Chemical Constituents of the Egg Cases of Tenodera angustipennis (Mantidis ootheca) with Intracellular Reactive Oxygen Species Scavenging Activity

As a traditional medicine with potential antioxidant effects, Tenodera angustipennis egg cases (Mantidis ootheca) are a potential source of new bioactive substances. Herein, three new N-acetyldopamine derivatives, namely, (+)-tenoderin A (1a), (−)-tenoderin A (1b), and tenoderin B (2), along with thirteen known compounds (3–15), were isolated from a 70% EtOH extract of T. angustipennis egg cases. Compound 1 was isolated as a racemic mixture, and two enantiomers (1a and 1b) were successfully separated by chiral-phase preparative HPLC. The chemical structures of the new compounds were established by NMR spectroscopy and high-resolution electrospray ionization mass spectrometry, and the absolute configurations of enantiomers 1a and 1b were determined by electronic circular dichroism spectroscopy. All the new compounds exhibited antioxidant activities with IC50 values of 19.45–81.98 μM, as evaluated using free-radical scavenging assays, with the highest activity observed for compound 2. In addition, compounds 1a, 1b, and 2 exhibited inhibitory activities on intracellular reactive oxygen species generation.


Introduction
Mantidis ootheca, which refers to egg cases of the Mantidae family, such as Tenodera angustipennis Saussure, Hierodula patellifera Serville, Statilia maculate Thunberg, and Tenodera sinensis Saussure, has been used as a source of traditional medicines in East Asia, including Korea and China [1]. Mantidis ootheca has been used in traditional medicines to treat incontinence, lumbago, spermatorrhea, acidosis, renal failure, and leukorrhea [2], and has been reported to have various biological activities, including anti-inflammatory, antidiuretic, anticancer, and antioxidant activities, as well as vascular relaxant effects [3][4][5][6]. Although various pharmacological studies on Mantidis ootheca have been reported, only a few studies have investigated its chemical constituents. The chemical constituents of Mantidis ootheca have been revealed to include fatty acids and phenols that exhibit pharmacological properties, such as antibacterial, antibiofilm, antioxidant, and anti-atherosclerotic effects [7][8][9]. Notably, previous studies have indicated that commercially available Mantidis ootheca products contain the egg cases of several mantis species within one package [10]. As such mixtures may be used for research, the exact identification of research samples is of particular importance.

Insect Material
The insect material (Mantidis ootheca) was purchased from a medicinal herb company (Gwangmyeongdang Co., Ulsan, Korea) in July 2019, authenticated, and deposited in the Korean Herbarium of Standard Herbal Resources (Index Herbarium code KIOM, specimen no. 2-20-0223) at the Korea Institute of Oriental Medicine. To prepare the sample for this study, the egg cases of T. angustipennis were identified and selected based on key morphological characteristics, such as shape, color, texture, and angle of the distal end [10]. All materials had a fusiform shape, a tapered distal end with angles of 22 • -50 • , and a lusterless and brownish textured external wall ( Figure S16).

Computational Methods
ECD calculations were performed for 1a and 1b. A conformer distribution was constructed using the Spartan'14 software (Wavefunction, Inc., Irvine, CA, USA) with a Merck molecular force field. The conformers were optimized at the DFT [B3LYP/6-31+G(d,p)] level, and ECD calculations were performed at the TDDFT (CAM-B3LYP/SVP) level with a CPCM solvent model in MeOH using the Gaussian 09 software (Gaussian, Inc., Wallingford, CT, USA).

DPPH and ABTS Radical Scavenging Activities
The DPPH (Sigma-Aldrich, St. Louis, MO, USA) and ABTS (Sigma-Aldrich) freeradical scavenging activities were measured according to previous studies [12,13]. All measurements were independently repeated three times. The DPPH and ABTS scavenging activities were calculated as follows: DPPH or ABTS scavenging activity (%) = [A(free radical) − A(standard)]/A(free radical), where A is the absorbance value of DPPH at 517 nm or ABTS at 734 nm.

