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Sesquiterpenoids of diverse types from the rhizomes of Acorus calamus

Six new and fourteen known sesquiterpenoids of diverse types were isolated from the rhizomes of Acorus calamus.

Our previous study on the 95% aqueous ethanol extract of A. calamus reported nine new sesquiterpenoids (calamusins A-I) and their bioactivities. 16 During the continued investigation, six new (1)(2)(3)(4)(5)(6) and fourteen known sesquiterpenoids of diverse types were characterized. The isolation and structure elucidation of these compounds are described herein, as well as the in vitro neuroprotective assays of the new compounds (Fig .1).

Results and discussion
Calamusin L (1) was assigned the molecular formula C 15 (Table 3) and DEPT spectra of 1 exhibited 15 carbon resonances corresponding to four methyls, two methylenes, two methines (one aliphatic, d C 35.3; one oxygenated, d C 74.7), and seven quaternary carbons (two oxygenated ones at d C 85.2 and 80.1; an aliphatic one at d C 51.9; two olenic ones at d C 149.2 and 146.1; an a,b-unsaturated keto carbonyl group at d C 200.8; an saturated keto carbonyl group at d C 213.7). Analysis of 1 H-1 H COSY spectrum revealed the presence of two discrete spin systems (Fig. 2) corresponding to a -CH-CH 2unit (C9-C10) and a CH 3 -CH-CH 3 moiety (C12-C11-C13, an isopropyl group). The HMBC correlations (Fig. 2) were displayed from H-14/10/6 to C-5/4, Me-15 to C-7/8/9, and Me-13/Me-12 to C-1, allowing the establishment of an acorane-type sesquiterpene skeleton, which exists extensively in genus Acorus. 16,30 Furthermore, NOESY correlations ( Fig. 2) (Fig. 2). Additionally, coupling constant of Me-15 (1.2 Hz) in the 1 H NMR spectrum and the weak cross signal of 1 H-1 H COSY spectrum between Me-15 and H-3 suggested an allylic system of C15-C4-C3 is present in the structure. The HMBC correlations (Fig. 2) from Me-15 to C-5, H-8 to C-10, H-9 to C-1/14, and H-3 to C-1 suggested C-5 is linked with C-4, C-10 is linked with C-9, C-10/ Me-14 are linked with C-10, and C-1 is linked with C-2. The above data, similar to those of tatarinowin C, 32 suggested that compound 2 possessed a cadinane-type sesquiterpene carbon skeleton. Analysis of the chemical shis and degrees of unsaturation indicated that C-1, C-5, and C-10 are all linked with  hydroxy groups. The combined analysis of coupling constant and NOESY spectrum allowed the determination of the relative conguration of 2. The rather small coupling constant (3.5 Hz) between H-5 and H-6 indicated that the two protons are cisoriented (b-oriented). Furthermore, the NOESY correlations ( Fig. 2) of H-5 with H-11/Me-13 suggested the isopropyl group is b-oriented, and H-7 is a-oriented. In addition, the NOESY correlations of H-7 with 1-OH/Me-14 suggested 1-OH and Me-14 are both a-oriented. According to the octant rule of n / p* transition of a,b-unsaturated cyclohexanone unit, the absolute conguration of 2 was determined to be 1S,5S,6R,7S,10S on the basis of the positive Cotton effect observed in the CD spectrum at 343 nm (D3 +1.03). 33 Calamusin N (3) was assigned the molecular formula C 15 (Fig. 2) of 3 suggested the presence of C9-C8-C7-C11(C12)-C13 and C1-C2-C3 structural moieties. Additionally, the observed HMBC (Fig. 2) correlations between H-15 and C-4/C-3, H-5 and C-4/C-15, H-11 and C-6, H-8 and C-10, and Me-14 and C-1/9/10 suggested C-4 is linked with C-3/5/15, C-6 is linked with C-7, C-10 is linked with C-9, and C-10 is attached to C-1 and C-14, allowing the establishment of a cadinene-type sesquiterpene carbon skeleton. The NOESY spectrum showed correlations (Fig. 2) of H-5 and H-11, H-7 and H-8a (d 1.52), H-8a and Me-14, Me-14 and H-1, which indicated 5-OH/Me-14/H-1 are a-oriented, the isopropyl group at position 7 and 10-OH are b-oriented. As the structural analysis, H-5 and H-7 should be closely spatially if 6-OH was aoriented, but the correlation was not appeared between the above two in the NOESY spectrum, indicating 6-OH is boriented.
Determination of the absolute conguration of 3 was implemented by comparison of the experimental and calculated specic optical rotation (SOR) of the two stereoisomers. The SOR of each conguration of 3 was calculated using DFT theory method at the pbe1pbe/aug-cc-pVTZ level. Finally, the calculated SOR of (1S,5R,6R,7S,10S)-3 ([a] D +41.9) had the same trend as the experimental vale ([a] D +23.4, MeOH), while the calculated data of (1R,5S,6S,7R,10R)-3 ([a] D À42.8) had the opposite sign to the experimental value, indicating the absolute conguration of 3 was 1S,5R,6R,7S,10S.
The molecular formula of calamusin O (4) was determined to be C 15 (Table 3) and DEPT-135/90 spectra displayed 15 signals corresponding to four methyl groups, three methylenes, three methines (two   (Fig. 2
Calamusin Q (6)  A resonance corresponding to an oxygen-bearing methine was also observed [d H 4.14 (m), H-3] in 1 H NMR spectrum. In addition, it showed resonance signals assignable to several aliphatic methylenes and methines between d H 2.05 and 3.00. 13 C NMR (Table 3) and DEPT-135/90 spectra showed 17 carbon signals corresponding to ve methyl groups, four methylenes (an oxygenated one at d C 60.7), two methines (an oxygenated one at d C 81.1), and six quaternary carbons (a ketone carbonyl group at d C 207.7; an ester carbonyl group at d C 173.1; a double band at d C 151.1 and 101.5; an oxygenated one at d C 74.7). Additionally, the olenic carbon C-11 must be linked with an oxygen atom according to its chemical shi (d c-11 151.1). The cross signals observed in 1 H-1 H COSY spectrum indicated the presence of structural units of C16-C17, C8-C7-C10, and C2-C3. The HMBC spectrum displayed cross peaks of Me-13 and C-14/12/ 11, H-16 and C-9, H-8 and C-1, H-7 and C-2/6, Me-15 and C-3/ 4/5, -OH and C-4, H-6 and C-4/C-5, and H-6 and C-11 indicating Me-13/Me-14 is attached to C-12, the ethoxyl group is linked with C-9, C-1 is linked with C-7/C-2/C-6, C-4 is linked with C-3/5/15, the hydroxyl group is linked with C-4, C-5 is linked with C-6, and C-11 is attached to C-1. Furthermore, the HMBC correlation from H-3 to C-11 and the chemical shis of C-3 (d C 81.1) and C-11(d C 151.1) indicated that an oxygen bridge connected C-3 and C-11.
The NOESY correlation between the protons of Me-13 and Me-15 suggested Me-15 and the oxygen bridge are on the same side of the cyclohexanone unit, then the hydroxyl group and C-7 are on the other side. A positive Cotton effect occurred at 302 nm (D3 +0.65) in CD spectrum indicated the absolute congurations of the chiral centers at the cyclohexanone unit are 1S,3R,4S, according to the n / p* transition of saturated cyclopentanone. 31 According to the empirical isoprene rule, compound 6 can't be considered as a sesquiterpene. However, as our conjecture, the biogenic precursors of 6 is a previously reported acorane type sesquiterpenoid, calamusin D, 16 in which a series of oxidation, dehydration, oxidative cleavage, and ethylation reactions occurred to yield compound 6 (Fig. 4). As this conjecture, the absolute conguration of C-7 in compound 6 should be S, the same as C-4 in compound calamusin D. This was supported by NMR chemical shis calculations of both the possible isomers 6a (1S,3R,4S,7S) and 6b (1S,3R,4S,7R) using the GIAO method at the mPW1PW91/6-311G(d,p) level in acetone. The calculated 1 H and 13 C NMR chemical shis of the two possible isomers were compared with the experimental values of 6 by utilizing DP4+ probability analysis, which showed that 6a was predicted to be correct with probabilities of 100% for the 1 H NMR data, 93.4% for the 13 C NMR data, and 100% for their combination. The absolute conguration of 6 was thus proposed to be 1S,3R,4S,7S.
The known compounds were identied by comparison of their spectroscopic data with reported data. They were hedytriol (7), 34 , (À)-1b,4b,7a-trihydroxyeudesmane (8), 35 oplodiol (9), 36 6eudesmene-1b,4b-diol (10), 37 4b,5a,10b-trihydroxycadinan (11), 38 43 1,10-seco-4x-hydroxy-muurol-5-ene-1,10-diketone (19), 44 and 2-hydroxyacorenone (20). 45 Compounds 1-6 were evaluated for their neuroprotective effect on PC12 cells induced by serum withdrawal, rotenone, and OGD (Oxygen and Glucose Deprivation) in vitro with MTT method. As shown in Table 4, compounds 1 and 6 increased the cell survival rate of the OGD-treated group moderately, while compounds 3 and 5 decreased the cell survival rate of the serum withdrawal-treated PC12 cells moderately, and none of the compounds showed activity in the rotenone-treated models at 10 mM. Few investigations have been taken previously about neuroprotective activity and the corresponding mechanisms of sesquiterpenes from A. calamus. However, a study by Li et al. 19 indicated the sesquiterpenes, calamusin D (with a same carbon skeleton of compound 1) and acoric acid (with a same carbon skeleton of compound 6), showed signicant cell proliferation activity on the human neuroblastoma cell line SK-N-BE (2), suggesting the both possessed neuroprotective activity. Therefore, more sesquiterpenes of diverse types, such as acorane-and 1,2-secoacorane-types, should be evaluated for their neuroprotective activity, and what's more, the action mechanisms and structure-activity relationships should be investigated in the future to discover the drug leads to treat neurodegenerative diseases.

