Li-Yuan Zhang, You-Heng Gao*, Ling-Zhi Ren , Ming-Zhu Wan, Teng-Hua Wang

ABSTRACT
This report is related to phytochemical investigation about a Chinese fern Lepidogrammitis drymoglossoides. Based on spectroscopic analysis including UV, MS, 1H and 13C-NMR as well as their chemical and physical properties, their structures were identified to be β-ecdysterone (1), stigmasterol (2), physcion (3), emodin (4), umbelliferone (5), scoparone (6), aesculetin (7), caffeic acid (8), ferulic acid (9), protocatechuic acid (10), pyrocatechualdehyde (11), gallic acid (12), 4-hydroxybenzoic acid methyl ester (13), docosanyl tetracosanoate (14). Compounds 4-6, 9, 12-14 were isolated from the genus Lepidogrammitis for the first time.
Keywords: Polypodiaceae; Lepidogrammitis; Lepidogrammitis drymoglossoides; anthraquinones; coumarins; phenolic acids
1. Subject and source
Lepidogrammitis drymoglossoides (Baker) Ching is a member of Polypodiaceae family, which is widely distributed in the south of Yangtze River basin and other provinces in southern China, especially in Guizhou, Fujian, Hubei, Guangdong, Guangxi provinces. The whole grass of Lepidogrammitis drymoglossoides was collected from Xinhui City, Guangdong province, P. R. China, in October 2011. The specie was identified by Prof. Chao-Mei Pan, Department of Pharmaceutical Botany, Guangzhou University of Chinese Traditional Medicine. A voucher specimen (LY 20111023) has been deposited with the Laboratory of Phytochemistry, Department of Phytochemistry, Guangzhou University of Chinese Traditional Medicine, Guangdong, P. R. China. The whole grass was smashed by an electrical grinder and dried in a drying oven under the temperature of 60 before extraction.
2. Previous work
The medicinal plant is extensively used in the treatment of pharyngalgia, pulmonary tuberculosis, rheumatism arthritis, lymphnoditis, cholecystitis, urolithiasis, traumatic injury, hypertension and furunculosis bloated by the ethnic minorities of Miao, Yao, and Tong (He and Xu, 2007; The Editorial Committee of Chinese Materia Medica, 1999).
Previous works revealed that the main chemical component of Lepidogrammitis drymoglossoides is steroids and phenolic acids, triterpenes, anthraquinones and amides are also included (Li Zhifu, 2011). However, little is known concerning the phytochemistry of the specie, especially the bioactive substances, and yet no method has been proposed for the standardization of this herb, which limits the exploitation and commercialization of these medicinal plants.
3. Present work
The dried whole grass of L. drymoglossoides (21 kg) was extracted with 95% EtOH two times (each for 2 h) by usingheatingcircumfluencemethod. The extracts were concentrated under reduced pressure to a crude extract, which was further dissolved in water and extracted successively with petroleum ether (P), chloroform (CHCl3), EtOAc and n-BuOH to yield 239.5 g, 132 g, 56 g and 122.5 g of the corresponding extracts, respectively .
The petroleum ether fraction (100 g) was chromatographed over silica gel column using P/EtOAc (100:1-0:100) to give 8 fractions (Fr.1-8) based on TLC analysis. Fr.4 was separated on silica gel CC by eluting with P/EtOAc (30:1), and then was purified repeatedly by Sephadex LH-20 column with CHCl3/MeOH (1:1) to obtain 2 (18.4 mg).
The chloroform fraction (100 g) was subjected to a silica gel column chromatography (CC) with the gradient (P/EtOAc 100:0–0:100) as eluants to afford 9 fractions (Fr.1-9) based on TLC analysis. Fr.1 was separated on silica gel CC by eluting with P/EtOAc (100:1-0:100), and then was purified by Sephadex LH-20 column with CHCl3/MeOH (2:1) to obtain 3 (18.4 mg).Fr.2 was submitted to silica gel CC eluted with P/EtOAc gradient (100:1, 50:1, 0:1) to give 5 sub-fractions (Fr.C1-C5). Fr.C2 was purified by Sephadex LH-20 column with CHCl3/MeOH (1:1) as eluant to yield 14 (102 mg). Fr.9 was separated on silica gel CC by eluting with P/EtOAc (10:1, 5:1, 1:1, 0:1), and then was further separated by semi-preparative RP-HPLC with mobile phase of CH3CN/H2O (55:45) to afford 4 (mg).

