Anti-Helicobacter pylori, Anti-Inflammatory, Cytotoxic, and Antioxidant Activities of Mace Extracts from Myristica fragrans

The aril (mace) of Myristica fragrans, known as Dok-Chan, is a spice that has long been used for treating stomach discomfort, peptic ulcer, and nausea. It is an ingredient in many remedies in Thai traditional medicine, e.g., Ya-Hom-Thep-Bha-Jit, Ya-Hom-Nao-Wa-Kot, and Ya-That-Bun-Job, which are used to treat dyspepsia and other gastrointestinal tract symptoms. The aqueous and ethanolic extracts of mace were used for all tests. Anti-H. pylori activities were determined by the disc diffusion method and agar dilution. Anti-inflammatory activity was determined by the LPS-induced nitric oxide (NO) inhibition in a RAW264.7 cell line, and cytotoxicity was determined against gastric cancer cell lines (Kato III) using the sulphorhodamine B (SRB) assay. The DPPH radical scavenging and ABTS radical cation decolorization assays were used to determine the antioxidant activities. The result found that the ethanolic extract of mace exhibited antimicrobial activity against H. pylori ATCC 43504 and six clinical strains with MIC values of 125–250 μg/ml. The aqueous extract MICs against H. pylori ATCC reference strain and six clinical strains were 500 μg/ml compared with 0.5 μg/ml for the positive control, clarithromycin. The inhibitory effect of LPS-induced NO release and cytotoxic activity of the ethanolic extract had IC50 values of 82.19 μg/ml and 26.06 μg/ml, respectively, and the EC50 values for the DPPH and ABTS antioxidant assays were 13.41 μg/ml and 12.44 μg/ml, respectively. The mace extract also had anticancer properties. In conclusion, the ethanolic mace extract had anti-H. pylori, anti-inflammatory, antioxidant, and anticancer activities. These data support further preclinical and clinical investigation to see if the mace extract could have a role in treating patients with dyspepsia, peptic ulcers, and possibly gastric cancer.


Introduction
e common gastrointestinal (GI) conditions include acid reflux, dyspepsia, nausea, vomiting, peptic ulcer, abdominal pain, flatulence, diarrhea, elicobacter pylori (H. pylori) infection, and gastrointestinal cancer [1]. Inflammation is the initial immune reaction in response to cells against pathogens, chemicals, and foreign antigens. NO plays a central role in the regulation of cytokines in the acute inflammatory response and cancer [2]. Increased level of cytokines and oxidants also promote chronic inflammation that can cause DNA damage and the development of cancer [3]. In GI patients, H. pylori infection causes gastritis and gastric ulcer which predispose to gastric cancer [4][5][6].
Myristica fragrans (MF) is an evergreen tree whose fruit is used to produce mace (aril) and nutmeg. Both are used commonly for treating stomach discomfort, peptic ulcer, flatulence, and anxiety. Mace and nutmeg are components of many remedies in ai traditional medicine, e.g., Ya-Homep-Bha-Jit, Ya-Hom-Nao-Wa-Kot, and Ya-at-Bun-Job, which are used to treat gastrointestinal symptoms [7]. Moreover, they are also used for the same properties in ayurvedic, Chinese medicine, and Tibetan traditional medicine [8,9].
Nutmeg has antioxidant, antimicrobial, antidiabetic, cytotoxic, anti-inflammatory, and antidepressant activities [10][11][12][13][14][15][16]. By contrast, there are few data on the properties of mace, which is well known as Dok-Chan in ailand. Bhamarapravati et al. [17] reported that dihydroguaiaretic acid (DGA) had antimicrobial activity against H. pylori. Neolignans and phenolic compounds, present in the methanolic extract of mace, showed inhibitory effects on mediators in the inflammatory pathway and antiproliferative effects [18]. DNA barcoding of M. fragrans (mace) was investigated for the identification of M. malabarica adulteration [19]. e safety and efficacy of mace powder combined with pelvic floor muscle training were assessed in a placebo-controlled trial in patients with urinary incontinence. ose receiving mace had greater improvement compared to the placebo group [20].

