Identification of Miscanthus floridulus as a promising anti-Alzheimer’s disease and antidiabetic agent through bioactivity evaluations and chemical composition analyses

2.1 Plant materials

Whole grasses of five species were collected from Anhui Province, China, in July 2022. The grasses were identified as E. crus-galli, M. floridulus, S. fertilis, B. japonicus, or E. indica. (Fig. 1) by Dr. Hui Wang from Anhui Normal University. Voucher samples (EC20220708, MF20220708, SF20220708, BJ20220708, and EI20220708) were deposited in the herbarium of the School of Medicine of Yangzhou University.

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Fig. 1

Pictures of five Poaceae plants. (A) Miscanthus floridulus. (B) Sporobolus fertilis. (C) Bromus japonicus. (D) Eleusine indica. (E) Echinochloa crus-galli.

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2.2 Sample preparation

The whole grasses of E. crus-galli, M. floridulus, S. fertilis, B. japonicus, and E. indica were pulverized into powders and extracted with 95 % EtOH four times at 85 °C for 2 h each time by the hot reflux method to obtain the corresponding crude extracts. For LC–IT–TOF–MS analysis, approximately 10.0 mg of each extract was sonicated for 30 min at 100 Hz, followed by filtration through a 0.22 μm Millipore filter before use. The 18 chemical compounds were weighed and dissolved in 70 % methanol. Then, an assimilated standard sample was obtained by mixing the single standard solutions.

2.3 Chemicals and reagents

Quercetin, luteolin, apigenin, diosmetin, baicalein, and gallic acid were purchased from Must Biotechnology Co., Ltd. (Chengdu, China). Quinic acid, malic acid, trans-5-O-caffeoylquinic acid, 4-hydroxycinnamic acid, isoorientin, rutin, isovitexin, isoquercetin, cynaroside, eriodictyol, naringenin, kaempferide, and emodin were purchased from Pufei De Biotechnology Co., Ltd. (Chengdu, China). α-Glucosidase, acarbose, and α-amylase were purchased from Sigma–Aldrich Chemical Co. (St. Louis, MO, USA). Vitamin C, 2,2-diphenyl-1-picrylhydrazyl (DPPH), and 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were purchased from Bomei Biotechnology Co., Ltd. (Hefei, China). Electric eel AChE, horse serum BChE, acetylthiocholine iodide, S-butyrylthiocholine iodide, and 5′,5-dithio-bis(2-nitrobenzoic acid) (DTNB) were purchased from Yuanye Biotechnology Co., Ltd. (Shanghai, China). Sodium dodecyl sulfate (SDS), phosphate-buffered saline (PBS), and galanthamine were purchased from Solarbio Co., Ltd. (Beijing, China). 4-Nitrophenyl-α-D-glucopyranoside (PNPG) was purchased from Macklin Biotechnology Co., Ltd. (Shanghai, China).

2.4 Determination of the total phenol and total flavonoid contents

The total phenol contents of EEC, EMF, ESF, EBJ and EEI were determined by the Folin–Ciocalteu method (Qneibi et al., 2021). Folin-Ciocalteu solution (2.5 mL, 0.1 mM) was added to 2.5 mL of the sample solution and incubated for 6 min. Then, 150 μL of Na₂CO₃ solution (200 mg/mL) was added to the mixed solution, followed by a reaction at room temperature for 2 h. The absorbance was measured at 765 nm by an EnSpire Multilabel Reader (PerkinElmer). The total phenol content was calculated with reference to a standard curve for gallic acid (Abs = 0.0104 [GA] + 0.037, R² = 0.9993). The results are expressed as gallic acid equivalents (mg GAE/g dry weight of extract).

The total flavonoid contents of EEC, EMF, ESF, EBJ and EEI were determined using a UV–visible spectrophotometer. The sample solution (1 mL) was mixed with 4 mL of 30 % EtOH and 0.3 mL of NaNO₂ (50 mg/mL). After 6 min of incubation, 300 μL of 10 % Al(NO₃)·9H₂O was added, and the mixture was incubated for another 6 min. Then, 4.0 mL of NaOH solution (100 mg/mL) was added to the mixture, and 60 % EtOH was added to reach a volume of 10.0 mL. The absorbance was measured at 510 nm after 30 min. The total flavonoid content of the sample was calculated according to a standard curve generated from freshly prepared rutin solutions of different concentrations, ranging from 10–50 μg/mL (Abs = 0.755 [R] + 0.0435, R² = 0.9996). The results are expressed as rutin equivalents (mg RUE/g dry weight of extract).