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The EtOAc fraction (50 g) was subjected to a silica gel CC with using gradient mixtures of CHCl3/MeOH (100:0-0:100) as eluants to afford 10 fractions (Fr.1-10) based on TLC analysis. Fr.2 was submitted to silica gel CC eluted with CH2Cl2/MeOH gradient (100:0-0:100), and then was purified by Sephadex LH-20 column with CHCl3/MeOH (2:1) to obtain 6 (18.4 mg). Fr.3 was subjected to a silica gel CC with using gradient mixtures of CH2Cl2/MeOH (100:0-0:100) as eluants to produce 13 (18.4 mg). Fr.4 was separated on silica gel CC by eluting with CH2Cl2/MeOH (200:1-0:100), and then was purified by Sephadex LH-20 column with CHCl3/MeOH (1:1) to obtain 11 (18.4 mg). Fr.5 was subjected to silica gel CC eluted with CH2Cl2/MeOH (70:1-0:1) to give 4 sub-fractions (Fr.E1-E5). Fr.E2-3 was resubjected to silica gel CC eluting with CH2Cl2/MeOH (50:1-0:1), and then was purified by Sephadex LH-20 column with CHCl3/MeOH (1:1) to obtain 5 (18.4 mg). Fr.E4 was resubmitted sequentially to silica gel CC eluting with CH2Cl2/MeOH (50:1-0:1), Sephadex LH-20 column with CHCl3/MeOH (1:1) as eluent repeatedly, and preparative TLC with mobile phase of CHCl3/MeOH/ CH2O2 (20:1:0.2) to afford 7 (18.4 mg). Fr.6 was separated on D101 macroporous resin (MeOH/H2O, 0:100, 50:50, 70:30, 100:0) to yield four main fractions(Fr.E1-4). Fr.E1 was then seperated on a reversed-phase C18 column (MeOH/H2O, 0:100-100:0) to give 4 sub-fractions (Fr.ER1-4). Fr.ER1 was subjected to silica gel CC eluted with CHCl3/MeOH (30:1, 20:1, 10:1, 0:1) to give 4 sub-fractions (Fr.ERC1-4). Fr.ERC3 was chromatographed over silica gel column using CH2Cl2/MeOH (30:1-0:1) and followed by Sephadex LH-20 CHCl3/MeOH (1:1) as eluent to produce compound 9 (18.4 mg) and 10 (mg). Fr.ERC4 was further separated by semi-preparative RP-HPLC with mobile phase of CH3CN/H2O-0.3%CH2O2 (20:80) to afford compound 8 (18.4 mg). Fr.8 was separated by D101 macroporous resin (MeOH/H2O, 0:100, 30:70, 50:50, 70:30, 100:0) to yield 5 fractions (Fr.ED1-5). Fr.ED2-3 was submitted successively to silica gel CC eluting with CH2Cl2/MeOH (50:1-0:1), silica gel CC eluting with CH2Cl2/MeOH (30:1-0:1), reversed-phase C18 column (MeOH/H2O, 0:100-100:0), Sephadex LH-20 column with CHCl3/MeOH (1:1) to afford 1 (18.4 mg) and 12 (mg).
By comparison of the 1H and 13C NMR spectra data with those reported, compounds 1-14 (Fig. 1) were identified to be β-ecdysterone (1) (Wu et al., 2006), stigmasterol (2) (Wu et al., 2008), physcion (3) (Qin and Liang, 2013), emodin (4) (Wei et al., 2012), umbelliferone (5) (Zheng et al., 2011), scoparone (6) (Chen et al., 2012), aesculetin (7) (Qian et al., 2007), caffeic acid (8) (Huang et al., 2013), ferulic acid (9) (Wei et al., 2014), protocatechuic acid (10) (Zheng et al., 2006 ), pyrocatechualdehyde (11) (Zhou and Yang, 2008), gallic acid (12) (Li et al., 2012), 4-hydroxybenzoic acid methyl ester (13) (Zhang et al., 2011), and docosanyl tetracosanoate (14) (Zhang Qian, 2010).

