**The Research of Lygodium**

Zhang Guo-Gang, He Ying-Cui, Liu Hong-Xia, Zhu Lin-Xia and Chen Li-Juan *College of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shengyang, China* 

## **1. Introduction**

76 Drug Discovery Research in Pharmacognosy

[20] Centonze D, Battistini L, Maccarrone M (2008 ). "The endocannabinoid system in

[23] Union movie. Business behind getting high. Quote from Dr. Lester Grinspoon MD |

[24] Annas, George J. (1997). "Reefer Madness — The Federal Response to California's Medical-Marijuana Law". New England Journal of Medicine 337 (6): 435–9. [25] John A. Benson, Jr., MD, Janet E. Joy, PhD, and Stanley J. Watson, Jr., MD, PhD, co-

[26] Scuderi C, Filippis DD, Iuvone T, Blasio A, Steardo A, Esposito G Cannabidiol in

[27] Morel LJ, Giros B, Daugé V (2009). "Adolescent Exposure to Chronic Delta-9-

[29] M. Springs1,J. R. Wells2,G. C. Reaction rates of ozone and terpenes adsorbed to model

[31] KP, Glaser ST, Gatley SJ.Handb Exp Pharmacol. 2005;(168):425-43. Imaging of the brain

Des. 14 (23): 2370–42. [21] Hornby et al.Cases Journal 2009, 2:7487 [22] Hornby, et al. Cases Journal 2010, 3:7

Technology:

2008 Oct 9.

Professor Emeritus, Harvard Medical School.

Neuropsychopharmacology 34 (11): 2469–76.

10.1111/j.1600-0668.2010.00707.x

cannabinoid system.Lindsey

[30] Memidex/WordNet - euphoria

[28] Dr. Donald Abrams Compares Cannabis To Opiate-based Medicines http://www.youtube.com/watch?v=cGPtirNqGlM

peripheral lymphocytes as a mirror of neuroinflammatory diseases". Curr. Pharm.

writers of the Mar. 1999 Institute of Medicine report titled "Marijuana and Medicine: Assessing the Science Base," wrote the following in their Mar. 22, 1999 article titled "From Marijuana to Medicine," published in Issues in Science and

medicine: a review of its therapeutic potential in CNS disorders. Phytother Res

Tetrahydrocannabinol Blocks Opiate Dependence in Maternally Deprived Rats".

indoor surfaces Morrison1Article first published online: 7 FEB 2011DOI:

Lygodium is the dry root and rhizome of *Lygodium japonicum* (Thunb.)Sw. which belongs to the family Lygodiaceae. *Lygodium* is the only genus of Lygodiaceae comprises 45 species throughout the world. In China, there are 10 species of *Lygodium* distributed in the southwest and south China, and five species of them had been used for Chinese herbs medicine to treat hepatitis and dysentery[1]. They are named Lygodium, Lygodium of hainan, Crankshaft Lygodium, Angustifolia Lygodium, Pinnately lobed Lygodium, Willowlike leaves Lygodium, Reticulata Lygodium, yunnan Lygodium, Lobular Lygodium, Palm leaf Lygodium.

Fig. 1. Leaves and branches of *Lygodium japonicum* (Thunb.)Sw.

The Research of Lygodium 79

Friedelin 2

O

Rha

α

Kaempferol-3-O-

22-hydroxyhopane

Glc-O

O

O


OH

OH

O

HO

Daucosterol

β


Rha

α

HO

HO

α

OH



COOH

**2.1.2 Triterpenes** 

O

**2.1.3 Flavonoids** 

**2.1.4 Phytosterols** 

Fig. 2. Dried powder of *Lygodium japonicum* (Thunb.)Sw.

## **2. Chemical composition**

Currently, the research on the active components in the Lygodium were done on the underground parts. We summarize the structure and classification of these compounds from *lygodium*.

## **2.1 The main components of the Lygodium root[3]**

#### **2.1.1 Ecdysteroside**

Capitasterone-3-Oβ

Lygodiumsteroside B\* Makisterone C


#### **2.1.2 Triterpenes**

78 Drug Discovery Research in Pharmacognosy

Currently, the research on the active components in the Lygodium were done on the underground parts. We summarize the structure and classification of these compounds

HO

O


HO

HO

Lygodiumsteroside B\* Makisterone C

Glc

O

O

OH

O

O

OH

OH OH

OH

OH

Fig. 2. Dried powder of *Lygodium japonicum* (Thunb.)Sw.

**2.1 The main components of the Lygodium root[3]** 

OH OH

OH

β

OH OH

OH

**2. Chemical composition** 

from *lygodium*.

HO

O

HO

O

Glc

Glc

O

O

Capitasterone-3-O-

**2.1.1 Ecdysteroside** 

α-hydroxyursolic acid

#### **2.1.3 Flavonoids**

Kaempferol-3-Oα-L- rhamnopyranoside-7-Oα- L-rhamnopyranoside

#### **2.1.4 Phytosterols**

Daucosterol

The Research of Lygodium 81

Nicotflorin Tilianine

OH

HO

Kaempferol- α Kaempferol-1-rhamnopyranoside

HO

Caffeic acid P-coumaric acid

HO

O

O

OH

COOH

O

OH

O

O

O

OH OH

OH

OH

OH

O

OH

O O

OH

OH OH

HO

OH

O

O

O O

O

O

OH

OH

OH

**2.2.2 Phenylpropanoids** 

OH

O

O

O

O

O

OH OH

OH

OH

6-O-p-coumaroyl-D-glucopyranose 6-O-caffeoyl-D-glucopyranose

OH

O

OH

OH <sup>O</sup>

OH

OH

OH OH

O O

OH

OH

HO

HO

HO

HO

#### **2.1.5 Glycosides**

3,4-dihydroxybenzoic acid 4-O- (4′-O-methyl)β-D-glucopyranoside

#### **2.1.6 Organic acids**

#### **2.1.7 Naphthoquinone**

2-isopropyl-7-methly-6-hydroxyα-(1,4) naphthoquinone\*

#### **2.2 The main components of the root of Lygodium from n-butanol layer[2][3]**

#### **2.2.1 flavonoids**

Linaribn Dosinin

<sup>O</sup> <sup>O</sup>

Succinic acid Methylmalic acid

O

O

α

OH

Linaribn Dosinin

CH3


O

OH

O O

OH OH

OH

O

<sup>O</sup> <sup>O</sup>

O

OH OH

OH

H3C CH3

OH

OH OMe

HO

3,4-dihydroxybenzoic acid 4-O- (4′-O-methyl)-

COOH

COOH

HO

2-isopropyl-7-methly-6-hydroxy-

O

<sup>O</sup> <sup>O</sup>

O

OH

**2.2 The main components of the root of Lygodium from n-butanol layer[2][3]**

COOH

OH

β

HOOC


OH

COOMe

**2.1.5 Glycosides** 

**2.1.6 Organic acids** 

**2.1.7 Naphthoquinone** 

**2.2.1 flavonoids** 

O O

OH OH

OH

O

OH

OH

O O OH O OH OH O O OH OH OH OH O

OH

Nicotflorin Tilianine

Kaempferol- α Kaempferol-1-rhamnopyranoside

**2.2.2 Phenylpropanoids** 

#### Caffeic acid P-coumaric acid

O O O OH OH OH OH

6-O-p-coumaroyl-D-glucopyranose 6-O-caffeoyl-D-glucopyranose

HO

The Research of Lygodium 83


Air-dried roots of *L. japonicum* (Thunb.) Sw.(4 kg) were crushed and extracted twice under reux with 70% EtOH. Evaporation of the solvent under reduced pressure delivered the 70% EtOH extract (around 280 g). The extract was partitioned successively with CHCl3, AcOEt and n-BuOH. The n-BuOH-soluble fraction(50.0 g) was further eluted on a silica gel column using gradient elution with CHCl3–MeOH (100:1–1:1) to give ten fractions. Fraction 2 (2.3 g) was subjected to another silica gel column chromatography eluted with petroleum ether (PE)–EtOAc (20:1–1:1) to afford a further ve fractions (frs. 2-1 to 2-5). Fraction 2-2 was puried twice by Sephadex LH-20 eluted with MeOH to give the new

Melting points were determined on an X4-A micro-melting point apparatus and were uncorrected. ESI–MS spectra were measured on an Agilent 1100 LC-MSD-Trap-SL, and HR– ESI–MS spectra were measured on an Bruker Dal- tonics MicroTOFQ. NMR spectra were measured on a Bruker ARX-600 and 300 NMR spectrometer with tetra-methylsilane (TMS) as the internal reference and chemical shifts are expressed with δ (ppm). UV spectra were recorded on a Shimadzu UV-2201 spectrometer. IR spectra were recorded on a Bruker IFS-55 spectrophotometer. TLC was performed on silica gel GF254 (10–40 lm; Qingdao,China). Separations were performed by Semiprep-HPLC named Shimadzu SPD-10A apparatus

The new compound,yellow powder, melting point:193–194℃. The molecular formula was determined as C14H14O3 by HR–ESI–TOF–MS (m/z 231.1004[M + H]+, calcd. 231.1016), along with 1H-NMR and 13C-NMR data. The UV spectrum displayed absorption bands at 207, 267 and 347 nm, closely resembling that of 1,4-naphthoquinones. The 13C-NMR spectrum revealed 14 carbon resonances; in the low field area of it, two were assigned as carbonyl carbons, eight were assigned as aromatic carbons. However, in the high field area of 13C-NMR spectrum, there were four carbon resonances all that assigned as sp3 carbons. By observing these data of

<sup>H</sup> <sup>H</sup>

OC

<sup>H</sup> <sup>O</sup>

COOCH3

HO

H

<sup>H</sup> <sup>H</sup>

12α

**3.1.1 Extraction and isolation** 

compound 1(9mg).

