PHYTOTHERAPY RESEARCH Phytother. Res. 29: 80–85 (2015) Published online 23 September 2014 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ptr.5229

A Novel Diterpene Skeleton: Identification of a Highly Aromatic, Cytotoxic and Antioxidant 5-Methyl-10-demethyl-abietane-type Diterpene from Premna serratifolia Solomon Habtemariam1* and George K. Varghese2 1

Pharmacognosy Research Laboratories, Medway School of Science, University of Greenwich, Chatham-Maritime, Kent ME4 4TB, UK Department of Botany, CMS College, Kottayam, Kerala, India

2

Premna serratifolia Linn. (syn: . P. corymbosa (Burm. f.) Merr., P. integrifolia L. and P. obtusifolia R. Br.) is a member of the Verbenaceae family that is extensively used in the Ayurvedic system of medicine in India. As part of our continuous pharmacological and phytochemical studies on medicinal plants, we have screened the methanolic extracts of leaves, root bark (RB) and root wood of P. serratifolia for cytotoxic activity against two cancer cell lines: SHSY-5Y neuroblastoma and B16 melanoma cells. The RB extract that showed promising activity was fractionated using solvents of increasing polarity followed by a combination of Sephadex LH-20 column and Combiflash chromatography as well as HPLC to afford the active principle. Comprehensive spectroscopic analysis including 1D and 2D NMR (COSY, HMQC, HMBC, NOESY) and MS analysis revealed the identity of the isolated compound as 11,12,16-trihydroxy-2-oxo-5-methyl-10-demethyl-abieta-1[10],6,8,11,13-pentene that appears to be a novel compound based on a new diterpene skeleton. The cytotoxic activity of the isolated compound was 21 and 23 times higher than the crude extract against the SHSY-5Y and B16 cells, respectively. The novel compound also possesses in vitro antioxidant effects as evidenced by the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging effect where an IC50 value of 20.4 ± 1.3 μM was obtained. In comparison, the positive control, caffeic acid, showed an IC50 value of 14.4 ± 1.6 μM. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: Premna serratifolia; Verbenaceae; novel diterpene skeleton; cytotoxic; antioxidant.

INTRODUCTION Premna serratifolia Linn. (syn: P. corymbosa (Burm. f.) Merr., P. integrifolia L. and P. obtusifolia R. Br.), is a small tree or shrub of the Verbenaceae family that is extensively used in the Indian traditional medicine formulations such as Dashmoola kwatha, Chyanprashavleh, Haritakiavleh, Ayushyavardhaak tel and Narayana taila (API, The Ayurvedic Pharmacopoeia of India, 2007; Jothi et al., 2010). Amongst the various medicinal claims of the roots of plant are those related to treating diabetes, chyluria, gonorrhoea, inflammation, swelling, bronchitis, dyspepsia, headache, liver disorder, piles, constipation and fever (API, The Ayurvedic Pharmacopoeia of India, 2007). Previous pharmacological studies on the plant have shown that the plant possesses anti-coagulant (Gopal and Purushothaman, 1984), anti-inflammatory (Karthikeyan and Deepa, 2011), anti-arthritic (Karthikeyan and Deepa, 2010a; Rajendran and Krishnakumar, 2010), antinociceptive (Karthikeyan and Deepa, 2010b), antioxidant (Gokani et al., 2011; Bose et al., 2013), hypoglycemic (Alamgir et al., 2001; Dash et al., 2005; Kar et al., 2003), gastroprotective (Jothi et al., 2010), anti-microbial * Correspondence to: Solomon Habtemariam, Pharmacognosy Research Laboratories, Medway School of Science, University of Greenwich, Chatham-Maritime, Kent ME4 4TB, UK. E-mail: [email protected]

Copyright © 2014 John Wiley & Sons, Ltd.