1 2 3: R=CH3
4: R=H

5: R1=H, R2=H 8: R=H 10_R1=H, R2=OH 13
6: R1=OCH3, R2=CH 9: R=CH3 11: R1=H, R2=H
7: R1=OH, R2=H 12: R1=OH, R2=OH

14
Fig.1. 14 compounds from Lepidogrammitis drymoglossoides.
4. Chemotaxonomic significance
The main chemical component types of L. drymoglossoides in previously research are steroids and phenolic acids. This current research discusses the isolation and identification of a series of chemical constituents from L. drymoglossoides, compounds 1-14 were classified as steroids (1, 2), anthraquinones (3, 4), coumarins (5-7), phenolic acids (8-13) and fatty acids (14). Among them, compounds 4, 5, 6, 9, 12, 13, 14 were isolated from this species for the first time. In addition, compounds3, 4, 5, 6, 7, 9, 12, 13, 14 have not been reported in any species of the genus Lepidogrammitis. And also this is the first report of compounds 3, 4, 5, 6, 7, 9, 12, 13, 14 from the family Polypodiaceae.
There are about 5 species in the genus Lepidogrammitis which are both native to China, and only one specie spreads to India, Burma, Laos and Vietnam (Kunming Institute of Botany, Chinese Academy of Sciences, 2005). L. drymoglossoides mentionedabove is the only one that be studied up to now. To the best of my knowledge, other species in genus Drynaria, Pyrrosia Mirbel and Lepisorus from the family Polypodiaceae have been studied more in recent years home and abroad. Drynaria mainly contains flavones, triterpenoids and phenylpropanoids. Thechemicalcomponentsisolated from Pyrrosia Mirbel include mainlyflavonoid glycosidesand xanthanes. Several of the compounds isolated in this study have been previously isolated and identified from genus mentionedabove. For example, ecdysterone (1) were isolated from L. marginatus, L. thunbergianus, L. longus, L. contortus, L. macrosphaerus, L. albertii and L. ussuriensis (Zhang et al., 2012; Xu Dongying, 1997; Yao et al., 2004; Zhang Xiaojing, 2012; Choi et al., 1999). Stigmasterol (2) has been identified from P. calvata (Zheng et al., 1999) and P. gralla (Zheng et al., 1998) while caffeic acid (8), protocatechuic acid(9) and protocatechuic acid(10) have been obtained from R. Drynariae, P. petiolosa and P. sheareri (Han and Wang, 1984; ShanghaiFirstMedical College, 1973; Liang et al., 2010; Wang et al., 2003). But there are no reports of physcion (3), emodin (4), umbelliferone (5), scoparone (6), aesculetin (7) in the family Polypodiaceae. In addition, coumarins is a rare constituent in ferns though previously obtained from Polypodium hastatum Thunb., a fern of the same family Polypodiaceae (Yao et al., 2012). Identification of these class of anthraquinones (3, 4) and coumarins (5-7) indicates that they would be used as chemotaxonomic markers for the species L. drymoglossoides within the family. And this investigation may supply characteristic constituents in this genus. But more phytochemistry studies on it are needed.
Acknowledgements
This project was financially supported by a grant from Zhongshan Science and Technology Planning Project of Guangdong province (No. 20113A082).
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[1]Corresponding authors. Tel.:+86 20 39358083.
E-mail addresses: gaoyouheng@gzhtcm.edu.cn(Y.-H. Gao).