Detection of Intracellular ROS
The HUVECs used in the ROS scavenging experiments were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in α-MEM medium, and 20% FBS in a humid atmosphere of 5% CO 2 at 37 • C.
To confirm the antioxidant effect of each compound against oxidative stress, the cells were treated with each sample at a concentration of 5, 10, 50 or 100 µM and hydrogen peroxide (1 mM), cultured for 24 h, washed with PBS, and treated with 100 µM DCFDA (ThermoFisher, Waltham, MA, USA) for 30 min at 37 • C. Reacted. After DCFDA was removed, and the amount of DCFDA remaining in the cells was observed using a fluorescence microscope. The fluorescence intensity at an excitation wavelength of 485 nm and an emission wavelength of 520 nm was measured using a microplate reader.
The antioxidant activity of the extract, as measured using DPPH and ABTS radical scavenging assays, was significant (81.99 ± 1.98% and 99.74% ± 0.13%, respectively, at 100 μg/mL) (Table S1). Furthermore, all the isolated compounds were screened for antioxidant activity at a concentration of 100 μM using DPPH and ABTS radical scavenging assays. DPPH and ABTS radical scavenging activities were observed for compounds 1a, 1b, 2, 3, 4, and 9 (Table S1). In particular, new compounds 1a, 1b, and 2 displayed antioxidant activities with IC50 values between 19.45 and 81.98 μM. Among the new compounds, the antioxidant activity of compound 2 was the highest. In addition, although compounds 1a and 1b were enantiomers, there was a difference in their antioxidant activities (Table 2). Compound 1b exhibited stronger antioxidant effects than 1a, and the ratios of their IC50 Compound 2 was obtained as a brown gum, and its elemental formula was determined to be C 17 H 17 NO 5 using HRESIMS analysis. The 1D NMR data revealed the presence of a N-acetyldopamine (3) moiety as well as a 1,4-disubstituted benzene ring (δ H 7.68 (2H, d, J = 8.2 Hz, CH 3 -2 and 6 ) and 6.84 (2H, d, J = 8.3 Hz, CH 3 -3 and 5 ); δ C 134.3 (C-2 and 6 ) and 116.2 (C-3 and 5 )) and a ketone group (δ C 199.1 (C-7 )) ( Table 1). A detailed analysis of the NMR data showed that compound 2 was similar to periplanetol A [15], except for the absence of a hydroxy group at C-5 and the presence of an aromatic methine signal (δ H 6.84, H-5 ) for compound 2. These results are supported by the COSY correlations between H-5 /H-6 and the HMBC correlations between H-5 / C-4 (δ C 164.0), C-3 (δ C 116.2), and C-1 (δ C 131.3) (Figure 2). Accordingly, the structure of new compound 2 was elucidated, and this compound was given the trivial name tenoderin B.
The antioxidant activity of the extract, as measured using DPPH and ABTS radical scavenging assays, was significant (81.99 ± 1.98% and 99.74% ± 0.13%, respectively, at 100 µg/mL) (Table S1). Furthermore, all the isolated compounds were screened for antioxidant activity at a concentration of 100 µM using DPPH and ABTS radical scavenging assays. DPPH and ABTS radical scavenging activities were observed for compounds 1a, 1b, 2, 3, 4, and 9 (Table S1). In particular, new compounds 1a, 1b, and 2 displayed antioxidant activities with IC 50 values between 19.45 and 81.98 µM. Among the new compounds, the antioxidant activity of compound 2 was the highest. In addition, although compounds 1a and 1b were enantiomers, there was a difference in their antioxidant activities (Table 2). Compound 1b exhibited stronger antioxidant effects than 1a, and the ratios of their IC 50 values were 1:1.8 (DPPH) and 1:1.3 (ABTS). These results suggest that the difference in antioxidant activity between the two enantiomers is due to the chirality of C-8 . In addition, to determine whether the antioxidant activities were associated with protective effects in H 2 O 2 -treated human umbilical vein/vascular endothelium cells (HU-VECs), intracellular 2 ,7 -dichlorofluorescin diacetate (DCFDA) levels were measured. Oxidative-stress-induced cell damage has been implicated in various types of disease. H 2 O 2 is a major ROS produced intracellularly during pathological processes and causes oxidative injury. H 2 O 2 has been extensively used as an inducer of oxidative stress for in vitro models [27]. Therefore, H 2 O 2 was selected to promote oxidative stress in the current investigation. In this study, as shown in Figure 5, H 2 O 2 significantly increased the intracellular DCFDA level. In this assay system, compound 2 showed a potent antioxidant activity in a dose-dependent manner and compounds 1a and 1b also showed significant antioxidant activity, which presented the identical activity pattern supporting the results in the free-radical scavenging assay.
Biomolecules 2021, 11, x FOR PEER REVIEW 8 of 10 values were 1:1.8 (DPPH) and 1:1.3 (ABTS). These results suggest that the difference in antioxidant activity between the two enantiomers is due to the chirality of C-8ʹ. In addition, to determine whether the antioxidant activities were associated with protective effects in H2O2-treated human umbilical vein/vascular endothelium cells (HUVECs), intracellular 2′,7′-dichlorofluorescin diacetate (DCFDA) levels were measured. Oxidative-stress-induced cell damage has been implicated in various types of disease. H2O2 is a major ROS produced intracellularly during pathological processes and causes oxidative injury. H2O2 has been extensively used as an inducer of oxidative stress for in vitro models [27]. Therefore, H2O2 was selected to promote oxidative stress in the current investigation. In this study, as shown in Figure 5, H2O2 significantly increased the intracellular DCFDA level. In this assay system, compound 2 showed a potent antioxidant activity in a dose-dependent manner and compounds 1a and 1b also showed significant antioxidant activity, which presented the identical activity pattern supporting the results in the free-radical scavenging assay.

Conclusions
In conclusion, this study on the chemical constituents of T. angustipennis egg cases (Mantidis ootheca) revealed sixteen compounds, including three new N-acetyldopamine derivatives (1a, 1b, and 2) and thirteen known compounds (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). Two enantiomers (1a and 1b) were successfully separated by chiral-phase preparative HPLC, and the absolute configurations were determined by ECD spectroscopy. All the isolated compounds were evaluated for antioxidant activity, and the new compounds appeared to show antioxidant effects in HUVECs. These findings not only reveal various chemical constituents in the egg cases of T. angustipennis, they also provide guidance for clarifying the pharmacodynamic basis of the antioxidant effects of Mantidis ootheca.