(Rajendran and Basha, 2010), and cardioactivity (Rajendran et al., 2008) properties. A number of diterpenes of biological significance belonging to the abietane (Salae et al., 2012), labdane and clerodane (Chin et al., 2006; Habtemariam et al., 1990, 1991, 1992; Habtemariam, 1995, 2003), icetexane (Ayinampudi et al., 2012; Hymavathi et al., 2009; Suresh et al., 2011a), kaurene (Padla et al., 2012) and pimarane, rosane and other structural classes (Rayanil et al., 2013; Salae et al., 2012) have been isolated from the genus Premna. Although their biological activity is yet to be demonstrated, the identification of three novel diterpenoids from the methanolic extract of root bark (RB) of P. serratifolia has also been documented in recent years (Yadav et al., 2010). Our studies on the root wood (RW) of the same plant (P. serattifolia) did not yield diterpenes but rather a biologically active glucoside derivative, acteoside, was isolated in large quantities (Bose et al., 2013). In view of most of the diterpene classes isolated from Premna possess cytotoxic activities, we screened the leaves, RW and RB extracts of P. serratifolia against two cancer cell lines. From the RB extract that showed cytotoxic activity, we herewith report the identification of a novel bioactive compound based on a new class of diterpene skeleton. The isolation and structural determination of the novel compound as 11,12,16-trihydroxy-2-oxo-5-methyl-10-demethyl-abieta-1[10],6,8,11,13pentene (1) along with its cytotoxic and antioxidant activities are discussed. Received 23 April 2014 Revised 4 August 2014 Accepted 29 August 2014

A NOVEL DITERPENE SKELETON IDENTIFIED FROM PREMNA SERRATIFOLIA

MATERIALS AND METHODS General phytochemical analysis methodology. 1H NMR, 13 C NMR and 2D-NMR (COSY, NOESY, HMQC and HMBC) spectra were obtained on a JEOL 500 MHz instrument. Homonuclear 1H connectivities were determined by using the COSY experiment. One bond 1 H–13C connectivities were determined with HMQC whilst two-bond and three-bond 1H–13C connectivities were determined by HMBC experiments. Chemical shifts were reported in δ (ppm) using the solvent (CDCl3) standard and coupling constants (J) were measured in Hertz. A Waters Synapt G2 TOF mass spectrometer (Waters, UK) with an electrospray ionisation probe was used to acquire data over a mass range of 50–800 u. A lock spray correction was applied to each acquired data set using Leu-Enk. The mass pectrometer was coupled with an Acquity UPLC system comprising of Acquity UPLC BEH C18 1.7 μm (2.1 × 50 mm) column and Waters TUVat 220 nm detector. Samples dissolved in methanol (0.1 mg/mL) were injected into the LC-mass spectrometer operating in a positive ion mode. Optical activity was recorded using CETI WZZ-2S Automatic Polarimeter (Medline Scientific Ltd, Oxfordshire, UK). Sephadex LH-20 (Sigma-Aldrich) was used as an adsorbent for open column chromatography.

HPLC analysis. An Agilent 1200 series gradient HPLC system composed of degasser (G1322A), quaternary pump (G1322A), auto sampler (G1329A), thermostat column compartment (G1316A) maintained at 25 °C, and a diode array detector (G1315D) was used. For analytical scale study, concentrations of standard samples and plant extracts were injected (20 μL) onto a reverse phase column (Agilent – Eclipse Plus C18, 5 μm, 4.9 × 150 mm). The mobile phase was a mixture of water (A) and methanol (B). The composition of the mobile phase at a flow rate of 1 mL/min was rising from 50% to 90% B over a period of 50 min. For preparative scale study, a 100 mg/ml solution of semi-purified compounds was made in methanol. A 0.2 mL sample was injected at a time onto a preparative-scale reverse phase column (Agilent – Eclipse Plus C18, 5 μm, 9.4 × 150 mm) and HPLC peaks/fractions collected.

Plant material. P. serratifolia locally known as Chemparathy Munja and morphologically of serrated nature (P. serratifolia with serrate leaves) growing for several years in our experimental field (Kadakasseril experimental garden, Ayanivelikulangara south, Karunagapally, Kollam District, Kerala, India 09°02’ 020, North latitude and 076° 31. .972, East longitude (altitude ± 12 m) was used. A voucher specimen numbered 2517 was kept in the CMS Herbarium, CMS College, Kottayam, Kerala, India. Processing and extraction of the plant material. The leaves, RB and RW tissues of the underground roots were separated, air dried and grounded. The powdered leaves (250 g), RB (260 g), and RW (280 g) were extracted twice by soaking in methanol (3 L) for 2 weeks. Removal of the solvent under reduced pressure yielded 12, 11 and 10 g yield for the leaves, RB and RW tissues, respectively. Copyright © 2014 John Wiley & Sons, Ltd.