**3.1.2 Apparatus** 

OC

<sup>H</sup> <sup>H</sup> <sup>O</sup>

H

OH

**3.1 New naphthalene ketone from the root of** *Lygodium japonicum***[5]** 

equipped with UV detector under ODS column (i.d. 10 mm 9 200 mm).

**3.1.3 Physical data of the new compound 1** 

COOCH3

**3. New compounds from** *Lygodium japonicum*

## **2.2.3 Phenolic acids[4]**

#### **2.2.4 Sterols**

A new steroidal saponins was Isolated from the root of Lygodium, which is (24- *R*) stigmastan-3*β*, 5*α*,6*β*-triol-3-*O*-*β*-*D*-glucopyranoside, in addition to daucostero1 and β-sitostero1.

#### **2.2.5 Others**

Hexadecanoic acid 2, 3-dihydroxy, propyl ester, hexacosanoic acid, 1-hentriacontano1, pentacosanoic acid, palmitic acid, linoleic acid, (6S, 9R) -6 - hydroxy -3- ketone - Violet alcohol-D-β-9-O-glucoside (roseoside) and so on.

#### **2.2.6 Diterpenoids**

H <sup>H</sup> <sup>O</sup> OC COOCH3

A new steroidal saponins was Isolated from the root of Lygodium, which is (24- *R*) stigmastan-3*β*, 5*α*,6*β*-triol-3-*O*-*β*-*D*-glucopyranoside, in addition to daucostero1 and

Hexadecanoic acid 2, 3-dihydroxy, propyl ester, hexacosanoic acid, 1-hentriacontano1, pentacosanoic acid, palmitic acid, linoleic acid, (6S, 9R) -6 - hydroxy -3- ketone - Violet

Gibberellin A9 methyl ester Gibberellin A73 methyl ester

Gibberellin A12 methylester GA20-Me

COOH

OH

H

H

COOCH3

<sup>H</sup> OH <sup>O</sup>

COOCH3

CH2

<sup>H</sup> <sup>O</sup>

OC

OC

Vanillic acid

OCH3

**2.2.3 Phenolic acids[4]** 

COOH

<sup>O</sup> <sup>O</sup>

3,4-Dihydroxybenzoic acid 4-O- (4'-O-methyl)-β-D-glucopyranoside

alcohol-D-β-9-O-glucoside (roseoside) and so on.

<sup>H</sup> <sup>H</sup> <sup>O</sup>

COOCH3

CO2CH3

H

H H

H <sup>H</sup>

CH2

OH

OH OMe

HO

**2.2.4 Sterols** 

β-sitostero1.

**2.2.5 Others** 

**2.2.6 Diterpenoids** 

<sup>H</sup> <sup>H</sup>

OC

OH

#### **3. New compounds from** *Lygodium japonicum*

#### **3.1 New naphthalene ketone from the root of** *Lygodium japonicum***[5]**

## **3.1.1 Extraction and isolation**

Air-dried roots of *L. japonicum* (Thunb.) Sw.(4 kg) were crushed and extracted twice under reux with 70% EtOH. Evaporation of the solvent under reduced pressure delivered the 70% EtOH extract (around 280 g). The extract was partitioned successively with CHCl3, AcOEt and n-BuOH. The n-BuOH-soluble fraction(50.0 g) was further eluted on a silica gel column using gradient elution with CHCl3–MeOH (100:1–1:1) to give ten fractions. Fraction 2 (2.3 g) was subjected to another silica gel column chromatography eluted with petroleum ether (PE)–EtOAc (20:1–1:1) to afford a further ve fractions (frs. 2-1 to 2-5). Fraction 2-2 was puried twice by Sephadex LH-20 eluted with MeOH to give the new compound 1(9mg).

#### **3.1.2 Apparatus**

Melting points were determined on an X4-A micro-melting point apparatus and were uncorrected. ESI–MS spectra were measured on an Agilent 1100 LC-MSD-Trap-SL, and HR– ESI–MS spectra were measured on an Bruker Dal- tonics MicroTOFQ. NMR spectra were measured on a Bruker ARX-600 and 300 NMR spectrometer with tetra-methylsilane (TMS) as the internal reference and chemical shifts are expressed with δ (ppm). UV spectra were recorded on a Shimadzu UV-2201 spectrometer. IR spectra were recorded on a Bruker IFS-55 spectrophotometer. TLC was performed on silica gel GF254 (10–40 lm; Qingdao,China). Separations were performed by Semiprep-HPLC named Shimadzu SPD-10A apparatus equipped with UV detector under ODS column (i.d. 10 mm 9 200 mm).

#### **3.1.3 Physical data of the new compound 1**

The new compound,yellow powder, melting point:193–194℃. The molecular formula was determined as C14H14O3 by HR–ESI–TOF–MS (m/z 231.1004[M + H]+, calcd. 231.1016), along with 1H-NMR and 13C-NMR data. The UV spectrum displayed absorption bands at 207, 267 and 347 nm, closely resembling that of 1,4-naphthoquinones. The 13C-NMR spectrum revealed 14 carbon resonances; in the low field area of it, two were assigned as carbonyl carbons, eight were assigned as aromatic carbons. However, in the high field area of 13C-NMR spectrum, there were four carbon resonances all that assigned as sp3 carbons. By observing these data of

The Research of Lygodium 85

OH OH

OH

lygodiumsteroside B

The air-dried roots of *L. japonicum* (Thunb.) Sw were crushed and extracted twice using reflux with 70% ethanol; the solution was concentrated under reduced pressure to obtain the residue, and then the residue was extracted with MeOH. The MeOH-soluble fraction (100 g) was isolated by column chromatography on silica gel and gradient elution with CHCl3:MeOH (50 : 1 to 1 : 1) gave 14 fractions. Fraction 8 was isolated by semipreparative ODS column using MeOH:H2O (65 : 35) as eluent to afford the new compound 2 (13mg).

Melting point: X-4 micro melting point determination apparatus (uncorrected). ESI–MS spectra: LC–MSD-Trap-SL. HR–ESI–MS spectra: Bruker MicroTOFQ.1H NMR(600MHz) and

Shimadzu SPD-10A apparatus equipped with UV detector under ODS column (i.d.

O

<sup>H</sup> <sup>O</sup>

O O

OH

OH

–Vis. Semiprep-HPLC:

13 C NMR (150MHz): Bruker ARX-600. UV: Shimadzu UV 260 UV -

1. 1H-NMR and 13C-NMR data for the new compound 2

HO

O

H

OH

Fig. 4. The key HMBC correlations of the new compound 2.

OH OH

HO

**3.2 The new compound 2 from the roots of** *Lygodium japonicum***[6]** 

O

HO

O

Glc

**3.2.1 Extraction and isolation** 

**3.2.2 Apparatus** 

10mm\*200mm).

**3.2.3 The spectrum of new compound** 

13C-NMR spectrum, nucleus of naphthoquinone was revealed. All protonated carbons were assigned by analysis of the HSQC spectrum (Table 1).The 1H-NMR spectrum showed signals of two aromatic protons atδ7.30 (1H, s, H-5), 7.77 (1H, s, H-8) and one aromatic methyl proton at d 2.24 (3H, s, 7-CH3) that were assigned by analyzing HMBC spectrum (Table 1; Fig. 1). Additionally, δ1.12 (6H, d, J = 6.8 Hz,H-12, H-13) and 6.68 (1H, s, H-3) correlated, respectively, with d: δ26.6 (C-11), δ156.6 (C-2) in the HMBC spectrum and δ 3.09 (1H, m, H-11) correlated with δ21.4(C-12 and 13), 156.6 (C-2), 131.6 (C-3), 183.4 (C-1) all that revealed the presence of isopropyl and it connected C-2 of quinone ring. Other detailed correlations in the HMBC spectrum see Table 1. All these spectroscopic data discussed above showed compound 1 as 6-hydroxy-2-isopropyl-7-methyl-1,4- naphthoquinone.


