Fitoterapia 81 (2010) 610–613

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Fitoterapia

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A new diterpene from the leaves of Andrographis paniculata Nees

Chong Xu a, Gui Xin Chou b, Zheng Tao Wang a,⁎a The MOE Key Laboratory for Standardization of Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine,Shanghai 201210, PR Chinab Shanghai R&D Center for Standardization of Chinese Medicines (SCSCM), Shanghai 201210, PR China

a r t i c l e i n f o

⁎ Corresponding author. Tel.: +86 21 51322507; faE-mail address: wangzht@hotmail.com (Z.T. Wang

0367-326X/$ – see front matter © 2010 Elsevier B.V.doi:10.1016/j.fitote.2010.03.003

a b s t r a c t

Article history:Received 27 September 2009Accepted in revised form 21 February 2010Available online 15 March 2010

Phytochemical investigation of the ethanol extract of the leaves of Andrographis paniculata yieldedone novel diterpene (13R, 14R) 3, 13, 14, 19-tetrahydroxy-ent-labda-8 (17), 11-dien-16, 15-olide1 which has an uncommon cis-diol groups in the lactone moiety, and 3, 19-isopropylidene-14-deoxy-ent-labda-8 (17), 13-dien-16, 15-olide2, probably anartifactditerpene, togetherwith eightknown diterpenoids 3–10. The structures of these compounds were determined on the basis ofspectralmethods. The structure and stereochemistry of1was confirmedbyX-ray crystallographicanalyses.

© 2010 Elsevier B.V. All rights reserved.

Keywords:Andrographis paniculataent-labdane diterpenoidsX-ray crystallographic analyses

1. Introduction

Andrographis paniculata Nees (Acanthaceae) is a medicinalherbwidelyused for centuries in India, China andSoutheastAsiaas a antibacterial [1], anti-inflammatory [2–7], anti-malarial [8],antithrombotic [9], hepatoprotective [10], anticancer [11,12],immunostimulant [11] agent. It is also claimed effective to treatdiabetes [13] and HIV-I [14]. Previous phytochemical studies onthis herb have resulted in the isolation of about 50 labdanediterpenoids as well as 30 flavonoids [15–18].

In the present study, we report one new diterpene (13R,14R) 3, 13, 14, 19-tetrahydroxy-ent-labda-8 (17), 11-dien-16,15-olide 1 and a artifact 3, 19-isopropylidene-14-deoxy-ent-labda-8 (17), 13-dien-16, 15-olide 2 (Fig. 1) together with8 knownditerpenoids. The structureswere identifiedmainly by1D and 2D NMR. The stereochemistry of 1 was determined byX-ray crystallographic analyses.

2. Experimental

2.1. Generals

Optical rotations: KRÜSS P8000-T. CD: Jasco J-810 Chirop-tical spectrometer. IR: ThermoNICOLET 380 FT-IR.UV: TV-1901

x: +86 21 51322519.).

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Atomic absorption spectrophotometer. NMR: Bruker 500ultrashield spectrometer. ESI–MS: Thermo Finnigan SurvyorLCQ DECA XP Plus spectrometer. HR–ESI–MS:Waters ACQUITYUPLC Micromass Q-TOF micro. X-ray crystallographic analysis:Bruker-SMART CCD area detector diffractometer.

2.2. Plant material

The aerial parts of Andrographis paniculata were pur-chased from Guangxi province and identified by associateprofessor Li Hong Wu of Shanghai University of TraditionalChinese Medicine. A voucher specimen (No. s01072301) wasdeposited in the herbarium of Institute of Chinese MateriaMedica, Shanghai University of TCM.

2.3. Extraction and isolation

The dried powder of the leaves of Andrographis paniculata(10 kg) was extracted with 85% ethanol (40 L) for 3 times andfiltered. The filtrate which was suspended in water waspartitioned by ethyl acetate for 6 times. Then the combinedextracts were concentrated in vacuum and degreased byactivated carbon. This filtrate was evaporated (yield 400 gextract) and subjected to silica gel column chromatography(4 kg)usinga gradientmixture of petroleumether (60–90 °C)—acetone 9:1, 4:1, 7:3 and 3:2 as eluent to afford 10 frac-tions. Repeated chromatography of the fractions followed by

Fig. 1. Selected HMBC and NOSEY correlations of compounds 1 and 2.

Fig. 2. ORTEP drawing for compound 1.

