The Potential Antioxidant Activity and Characterization of Bioactive Compounds of Stahlianthus involucratus

Stahlianthus involucratus (S. involucratus) has anti-inflammatory, antinociceptive, and antipyretic activities; however, there are no literature reports on its antioxidant capacity. This study presents a comparative assessment of the polyphenols contents, flavonoids contents, and antioxidant activity of the aqueous and methanol extracts of S. involucratus (ASI and MSI). Moreover, the expression of oxidative stress-related genes in H2O2-induced H9c2 cells pretreated with the MSI was measured by RT-qPCR, and furthermore, MSI were characterized by UHPLC-Q-Orbitrap-MS/MS. The results indicated that the MSI had higher antioxidant contents and antioxidant capacity, and MSI could inhibit H2O2-induced oxidative stress in H9c2 cells by activating the Nrf2/HO-1 pathway. UHPLC-Q-Orbitrap-MS/MS characterized 15 phenolic compounds from the MSI. In conclusion, S. involucratus has the potential antioxidant capacity.


Introduction
S. involucratus, belonging to Zingiberaceae [1], as a Traditional Chinese Medicine (TCM), was mainly used for the treatment of bruises and rheumatic arthralgia [2]. The plants in Zingiberaceae family were enriched with bioactive compounds; the previous reporters had confirmed the phytochemical and pharmacological properties of Zingiberaceae [3]. The ethanol extracts of S. involucratus had anti-inflammatory, antinociceptive, and antipyretic activities [4,5]. The extraction of Elettaria cardamomum, Curcuma longa, Zingiber officinale, and Alpinia officinarum had the scavenging activity of the cationic ABTS radical and ferric-reducing antioxidant power-FRAP [6]. The standardized extraction from the rhizomes of ginger defended a rat liver from cancer by decreasing oxidative and inflammatory injury [7]. The reporter verified that cardamonin from the Alpinia plant inhibits oxidative stress, apoptosis, and inflammatory responses in the mouse heart by improving NRF2 signaling [8]. Excessive secretion of free radicals causes oxidative damage to cells and tissues, induces oxidative stress, and further leads to inflammatory responses and damages the immune system [9]. Nuclear factor-related gene 2 (NRF2), a transcription factor, is thought to be the dominant factor in the regulation of cellular oxidative stress [10]. The activated NRF2 binds to antioxidant response elements (ARE) to regulate downstream genes and then defend against damage caused by oxidative stress.
Oxidative stress, a negative effect of free radicals in the body, is associated with various diseases, including inflammatory response, cell damage, tissue sclerosis, and cancer [11]. Natural plant extracts are rich in bioactive components such as phenols, flavonoids, and glycosides. Modern research has demonstrated that all these compounds have antioxidant, anti-inflammatory, and antitumor activities and can act directly on inflammatory and tumor cells without affecting the viability of normal cells [12,13].
This study presents a comparative assessment of the polyphenols contents, flavonoids contents, and antioxidant activity of ASI and MSI, and furthermore, the expression of Nqo1, Ho-1, Gclc, Gclm, Gst, and Nfe212 in H 2 O 2 -induced H9c2 cells pretreated with the MSI was examined by RT-qPCR, and then the phytochemical compounds from MSI were isolated and characterized by UHPLC-Q-Orbitrap-MS/MS.

ASI Preparation.
Thirty grams of dried powder with 600 mL distilled water was decocted at 100°C for half an hour in a water heater, and the mixture was then filtered with a gauze. The supernatant was obtained by centrifugation. Repeat the above extraction procedure. The supernatants consolidated were dried by a freeze-drying method and stockpiled in the refrigerator at -20°C.

MSI Preparation.
Thirty grams of dried powder with 600 mL methanol was extracted for an hour in an ultrasonic bath, and the mixture was then filtered with a filter paper. Repeat the above extraction procedure. The consolidated supernatants were concentrated and dried by a freezedrying method and stockpiled in the refrigerator at -20°C.

Physicochemical and Antioxidant Analysis
2.3.1. Estimation of Total Phenolic Content (TPC). The solution, a mixture of 25 μL of the sample solution and 25 μL of Folin's reagent, was kept at 25°C for 5 min in the dark, and next, 25 μL of 10% (w : w) sodium carbonate solution and 200 μL distilled water were added. The absorbance was detected at 765 nm using a UV spectrophotometer. The total phenolic content was expressed as μg gallic acid equivalents per gram extract (μg GAE/mg of extract).

Estimation of Total Flavonoid Content (TFC).
The solution, a mixture of 80 μL of the sample solution, 80 μL 2% (w : w) aluminum chloride solution, and 100 μL sodium acetate (50 g/L), was kept at 25°C for 2.5 h in the dark. The absorbance was detected at 415 nm using UV spectrophotometer. The total flavonoid content was expressed as μg quercetin equivalents per gram extract (μg QE/mg of extract).