Table 1. 1H and 13C data for New naphthalene ketone (300and 75MHz,in DMSO- *d6*).

Fig. 3. The key HMBC correlations of the new compound 1.

13C-NMR spectrum, nucleus of naphthoquinone was revealed. All protonated carbons were assigned by analysis of the HSQC spectrum (Table 1).The 1H-NMR spectrum showed signals of two aromatic protons atδ7.30 (1H, s, H-5), 7.77 (1H, s, H-8) and one aromatic methyl proton at d 2.24 (3H, s, 7-CH3) that were assigned by analyzing HMBC spectrum (Table 1; Fig. 1). Additionally, δ1.12 (6H, d, J = 6.8 Hz,H-12, H-13) and 6.68 (1H, s, H-3) correlated, respectively, with d: δ26.6 (C-11), δ156.6 (C-2) in the HMBC spectrum and δ 3.09 (1H, m, H-11) correlated with δ21.4(C-12 and 13), 156.6 (C-2), 131.6 (C-3), 183.4 (C-1) all that revealed the presence of isopropyl and it connected C-2 of quinone ring. Other detailed correlations in the HMBC spectrum see Table 1. All these spectroscopic data discussed above showed compound

1 as 6-hydroxy-2-isopropyl-7-methyl-1,4- naphthoquinone.

δ<sup>C</sup> δH, mult

HO

H3C

Fig. 3. The key HMBC correlations of the new compound 1.

1 183.5 2 156.6

4 185.2

6 160.9 7 131.8

9 124.1 10 131.7

C No. HSQC HMBC

3 131.6 6.68 (1H, s) C2/C11

5 110.3 7. 30 (1H, s) C6/C9/C4

CH3-7 16.2 2.24 (3H, s) C6/C8 11-CH- 26.6 3.09 (1H, m) C12/ C13/C2 12-CH3 21.4 1.13 (6H, d, *J*=6.8 Hz) C11/C2 13-CH3 21.4 1.11 (6H, d, *J*=6.8 Hz ) C11/C2

Table 1. 1H and 13C data for New naphthalene ketone (300and 75MHz,in DMSO- *d6*).

5 4

10

H

6

H

<sup>7</sup> <sup>8</sup> <sup>9</sup>

O

O CH3

2 3

1

H

CH3

H

8 129.5 7. 77 (1H, s) C1/C10/C7 (CH3)/C6

## OH OH HO O O OH Glc

#### **3.2 The new compound 2 from the roots of** *Lygodium japonicum***[6]**

lygodiumsteroside B

#### **3.2.1 Extraction and isolation**

The air-dried roots of *L. japonicum* (Thunb.) Sw were crushed and extracted twice using reflux with 70% ethanol; the solution was concentrated under reduced pressure to obtain the residue, and then the residue was extracted with MeOH. The MeOH-soluble fraction (100 g) was isolated by column chromatography on silica gel and gradient elution with CHCl3:MeOH (50 : 1 to 1 : 1) gave 14 fractions. Fraction 8 was isolated by semipreparative ODS column using MeOH:H2O (65 : 35) as eluent to afford the new compound 2 (13mg).

#### **3.2.2 Apparatus**

Melting point: X-4 micro melting point determination apparatus (uncorrected). ESI–MS spectra: LC–MSD-Trap-SL. HR–ESI–MS spectra: Bruker MicroTOFQ.1H NMR(600MHz) and 13 C NMR (150MHz): Bruker ARX-600. UV: Shimadzu UV 260 UV - –Vis. Semiprep-HPLC: Shimadzu SPD-10A apparatus equipped with UV detector under ODS column (i.d. 10mm\*200mm).

#### **3.2.3 The spectrum of new compound**

1. 1H-NMR and 13C-NMR data for the new compound 2

Fig. 4. The key HMBC correlations of the new compound 2.

The Research of Lygodium 87

Lygodiumsteroside B, white powder, m.p. 294–295℃, gave positive responses to Liebermann–Burchard and Molish reactions, which suggested a steroid glycoside structure. The sugar was identified as glucose by co-TLC with authentic sample after acid hydrolysis. The molecular formula was established to be C34H56O11 based on HR–ESI–MS([M+H]+,m/z 639.3701, Calcd for C34H55O11 639.3750). Additionally, the UV spectrum showed a maximum at243 nm[Check this typing] for anα,β-unsaturated carbonyl group.The1H NMR(600MHz,C5D5N) spectrum showed an olefinic proton at δ6.23 (1H,brs) and six methyl signals atδ1.58 (3H, s), 1.20 (3H, s), 0.88 (3H, s), 0.83(3H, d, J¼6.6Hz), 0.80 (3H, d, J¼6.6Hz), 0.76 (3H, d, J¼6.6Hz). The13C –NMR (150MHz,C5D5N) spectrum showed six methyl signals and a typicalα, β-unsaturated carbonyl group signals at δ 203.0 (C-6), 166.3 (C-8) and 121.7 (C-7). The HMBC spectrum, showed the long-range correlations betweenδ 6.23 (1H, brs, H-7) andδ 34.3(C-9), 51.4 (C-5), 84.2 (C-14), the correlations between methyl proton signal at δ 0.88 (3H, s,H-19) and the carbon signals at δ38.7 (C-1), 39.1 (C-10), 51.4 (C-5) and 67.5 (C-2) could also be observed. In addition, the correlations between methyl proton signal at δ1.20 (3H, s,H-18) and the carbon signals at δ 32.0 (C-12), 48.1 (C-13), 50.0 (C-17) and 84.1(C-14) could also be found. Thus, the ecdysteroid-type skeleton was identified. In the HSQC spectrum, δ 4.07 (1H, brd, H-2) had the correlation withδ 67.5 (C-2) and δ 4.28(1H, brs, H-3) had the correlation with (1H, brs, H-3) had the correlation with (1H, brs, H-3) had the correlation with(1H, brs, H-3) had the correlation withδ77.7 (C-3). δ1.58 (3H, s,H-21) showed the correlation withδ50.0 (C-17), δ 74.2 (C-22 and δ76.9 (C-20) in the HMBC. So the signals of the five hydroxyl carbons C-2, C-3, C-14, C-20, C-22 were evident. Additionally, δ0.83 (3H, d,H-28) showed correlation withδ1.93 (1H,m,H-24) in the1H–1H COSY, and the HMBC spectrum showed the correlations betweenδ0.83 (3H, s,H-28) andδ18.7 (C-27),33.7 (C-25), 35.6 (C-24), 36.9 (C-23). These facts

Compared with polyporusterone A (Ishida et al., 1999; Ohsawa, Yukama, Takao,Murayama, & Bando, 1992), the chemical shifts of C5–C22 were very similar; this fact suggested that the positions of the substituents on the steroid rings and side-chain of the new compound were identical with polyporusterone A except for C24, and the configuration of hydroxyls were 14α,20R,22R. Since the signals at 33.7 (C-25), 20.3 (C-26), 18.7 (C-27) were different from the corresponding values of polyporusterone A, the configuration of C-24 could be different. Compared with the compound schizaeasterone A (Fuchino et al., 1997), which has 24R configuration, the chemical shifts of C20–C28 were very similar to those of schizaeasterone A. Moreover, in the NOESY spectrum, a cross peak was observed betweenδ3.91 (1H, overlap, H-22) andδ0.83 (3H, d,H-28). So all these facts indicated that the configuration of C-24 of compound 1 was R (Figure 2). The NOESY spectrum also showed the correlation between the proton signal atδ4.07 (1H, brd, H-2) andδ4.28 (1H, brs, H-3), δ1.65

Since the signal at δ77.7, which could assignable to C-3, was downfield shifted by 9 ppm, and the signal atδ30.6 (C-4) was upfield shifted, glycosylation was present atC-3. The chemical shifts of the sugar moiety in 13C -NMR (δ104.2, 74.7, 78.7, 71.6, 78.5,62.6) also

3. NMR Analysis of the new compound 2

indicated that a methyl group (C-28) was attached to C-24.

(1H,m,Hα-4), so the relative configuration was confirmed to be2β,3β.


Table 2. 1H and 13C NMR data for lygodiumsteroside B (600 and 150 MHz, in C5D5N).