2.5 Evaluation of in vitro antioxidant activity

DPPH/ABTS assays were performed to determine the in vitro antioxidant capacities of EEC, EMF, ESF, EBJ and EEI via methods described in previous studies (Xu et al., 2020, Zhang et al., 2021). The samples (100 µL) were mixed with an equal volume of DPPH/ABTS radical solution. After reacting at 37 °C in the dark for 30 min (DPPH) or 5 min (ABTS), the absorbance of each sample was measured at 517 nm (DPPH) or 734 nm (ABTS). The sample and EtOH (both 100 µL) were mixed in the control group. DPPH/ABTS and EtOH (both 100 µL) were mixed in the blank group. The sample concentrations in the DPPH assay were 400, 200, 100, 50, and 25 μg/mL. The concentrations used in the ABTS assay were 2000, 1000, 500, 250, and 125 μg/mL. Vitamin C served as the positive control. The free radical scavenging rates were calculated using the following equation: Free radical scavenging rate = [1 − (OD₁ − OD₂)/OD₃] × 100 % where, OD₁, OD₂, and OD₃ are the absorbances of the sample and DPPH/ABTS, sample and EtOH, and DPPH/ABTS and EtOH, respectively.

2.6 AChE/BChE inhibition assay

The AChE/BChE-inhibitory activity was determined according to methods described previously (Zou et al., 2021, Liu et al., 2022). PBS solution (100 μL, pH 8.0), acetylthiocholine iodide or butyrylthiocholine iodide (20 μL, 1.2 mM), AChE or BChE (20 μL, 0.05 U/mL), and sample solution (20 μL) were added in turn to a 96-well plate and then incubated at 37 °C for 30 min. SDS (20 μL, 4 %) was added to the well to terminate the reaction, followed by the addition of DTNB (20 μL, 0.6 mM). Afterward, the absorbance of each sample was immediately measured at 517 nm. Galanthamine served as the positive control. The inhibition rate of the sample was estimated using the following formula: Inhibition rate = [1 − (OD₁ − OD₂)/(OD₃ − OD₄)] × 100 % where, OD₁, OD₂, OD₃, and OD₄ are the absorbance of the sample, the absorbance of the sample without AChE/BChE, the absorbance of AChE/BChE, and the absorbance in the absence of sample and AChE/BChE, respectively.

2.7 α-Glucosidase inhibition assay

The α-glucosidase assay was performed following protocols described in previous publications (Xu et al., 2020, Liu et al., 2022). Sample solution (24 μL), PBS solution (96 μL, 0.1 mol/L), and α-glucosidase solution (10 μL) were added to the wells. The mixture was reacted at 37 °C for 15 min, and then PNPG solution (30 μL) was added to the mixture. After reacting at 37 °C for another 20 min, the reaction was terminated by adding 80 μL of Na₂CO₃ solution (0.2 mol/L). The absorbance of each sample was measured at 405 nm. Acarbose was used as the positive control. The equation used in the AChE/BChE-inhibitory assays was used to calculate the α-glucosidase inhibition rate.

2.8 α-Amylase inhibition assay

The α-amylase assay was performed following protocols described in previous publications (Meng et al., 2016, Liu et al., 2022). Sample solution (50 μL) and α-amylase solution (50 μL) were mixed in a glass test tube. After reacting in a water bath at 37 °C for 10 min, starch solution (50 μL) was added to the mixture, which was then allowed to react at 37 °C for another 10 min. Afterward, 1 mL of 3.5-dinitrosalicylic acid solution was added to the tube, and the final mixture was reacted in boiling water for 10 min, followed by cooling to room temperature. The absorbance of each sample was measured at 540 nm. The equation for the AChE/BChE-inhibitory assays was used to calculate the percentage of α-amylase inhibition.

