Three New Phthalide Glycosides from the Rhizomes of Cnidium officinale and Their Recovery Effect on Damaged Otic Hair Cells in Zebrafish

The extract from Cnidium officinale rhizomes was shown in a prior experiment to markedly recover otic hair cells in zebrafish damaged by neomycin. The current study was brought about to identify the principal metabolite. Column chromatography using octadecyl SiO2 and SiO2 was performed to isolate the major metabolites from the active fraction. The chemical structures were resolved on the basis of spectroscopic data, including NMR, IR, MS, and circular dichroism (CD) data. The isolated phthalide glycosides were assessed for their recovery effect on damaged otic hair cells in neomycin-treated zebrafish. Three new phthalide glycosides were isolated, and their chemical structures, including stereochemical characteristics, were determined. Two glycosides (0.1 μM) showed a recovery effect (p < 0.01) on otic hair cells in zebrafish affected by neomycin ototoxicity. Repeated column chromatography led to the isolation of three new phthalide glycosides, named ligusticosides C (1), D (2), and E (3). Ligusticoside C and ligusticoside E recovered damaged otic hair cells in zebrafish.


Introduction
Cnidium officinale Makino (Apiaceae) is a perennial flowering plant extensively cultivated in East Asian countries [1]. The rhizomes of C. officinale have been used traditionally in Korea, China, and Japan to treat female sexual disorders such as oligomenorrhea, hypomenorrhea, and amenorrhea by improving blood circulation, but also to relieve pain and inflammation [2,3]. Extracts of Cnidium officinale rhizome are reported to have antioxidant [4], anti-inflammatory [5,6], anticancer [5], blood circulation improvement [7], analgesic [6], anticonvulsive [8], and sedative [9] effects. A variety of components were identified as the major metabolites of Cnidium officinale rhizome, such as phthalides, phenyl alkanoids, triterpenoids, and polyacetylenes, etc. Among them, phthalide derivatives are the most important components and are reported to be key materials participating in several pharmacological activities of C. officinale rhizome [10]. Our advance experiment showed that the n-BuOH fraction from C. officinale rhizome recovered otic hair cells in zebrafish damaged by neomycin treatment. In a subsequent study presented herein, repeated column chromatography (CC) for the n-BuOH fraction yielded three new phthalide glycosides, the chemical structures of which, including stereostructures, were determined Molecules 2021, 26, 7034 2 of 8 without ambiguity based on several spectroscopic data, that is, NMR, IR, MS, and circular dichroism spectroscopy (CD). Only a few glycosides have been isolated from plants. However, the phthalide glycosides with a double bond at C-6 and C-7 in the aglycone moiety have never been reported, to date. The phthalide glycosides were also evaluated for their recovery effect on damaged otic hair cells.

Structure Determination of Three New Phthalide Glycosides
Aqueous EtOH was used to obtain an extract of the rhizomes of Cnidium officinale, and the concentrate was fractionated using EtOAc, n-BuOH, and H 2 O. The n-BuOH fraction (Fr) was treated using Sephadex LH-20, silica gel (SiO 2 ), and octadecyl SiO 2 (ODS) as the stationary phase for column chromatography, resulting in the isolation of three new phthalide glycosides. Their chemical structures, including their absolute stereochemistry, were revealed based on MS, IR, NMR, and CD data.
To date, lots of phthalides have been isolated from Cnidium officinale or Angelica gigas. However, less than 10 phthalide glycoside have been reported. Among them, most glycosides have a double bond at C-7 and C-8 or at C-8 and C-9. Ligusticosides C-E isolated in this study are the first occurrence for phthalide glycosides to have a double bond at C-6 and C-7 in the aglycone moiety.