2. Other spectrum data for the new compound 2

The new compound 2(lygodiumsteroside B): white powder, m.p. 294–295℃,UV max (MeOH): 243 nm; ESI–MS:m/z 675.5 [M+Cl]- , m/z 639.7 [M+H]+; HR–ESI–MS: m/z 639.3701 [M+H]+(Calcd forC34H55O11 639.3750);1H-NMR (600MHz, in C5D5N) and 13C-NMR (150MHz, inC5D5N), see table 2 .

1.74(brd, *J*=12.6Hz), 2.02(m) 4.07(br.dt, *J*=10.8 Hz), 4.28(brs) 1.65, 2.20(each m) 2.93(m) ⎯ 6.23(brs) ⎯ 3.53(t, *J*=8.4 Hz) ⎯ 1.67, 1.80(each m) 2.02(m),2.58(dt, *J*=4.2 and 12.6 Hz) ⎯ ⎯ 1.92, 2.17(each m) 2.08, 2.45(each m) 2.89(m) 1.20(s) 0.88(s) ⎯ 1.58(s) 3.90(m) 1.45, 1.63(each m) 1.93(m) 1.46(m) 0.80(d, *J*=6.6 Hz) 0.76(d, *J*=6.6 Hz) 0.83(d, *J*=6.6 Hz) 4.92(d, *J*=7.8 Hz) 4.03(overlap) 4.20(overlap) 4.21(m) 3.92(m) 4.32(m),4.52(brd, *J*=10.8 Hz)

C-2,C-3,C-9,C-10,C-19

C-4,C-9

C-5,C-9,C-14

C-8,C-9,C-12,C-13 C-9,C-11,C-18

C-13,C-16 C-17 C-13,C-15,C-16,C-18 C-12,C-13,C-14,C-17 C-1,C-2,C-5,C-10

C-17, C-20, C-22 C-20 C-21,C-24,C-28

C-23,C-26,C-27 C-24,C-25,C-27 C-24,C-25,C-26 C-23,C-24,C-25,C-27 C-3 C-3′ C-2′, C-4′ C-5′, C-6′ C-4′, C-5′ C-4′, C-5′

, m/z 639.7 [M+H]+; HR–ESI–MS: m/z 639.3701

C HMQC HMBC

Table 2. 1H and 13C NMR data for lygodiumsteroside B (600 and 150 MHz, in C5D5N).

The new compound 2(lygodiumsteroside B): white powder, m.p. 294–295℃,UV max

[M+H]+(Calcd forC34H55O11 639.3750);1H-NMR (600MHz, in C5D5N) and 13C-NMR

2. Other spectrum data for the new compound 2

(MeOH): 243 nm; ESI–MS:m/z 675.5 [M+Cl]-

(150MHz, inC5D5N), see table 2 .

δC(ppm) δH(ppm)

38.7 67.5 77.7 30.6 51.4 203.0 121.7 166.3 34.3 39.1 21.1 32.0 48.1 84.2 31.9 21.4 50.0 18.0 24.1 76.9 21.6 74.2 36.9 35.6 33.7 20.3 18.7 15.2 104.2 74.7 78.7 71.6 78.5 62.6

#### 3. NMR Analysis of the new compound 2

Lygodiumsteroside B, white powder, m.p. 294–295℃, gave positive responses to Liebermann–Burchard and Molish reactions, which suggested a steroid glycoside structure. The sugar was identified as glucose by co-TLC with authentic sample after acid hydrolysis. The molecular formula was established to be C34H56O11 based on HR–ESI–MS([M+H]+,m/z 639.3701, Calcd for C34H55O11 639.3750). Additionally, the UV spectrum showed a maximum at243 nm[Check this typing] for anα,β-unsaturated carbonyl group.The1H NMR(600MHz,C5D5N) spectrum showed an olefinic proton at δ6.23 (1H,brs) and six methyl signals atδ1.58 (3H, s), 1.20 (3H, s), 0.88 (3H, s), 0.83(3H, d, J¼6.6Hz), 0.80 (3H, d, J¼6.6Hz), 0.76 (3H, d, J¼6.6Hz). The13C –NMR (150MHz,C5D5N) spectrum showed six methyl signals and a typicalα, β-unsaturated carbonyl group signals at δ 203.0 (C-6), 166.3 (C-8) and 121.7 (C-7). The HMBC spectrum, showed the long-range correlations betweenδ 6.23 (1H, brs, H-7) andδ 34.3(C-9), 51.4 (C-5), 84.2 (C-14), the correlations between methyl proton signal at δ 0.88 (3H, s,H-19) and the carbon signals at δ38.7 (C-1), 39.1 (C-10), 51.4 (C-5) and 67.5 (C-2) could also be observed. In addition, the correlations between methyl proton signal at δ1.20 (3H, s,H-18) and the carbon signals at δ 32.0 (C-12), 48.1 (C-13), 50.0 (C-17) and 84.1(C-14) could also be found. Thus, the ecdysteroid-type skeleton was identified. In the HSQC spectrum, δ 4.07 (1H, brd, H-2) had the correlation withδ 67.5 (C-2) and δ 4.28(1H, brs, H-3) had the correlation with (1H, brs, H-3) had the correlation with (1H, brs, H-3) had the correlation with(1H, brs, H-3) had the correlation withδ77.7 (C-3). δ1.58 (3H, s,H-21) showed the correlation withδ50.0 (C-17), δ 74.2 (C-22 and δ76.9 (C-20) in the HMBC. So the signals of the five hydroxyl carbons C-2, C-3, C-14, C-20, C-22 were evident. Additionally, δ0.83 (3H, d,H-28) showed correlation withδ1.93 (1H,m,H-24) in the1H–1H COSY, and the HMBC spectrum showed the correlations betweenδ0.83 (3H, s,H-28) andδ18.7 (C-27),33.7 (C-25), 35.6 (C-24), 36.9 (C-23). These facts indicated that a methyl group (C-28) was attached to C-24.

Compared with polyporusterone A (Ishida et al., 1999; Ohsawa, Yukama, Takao,Murayama, & Bando, 1992), the chemical shifts of C5–C22 were very similar; this fact suggested that the positions of the substituents on the steroid rings and side-chain of the new compound were identical with polyporusterone A except for C24, and the configuration of hydroxyls were 14α,20R,22R. Since the signals at 33.7 (C-25), 20.3 (C-26), 18.7 (C-27) were different from the corresponding values of polyporusterone A, the configuration of C-24 could be different. Compared with the compound schizaeasterone A (Fuchino et al., 1997), which has 24R configuration, the chemical shifts of C20–C28 were very similar to those of schizaeasterone A. Moreover, in the NOESY spectrum, a cross peak was observed betweenδ3.91 (1H, overlap, H-22) andδ0.83 (3H, d,H-28). So all these facts indicated that the configuration of C-24 of compound 1 was R (Figure 2). The NOESY spectrum also showed the correlation between the proton signal atδ4.07 (1H, brd, H-2) andδ4.28 (1H, brs, H-3), δ1.65 (1H,m,Hα-4), so the relative configuration was confirmed to be2β,3β.

Since the signal at δ77.7, which could assignable to C-3, was downfield shifted by 9 ppm, and the signal atδ30.6 (C-4) was upfield shifted, glycosylation was present atC-3. The chemical shifts of the sugar moiety in 13C -NMR (δ104.2, 74.7, 78.7, 71.6, 78.5,62.6) also

The Research of Lygodium 89

4. Attached figure:

Fig. 5. Lygosteroside B (1H-NMR).

Fig. 6. Lygosteroside B (1H-NMR).

confirmed the presence of glucose. The HMBC correlation was observed between the anomeric proton signal at δ4.92 (1H, d,H-10) and the carbon signal atδ77.7due to C-3 of the aglycone moiety. The anomeric configurations of glucose were determined to be on the basis of the JH–H values (J¼7.8Hz).Therefore, the structure of 2 was elucidated as 2β,3β,14α, 20R, 22R - pentahydroxy-24R-methly-5-cholest-7-en-6-one-3-O-β-Dglucopyranoside, and named lygodiumsteroside B


Table 3. 1H and 13C NMR data for polyporusterone A (600 and 150 MHz, in C5D5N).

#### 4. Attached figure:

88 Drug Discovery Research in Pharmacognosy

confirmed the presence of glucose. The HMBC correlation was observed between the anomeric proton signal at δ4.92 (1H, d,H-10) and the carbon signal atδ77.7due to C-3 of the aglycone moiety. The anomeric configurations of glucose were determined to be on the basis of the JH–H values (J¼7.8Hz).Therefore, the structure of 2 was elucidated as 2β,3β,14α, 20R, 22R - pentahydroxy-24R-methly-5-cholest-7-en-6-one-3-O-β-D-

C No. HMQC HMBC

Table 3. 1H and 13C NMR data for polyporusterone A (600 and 150 MHz, in C5D5N).