2.9 LC–IT–TOF–MS analysis

The analytical system (Kyoto, Japan) was composed of a Shimadzu LC–MS/MS system, which included two LC–20AD_XR pumps, a DGU–20A_3R degasser, an SIL–20AC_XR autosampler, and a hybrid ion trap-time of flight–mass spectrometer (IT–TOF–MS). A Waters Acquity UPLC HSS T3 column (2.1 × 100 mm, 1.8 μm, Milford, MA, USA) was connected to an electrospray ionization (ESI) interface and used for chromatographic separations. The mobile phase consisted of solvents A (0.1 % formic acid in water) and B (acetonitrile) and was used for gradient elution. The elution method was as follows: 0–5 min, 95–85 % A; 5–13 min, 85–72 % A; 13–23 min, 72–5 % A; 23–25 min, 5 % A; and 25–30 min, 5–95 % A. The temperature of the column was controlled at 40 °C, the flow rate was set to 0.2 ml/min, and the injection volume was set to 3 µl.

The following mass spectrometric parameters were used: nitrogen was used as the nebulizing gas and drying gas, with a nebulizing gas flow rate of 1.45 L/min and a drying gas pressure of 100 MPa. Furthermore, a curved desolvation line (CDL) voltage was set at a constant level, with a CDL temperature of 200 °C, a block heater temperature of 200 °C, an interface voltage of 1.40 kV, a detector voltage of 1.45 kV and an IT vacuum of 1.9 × 10⁻² Pa. The collision energy during collision-induced dissociation (CID) was maintained at 50 %, and 100 ms was set as the ion accumulation time. The samples were analyzed in both positive and negative ion modes over a m/z range of 100 to 1000 Da via LCMS solution software (Version 3, Shimadzu).

2.10 Statistical analysis

One-way ANOVA followed by Dunnett’s test was used to compare differences among groups. All the statistical analyses were carried out using SPSS Version 25.0 for Windows (IBM SPSS Inc., Chicago, IL), and P < 0.05 was considered statistically significant. For quantitative analyses, all data are presented as the mean ± S.E.M. Bioactivity and chemical profile experiments were repeated at least 3 times to confirm their reproducibility.

3.1 Total phenol and total flavonoid contents

The results are shown in Table 1. The total phenol contents of the EEC, EMF, ESF, EBJ and EEI ranged from 27.92 ± 0.60 to 74.34 ± 2.84 GAE/g. The order of total phenol content from high to low was as follows: EMF > EBJ > ESF > EEI > EEC. Moreover, the total flavonoid contents of the EEC, EMF, ESF, EBJ and EEI were in the range of 71.63 ± 1.23 to 147.79 ± 3.18 mg RUE/g. The order of total flavonoid content from high to low was as follows: EMF > EBJ > EEI > EEC > ESF. Among all five tested samples, EMF contained the highest phenol and flavonoid contents.

3.2 Antioxidant assays

The antioxidant activity is presented as IC₅₀ values. The smaller the IC₅₀ value is, the stronger the antioxidant activity. According to the data presented in Table 2 and Fig. 2, the IC₅₀ values of the DPPH and ABTS radicals scavenged by EEC, EMF, ESF, EBJ and EEI ranged from 23.18 ± 0.36 to 168.81 ± 2.60 μg/mL and from 261.07 ± 1.51 to 1591.86 ± 54.57 μg/mL, respectively. EMF exhibited the strongest DPPH scavenging ability. In the ABTS assay, the inhibitory activities of EMF and EBJ were almost equal, and EBJ had the strongest ABTS scavenging ability. All the samples scavenged the DPPH radical much more efficiently than the ABTS radical.

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Fig. 2

Multiple activities of five different extracts. (A and B) DPPH and ABTS radical scavenging activities of 95 % ethanol extracts of E. crus-galli (EEC), M. floridulus (EMF), S. fertilis (ESF), B. japonicus (EBJ) and E. indica (EEI). (C and D) AChE and BChE inhibitory activities of EMF, ESF and EBJ. (E) α-Glucosidase inhibitory activity of EMF. (F and G) DPPH and ABTS radical scavenging activities of vitamin C. (H and I) AChE and BChE inhibitory activities of galatamine. (J and K) α-Glucosidase and α-amylase inhibitory activities of acarbose. The data are presented as the means ± S.E.M.s.