611C. Xu et al. / Fitoterapia 81 (2010) 610–613

preparative TLC resulted in the isolation of 1 32 mg (eluted fromfraction E by DCM: MeOH 50:1, recrystallized from methanolat room temperature), 2 20 mg (eluted from fraction B bypetroleum ether: ethyl acetate 5:1; further purified by prepar-ative TLC silica gel plate, mobile phase chloroform: methanol30:1, Rf=0.6 ), andrographolide (3) 20 g, neoandrographolide(4) 10 g, andrograpanin (5) 20 mg, 14-deoxyandrographolide(6) 400 mg, 14-deoxy-11,12-didehydroandrographolide (7)80 mg, 14-deoxyandrographoside (8) 1 g, andrographoside(9) 100 mg and 8-methylandrographolide (10) 15 mg.

3. Results and discussion

Compound 1, was obtained as colorless needles, m.p. 204–205 °C, [α]D25 +55.1 (c 0.08, MeOH), its molecular formulawas established as C20H30O6 by HR–ESI–MS ([2 M+Na]+

found: m/z 755.4019, C40H60NaO12+; calc. 755.3982). The IR

spectrum showed the presence of hydroxyl, γ-lactone andexomethylene functions at 3431, 1768, and 891 cm−1

respectively. The 1H-NMR and 13C-NMR spectral data werevery similar to those of 14-deoxy-11, 12-didehydroandro-grapholide, except for the disappearance of two olefiniccarbons at C-13 and C-14 and appearance of two new oxygen-bearing carbons at δ 74.4 (d, C-14) and 78.5(s, C-13)suggested that the double bond was oxygenized. Furtherstructural information was provided by 2D NMR. In the HSQCspectrum, signal at δ 78.5 was nonprotonated ascribed to C-13 and δ 4.63 correlated with C-14 at δ 74.4 could beassignable to H-14. Besides, δ 4.63 signal showed cross-peakswith C-12, C-13, C-15 and C-16 signals from the HMBC,further confirmed the assignment. From the above data, com-pound 1 was deduced to be 3, 13, 14, 19-tetrahydroxy-ent-labda-8 (17), 11-dien-16, 15-olide. The stereochemistry of1 was determined by NOESY and X-ray crystallographicanalysis. The NOESY spectrum of 1 showed correlations ofH-9/H-11, H-12, and H-14/H-11, H-12, implied α configura-tion of 14-OH. CD spectrumgave a negative cotton effect curve(CD (MeOH) nm: 196 (10.1759), 229 (−5.4233), 299(0.7186)) as that of andrographolide, the main componentof the plant [19]. But according to Beecham's rule, the stereoconfiguration of C-13 still couldn't be identified. X-raycrystallographic analysis of 1 revealed that the C-13 and C-

14 should be 13R, 14R in the ORTEP diagram (Fig. 2, detail datasee Appendix A.). The minimal energy analysis by using aprogram of ChemBio 3D Ultra 11.0 also showed that the cis-13α,14α-dihydroxyl groups or an 13α,14α-epoxy structure isin superior conformation, probably due to the effect of five-member lactone ring. The biogenetic route was proposed as

Fig. 3. Proposed derivations of compound 1.

612 C. Xu et al. / Fitoterapia 81 (2010) 610–613

shown in Fig. 3. This 13,14-dihydroxylation in the lactonemoiety is a new discovery in labdane diterpenoids.

Compound 2 was obtained as a pale yellow oil, [α]D25 40.6(c 0.17, CHCl3), displayed a quasimolecular ion peak at m/z397.2361 in the positive HR–ESI–MS attributed to the [M+Na]+ (C23H34NaO4

+; calc. 397.2355), together with 1H-NMRand 13C-NMR data, these information suggested its molec-ular formula to be C23H34O4. The IR spectrum of 2 showedabsorption bands at 3420 (hydroxyl group), 1745 (α, β-unsaturated-γ-lactone), and 909 (exomethylene)cm−1. The

Table 11H and 13C-NMR data of compounds 1 (C5D5N) and 2 (CDCl3).