1.76(m), 2.10(m) 4.10(br.dt, *J*=11.4 Hz), 4.30(overlap) 1.73, 2.20(each m) 2.93(m) ⎯ 6.23(d, *J*=1.8 Hz) ⎯ 3.55(t, *J*=8.4 Hz) ⎯ 1.65, 1.83(each m) 2.02(m),2.58(dt, *J*=4.2 and 12.6 Hz) ⎯ ⎯ 1.92, 2.15(each m) 2.08, 2.44(each m) 2.91(m) 1.19(s) 0.87(s) ⎯ 1.58(s) 3.81(brd, *J*=10.8 Hz) 1.47(m) 1.40, 1.70(each m) 1.54(m) 0.81(d, *J*=6.0 Hz) 0.82(d, *J*=6.0 Hz) 4.90(d, *J*=7.8 Hz) 4.03(m) 4.20(m) 4.18(m) 3.93(m) 4.32(m),4.53(brd, *J*=10.2 Hz)

C-2,C-3,C-9,C-10,C-19

C-1,C-5,C-9,C-19 C-1,C-4,C-6,C-9

C-5,C-9,C-14

C-1,C-11,C-19

C-8,C-9,C-13 C-11,C-13,C-17,C-18

C-13,C-16 C-17 C-11,C-13,C-15,C-18,C-22 C-12,C-13,C-14,C-17 C-1,C-2,C-5,C-9

C-17,C-22,C-23 C-21,C-24 C-21,C-24,C-25,C-26,C-27 C-23,C-26,C-27

> C-23,C-24,C-27 C-23,C-24,C-26 C-3 C-3′ C-2′, C-4′ C-5′, C-6′ C-4′, C-5′ C-4′, C-5′

δC(ppm) δH(ppm)

glucopyranoside, and named lygodiumsteroside B

38.7 67.5 76.8 30.3 51.4 203.1 121.7 166.4 34.3 39.0 21.1 32.0 48.1 84.2 31.8 21.5 50.1 17.9 24.1 77.7 21.6 76.8 28.2 37.2 30.6 23.4 22.4 104.2 74.7 78.7 71.6 78.5 62.6

Fig. 5. Lygosteroside B (1H-NMR).

Fig. 6. Lygosteroside B (1H-NMR).

The Research of Lygodium 91

Fig. 9. Lygosteroside B (HMBC).

Fig. 10. Lygosteroside B (HMBC).

Fig. 7. Lygosteroside B (HSQC).

Fig. 8. Lygosteroside B (HSQC).

Fig. 7. Lygosteroside B (HSQC).

Fig. 8. Lygosteroside B (HSQC).

Fig. 9. Lygosteroside B (HMBC).

Fig. 10. Lygosteroside B (HMBC).

The Research of Lygodium 93

Fig. 13. Lygosteroside B (NOESY).

Fig. 14. Lygosteroside B (NOESY).

Fig. 11. Lygosteroside B (HMBC).

Fig. 12. Lygosteroside B (1H-1H COSY).

Fig. 11. Lygosteroside B (HMBC).

Fig. 12. Lygosteroside B (1H-1H COSY).

Fig. 13. Lygosteroside B (NOESY).

Fig. 14. Lygosteroside B (NOESY).

The Research of Lygodium 95

MeOH–H2O system, giving two fractions. Fraction 2 (1.3 g) was isolated by a semipreparative ODS column using MeOH–H2O (65:35) as the eluent to afford lygodium A (9

Melting points were determined on an X4-A micro-melting point apparatus and were uncorrected. ESI-MS spectra were measured on an Agilent1100 LC-MSD-Trap-SL, and HR-ESI-MS spectra were measured on a Bruker Daltonics MicroTOFQ. NMR spectra were measured on a Bruker ARX-600 NMR spectrometer with tetramethylsilane (TMS) as the internal reference and chemical shifts are expressed with δ (ppm). UV spectra were recorded on a Shimadzu UV-2201 spectrometer. IR spectra were recorded on a Bruker IFS-55 spectrophotometer. TLC was performed on silica gel GF254 (10–40 l; Qingdao, China).Separation was performed by semiprep HPLC using Shimadzu SPD-10A apparatus

mg) and a kown compound ponastteroside A(30 mg), respectively.

equipped with a UV detector under an ODS column (i.d. 10 mm \* 200 mm).

C No. HMQC HMBC

1.72(t, *J*=12.6 Hz), 2.05(m) 4.06(br.dt, *J*=11.4 Hz), 4.29(brs) 1.66, 2.16(each m) 2.90(m) ⎯ 6.18(brs) ⎯ 3.50(brt) ⎯ 1.64, 1.77(each m) 1.86, 2.57(each m) ⎯ ⎯ 1.86, 2.12(each m) 2.05, 2.39(each m) 2.93(m) 1.09(s) 0.86(s) ⎯ 1.44(s) 4.44(dd, *J*=11.5Hz and 2.5Hz) C-2,C-3,C-9,C-10,C-19

C-4,C-9

C-5,C-9,C-14

C-8,C-9,C-10,C-12 C-9,C-11,C-13,C-18

C-14,C-16,C-17 C-17 C-13,C-16,C-18 C-12,C-13,C-14,C-17 C-2,C-5,C-9,C-10

C-17, C-20, C-22 C-23,C-24

1. NMR data (nuclear magnetic resonance) of lygodiumsteroside A

δC(ppm) DEPT δH(ppm)

**3.3.2 Apparatus** 

**3.3.3 The spectrum of new compound** 

38.0 (t) 66.8 (d) 77.1 (d) 29.9 (t) 50.7 (d) 202.3 (s) 121.1 (d) 165.4 (s) 33.5 (d) 38.4 (s) 20.3 (t) 31.2 (t) 47.2 (d) 83.4 (s) 31.1 (t) 20.7 (t) 49.2 (d) 17.3 (q) 23.4 (q) 75.1 (s) 20.7 (q) 85.3 (d)

Fig. 15. Lygosteroside B (ESI).

#### **3.3 The new compound 3 from** *Lygodium* **[7]**

lygodiumsteroside A

#### **3.3.1 Extraction and isolation**

The air-dried roots of *L. japonicum* (Thunb.) Sw. (4 kg) were crushed and extracted twice under reux with 70% EtOH. The solution was concentrated under reduced pressure to obtain the residue, and then the residue was extracted with MeOH. The MeOH-soluble fraction (100 g) was isolated by column chromatography on silica gel using gradient elution with CHCl3–MeOH (50:1 to 1:1), which gave 14 fractions. Fraction 9 (10 g) was subjected to silica gel column chromatography using CHCL3–MeOH(40:1 to1:1) in gradient to give fractions 1–4. Fraction 4 (3.7 g) was chromatographed on an ODS column eluting with

OH

O

O

OH

lygodiumsteroside A

The air-dried roots of *L. japonicum* (Thunb.) Sw. (4 kg) were crushed and extracted twice under reux with 70% EtOH. The solution was concentrated under reduced pressure to obtain the residue, and then the residue was extracted with MeOH. The MeOH-soluble fraction (100 g) was isolated by column chromatography on silica gel using gradient elution with CHCl3–MeOH (50:1 to 1:1), which gave 14 fractions. Fraction 9 (10 g) was subjected to silica gel column chromatography using CHCL3–MeOH(40:1 to1:1) in gradient to give fractions 1–4. Fraction 4 (3.7 g) was chromatographed on an ODS column eluting with

Fig. 15. Lygosteroside B (ESI).

**3.3 The new compound 3 from** *Lygodium* **[7]** 

HO

O

Glc

**3.3.1 Extraction and isolation** 

O

MeOH–H2O system, giving two fractions. Fraction 2 (1.3 g) was isolated by a semipreparative ODS column using MeOH–H2O (65:35) as the eluent to afford lygodium A (9 mg) and a kown compound ponastteroside A(30 mg), respectively.

#### **3.3.2 Apparatus**

Melting points were determined on an X4-A micro-melting point apparatus and were uncorrected. ESI-MS spectra were measured on an Agilent1100 LC-MSD-Trap-SL, and HR-ESI-MS spectra were measured on a Bruker Daltonics MicroTOFQ. NMR spectra were measured on a Bruker ARX-600 NMR spectrometer with tetramethylsilane (TMS) as the internal reference and chemical shifts are expressed with δ (ppm). UV spectra were recorded on a Shimadzu UV-2201 spectrometer. IR spectra were recorded on a Bruker IFS-55 spectrophotometer. TLC was performed on silica gel GF254 (10–40 l; Qingdao, China).Separation was performed by semiprep HPLC using Shimadzu SPD-10A apparatus equipped with a UV detector under an ODS column (i.d. 10 mm \* 200 mm).

