Novel phenylpropanoids and isoflavone glycoside are isolated and identified from the carob pods (Ceratonia siliqua L.)

Abstract Two new phenylpropanoids (1 and 2) and one new isoflavone glycoside (3), along with nine known compounds (4 − 12), were isolated from the pod of Ceratonia siliqua L. Their chemical structures were elucidated based on extensive spectroscopic analyses (1 D and 2 D NMR, UV, IR, and HRESIMS) and compared with the literature data. In addition, all isolated compounds were evaluated in vitro for inhibitory activity against acetylcholinesterase (AChE). Compounds 4, 5, and 12 showed inhibitory activity against acetylcholinesterase (AChE) with IC50 values ranging from 15.0 to 50.2 μM. Graphical Abstract


Introduction
The dried bean pod of Ceratonia siliqua L., also called carob tree belonging to the genus Leguminosae (Fabaceae) family, is an evergreen tree mainly distributed in Mediterranean region, such as Italy, Spain, Morocco and Portugal (Stavrou et al. 2018). The mature pod of C. silique having the length of 10-25 cm consists of about 10% seeds and 90% pulp, and which has been found to contain various constituent, including carbohydrates (40-60%), dietary fibers (27-50%), polyphenols, protein, lipids and minerals, etc. (Rtibi et al. 2017). The categories of polyphenols being identified from C. silique are including phenolic acids, flavonoids, tannins, lignans and stilbenes. The pods can be included as animal feed. Due to its high content of sugar and no toxicological sign, the pod of C. silique is commonly employed in food industry as locust bean gum, and which is also consumed as candy for children in ancient times or in emergency situations, e.g., war (Owen et al. 2003). The roasted and ground pods are having similarity in flavor to cocoa, and therefore the pod is often being included as a chocolate or cocoa substitute. The production is about 315,000 tons per year in the world.
In addition, the pod of C. silique has been employed as folk medicine in the treatment of diarrhoea, heartburn, vomiting, obesity, high cholesterol and pregnancy conditions (Custodio et al. 2011). The pharmacological properties of C. silique pod have been proposed in anti-inflammatory, antioxidant, anti-microbial, anti-ulcer, anti-diarrheal and anti-diabetic, anti-depressant, etc (Agrawal et al. 2011;Cust odio et al. 2015;Rtibi et al. 2017). Several lines of evidence have shown multiple pharmacological activities of C. silique are linking to polyphenols (Owen et al. 2003). In order to discover the biologically active ingredients, the objective of the present study was to investigate the chemical constituents of C. silique pod. Two new phenylpropanoids and one new isoflavone glycoside, together with nine known compounds, were identified. Herein, the isolation and structural elucidation of the isolated compounds, as well as their inhibitory activity against acetylcholinesterase (AChE), were described.
Compound 2, yellow powder, was determined to access to the molecular formula C 21 H 30 O 10 , as deduced from positive-ion HRESIMS (m/z 465.1983 [M þ Na] þ ), with 7 indices of hydrogen deficiency (Supplementary material, Figure S9). The 1 H and 13 C NMR data (Supplementary material, Table S2) of 2 were similar to those of syringin  (Kiem et al. 2003) with the exception of an extra isobutyryl group. Analysis of its 1 D and 2 D NMR data indicated that the hydroxy attached to C-9 in syringin was replaced by isobutyryl group in 2. This deduction was verified by HMBC correlations (Supplementary material, Figure S15) from H 3 -3 0 to C-1 0 /C-4 0 , H-2 0 to C-1 0 /C-4 0 , H 2 -9 to C-1 0 . The measurement of the optical rotation value of 2 exhibited the presence of Dglucose. The relative configuration of D-glucose was determined as b-type based on the coupling constant of glucose terminal hydrogen d H 4.89 (1H, d, J ¼ 7.8 Hz, H-1 00 ). Thus, the structure of 2 was established as 9-isobutyryl syringing.
Compound 3 was obtained as yellow powder, with the molecular formula of C 23 H 24 O 11 as determined on the basis of the positive-ion HRESIMS (m/z 499.1266 [M þ Na] þ ) (Supplementary material, Figure S16), demonstrating 12 indices of hydrogen deficiency. The 1 H and 13 C NMR data (Supplementary material, Table S3) exhibited characteristic resonances of isoflavone skeleton, like 4 0 ,6-dihydroxy-7,8-dimethoxyisoflavone (Wang et al. 2015). The main differences were that a set of glucose residue group was connected to C-6 in 3 instead of hydroxyl, which was supported by the HMBC correlations (Supplementary material, Figure S22) from H-5 to C-4/C-6/C-7/C-8/ C-9 and H-1 00 to C-6 as well as NOESY correlation (Supplementary material, Figure S23) of H-1 00 with H-5. Besides, the O-methyl group connected to C-7 and hydroxy attached to C-4 0 in 4 0 ,6-dihydroxy-7,8-dimethoxyisoflavone was replaced by hydroxy and Omethyl group in 3, respectively. This deduction was verified by the HMBC correlations from H-5 to C-7 and H-2 0 /H-6 0 /OCH 3 -4 0 to C-4 0 (Supplementary material, Figure S22). The measurement of optical rotation value of 3 revealed the presence of D-glucose. The relative configuration of D-glucose was determined as b-type based on the coupling constant of glucose terminal hydrogen d H 5.09 (1H, d, J ¼ 7.8 Hz, H-1 00 ). Thus, the structure of 3 was established as 7-hydroxy-8,4 0 -dimethoxyisoflavone-6-O-b-Dglucopyranoside.
Compounds 1-12 were evaluated in vitro for their inhibitory activities against AChE by a modified Ellman's method. BW284C51 (10 lM) was used as a positive control, and AChE inhibition rate was $61.8%. Compound 5 exhibited robust AChE inhibition having an IC 50 value at $15.0 lM, and compounds 4 and 12 showed mild AChE inhibition having IC 50 values at $50.2 lM and $45.7 lM, respectively. The results were shown in Figure S24 in Supplementary material.

