Labdane-type Diterpenoids fromLeonurus heterophyllus and TheirCholinesterase Inhibitory Activityptr_3307 611..614

Tran Manh Hung,1,2* Tran Cong Luan,3 Bui The Vinh,3 To Dao Cuong1 and Byung Sun Min1

1College of Pharmacy, Catholic University of Daegu, Gyeongbuk 712-702, Korea2Department of Pharmacognosy, Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan3Research Center of Ginseng and Medicinal Materials, 41 Dinh Tien Hoang, Ho Chi Minh City, Vietnam

In the course of screening plants used in natural medicines as memory enhancers, a 70% ethanol extract of theaerial parts of Leonurus heterophyllus showed significant AChE inhibitory activity. Bioassay-guided fraction-ation and repeated column chromatography led to the isolation of a new labdane-type diterpenoids (1), namedleoheteronin F, and six known compounds (2–7). The chemical structures of isolated compounds were elucidatedbased on extensive 1D and 2D NMR spectroscopic data. The isolates 1–7 were investigated in vitro for theiranticholinesterase activity using mouse cortex AChE enzyme. Leoheteronin A (5) and leopersin G (7), whichpossess a 15,16-epoxy group at the side chain, were found to be potent in the inhibition of AChE. Copyright ©2010 John Wiley & Sons, Ltd.

Keywords: Leonurus heterophyllus; Lamiaceae; leoheteronin; leopersin; acetylcholinesterase.

INTRODUCTION

Alzheimer’s disease (AD) is the most common age-related neurodegenerative disease with many cognitiveand neuropsychiatric manifestations that result in pro-gressive disability and eventual incapacitation. Adecrease of acetylcholine in the brain of patients withAD appears to be a critical element in producingdementia (Becker et al., 1988). Loss of cholinergic cells,particularly in the basal forebrain, is accompanied byloss of the neurotransmitter acetylcholine. Oneapproach is to inactivate acetylcholinesterase, theenzyme that cleaves synaptic acetylcholine and termi-nates neuronal signaling. Acetylcholinesterase (AChE)inhibitors increase the availability of acetylcholine incentral cholinergic synapses and are the most promisingcurrently available drugs for the treatment of AD (Gia-cobini, 2000). The AChE inhibitors from general chemi-cal classes such as physostigmine, tacrine, donepezil,galanthamine, huperzine A and heptylphysostigminehave been tested for the symptomatic treatment of AD.Although there have been a number of reports on thedesign and development of synthetic AChE inhibitors, itis still necessary to have other studies reporting AChEinhibitors derived from medicinal plants (Oh et al.,2004; Houghton et al., 2006).

In our screening programme to search for AChEinhibitors from plants, a 70% EtOH extract of the aerialparts of Leonurus heterophyllus SW (Lamiaceae) exhib-ited significant inhibitory activity. L. heterophyllus isknown as ‘Ich mau thao’ (motherwort) in Vietnamese

traditional medicine. The dried aerial parts of this plant(Herba Leonuri) are applicable in the treatment ofmenstrual pain and childbirth, high blood pressure,blood stasis, heart disorders, dysentery (Do, 2001) andfor anticancer (Chinwala et al., 2003). The dried ripefruits (Fructus Leonuri) are used to treat edema and asa diuretic (Do, 2001). Previously, labdane-type diterpe-noids (Hon et al., 1991, 1993; Giang et al., 2005a, 2005b;Cai et al., 2006), sterols (Giang et al., 2005a), flavonoids(Giang et al., 2005a; Cong et al., 2005) and cyclic non-apeptides (Morita et al., 2006) were isolated from L.heterophyllus. Prehispanolone, one of the labdane diter-penoids isolated from this plant, was shown to be anovel platelet activating factor receptor antagonist (Leeet al., 1991). However, components with anticholine-sterase activity have not been investigated yet. Thepresent study reports the isolation and identification ofa new labdane-type diterpenoids (1) and six known ones(2–7) with regard to the active principles in AChEactivity.

MATERIALS AND METHODS

Plant material. The aerial parts of Leonurus hetero-phyllus were purchased at Dong Xuan Oriental her-barium market in May 2004 in Hanoi, Vietnam. Theplant was botanically identified by Professor Vu VanChuyen, Hanoi University of Pharmacy, where thevoucher specimen was deposited (2004–5).

