S. typhimurium (MIC 1.5 µg/mL), S. kentucky, B. subtilis, E. faecalis, E. durans, E. aerogenes, E. faecium, E. coli, K. pneumoniae, L. innocua, S. enteritidis, P. aeruginosa, S. infantis, and C. albicans, with MIC values ranging from 3.1 to 12.5 µg/mL. However, the extract showed weak activity against S. epidermidis (MIC 100 µg/mL) and no antibacterial activity against S. aureus [27]. In another report, no inhibition was observed for nutmeg seed extract against MRSA, vancomycin-resistant Enterococci, or P. aeruginosa or K. pneumonia; however, it was active against C. albicans and E. coli at 200 μg/mL [14].
The higher antibacterial activity of the MFC extract appeared to be primarily due to its higher content of bioactive compounds. Phenols have received considerable attention because of their scavenging ability [28], antioxidant potential [29], antiviral, antibacterial, anticancer, anti-inflammatory, and anti-allergic activities [30,31]. Plants rich in phenolic compounds are used in the food industry because they lower the risk of health disorders, prolong shelf life, and preserve the nutritional quality of food [32]. The DPPH assay is commonly employed for the investigation of antioxidant potential, and antioxidant extracts that exhibit scavenging activity may contribute to the inhibition of oxidative stress [33]. In our experimental setup, we found a concentration-dependent reduction in DPPH because of the scavenging activities of the MCF extract ( Figure 1). However, the extract was not more effective than the positive control, gallic acid (IC 50 = 1.49 and 81.2 μg/mL, respectively). Results of this research suggested that the radical scavenging capacity of the MFC extract may be due to its content of phenolic acids and flavonoids capable of donating hydrogen atoms. Thus, the therapeutic properties of M. fragrans mace could be attributed to the phenolic content present. Flavonoids constitute a special class of phenolic compounds and are known to scavenge or delay oxidation by shifting a single electron to OH − and O 2˙r adicals [34]. Several reports confirmed the antioxidant activity of different parts of M. fragrans [35,36].
Researchers have used HUVEC lines to assess the cytotoxicity of test samples against normal cells [37,38]. Similar methods have also been utilized in high-throughput tests for drug discovery [39]. The MFC extract showed concentration-dependent inhibition against a non-cancer HUVEC line (IC 50 = 49.18 µg/mL). The United States National Cancer Institute designated that plant crude extracts are safe if the LC 50 is ≥20 µg/mL and cytotoxic if the LC 50 is ≤20 µg/mL [40]. As the MFC extract had an LC 50 of >20 µg/mL, it may be regarded as safe for use.
The SI was assessed for the MFC extract using the MIC for the selected microbes ( Table 2). Generally, lower  SI values reflect that extracts are not suitable for use as antimicrobials owing to metabolic toxic effects [41].
When the SI value is >1, an extract may be considered safer to mammalian cells than to bacteria [41,42]. From the results of this investigation, the MFC extract had an SI value greater than 1 for all selected pathogens, indicating that the antimicrobial extract is not merely composed of by-products of metabolism. The MFC extract can be considered safe if used to treat pathogens when the SI values against those pathogens are greater than 1.
Our results also showed that the MFC extract was safe and that the LD 50 value was higher than 2,000 mg/kg. Our result is in accordance with the study of a previous report that showed no apparent toxicity of M. Fragrans acetone mace extract in rats [43]. In contrast, mice treated orally with mace ethanol extract (0.3 mg/day for 7 days) of M. Fragrans showed a significant increase in creatine phosphokinase level, no change in catalase activities, and showed morphological perturbation in mice's liver [44].
Assessing acute toxicity is an initial experiment that provides a basis for classification and labeling [45]. In our acute toxicity assay, the extract up to 2,000 mg/kg caused no toxicity in the treated mice, and the LD 50 value could not be calculated, as it was larger than the largest dose recommended by OECD guidelines [22] (2,000 mg/kg). No mortality, behavioral, or toxicity symptoms were observed. It was therefore assumed that the LD 50 value of the extract was higher than 2,000 mg/kg and that the extract can be categorized as unclassified material.
Our result was similar to the report of Ali and his group where the HC of the seed essential oil of M. fragrans revealed maximum HC at 10 mg/mL for human erythrocytes [46]. The hemolytic assay is often employed in studies of membrane-active novel xenobiotics and antimicrobial agents. The membrane of the RBC is composed of lipids and proteins. Transmembrane proteins provide the cell structure and mechanical properties and facilitate ion flow, which can have a great impact on the cellular sensitivity to toxic extracts [47]. Plant extracts interact with different cellular systems and may cause cell damage. The purpose of the hemolytic test was to assess the safety of the extract in blood. The hemolytic assay was performed because the extract may have possessed biological HC and thus may not have been suitable for pharmacological formulation. The lysis effect of the MFC extract may be due to a direct effect on the membranes of the RBCs. The membrane of the human RBC is structurally similar to that of mammals such as mice, rats, rabbits, dogs, monkeys, and horses [48,49]. However, the difference between the HC of human and mouse erythrocytes could be attributed to the variation in the composition between species [50]. Further investigation is required to confirm our results using different animal models.

Conclusion
This study provides information on the antimicrobial and toxicological properties of the MFC extract, which is traditionally prescribed by herbalists. The extract inhibited the growth of all Gram-positive bacteria tested, as well as of C. albicans. The SI of the extract was greater than 1. In the assessment of acute toxicity, administration of the MFC extract produced no major toxicological effects after administration of 2,000 mg/kg BW. However, further study is needed to isolate the active compounds, study their biological properties, and assess their safety and efficacy in humans.  Ethical approval: The conducted research is not related to either human or animal use.
Data availability statement: All data generated or analyzed during this study are included in this published article.