ABTS Analysis.
The ABTS reaction solution consisting of the potassium persulfate (0.7 mg/L) and the ABTS (4 mg/L) at 1 : 1 was kept at 25°C for 18 h in the dark. The solution, a mixture of 10 μL of the sample extraction and 290 μL of ABTS reaction solution, was added to 96-well plates and incubated at 25°C for 15 min. The absorbance was detected at 734 nm using a UV spectrophotometer.
The results were expressed as μg ascorbic acid equivalents per gram extract (μg AAE/mg of extract).

DPPH Analysis.
The solution, a mixture of 40 μL of the aqueous extraction and 260 μL of DPPH reaction solution (40 mg/L), was added into the plates and incubated at 25°C for 30 min. The absorbance was detected at 517 nm using a UV spectrophotometer. The results were expressed as μg ascorbic acid equivalents per gram extract (μg AAE/mg of extract).

Cell
Culture. The H9c2 cell line was purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). The H9c2 cells were cultivated within the DMEM (Thermo Fisher Scientific, MA, USA) containing 10% fetal bovine serum (FBS, Thermo Fisher Scientific, MA, USA), 100 μg/mL streptomycin, and 100 U/mL penicillin. Afterward, cells were subjected to incubation under 37°C and 5% CO 2 conditions. When cells reached confluence, PBS was used to wash cells for two times and the serum-free medium was used for further cell culture prior to the subsequent experiment.
2.5. Cell Viability. The cell viability was determined by the MTT method. In 96-well plates, the cells were grown in the complete medicated DMEM medium (0, 1, 5, 25, 50, 100, 250, 500, and 1000 μg/mL of MSI) at 37°C with 5% CO 2 for 24 h, 10 μL MTT (5 mg/mL) was added into the plates; next, the plates were incubated for 4 h. Adding the 150 μL DMSO after the medium was waived. The microplate reader (Thermo Varioskan LUX, MA, USA) was used to measure the absorbance (OD) value at 490 nm.
2.6. Assay of ROS Production. Cultured H9c2 cells were treated with indicated agents for 16 h, and the intracellular ROS generation was determined by measuring the oxidative conversion of cell permeable DCFH-DA to fluorescent dichlorofluorescein. Images were viewed by a fluorescence microscope (Leica, Heerbrugg, Germany) at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

Total RNA Isolation and Quantification.
In real-time PCR experiment, the cells were seeded in a 6-well plate and the optimal cell number is 1 × 10 6 cells per well. H9c2 cells were incubated with 200 μM H 2 O 2 at 37°C with 5% CO 2 for 4 h and then incubated the complete medicated DMEM medium (25,50, and 100 μg/mL of MSI) at 37°C with 5% CO 2 for 12 h. Total RNA was obtained by a Trizol method; further the purity and concentration of total RNA were determined. The expression of target genes was 2 BioMed Research International measured using Real-time Quantitative PCR (RT-qRCR) after the total RNA was reverse-transcribed to cDNA. The housekeeping gene was Gapdh gene. The sequences of the target genes are listed in Table 1. The results were analyzed using the 2 -ΔΔCt method.

Metabolomics Profiling by UHPLC-Q-Orbitrap-MS/MS
Technique. In the present literature, the compounds from S. Involucratus, including ergotane dimer, sesquiterpenes, cardanane dimer, and alkaloids, were isolated and characterized by UPLC-MS [14,15]. Ultrahigh performance liquid chromatography (UHPLC) coupled with an electrospray ionization tandem mass spectrometry (ESI-MS-MS) is a common technique for the isolation and identification of natural products [16]. The Q-Orbitrap, a high-resolution mass spectrometer, allows molecular weight and fragment information. UHPLC-Q-Orbitrap-MS/MS are used to identify the metabolites in herbal medicines [17,18]. The compounds in TCM were characterized by combining parent ions with fragment ions. These means were extensively applied to phytochemical analysis as a result of accuracy, efficiency, and celerity. MSI were analyzed by UHPLC-MS/MS spectrum consisting of Thermo Scientific™, Ultimate™ 3000RS, Thermo Scientific™, Q Exactive™, Hbrid Quadrupole-Orbitrap mass spectrometer, ESI source, and RP-C18 column (150 × 2:1 mm, 1. The condition of the mass spectrometer was as follows: scan range 150-2000 m/z; aux gas heater temperature 350°C; capillary temperature 300°C; spray voltage 3.8 kV; and sheath gas pressure 40 Arb. The aux gas and sheath gas were high purity nitrogen gas (purity ≥ 99:999%), and the collision gas was high purity argon gas (purity ≥ 99:999%). Full-mass and dd-MS 2 data in positive and negative modes were acquired at 70,000 and 17,500 FWHM (full width half maximum), respectively.

Statistical
Analysis. The results were tested by one-way analysis of variance (ANOVA); P < 0:05 was regarded as significant and evaluated with Tukey's test by SPSS 20.0 software. The data acquired by UHPLC-MS/MS spectrum was processed by CD2.1 (Thermo Fisher Scientific), and then the compounds were characterized according to precursor ions and fragment ions compared with the chemical database like mzCloud, mzVault, and ChemSpide.