Plant Materials and Preparation of the Extracts.
Dried arils of Myristica fragrans (Mace) were purchased from a herbal shop in January 2017 in Nakhon Pathom province, ailand. Further botanical identification was conducted at the Department of Applied ai Traditional Medicine, Faculty of Medicine, ammasat University. A voucher specimen was deposited in the Herbarium of the Southern Center of Traditional Medicine, Faculty of Pharmaceutical Sciences, Prince of Songkla University, ailand (SKP 121 13 06 01).
Mace was washed, dried, and grounded before extraction. e aril powder was boiled with water for 30 min and filtered using Whatman paper # 1 and then put in the freezer and dried by a lyophilizer to obtain the aqueous extract. For maceration, the powder was soaked with 95% EtOH for three days, filtered and evaporated twice, and then soaked three times to obtain the ethanolic extracts. Both aqueous and ethanolic extracts were refrigerated at− 20°C before use.

Determination of Anti-H. pylori Activity by Disc Diffusion
Method and Agar Dilution Technique. Helicobacter pylori ATCC 43504 and six isolates from patients were cultured on Columbia agar supplemented with 5% sheep's blood and incubated at 37°C under microaerobic conditions (N 2 , 85%; O 2 , 5%; CO 2 , 10%) using a gas-generating kit. H. pylori were subcultured every 3 days in an anaerobic jar. e agar disc diffusion technique followed the method by Ogata et al. [21]. Filter paper discs (6 mm in diameter) were impregnated of the extracts (conc. 1 mg/disc). Airdried discs were placed onto the inoculated Columbia 5% sheep blood agar and incubated at 37°C under microaerobic conditions for 3 days. e triplicate inhibition zones (clear zone) were calculated by measuring the diameter (mm). Clarithromycin (conc. 10 μg/ml) was used as the positive control.
e minimum inhibitory concentration (MIC) was determined by the agar dilution method following the Clinical and Laboratory Standards Institute (CLSI) guidelines [22]. e extracts were serially diluted 2-fold in Columbia agar containing 5% sheep's blood and then transferred separately into Petri dishes. e final concentrations of the extracts in the culture medium ranged from 31.25 to 500 μg/ml. A 72 h bacterial colony of H. pylori was harvested and suspended in brain heart infusion (BHI). Bacterial suspensions were prepared to approximately 2.0 of the McFarland standard. Bacterial suspension (3 μl) per spot was replicated on each plate followed by incubating at 37°C for 72 h under microaerobic conditions. e MIC was defined as the lowest concentration of extract which resulted in no visible growth.

Determination of Anti-Inflammatory Activity by Nitric
Oxide-Inhibitory Effect. Mouse macrophage leukemia-like cells (RAW264.7, ATCC TIB-71 TM) were induced by lipopolysaccharide (LPS) to release inflammatory mediators including nitric oxide. e Griess reagent was used to determine nitrite, which is a stable end product of NO in cell culture supernatants. e inhibitory effect on NO 2 Evidence-Based Complementary and Alternative Medicine production was evaluated using the method of Dechayont et al. [23]. Cytotoxicity was also determined using the MTT method. e supernatant in 96-well plates was detected at a wavelength of 570 nm. e percentage of inhibition was calculated using the following formula: (1) IC 50 values were calculated form the % inhibition of concentrations using the PRISM program.

Determination of Cytotoxic Activity by Sulphorhodamine B (SRB)
Assay. Cytotoxic activity was tested using the SRB assay following the method by Dechayont et al. [24]. Gastric cancer cell lines, Kato III, were cultured in the RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin with CO 2 incubator at 37°C. e monolayer of cell culture in the flask was trypsinized, and the density was provided 5 × 10 3 cells/well for testing. After various concentrations of extracts (1, 10, 50, and 100) μg/ml were seeded into the 96 well-plate, they were then placed in the same condition. e cells were dyed with SRB, and the viability of the cells was assessed at a wavelength of 492 nm. e percentage of inhibition was calculated by the following formula: [(abs. control − abs. sample)/abs. control] × 100.