Extraction and isolation. Dried aerial parts of L. het-erophyllus (1.0 kg) were extracted with hot 70% EtOH(3 ¥ 3 L) under reflux, and the combined extract wasremoved under vacuum to afford a viscous residue (45.9 g). The crude extract was suspended in water and

* Correspondence to: Dr Tran Manh Hung, Department of Pharmacog-nosy, Institute of Natural Medicine, University of Toyama, 2630 Sugitani,Toyama 930–0194, Japan.E-mail: tmhung1810@yahoo.com

PHYTOTHERAPY RESEARCHPhytother. Res. 25: 611–614 (2011)Published online 27 October 2010 in Wiley Online Library(wileyonlinelibrary.com) DOI: 10.1002/ptr.3307

Copyright © 2010 John Wiley & Sons, Ltd.

Received 18 June 2010Revised 25 August 2010

Accepted 07 September 2010

extracted successively with n-hexane, ethyl acetate(EtOAc) and butanol (BuOH). The n-hexane-solublefraction was concentrated under diminished pressure,and the residue (11.2 g) was purified by open columnchromatography over silica gel eluted with a mixture ofhexane–EtOAC (80:1 to 0:1) to afford ten fractions(H-1–H-10) based on the TLC profile. Fraction H-3 wassubjected to a silica gel column using CHCl3–MeOH(90:1) to yield 2 (38.5 mg) and 3 (10.1 mg). Fraction H-4was chromatographed on a silica gel column using stepgradient elution with hexane–acetone (30:1 and 20:1)and then followed by preparative TLC (silica gel F254

plates) to give compounds 1 (5.5 mg) and 6 (15.7 mg).Fraction H-5 was subjected to a Sephadex LH–20column eluted with CHCl3–MeOH (60:40) to affordthree subfractions (H-5-1–H-5-3). Compounds 5 (18.5 mg) and 7 (8.4 mg) were obtained from the H-5-2fraction, using a silica gel column with hexane–acetone(15:1) as eluant. Separation of subfraction H-5-3 on asilica gel column eluted with hexane–EtOAc gradient(13:1 and 10:1) gave 4 (28.1 mg).

Leoheteronin F (1): white amophous powder; α[ ]D25 -

2.5 (c 0.10, CHCl3), IR (KBr) vmax: 3420, 1705, 1674 cm-1;HR-ESI-MS: m/z 323.2591 [M+H]+ (calcd for C20H35O3

323.2597); 1H and 13C NMR data are shown in Table 1.

Acetylcholinesterase (AChE) activity assay. AdultSprague–Dawley male rats, weighing 200–250 g, werepurchased from the Daehan Biolink Co., Ltd. Theanimals were housed, allowed access to water and foodad libitum, and maintained in a constant temperature (25� 1°C) and humidity (60 � 10%) environment under a12 h light/dark cycle (light on 07.30–19.30 h). Animaltreatment and maintenance were carried out in accor-dance with the Principles of Laboratory Animal Care(NIH publication No. 85-23, revised 1985) and theAnimal Care and Use Guidelines of Catholic Universityof Daegu, Korea. The rats were decapitated, the cortex

was rapidly dissected from the brain on ice and thenweighed and homogenized in five volumes of cold 75 mmsodium phosphate buffer (pH 7.4). Homogenates werecentrifuged at 13000 ¥ g for 30 min at 4°C; the superna-tants selected as AChE sources were divided into ali-quots and stored at -20°C (Hung et al., 2008a, 2008b).The AChE activity was measured by the principle of theEllman method with some modifications (Ellman et al.,1961; Zhao and Tang, 2002). The tested compound wasinitially dissolved in 0.02% dimethyl sulfoxide (DMSO)and diluted to various concentrations in sodium phos-phate buffer (100 mm, pH 8.0) immediately before use.An aliquot of the diluted compound solution was thenmixed with sodium phosphate buffer (100 mm pH 8.0),acetylthiocholine iodide solution (75 mm) and Ellman’sreagent (10 mm 5,5′-dithio-bis [2-nitrobenzoic acid] and15 mm sodium bicarbonate) and reacted at room tem-perature for 30 min. The absorbance was measured at410 nm immediately after adding the enzyme source tothe reaction mixtures using a spectrophotometer (Shi-madzu UV-1240, Tokyo, Japan). Readings were taken at30 s intervals for 5 min.The concentration of compoundrequired to inhibit acetylcholinesterase activity by 50%(IC50) was calculated using an enzyme inhibition dose–response curve. Tacrine (Sigma) was used as a positivecontrol (Zhao and Tang, 2002).

RESULTS AND DISCUSSION

Repeated column chromatography led to the isolation ofseven compounds (1–7). The structures of the knownditerpenoids, leoheterin (2),hispanone (3),galeopsin (4),leoheteronin A (5), leoheteronin D (6) and leopersin G(7) were determined by comparing their physical andspectroscopic data with the literature values (Hon et al.,1991, 1993; Giang et al., 2005a, 2005b) (Fig. 1).