Recovery Effects for the Extract, Solvent Fractions, and Compounds 1-3 on Otic Hair Cells in Zebrafish Damaged by Neomycin Treatment
The recovery effects of the phthalide glycosides 1-3 on otic neuromast hair cells exposed to neomycin were evaluated. Treatment with the ototoxic drug severely damaged the hair cells, which significantly decreased in number (p < 0.001). Ligusticoside C (1) and ligusticoside E (3) significantly recovered the damaged hair cells (p < 0.01), while ligusticoside D (2) exhibited an effect without statistical significance ( Figure 3). Therefore, a phthalide glycoside-enriched fraction can be a promising source to prevent or treat the auditory pathological symptoms.  from the chemical shift in CMR. The characteristics of the stereostructure were revealed to be the same as those of compound 1 via the same methods as used previously. Therefore, the chemical structure, including the absolute stereochemistry, of compound 3 was determined as shown in Figure 2, and it was named ligusticoside E. Compound 2, a white powder, showed UV absorption at 254 and 365 nm and a deep brown color on TLC upon spraying with 10% sulfuric acid and heating. MF was determined to be C23H36O12 (calcd for C23H37O12, 505.2281) from a PM at m/z 505.2285 [M + H] + in HR-ESI-MS. The IR bands showed absorbance bands at 3356 (hydroxyl), 1747 (ester), and 1600 and 1456 cm −1 (double bond). From PMR and CMR, we concluded that the planar structure was the same as that of 3. In the NOESY spectrum of compound 2 (Figure 1), H-3 (δH 4.57) showed a correlation with H-8 (δH 3.60) and H-9 (δH 2.78-2.74), while H-10 (δH 2.18-2.12 and δH 2.05) showed a correlation with not H-4 (δH 4.15) but H-3, proving the relative stereostructure. The aglycone of compound 2, 2a, obtained by acid hydrolysis, exhibited the negative Cotton effect at 250 nm (Δε −1.66) and the positive Cotton effect at 218 nm (Δε 1.29), indicating the n-butyl group at C-3, the hydrogen at C-9, and the hydroxyl group at C-4 to have α-, β-, and β-positions, respectively [12,13]. Therefore, the chemical structure, including the absolute stereochemistry, of compound 2 was determined as shown in Figure 2,, and it was named ligusticoside D.
To date, lots of phthalides have been isolated from Cnidium officinale or Angelica gigas. However, less than 10 phthalide glycoside have been reported. Among them, most glycosides have a double bond at C-7 and C-8 or at C-8 and C-9. Ligusticosides C-E isolated in this study are the first occurrence for phthalide glycosides to have a double bond at C-6 and C-7 in the aglycone moiety.

Recovery Effects for the Extract, Solvent Fractions, and Compounds 1-3 on Otic Hair Cells in Zebrafish Damaged by Neomycin Treatment
The recovery effects of the phthalide glycosides 1-3 on otic neuromast hair cells exposed to neomycin were evaluated. Treatment with the ototoxic drug severely damaged the hair cells, which significantly decreased in number (p < 0.001). Ligusticoside C (1) and ligusticoside E (3) significantly recovered the damaged hair cells (p < 0.01), while ligusticoside D (2) exhibited an effect without statistical significance ( Figure 3). Therefore, a phthalide glycoside-enriched fraction can be a promising source to prevent or treat the auditory pathological symptoms.

Plant Materials
The Department of Herbal Crop Research, RDA, Eumseong, Korea, supplied the rhizomes of Cnidium officinale, and Dr. J.T. Jeong, Department of Herbal Crop Research, identified them. A voucher specimen (NPCL-20200023) was stored at the Natural Products Chemistry Laboratory of Kyung Hee University, Yongin, Korea.

General Experimental Procedures
The equipment and chemicals used for the isolation of the phthalide glycosides and structure determination of the isolated metabolites were selected by referring to the literature [14]. For the breeding and maintenance of the zebrafish to test the recovery effect on otic hair cells damaged by neomycin treatment, we followed the methods previously reported in the literature [15].

Plant Materials
The Department of Herbal Crop Research, RDA, Eumseong, Korea, supplied the rhizomes of Cnidium officinale, and Dr. J. T. Jeong, Department of Herbal Crop Research, identified them. A voucher specimen (NPCL-20200023) was stored at the Natural Products Chemistry Laboratory of Kyung Hee University, Yongin, Korea.

General Experimental Procedures
The equipment and chemicals used for the isolation of the phthalide glycosides and structure determination of the isolated metabolites were selected by referring to the literature [14]. For the breeding and maintenance of the zebrafish to test the recovery effect on otic hair cells damaged by neomycin treatment, we followed the methods previously reported in the literature [15].