Determination of Antioxidant Activities by DPPH Radical Scavenging Assay and ABTS Radical Cation Decolorization
Assay. DPPH radical scavenging activity was determined according to the modified method by Yamasaki et al. [25]. DPPH solution in absolute ethanol was freshly prepared before use and protected from light. e extracts were prepared in various concentrations (1, 10, 50, and 100 μg/ ml). A portion of the sample solution 0.1 ml was mixed with the DPPH solution (ratio 1 : 1) in 96-well plates and protected from light for 30 minutes at room temperature, and the absorbance was measured at 520 nm using a spectrophotometer.
e ABTS radical cation decolorization assay followed the method by Re et al. [26]. e solution was produced by reacting 7 mM ABTS stock solution in distilled water with 2.45 mM potassium persulfate to produce the ABTS •+ solution. e extracts were examined by the ABTS assay. A portion of the sample solution 0.02 ml was mixed with ABTS •+ solution (ratio 1 : 10) in 96-well plates and kept in the dark. e reaction was carried out for 6 min, and the absorbance was measured at 734 nm. BHT was used as a positive control in both antioxidant assays.

Statistical Analysis.
All experiments were carried out in triplicate and presented as mean ± SEM (standard error of the mean).

Preparation of the Extracts.
e aqueous extract (WMACE) and ethanolic extract (EMACE) yields were 6.74% and 28.60%, respectively. In ailand, nutmeg and mace are commonly combined together and used to be ingredients in the folk medicines which are not only oral administrative drugs but also inhalation drugs. e properties of them recorded in ai transcripts are quite similar to increasing blood circulation and balancing the digestive system [27]. e major compounds (85%) of chemical constituents of nutmeg and mace are volatile oils which is myristicin [28]. Nutmeg was reported in several activities including antioxidant, antimicrobial, antidiabetic, cytotoxic, anti-inflammatory, and antidepressant activities [10][11][12][13][14][15][16]. An adverse effect of myristicin in nutmeg on the central nervous system and psychiatric effect which include hallucinations and delusions are the harmful risks and should be made aware [29]. Recently, an overdose of powdered nutmeg was reported in one case study [30]. Although the FDA has no regulations regarding nutmeg, a suggested limit for powdered nutmeg usage is not more than one teaspoon (approx. 5 g) per day [31,32]. So, the researchers should find other compounds which would also show efficacy in pharmacology activities.