Compound 1 was isolated as a white powder with anoptical rotation of -2.5 (c 0.10, CHCl3). The molecularformula of 1 was deduced to be C20H34O3 on the basis ofthe peak at m/z 323.2591 [M + H]+ in HR–ESI–MS. TheIR spectrum showed absorption bands at 3420, 1705 and1674 cm-1, which are characteristic for hydroxy, ketonand olefinic functional groups. The 1H NMR spectrumdisplayed four singlet signals of methyl groups, includ-ing three tertiary methyls at d 0.85 (3H, s, H-18), 0.90(3H, s, H-19) and 1.12 (3H, s, H-20), and one olefinicmethyl at d 1.72 (3H, s, H-16). The proton signals at d1.15 (3H, d, J = 6.0 Hz, H-17) indicated the presence ofa secondary methyl group. The signals of an olefinicproton at d 5.48 (1H, t, J = 6.8 Hz, H-14), and a methineproton at d 2.28 (1H, q, J = 6.4 Hz, H-8) were alsoobserved.The other proton signals were assigned for sixmethylenes and one allylic group. Examination of the13C NMR and DEPT spectra confirmed the presence of20 carbon atoms. Among them, signals at d 210.4 and82.0 ppm were characteristic for a ketone (C-7) and acarbon bound to an oxygen atom (C-10); signals at d141.2 and 125.7 ppm were indicative of a quarternary(C-13) and an olefinic carbon (C-14), respectively. Thefull NMR assignments and connectivities of 1 weredetermined by the detailed analysis of its HMQC,HMBC and COSY spectra (Fig. 2). Its NMR data werecomparable to those of the other labdane-type diterpe-noids, leoheteronin D and leoheteronin E, which were

Table 1. 1H (400 MHz) and 13C NMR (100 MHz) assignments ofcompound 1 in CDCl3 (d ppm)

Position dH dC

1 1.54 (1H, m, Ha), 1.38 (1H, m, Hb) 33.52 1.62 (1H, m, Ha), 1.60 (1H, m, Hb) 18.63 1.40 (1H, m, Ha), 1.25 (1H, m, Hb) 41.54 34.05 2.05 (1H, dd, J = 14.2, 3.0 Hz) 46.66 2.47 (1H, dd, J = 14.2, 3.0 Hz, Ha)

2.32 (1H, t, J = 14.2 Hz, Hb)40.0

7 210.48 2.82 (1H, q, J = 6.6 Hz) 51.59 82.0

10 43.411 1.90 (2H, m) 23.712 1.98 (2H, t, J = 8.0 Hz) 43.513 141.214 5.48 (1H, t, J = 6.8 Hz) 125.715 4.27 (2H, dd, J = 6.8, 2.0 Hz) 60.116 1.72 (3H, s) 16.817 1.15 (3H, d, J = 6.6 Hz) 9.118 0.85 (3H, s) 33.919 0.90 (3H, s) 22.520 1.12 (3H, s) 25.7

612 T. M. HUNG ET AL.

Copyright © 2010 John Wiley & Sons, Ltd. Phytother. Res. 25: 611–614 (2011)

isolated from the same plant (Hon et al., 1993; Gianget al., 2005a). The HMBC correlations between methylprotons (d 1.15) and C-7 (d 210), C-8 (d 51.5), confirmedthat the methyl group was attached to C-8. The HMBCcorrelations between methyl protons (d 1.12, H-20), amethine proton (d 2.82, H-8) and C-9 (d 82.0), confirmedfor the location of a hydroxy group at C-9. Also, thecorrelations between H-8 (d 2.82), H-6 (d 2.32 and 2.47)and C-7 (d 210.4) determined the location of the ketonegroup on C-7. In adddition, significant HMBC correnla-tions were detected between methyl protons (d 1.71,H-16) and C-12 (d 43.5) and C-13 (d 141.2); betweenallyl alcohol proton (d 4.27, H-15) and C-13 and C-14 (d125.7); between both of the methyl groups at H-18 andH-19 and C-3 (d 41.5), C-4 (d 34.0), and C-5 (d 46.6)(Fig. 2).Thus, 1 was suggested to be a 9-hydroxylabd-13-ene diterpenoids (Giang et al., 2005a).The configurationof the double bond at the side chain was determined tobe E by observing the signal for both allylic protons at d