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3.3 Inhibitory activity of multiple enzymes

As shown in Table 3 and Fig. 2, EMF and ESF showed AChE-inhibitory activities, and their IC₅₀ values were 49.92 ± 1.44 and 64.52 ± 3.27 μg/mL, respectively. In the BChE assay, EMF, ESF and EBJ exhibited inhibitory activities, and their IC₅₀ values were 100.37 ± 2.48, 155.21 ± 4.34, and 238.42 ± 3.25 μg/mL, respectively. EMF ranked first in both assays. In the α-glucosidase assay, only EMF was found to exhibit inhibitory activity, and its IC₅₀ value was 50.97 ± 2.15 μg/mL. In addition, none of the five tested samples exhibited α-amylase-inhibitory activity.

3.4 LC–IT–TOF–MS analysis of EMF

Among the five samples, EMF had the highest total phenol/flavonoid contents and the strongest DPPH radical scavenging, dural anti-AChE and anti-BChE, and selective anti-α-glucosidase activities. The LC-IT-TOF-MS technique was employed to characterize the chemical profile of the EMF. Fig. 3 shows the total ion chromatograms (TICs) of EMF. The identification process for chemical compounds in EMF involved the following steps: (1) extraction of ion features from the raw LC-IT-TOF-MS data for each ion peak in TICs; (2) tentative characterization of the chemical compounds by comparing their ion features, retention times, and mass spectra with those of known chemical compounds in mass bank databases and relevant literature; and (3) to further confirm some identified compounds, their LC-IT-TOF-MS data was compared with those of their corresponding standard chemicals. As a result, a total of 44 compounds were identified, including 13 flavones, 8 flavonols, 5 flavanones, 2 other flavonoids, 13 organic acids, 2 anthraquinones and 1 benzopyran. Among these 44 compounds, 18 were confirmed through chemical standards. Notably, 21 compounds (xanthoxyletin, 3-hydroxy-3′-methoxyflavone, 5,7,2′,6′-tetrahydroxyflavanone, orientin, fisetin, vitexin, 5,7,3′,5′-tetrahydroxyflavanone, flavanomarein, isoquercetin, morin, swertisin, anthraglycoside B, naringenin chalcone, dalbergioidin, eriodictyol, rhamnetin, diosmetin, baicalein, 5,6,7-trihydroxy-3′,4′-dimethoxyflavone, kaempferide and emodin) were first identified from the genus Miscanthus, and 41 compounds (quinic acid, gluconic acid, xanthoxyletin, 3-hydroxy-3′-methoxyflavone, cis-aconitic acid, trans-aconitic acid, trans-3-O-caffeoylquinic acid, trans-5-O-caffeoylquinic acid, trans-4-O-caffeoylquinic acid, trans-3-O-feruloylquinic acid, trans-5-O-p-coumaroylquinic acid, 5,7,2′,6′-tetrahydroxyflavanone, trans-4-O-caffeoylshikimic acid, trans-5-O-feruloylquinic acid, orientin, 4-hydroxycinnamic acid, isoorientin, fisetin, rutin, vitexin, isovitexin, 5,7,3′,5′-tetrahydroxyflavanone, flavanomarein, isoquercetin, morin, swertisin, anthraglycoside B, naringenin chalcone, dalbergioidin, apigenin-8-C-pentose, tricin, eriodictyol, rhamnetin, luteolin, naringenin, apigenin, diosmetin, baicalein, 5,6,7-trihydroxy-3′,4′-dimethoxyflavone, kaempferide and emodin) were first identified from M. floridulus. The structures of these identified compounds are depicted in Fig. 4, and their detailed identification information can be found in Table 4. The MS/MS chromatograms of five typical identified compounds can be found in Fig. 5.

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Fig. 3

Typical total ion chromatograms (TICs) of EMF by LC–IT–TOF–MS. (A) positive mode; (B) negative mode.

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Fig. 4

Chemical structures of the 44 identified compounds.

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Fig. 5

MS/MS chromatograms of five typical identified compounds. (A) Cynaroside. (B) Rutin. (C) Eriodictyol. (D) Quinic acid. (E) Emodin.

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