Plant material
The rhizomes of A. calamus were purchased from a Chinese herb store in Anguo county, Hebei Province, China, and authenticated by Professor Lin Ma, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College. A voucher specimen (no. ID-S-2281) has been deposited at the Herbarium of the Institute of Material Medica,  p < 0.01 vs. control; *p < 0.05, **p < 0.01 vs. model. structures were averaged as described above, and DP4+ probability analysis was facilitated using an Excel sheet (DP4+) provided by Grimblat et al. 48 Neuroprotective assays The in vitro neuroprotective activity was tested by the method described previously. 49,50 Conclusions Six new sesquiterpenes, named calamusins L-Q (1-6), were isolated from the rhizomes of A. calamus, together with fourteen known ones. The new compounds and their absolute congurations were determined based on extensive spectroscopic analyses and computational methods. It was hypothesized that compound 6 was a secosesquiterpene, which was formed from calamusin D during the biosynthesis aer a series of oxidation, dehydration, oxidative cleavage, and ethylation reactions. Furthermore, compounds 1-6 were tested for their neuroprotective effects, and it was observed that compounds 1 and 6 increased the cell survival rate of the OGD-treated PC12 cells moderately, while compounds 3 and 5 decreased the cell survival rate of the serum withdrawal-treated PC12 cells moderately.

Conflicts of interest
There are no conicts to declare.