4.27 (2H, dd, 6.8, 2.0 Hz, H-15) since the Z-isomer wouldhave shown two distinct dd signals (Schmidt et al., 1995;Giang et al., 2005a). The relative stereochemistry of 1was determined by nuclear Overhauser enhancementand exchange spectroscopy (NOESY) (Fig. 2). The cor-relations between H-14 and H-12, between H-15 andH-16 provided the confirmation of the E-configurationof the double bond of side chain (Schmidt et al., 1993;Giang et al., 2005a). The NOESY cross-peak detectedbetween H-20 and H-11, and the lack of cross-peakbetween H-20 and H-17 suggested the sameb-orientation of the side chain, which was opposite tothe a-orientation of the hydroxy group at C-9.Addition-ally, the cross-peaks from H-8 to H-20, H-11, and H-6bconfirmed the relative stereochemistry of H-17 asa-orientation. Based on the above analyses, the struc-ture of 1 was established as 9a-hydroxylabd-13(E)-ene-15-ol, which was named as heteronin F.

To investigate the anticholinesterase activity of theisolated diterpenoids (1–7), rat brain cortex wasselected as a source of AChE enzyme (Zhao and Tang,2002). The results (IC50) are summarized in Table 2.Among the tested compounds, leoheteronin A (5) andleopersin G (7) showed potent activity in a dose depen-dent manner with IC50 values of 11.6 and 12.9 mm,respectively.The new compound, leoheteronin F (1) andleoheteronin D (6) showed moderate inhibitory activitywith IC50 values of 16.1 and 18.4 mm, respectively. Theother compounds showed weak activity with IC50 valuesranging from 38.5 to 42.7 mm. Tacrine was used as apositive control and exhibited inhibitory activity with anIC50 value of 170.2 nm.

OH

OHOH1

3 4 5

6

7

8910

1112

13

14

15

16

17

18 19

20

1

OHH

OH

2

OH

OH

3

O O

OH

OH

4

OH

5

O O

H

6

OH

OH

7

O

OAc O

OH

OH

O

Figure 1. Chemical structures of isolated compounds 1–7.

O

OHOH

COSYHMBC

H3C

CH3

CH3

HH

H

OHO CH3

H

OH

NOESY

Figure 2. Selected H–C long-range correlations in HMBC and1H–1H correlation in COSY spectrum of 1.

613ANTICHOLINESTERASE ACTIVITY OF LABDANE-TYPE DITERPENOIDS FROM L. HETEROPHYLLUS

Copyright © 2010 John Wiley & Sons, Ltd. Phytother. Res. 25: 611–614 (2011)

The AChE inhibitors increased the availability of ace-tylcholine in central cholinergic synapses and are themost promising currently available drugs for the treat-ment of Alzheimer diseases. In our previous reports, thenatural AChE inhibitors have been detected and tracedto the benzylisoquinoline alkaloids from Corydalisspecies (Hung et al., 2008a, 2008b), Stephania rotunda(Hung et al., 2010) and dibenzocyclooctadiene lignansfrom Schisandra chinensis (Kim et al., 2006; Hung et al.,2007). In this study, the isolated labbdane diterpenoidsshowed significant AChE inhibitory activities, however,

they are less effective than tacrine. These results are inaccordance with those of previous studies reporting thatthe tricyclic cis-clerodane diterpenoids possess potentialanticholinesterase activities (Calderon et al., 2001;Ahmad et al., 2005). Interestingly, the isolated diterpe-noids showed significant different activities relative totheir chemical structures. Leoheteronin A (5, IC50 =11.6 mm) was found to be the most potent compound,which possessed 8,13 diene and 15,16-epoxy groups on itsstructure. Also, leopersin G (7) having 13-dien,15,16-epoxy but not 8-dien, significantly inhibited the AChEactivity (IC50 = 12.9 mm). Leoheteronin F (1) and leohet-eronin D (6) substituted with 13(E)-ene at side chainwere somewhat less active than 5 and 7.The diterpenoidsbearing a furan ring at the side chain such as 2,3 and 4 hada 3–4 fold lower effect than the most active one (5).Theseresults indicate the important role for the substituent atside chain in determining the difference of the AChEinhibitory activity. Additionally, ketone, methyl andhydroxy groups, regardless of the C-7, C-8 and C-9 posi-tions, do not seem to be critical for this activity.Althoughmore studies need to be undertaken, the extract andthese isolated constituents of L. heterophyllus might bepotential AChE natural inhibitor sources.

Conflict of Interest

The authors have declared that there is no conflict of interest.

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Table 2. Inhibitory activity of isolated compounds on mousecortex AChE

Compound IC50 (mM)a

1 16.1 � 1.22 41.4 � 3.53 38.5 � 1.94 42.7 � 2.15 11.6 � 1.26 18.4 � 2.57 12.9 � 1.6Tacrineb 0.17 � 0.02

a Results are the mean of three replications.b Reference control.

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