#### **3.3.3 The spectrum of new compound**


1. NMR data (nuclear magnetic resonance) of lygodiumsteroside A

The Research of Lygodium 97

The molecular formula was determined as C35H54O12 by HR-ESI-MS (m/z 701.3309 [M+CL]-, calcd. 701.3309), along with 1H-NMR and 13C-NMR data. Additionally, the UV spectrum of compound 1 showed a maximum at λ = 243 nm and the IR spectrum exhibited absorption at 1,730 cm-1for an a, b-unsaturated carbonyl group. The 1H-NMR (600 MHz, C5D5N) spectrum showed an olenic proton at δ6.18 (1H, brs, H-7) and ve methyl signals at δ1.44 (3H, s, H-21), 1.28 (3H, d, J = 7.2 Hz,H-27), 1.09 (3H, s, H-18), 0.86 (3H, s, H-19), 0.67 (3H, t,J = 7.2 Hz,H-29). The 13C-NMR (150 MHz, C5D5N) showed ve methyl signals and a typical a, βunsaturated carbonyl group signals atδ 202.3 (C-6), 165.4 (C-8), and 121.1 (C-7), which revealed an ecdysteroid-type nucleus. It was also confirmed by the HMBC correlations of the new compound 3 . In the HSQC spectrum, δ4.06 (1H, brd,J = 11.4 Hz, H-2) andδ 4.29 (1H, brs, H-3) had direct correlation with δ 66.8 (C-2) and δ 77.1 (C-3), respectively, which combined with the information of long correlation in the HMBC spectrum—d 1.44 (3H, s, H-21) correlated with d 49.2 (C-17), d 85.2 (C-22), andδ 75.1 (C-20), respectively—all of these signals elucidated the presence of ve hydroxyl carbons C-2, C-3, C-14, C-20, and C-22. Additionally, δ 1.09 (3H, s, H-18) and δ1.44(3H, s, H-21), d 2.17 (1H, m, H-25) and δ1.28 (3H, d,J = 7.2 Hz, H-27), δ 2.93 (1H, m, H-17) andδ 2.05, 2.39(2H, each m, H-16) correlated mutually with each other in the 2D- COSY spectrum. The occurrence of α-βlactone ring in the side chain was evidenced by the carbonyl absorption at 1,730 cm-1in the IR and the δ 173.9 ppm in the13C-NMR. The HMBC spectrum showed the long correlation between the methyl proton signal at δ1.28 (3H, d,J = 7.2 Hz, H-27) and the carbon signals at δ39.5 (C-24),40.7 (C-25), and 173.9 (C-26). The correlation between the methyl proton signal at δ 0.67 (3H, t, J = 7.2 Hz, H-29) and the carbon signals at δ 25.9 (C-28), 39.5 (C-24), and the correlation between the methyl proton signal at δ2.19 (1H, m, H-25) and the carbon signals atδ 15.2 (C-27), 25.9 (C-28), and 39.5 (C-24) could be observed, respectively. Moreover , δ4.44 (1H, dd, J = 11.5 Hz and 2.5 Hz, H-22)also showed the correlation with δ29.0 (C-23), 39.5 (C-24)in the HMBC. Taken together, the structure of the side chain was identied. The HMBC correlation was observed between the anomeric proton signal at δ 4.89 (1H, d, H-10) and the carbon signal at d 77.1 due to C-3 of the aglycone moiety. This key long-range cross peak fixed the glycosidation position.The chemical shifts of the sugar moiety in 13C-NMR (δ103.5, 74.0, 78.0, 70.9, 77.8, 61.9) also confirmed the presence of B-glucopyranose. The anomeric configuration of glucose was determined to be on the basis of the JH–H values (J = 7.8 Hz) and d 4.09 (1H, brs, H-3) correlated with d 4.85 (1H, d, anomeric proton) in the NOE spectrum. (The NOESY spectra mentioned in this text was recorded in CD3OD because the key proton signals overlapped in C5D5N). The relative configuration of the new compound 3 was identified by the NOESY correlation between the proton signal atδ 4.09 (1H, brs, H-3) and δ3.85 (1H, brd, H-2), δ1.87 (1H, m, Ha-4). Careful comparison 13C-NMR data of the new compound 3 and a known compound, named capitasterone[8][9], revealed that the B, C, and D rings and the side chain were very similar to that of capitasterone. Furthermore, the stereochemistry of C-20 and C-22 of capitasterone had established R, in the NOE spectrum (in CD3OD) of he new compound 3, δ4.25 (1H, dd, J = 11.5 Hz and 2.5 Hz, H-22) had correlated with δ1.56 (1H, m, H-24);however, d 1.56 (1H, m, H-24) never correlated with d 2.17 (1H, m, H-25). All of these confirmed the stereochemistry of C24 and C25 as R and S, respectively. The 1H-NMR and13C-NMR signals of the aglycone moiety of he new compound 3 were found to be similar to that of capitasterone .Therefore, the structure of he

new compound 3 was elucidated safely as Lygodiumsteroside A (Fig. 15).


Table 4. 1H and 13C NMR data for lygodiumsteroside A (600 and 150 MHz, in C5D5N).

2. Correlative spectrum data:

Fig. 16. The key correlations of the new compound 3.

3. Analysis and conclusions of the new compound 3:

The new compound 3,white powder, mp.245–246℃, gave positive response to Liebermann– Burchard reaction and Molish reaction, suggesting a steroid glycoside structure. The sugar was identied as glucose by co-TLC with authentic sample after acid hydrolysis.

1.41, 2.05(each m) 1.41(m) 2.17(m) ⎯ 1.28(d, *J*=7.2Hz) 1.01,1.37(each m) 0.67(t, *J*=7.2Hz) 4.89(d, *J*=7.8Hz) 4.01(t-like) 4.18(overlap) 4.06(overlap) 3.90(t-like) 4.31(m), 4.49(brd, *J*=11.5Hz) C-24,C-25,C-28

C-24,C-27,C-28

C-24,C-25,C-26 C-23,C-24,C-29 C-24,C-28 C-3 C-3′ C-2′, C-4′ C-5′, C-6′ C-4′, C-5′ C-4′, C-5′

C No. HMQC HMBC

Table 4. 1H and 13C NMR data for lygodiumsteroside A (600 and 150 MHz, in C5D5N).

O

was identied as glucose by co-TLC with authentic sample after acid hydrolysis.

The new compound 3,white powder, mp.245–246℃, gave positive response to Liebermann– Burchard reaction and Molish reaction, suggesting a steroid glycoside structure. The sugar

<sup>H</sup> <sup>O</sup>

O

OH

OH

O

δC(ppm) DEPT δH(ppm)

29.0 (t) 39.5 (d) 40.7 (d) 173.9 (s) 15.2 (q) 25.9 (t) 9.6 (q) 103.5 (d) 74.0 (d) 78.0 (d) 70.9 (d) 77.8 (d) 61.9 (t)

23 24 25 26 27 28 29 C-1′ C-2′ C-3′ C-4′ C-5′ C-6′

2. Correlative spectrum data:

HO

O

H

Fig. 16. The key correlations of the new compound 3.