Results and Discussion
3.1. Estimations of TPC and TFC. The antioxidant capacity can be roughly determined by binding estimations of polyphenols and antioxidant assays. The aluminum trichloride method and Folin-Ciocalteu method were currently used for the determination of total flavonoids and total phenols, and the results of TFC and TPC were expressed as quercetin equivalents (QE)/g of dried extraction and gallic acid equivalents (GAE)/g dried extraction, respectively. Table 2 points out that the TPC and TFC of the MSI were 110:24 ± 1:01 mg GAE/g dried extraction and 12:30 ± 0:10 mg QE/g dried extraction, respectively, and were significantly higher than those of the ASI.

Antioxidant Activities (DPPH, ABTS, and FRAP).
There were many experiments to evaluate antioxidant activity in vitro. In this study, the DPPH, ABTS, and FRAP assays were used to validate the antioxidant activity of S. involucratus. Both DPPH and ABTS assays were detected by binding antioxidants to colored ionic free radicals, causing the color to fade. However, the FRAP assay was detected by the reduction of chelated ions by antioxidants, resulting in a color reaction. In short, a comprehensive analysis of the three assays can determine initially the antioxidant capacity. As described in Table 2, in the ABTS, DPPH, and FRAP assays, the result showed that the free radical scavenging capacity of the MSI was 89:62 ± 1:65, 21:09 ± 0:34, and 30:60 ± 0:27 mg TE/g dried extraction, respectively, and was higher than that of the ASI (P < 0:05), which indicated that the MSI had stronger antioxidant activity.

The MSI Activated Nrf2/HO-1 Pathway after H 2 O 2 -Induced H9c2
Cell. First, the cell viability was assessed and MSI showed no toxicity at the concentration ranging from 0.001 to 1 mg/mL, excluding the possibility that MSI influenced phenotypes via cytotoxicity (Figure 1(a)). Further, we evaluated the effects of MSI on ROS production by determining the ROS levels in vitro using commercial assay kit (Figure 1(b)). As anticipated, MSI effectively reduced ROS generation in cardiomyocytes, demonstrating its oxidative stress suppressing action.
ROS are the driving force for myocardial damage, and Nrf2 is a key orchestrator of the cell responses to oxidative stress and exerts a protective effect against oxidative damage. According to this, the expression of Nqo1, Ho-1, Gclc, Gclm, Gst, and Nfe212 was measured after H 2 O 2 -induced H9c2 cells treated with different concentrations of the MSI (25, 50, and 100 μg/mL) and shown in Figure 2(a)-2(f). For Gclc, Ho-1, Nqo1, and Nfe212 mRNA, the expression level was significantly increased in the administration group (25,50, and 100 μg/mL) compared to the model group (P < 0:05). Furthermore, compared with the model group, 100 μg/mL of MSI significantly increased the expression of Gclm, and 50 and 100 μg/mL of MSI significantly increased the expression of Gst (P < 0:05).  Figure 3. Fifteen compounds, whose score was higher than 80, were tentatively identified, and the relevant information is presented in Table 3.
A total of 13 phenols were tentatively detected.

Discussion
Oxidation is closely related to human diseases, and oxidation-induced damage is the most fundamental  BioMed Research International pathological process of the disease, so the exploration of natural antioxidants is vital [19]. Phenols, as natural antioxidant substances, scavenge free radicals by blocking the transmission process of free radicals and inhibit oxidative reactions by indirectly preventing the production of oxidative radicals complexing enzymes related to free radicals or metal ions [20]. This experiment provided a preliminary cognition of the antioxidant properties of S. involucratus.
In the comparison of the total polyphenols and total flavonoid contents of the ASI and MSI, the results showed that there are higher total polyphenol and total flavonoid contents in the MSI. Besides, in the ABTS, DPPH, and FRAP assays, the MSI has stronger free radical scavenging power and reduction capacity than the ASI. In addition, the extracts of natural products effectively inhibit cell damage by suppressing oxidative stress [21][22][23], and the Nrf2/HO-1 signaling pathway plays an important role in mitigating oxidative stress damage to cells and tissues [24], and HO-1 is one of the important endogenous protective enzymes of downstream of the Nrf2/HO-1 pathway [25], and the expression of HO-1 can inhibit oxidative reactions and thus protect cells and tissues from free radical damage [26]; the modern research had shown that natural product extracts are effective against H 2 O 2 -induced oxidative stress by activating Nrf2/HO-1 pathway [27,28]. In this study, MSI effectively reduced ROS generation in cardiomyocytes, demonstrating its oxidative stress suppressing action, and the expression of the regulatory genes (Nfe212, Hmox-1, and Nqo1) has increased significantly after H 2 O 2induced H9c2 cells pretreated with MSI; furthermore, the expression levels of Gclm, Gclc, and Gst mRNA have elevated. These results indicated that the MSI could inhibit oxidative stress by activating the Nrf2/HO-1 pathway.

Conclusion
In short, the results demonstrated that the antioxidants contents of MSI were more abundant than those of ASI, and the MSI inhibits H 2 O 2 -induced oxidative stress in H9c2 cells by activating the Nrf2/HO-1 pathway. These findings give guidance for the clinical use of S. involucratus.

Data Availability
The data used to support the findings of this study are included within the article.

Conflicts of Interest
The authors declare no conflict of interest.