Anti-H. pylori Activity by Disc Diffusion Method and Agar
Dilution Technique. By disc diffusion, the anti-H. pylori activity of the mace ethanolic extract, EMACE, was greater than WMACE for the ATCC reference strain and six clinical strains ( Figure 1); the MICs for the H. pylori ATCC 43504 isolate were 125 vs. 500 μg/ml for EMACE and WMACE, respectively (Table 1). Only the EMACE had activity against the CS24 strain with an MIC value of 250 μg/ml. ere are some research studies on chemical constituents and biological activities of mace [33]. Dihydroguaiaretic acid (DGA) was isolated from dried mace which was studied for anti-H. pylori activity [17]. e results showed that the diameter of the inhibition zone is 33.7 mm at the concentration 200 μg/ ml. Minimum inhibitory concentration (MIC) of DGA against the clinical strains was in range of 100-125 μg/ml [17]. Interestingly, EMACE showed effectiveness exhibited against four clinical strains and ATCC reference strain similar to DGA used in a previous study (MIC values of 125 μg/ml) [17]. ree compounds showed strong cytotoxic activity in an SRB assay against HT-29 colon cancer cell lines [18]. Moreover, Cuong et al. isolated the phenolic compounds from ethyl acetate soluble fraction of nutmeg. Malabaricone C showed strong inhibitory effect on LPS-induced production of NO in RAW264.7 cells and exhibited the LPS-induced COX-2 and iNOS expressions [15]. In addition, malabaricone C was also found from the organic-soluble extract from Myristica malabarica and Myristica cinnamomea (Myristaceace family), known for antimicrobial and strong scavenging activities [31,34]. Banerjee et al. reported a healing activity of malabaricone C against the indomethacin-induced gastric ulceration in mice, and it reduced the ulcer indices better than the positive control, omeprazole with 88.4% and 86.1%, respectively [35]. Other research studies evaluated maceneolignans A, verrucosin, and malabaricone C which were isolated from the methanol extract of mace and inhibited the release of β-hexosaminidase in rat basophilic leukemia cells (RBL-2H3) and also inhibited antigen-stimulated tumor necrosis factor-α production [36]. A decoction of pods of Cassia fistula Linn. as well as another decoction of arils of Myristica fragrans Houtt both significantly reduced pain for a greater length of time than mefenamic acid did in patients with dysmenorrhea. ese decoctions both improved patients' quality of life without causing any side effects [37].

Cytotoxic Activity against Gastric Cancer Cell Line (Kato III) by Sulphorhodamine B (SRB) Assay.
e results indicated that only EMACE exhibited significant in vitro cytotoxic activity against Kato III gastric cancer cells (IC 50 � 26.06 μg/ ml). e WMACE had no significant cytotoxicity (IC 50 > 100 μg/ml). Notably, several studies found that isolated malabaricone B and C were more effective than regular full-spectrum EMACE whilst some compounds were not [38]. An organic solvent such as methanol, chloroform, and acetone was commonly used for extraction in various studies. None of them extracted the mace based on traditional medicinal usage and demonstrated their GI properties. us, our research focused on approving the efficacy of   mace for GI treatment in ai traditional medicinal usage. is is a first report of mace extracts studied on gastric cancer cell lines. As EMACE showed to be effective in all activities, it is possible that the organic solvent can extract an active compound from mace better than water decoction. ese reports can support the potential of mace being used as the components of preparation for gastrointestinal symptoms treatment.

Antioxidant Activities by DPPH Radical Scavenging Assay and ABTS Radical Cation Decolorization Assay.
Antioxidant activities for EMACE and WMACE at concentrations of 100 μg/ml showed similar results with inhibition values of 85.11% and 72.96% in the DPPH radical scavenging assay.
e ethanolic extract showed greater antioxidant activities vs. BHT, the positive control, and the EC 50 values were 13.41 μg/ml and 16.02 μg/ml, respectively. In the ABTS assay, EMACE showed a higher percentage of inhibition at 100 μg/ml, whereas WMACE showed lower than 50%. EC 50 values are shown in Figure 2. From previous research, the acetone extract of mace exhibited a higher antioxidant activity in the DPPH assay compared to the TLC isolated fractions. On the other hand, all band fractions showed high radical scavenging in ABTS activities [39]. In the same way, the organic extract in our research (EMACE) showed a significant antioxidant activity in DPPH assay and ABTS assay when compare with WMACE.

Conclusion
e results revealed that the ethanolic extract, EMACE, was dominantly more effective in anti-H. pylori, anti-inflammatory, and cytotoxic activities than the aqueous extract, WMACE. Antioxidant activity of EMACE was slightly greater than WMACE. Our research supported the efficacy of using a mace for GI treatment. In future, it would be interesting to study experimentally induced ulcers and cytotoxicity mechanisms and point out the bioactive phytochemicals of mace ethanolic extract of the mace ethanolic extract. According to ai traditional medicine, mace is a component in the herbal preparations; however, the synergistic effect of the herbal combination in the preparation should be considered.
Data Availability e datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper. Evidence-Based Complementary and Alternative Medicine 5