3. Analysis and conclusions of the new compound 3:

OH

OH OH

HO

The molecular formula was determined as C35H54O12 by HR-ESI-MS (m/z 701.3309 [M+CL]-, calcd. 701.3309), along with 1H-NMR and 13C-NMR data. Additionally, the UV spectrum of compound 1 showed a maximum at λ = 243 nm and the IR spectrum exhibited absorption at 1,730 cm-1for an a, b-unsaturated carbonyl group. The 1H-NMR (600 MHz, C5D5N) spectrum showed an olenic proton at δ6.18 (1H, brs, H-7) and ve methyl signals at δ1.44 (3H, s, H-21), 1.28 (3H, d, J = 7.2 Hz,H-27), 1.09 (3H, s, H-18), 0.86 (3H, s, H-19), 0.67 (3H, t,J = 7.2 Hz,H-29). The 13C-NMR (150 MHz, C5D5N) showed ve methyl signals and a typical a, βunsaturated carbonyl group signals atδ 202.3 (C-6), 165.4 (C-8), and 121.1 (C-7), which revealed an ecdysteroid-type nucleus. It was also confirmed by the HMBC correlations of the new compound 3 . In the HSQC spectrum, δ4.06 (1H, brd,J = 11.4 Hz, H-2) andδ 4.29 (1H, brs, H-3) had direct correlation with δ 66.8 (C-2) and δ 77.1 (C-3), respectively, which combined with the information of long correlation in the HMBC spectrum—d 1.44 (3H, s, H-21) correlated with d 49.2 (C-17), d 85.2 (C-22), andδ 75.1 (C-20), respectively—all of these signals elucidated the presence of ve hydroxyl carbons C-2, C-3, C-14, C-20, and C-22. Additionally, δ 1.09 (3H, s, H-18) and δ1.44(3H, s, H-21), d 2.17 (1H, m, H-25) and δ1.28 (3H, d,J = 7.2 Hz, H-27), δ 2.93 (1H, m, H-17) andδ 2.05, 2.39(2H, each m, H-16) correlated mutually with each other in the 2D- COSY spectrum. The occurrence of α-βlactone ring in the side chain was evidenced by the carbonyl absorption at 1,730 cm-1in the IR and the δ 173.9 ppm in the13C-NMR. The HMBC spectrum showed the long correlation between the methyl proton signal at δ1.28 (3H, d,J = 7.2 Hz, H-27) and the carbon signals at δ39.5 (C-24),40.7 (C-25), and 173.9 (C-26). The correlation between the methyl proton signal at δ 0.67 (3H, t, J = 7.2 Hz, H-29) and the carbon signals at δ 25.9 (C-28), 39.5 (C-24), and the correlation between the methyl proton signal at δ2.19 (1H, m, H-25) and the carbon signals atδ 15.2 (C-27), 25.9 (C-28), and 39.5 (C-24) could be observed, respectively. Moreover , δ4.44 (1H, dd, J = 11.5 Hz and 2.5 Hz, H-22)also showed the correlation with δ29.0 (C-23), 39.5 (C-24)in the HMBC. Taken together, the structure of the side chain was identied. The HMBC correlation was observed between the anomeric proton signal at δ 4.89 (1H, d, H-10) and the carbon signal at d 77.1 due to C-3 of the aglycone moiety. This key long-range cross peak fixed the glycosidation position.The chemical shifts of the sugar moiety in 13C-NMR (δ103.5, 74.0, 78.0, 70.9, 77.8, 61.9) also confirmed the presence of B-glucopyranose. The anomeric configuration of glucose was determined to be on the basis of the JH–H values (J = 7.8 Hz) and d 4.09 (1H, brs, H-3) correlated with d 4.85 (1H, d, anomeric proton) in the NOE spectrum. (The NOESY spectra mentioned in this text was recorded in CD3OD because the key proton signals overlapped in C5D5N). The relative configuration of the new compound 3 was identified by the NOESY correlation between the proton signal atδ 4.09 (1H, brs, H-3) and δ3.85 (1H, brd, H-2), δ1.87 (1H, m, Ha-4). Careful comparison 13C-NMR data of the new compound 3 and a known compound, named capitasterone[8][9], revealed that the B, C, and D rings and the side chain were very similar to that of capitasterone. Furthermore, the stereochemistry of C-20 and C-22 of capitasterone had established R, in the NOE spectrum (in CD3OD) of he new compound 3, δ4.25 (1H, dd, J = 11.5 Hz and 2.5 Hz, H-22) had correlated with δ1.56 (1H, m, H-24);however, d 1.56 (1H, m, H-24) never correlated with d 2.17 (1H, m, H-25). All of these confirmed the stereochemistry of C24 and C25 as R and S, respectively. The 1H-NMR and13C-NMR signals of the aglycone moiety of he new compound 3 were found to be similar to that of capitasterone .Therefore, the structure of he new compound 3 was elucidated safely as Lygodiumsteroside A (Fig. 15).

The Research of Lygodium 99

Fig. 19. Lygosteroside A (DEPT).

Fig. 20. Lygosteroside A (HSQC, in C5D5N).

Fig. 17. Lygosteroside A (1H-NMR, in C5D5N).

Fig. 18. Lygosteroside A (13C-NMR, in C5D5N).

Fig. 17. Lygosteroside A (1H-NMR, in C5D5N).

Fig. 18. Lygosteroside A (13C-NMR, in C5D5N).

Fig. 19. Lygosteroside A (DEPT).

Fig. 20. Lygosteroside A (HSQC, in C5D5N).

The Research of Lygodium 101

Fig. 23. Lygosteroside A (HMBC, in C5D5N).

Fig. 24. Lygosteroside A (HMBC, in C5D5N).

Fig. 21. Lygosteroside A (HSQC, in C5D5N).

Fig. 22. Lygosteroside A (HMBC, in C5D5N).

Fig. 21. Lygosteroside A (HSQC, in C5D5N).

Fig. 22. Lygosteroside A (HMBC, in C5D5N).

Fig. 23. Lygosteroside A (HMBC, in C5D5N).

Fig. 24. Lygosteroside A (HMBC, in C5D5N).

The Research of Lygodium 103

Fig. 27. Lygosteroside A (NOESY, in MeOH).

Fig. 28. Lygosteroside A (ESI).

Fig. 25. Lygosteroside A (HSQC, in MeOH).

Fig. 26. Lygosteroside A (HSQC, in MeOH).

Fig. 25. Lygosteroside A (HSQC, in MeOH).

Fig. 26. Lygosteroside A (HSQC, in MeOH).

Fig. 27. Lygosteroside A (NOESY, in MeOH).

Fig. 28. Lygosteroside A (ESI).

The Research of Lygodium 105

are different.The paper aims at making a systematical research for the root of *Lygodium japonicum* (Thunb.)Sw. that is one specie of medicinal *Lygodium*. According to the existing literature, the main constituents of *Lygodium* are steroidal, flavonoids, organic acids and other substances, but it is no clear whether all these chemical constituents are effective

This paper summarize the research on chemical constituents and pharmacological actions of Lygodium, . From our systematic research on Lygodium, we have isolated and identified many kinds of compounds. In this paper, we focus on introduction of three new compounds: lygodiumsteroside A and lygodiumsteroside B and 2-isopropyl-7-methyl-6-

NMR、2D-NMR and HR-MS. We also generalize pharmacological actions and clinical

[1] Ren-chao Zhou,Shu-bin Li,The Rrsearch of Pteridophyta on antibacterial .Hunan

[2] Li-juan Chen , Guo-gang Zhang,Lin-xia Zhu.The research about the components of

[3] Li-xia Zhu ,Guo-gang Zhang,Li-juan Chen.The research about the components from N-

[4] C Ye, CL Fan, LH Zhang. A new phenolic glycoside from the roots of *Lygodium* 

[5] Lijuan Chen,Guogang Zhang, Jie He.New naphthoquinone from the root of *Lygodium*  japonicum(Thunb)Sw.[J] Journal of Natural medicine:2010(64):114-116. [6] Lixia Zhu,Guogang Zhang,shengchao Wang.A new compound from lygodium

[7] Linxia Zhu,Guogang Zhang,LIjuan Chen.A new ecdysteroside from Lygodium

[9] Huang X-C, Guo Y-W (2003) Ecdysteroids from the stems of Diploclisia glaucescens. Nat

[10] Tieguang tong,Ping Dong.Climbing Fern Spore[M].The practical science of traditional

[11] Ma CM, Nakamura N, Miyashiro H, et al. Screening of Chinese and Mongolian herbal

[12] Leihong Zhang, Wencai Ye.The research of chemical constituents and biological actions.[J].Natural Product Research and Development2007, (19):552-557: [13] Jiajun Liu,Jing wang.The experimental study on normalizing functioning of the

gallbladder .[J]. Anhui Medical Journal: 1987, 8 (1): 34-35.

c29 insect-moulting substance from cyathula capitata. Tetrahedron Lett 9:4929–

drugs for anti-human immunodeficiency virus type 1( HIV-1) activity[J].

japonicum(Thunb)SW[J].Natural product research :2009:1284-1289.

japonicum(Thunb)SW[J].Natural Product Reseach:2009(63):215-219. [8] Takemoto T, Nomoto K, Hikino Y, Hikino H (1968) Structure of capitasterone, a novel

newsletter of Traditional Chinese medicine: 1999,5 (1):13-14.

butanol layer of Lygodium japonicum [D]:2008:3-30.

Lygodium japonicum [D]:2008:3-26.

*japonicum*. Fitoperapia, 2007, 78: 600-601.


composition or not.

α

**7. References** 

applications of Lygodium.

4932.

Prod Res Dev 15:93–97.

Chinese medicinal herbs:439.

Phytotherapy Research, 2002, 16:186-189

hydroxy-

## **4. Pharmacological actions**

#### **4.1** Antibacterial action[10]

Inhibiting *Staphylococcus aureus*, *Pseudomonas aeruginosa, Salmonella typhi* and *shigella Flexneri*.