Position 1 2

δ (C) δ (H) δ (C) δ (H)

1 38.7 1.54 (br. d, J=13.5) 34.4 1.80–1.77 (m)1.14 (br. t, J=12.7) 1.27 (dt, J=12.8, 4.0)

2 28.9 1.93 (br. d, J=12.9) 25.0 2.01–1.96 (m)1.83⁎ 1.76–1.74 (m)

3 80.3 3.70–3.67⁎ 76.4 3.48 (dd, J=8.7, 3.7)4 43.5 – 38.5 –

5 55.0 1.26 (br. d, J=12.6) 52.4 1.24–1.21 (m)6 23.8 1.83⁎ 23.4 1.73–1.70⁎

1.48–1.41 (m) 1.27–1.25⁎

7 37.2 2.47–2.45⁎ 37.9 2.45–2.41 (m )2.11 (t, J=11.2) 1.94 (dt, J=12.0, 3.7)

8 149.6 – 147.2 –

9 60.8 2.47–2.45⁎ 56.1 1.63 (br. s)10 38.8 – 38.0 –

11 132.2 6.41 (dd, J=15.5, 9.9) 22.2 1.73–1.70⁎

1.68–1.65 (m)12 131.0 6.04 (d, J=15.6) 24.7 2.48–2.44 (m)

2.15–2.11 (m)13 78.5 – 134.7 –

14 74.4 4.65–4.63⁎ 143.9 7.11 (s)15 72.7 4.71⁎ 70.1 4.77 (t, J=1.8, 2H)

4.65–4.63⁎

16 177.9 – 174.3 –

17 108.8 4.88 (s) 107.5 4.89 (s)4.71⁎ 4.63 (s)

18 23.8 1.57 (s) 26.1 1.19 (s)19 64.4 4.51 (d, J=10.8) 63.9 3.96 (d, J=1.6)

3.70 (d, J=10.8) 3.16 (d, J=1.6)20 16.0 0.86 (s) 16.2 0.90 (s)21 99.0 –

22 27.2 1.41 (s)23 25.3 1.36 (s)

Asterisks (*) denotes overlapping signals.

characteristic 13C-NMR data indicated 2 as a labdane-typediterpene. An exomethylene (C-17, δ 107.5), a hydroxy-methylene group (C-19, δ 63.9), a methyl (C-18, δ 26.1) anda angular methyl (C-20, δ 16.2). The 1H and 13C-NMR datawere compared with those of 14-deoxyandrographolide. Twomore methyl groups were observed in 1H-NMR spectrum. Asignificant peak at δ 99.0 (C-21, HSQC showed it nonproto-nated along with the chemical shifts indicated it as a ketalcarbon.) was observed together with two more methyl sig-nals at δ 25.3 (C-23) and 27.2 (C-22). That illustrated an extraisopropylidene group was present. Furthermore, correlationsobserved from HMBC of C-21 with those of H-3, H-19, H-22and H-23 signals suggested that the isopropylidene moietymust be attached to C-3 and C-19 (Fig. 1). These evidencesindicated that the compound could be identified as 3, 19-isopropylidene-14-deoxy-ent-labda-8(17), 13-dien-16, 15-olide.

The NMR data of compounds 1 and 2 are provided inTable 1. The structures of known compounds were identifiedby comparison of their spectroscopic data with those inliterature [15], [20] and [21].

Appendix A. Supplementary data of X-ray crystallographicanalysis

The compound1was recrystallized frommethanol. A crystalwith approximate dimensions 0.426×0.347×0.311 mm wasused for analysis. Allmeasurementswere recorded on a Bruker-SMART CCD area detector diffractometer employing graphite-monochromated MoKα radiation (λ 0.71073 Å) at 293 K andoperating in the φ–ω mode. The structure was solved usingthe SHELXS program and refined with SHELXL-97. Crystal dataand refinement statistics: C20H30O6, Mr 366.44; orthorhombic,space group P212121 (Z=4), a=6.3539 (8)Å, b=14.1598 (17)Å, c=41.477 (5)Å, α=90°, β=90°, γ=90°; independent data,4199 (Rint=0.0651); θ range 1.52–26.00°, R (IN2σ (I))=0.0632,wR2=0.1662; largest peak and hole in difference map: 0.336and−0.259 e Å−3.

CCDC-729127 contains the supplementary crystallographicdata for this paper. These data can beobtained free of charge viahttp://www.ccdc.cam.ac.uk/data_request/cif (or from theCambridge Crystallographic Data Centre, 12 Union Road,Cambridge CB21EZ, UK; fax: +44-1223-336033; e-mail:deposit@ccdc.cam.ac.uk).

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