Plant material
The pod of C. siliqua was collected from Republic of Cyprus. The plant material was identified by one of the authors (Dr. Tina Ting-Xia Dong) according to their morphological characteristics. The voucher specimens (CL2018101) were deposited in Shenzhen Key Laboratory of Edible and Medicinal Bioresources, HKUST SRI, China.

AChE inhibition assay
The AChE inhibitory activity of the compounds was determined by the slightly modified Ellman's method (Ellman et al. 1961). In brief, 20 lL of test chemical were added to the reaction mixture with 20 lL of 0.05 U/mL AChE solution (from rat brain lysate) and 20 lL of 0.6 mM 5,50-dithiobis-2-nitrobenzoic acid (DTNB) in 96-well microplates, pH 7.4. The reaction was initiated by adding 20 lL of 1.2 mM ATCh and then quenched with addition of 20 lL of 4% sodium dodecyl sulfate (SDS) after incubation at 37 C for 30 min. Absorbance at 405 nm was recorded. BW284C51 was used as positive control. The inhibition rate (%) was calculated as following equation: Inhibition rate (%) ¼ [1 À (OD S À OD E )/OD C ] Â 100. (The OD S and OD C are, respectively, the optical density with and without the test compounds, and OD E is the optical density without AChE). Inhibition potency of the test compounds was expressed as IC 50 value, in which all assays were repeated in triplicate.

Conclusion
In this study, two new phenylpropanoids (1 and 2) and one new isoflavone glycoside (3), along with nine known compounds (4 À 12), were isolated from the pod of Ceratonia siliqua L. Among all these compounds, Compounds 4, 5, and 12 showed different levels of inhibitory activity against AChE with IC50 values of $50.2, $15.0 and $45.7 lM, respectively.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported by Special project of Foshan University of science and technology in 2019 (FSUST19-SRI10) and Shenzhen Science and Technology Innovation Committee (ZDSYS201707281432317; JCYJ20170413173747440; JCYJ20180306174903174).