#### **4.2** Antivirus[11]

Water extract and alcohol extract of Lygodium spores both can inhibit HIV-1 virus. The Concertration of water extract above 125ug/ml can wholly inhibit HIV-1 virus.

**4.3** Resistance to male hormone and effect on hair growth[12]

50% alcohol extract of Lygodium Spores can inhibit the activity of Testosterone 5-α reductase in vitro and activate hair follicle.

**4.4** Normalizing functioning of the gallbladder and dissolving stone[13]

**4.5** Liver protection[14]

The water extract of Lygodium (50ug/ml) can significantly reduce the GPT action of liver Cell cultures containing GaIN 5\*10~3mol/L,so it has effect on liver protection markably.

**4.6** Cure urinary impassability, tumescent feeling below umbilicus,

**4.7** Urgent pain:Lygodium powder drinking by licorice soup.

**4.8** Have a good effect on urinary tract infection, urinary calculus, nephritis edema, cold with fever ,urine content small and cardial, enteritis diarrhea.

## **5. Indications[10]**

**5.1** Stranguria marked by chyluria, stranguria caused by the passage of urinary stone, stranguria from urolithiasis and strangury due to heat. For dribling and painful micturition , there is powder of climbing fern spore from *standards* of *diagnosis* and *treament*: Climbing fern spore 6g,red poria 9g, umbellate pore-fungus 9g, white atractylodes rhizome 9g,peony root 9g, oriental water plantain rhizome 15g, talc 21g, pyrrosia leaf 3g. Grind these herbs into a fine powder. Take 9g each time. For stranguria marked by chyluria ,there is powder of climbing fern spore: Climbing fern spore 30g, talc 30g, licorice root 7.5g. Grind these herbs into a fine powder.Take 6g each time.

**5.2** Dampness in the spleen, general edema, distension in the abdomen can be treated with powder of fern spore from *Inventions of medicine:* Morning glory seed 45g, *kansui* root 15g climbing fen spore 15g, grind these herbs into a fine powder. Take 6g each time before meals.

#### **6. Conclusion**

*Lygodium japonicum* (Thunb.)Sw., which belongs to the genus *Lygodium* of the family Lygodiaceae, is the dry root and rhizome of *Lygodium japonicum* (Thunb.)Sw. There are different constituents in different parts of it and distribution of content of these constituents

Inhibiting *Staphylococcus aureus*, *Pseudomonas aeruginosa, Salmonella typhi* and *shigella*

Water extract and alcohol extract of Lygodium spores both can inhibit HIV-1 virus. The

50% alcohol extract of Lygodium Spores can inhibit the activity of Testosterone 5-α reduct-

The water extract of Lygodium (50ug/ml) can significantly reduce the GPT action of liver Cell cultures containing GaIN 5\*10~3mol/L,so it has effect on liver protection markably.

**4.8** Have a good effect on urinary tract infection, urinary calculus, nephritis edema, cold

**5.1** Stranguria marked by chyluria, stranguria caused by the passage of urinary stone, stranguria from urolithiasis and strangury due to heat. For dribling and painful micturition , there is powder of climbing fern spore from *standards* of *diagnosis* and *treament*: Climbing fern spore 6g,red poria 9g, umbellate pore-fungus 9g, white atractylodes rhizome 9g,peony root 9g, oriental water plantain rhizome 15g, talc 21g, pyrrosia leaf 3g. Grind these herbs into a fine powder. Take 9g each time. For stranguria marked by chyluria ,there is powder of climbing fern spore: Climbing fern spore 30g, talc 30g, licorice root 7.5g. Grind these herbs into a fine

**5.2** Dampness in the spleen, general edema, distension in the abdomen can be treated with powder of fern spore from *Inventions of medicine:* Morning glory seed 45g, *kansui* root 15g climbing fen spore 15g, grind these herbs into a fine powder. Take 6g each time before

*Lygodium japonicum* (Thunb.)Sw., which belongs to the genus *Lygodium* of the family Lygodiaceae, is the dry root and rhizome of *Lygodium japonicum* (Thunb.)Sw. There are different constituents in different parts of it and distribution of content of these constituents

Concertration of water extract above 125ug/ml can wholly inhibit HIV-1 virus.

**4.4** Normalizing functioning of the gallbladder and dissolving stone[13]

**4.6** Cure urinary impassability, tumescent feeling below umbilicus,

**4.7** Urgent pain:Lygodium powder drinking by licorice soup.

with fever ,urine content small and cardial, enteritis diarrhea.

**4.3** Resistance to male hormone and effect on hair growth[12]

**4. Pharmacological actions** 

ase in vitro and activate hair follicle.

**4.1** Antibacterial action[10]

*Flexneri*.

**4.2** Antivirus[11]

**4.5** Liver protection[14]

**5. Indications[10]**

powder.Take 6g each time.

meals.

**6. Conclusion** 

are different.The paper aims at making a systematical research for the root of *Lygodium japonicum* (Thunb.)Sw. that is one specie of medicinal *Lygodium*. According to the existing literature, the main constituents of *Lygodium* are steroidal, flavonoids, organic acids and other substances, but it is no clear whether all these chemical constituents are effective composition or not.

This paper summarize the research on chemical constituents and pharmacological actions of Lygodium, . From our systematic research on Lygodium, we have isolated and identified many kinds of compounds. In this paper, we focus on introduction of three new compounds: lygodiumsteroside A and lygodiumsteroside B and 2-isopropyl-7-methyl-6 hydroxyα- (1,4) naphthoquinone, as well as the data analysis of UV, IR, 1H-NMR, 13C-NMR、2D-NMR and HR-MS. We also generalize pharmacological actions and clinical applications of Lygodium.

#### **7. References**


**6** 

*México* 

**Natural Alkamides: Pharmacology,** 

*Centro de Investigaciones Químicas, Universidad Autónoma del Estado de Morelos,* 

Alkamides are a broad and expanding group of bioactive natural compounds found in at least 33 plant families. Despite the relatively simple molecular architecture of alkamides (fig. 1), these natural products show broad structural variability and an important range of biological activities, such as immunomodulatory, antimicrobial, antiviral, larvicidal, insecticidal, diuretic, pungent, analgesic, cannabimimetic and antioxidant activities. Additionally, alkamides are involved in the potentiation of antibiotics and the inhibition of prostaglandin biosynthesis,

Many plant species containing alkamides have been used in traditional medicine by different civilizations around the world. Many of the plants containing these natural products have been used in the treatment of toothaches and sore throats (Rios-Chavez et al., 2003). These compounds are present in different organs of the plant, such as roots (*Heliopsis longipes*, *Echinaceae purpurea*, *Achillea wilhelmsii*, A*cmella oppositifolia*, *Asiasarum heterotropoide, Cissampelos glaberrima*, etc.), leaves and stems (*Aristolochia gehrtii*, *Phyllanthus fraternus*, *Amaranthus hypochondriacus*, *Achyranthes ferruginea*, etc.), the pericarpium (*Zanthoxylum piperitum* and *Piper spp*.), the placenta of *Capsicum spp*., the fruits of *Piper longum*, the flowers of *Spilanthes acmella*, the seeds of the *Piper* species and tubers of *Lepidium meyenii*. It is believed that alkamides act as plant growth regulators, promoting or inhibiting the growth and formation of roots in a dose-dependent manner and showing a positive effect in plant

Structurally, natural alkamides commonly have an aliphatic, cyclic or aromatic amine residue, and a C8 to C18 saturated or unsaturated chain (including double or triple bonds, or both) acid, which can also be aromatic. The nature of the acid (carbon chain lengths, unsaturation level, stereochemistry, etc.) and the amine residues are characteristic of each family and genus of plants such that these characteristics serve as chemotaxonomic criteria (fig. 1). Because the nitrogen atom of alkamides is not part of a heterocyclic ring, these

Alkamides represent a class of lipidic compounds structurally related to animal endocannabinoids. Notably, based on the structural similarity of these compounds to

RNA synthesis and the arachidonic acid metabolism, among others.

biomass production (Campos-Cuevas, et al., 2008).

compounds are classified as protoalkaloids or pseudoalkaloids.

**1. Introduction** 

 **Chemistry and Distribution** 

María Yolanda Rios

*Col. Chamilpa, Cuernavaca, Morelos,* 

[14] Ruiying Fang,Zhongyao Shi.Observing ten kinds of traditional Chinese medicine on the function of resistance to toxic liver in using original generation to cultivate liver cells.[J] Modern pharmaceutical application : 1995, 12 (1): 5-7.