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DPPH radical scavenging. The antioxidant activity of test samples was measured by using our established microtitrebased DPPH assay (Habtemariam, 2007). Briefly, DPPH solution (0.1 mM, in methanol) was incubated with varying concentrations of test compounds for 20 min at room temperature and the absorbance of the resulting solution was read at 540 nm against a blank using Multiscan EX Reader (Thermo Labsystems, Altrincham, UK). Cell lines and culture conditions. The B16 mouse melanoma cells (Habtemariam and Dagne, 2010) and the neuroblastoma SHSY-5Y cells were obtained from the European Collection of Cell Cultures (Public Health England, Porton Down, Salisbury, UK) were used as representative cancer cell lines. Cell lines were maintained with RPMI 1640 medium supplemented with penicillin (50 U/mL), streptomycin (50 μg/mL) and 10% foetal bovine serum. Cells were cultured in humidified atmosphere of 95% air-5% CO2 at 37 °C. Cytotoxicity assay. Cells were seeded in 96-well plates at a density of 5000 cells per well and cultures were allowed to establish by incubating plates at 37 °C for 24 h. Various concentrations of the test compounds were then added and culture plates were further incubated for 48 h. Cell viability was assessed by measuring the fluorescence of Alamar BlueTM as described previously (Habtemariam and Jackson, 2007). Isolation of the principal cytotoxic compound. A portion of the crude extract (10 g) was suspended in 500 mL of deionised water and successively fractionated with solvents of ascending polarity: 3 times each of 500 mL petroleum ether (yield: 0.95 g), chloroform (yield: 1.6 g) and ethyl acetate (yield: 0.8 g). The chloroform fraction, which showed cytotoxic activity, was subjected to Sephadex LH-20 column (26 × 3.5 cm) fractionation with elution carried out using chloroform and methanol mixture (1:1). A total of 16 fractions of 20 mL each were collected and pooled into four major fractions based on their HPLC profile: I (60 mL), II (100 ml), III (60 mL), IV (100 mL). Fraction III was further subjected to Combiflash chromatography system as follows: A RediSep Rf flash Glold C18 column (100 g; Presearch, Hampshire, UK) attached to a Teledyne Isco flash chromatography system with a flow rate of 60 mL/min and a linear gradient of methanol (in water) starting from 50% to 90% over a period of 20 min and then increased 100% in 10 min. The chromatogram was monitored by observing absorbance at dual wavelengths of 214 and 254 nm. Fractions collected between 8 to 11 min were taken to dryness and further purified by the same system and also by HPLC to yield the active compound with the purity of over 98% (by HPLC). Statistical analysis. Data are presented as mean and SEM values from a minimum of four replicates and all experiments were repeated at least three times. Where appropriate, the significance of difference between two means was analysed by using unpaired t-test. 11,12,16-trihydroxy-2-oxo-5-methyl-10-demethyl-abieta-1 [10],6,8,11,13-pentene (1). Brown solid; [α]25 -354 :D Phytother. Res. 29: 80–85 (2015)

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(CHCl3, c 0.1).; UV (EtOH) λmax: 218, 249, 344 nm. IR (KBr) υmax = 3375 br, 2960, 1628, 1436, 1335, 1301, 1065, 753, 667 cm 1. 1H and 13C and 2D NMR, see Table 2; ESI-MS, see Fig. 2.

RESULTS AND DISCUSSIONS The methanol extracts of leaves, RW and RB of P. serratifolia were processed separately. Cytotoxic activity screening of the extracts was performed using two cancer cell lines: B16 melanoma and SHSY-5Y neuroblastoma cells. As shown in Fig. 1, a potent concentration-dependent cytototoxic effect was demonstrated for the RB extract whilst the leaves and RW extracts did not show activity up to the highest concentrations tested (Fig. 1). IC50 values obtained from three separate experiments are also shown in Table 1. Solvent-solvent fractionation followed by a combination of Sephadex LH-20 and Combiflash chromatography of the RB extract gave the cytotoxic active principle. In order to obtain a highly purified compound for analytical measurements such as MS and bioassays, preparative scale HPLC was also employed to isolate the active compound (1). The cytotoxic compound isolated had a deep brown colour. The 1H NMR spectrum amongst others showed three methyl singlets and one methyl doublet, two multiplet protons at δ 4.02 and 3.74, two distinctly coupled (17 Hz) aliphatic, and four aromatic protons of which

Figure 1. Cytotoxicity of P. serratifolia extracts and the isolated compound, 1, against B16 cells. Representative results on the concentration-dependent effects of test samples are shown. Data are mean and SEM values (n = 6) obtained for the leaves (LF), root wood (RW) and root bark (RB) extracts as well as compound 1.

two were ortho-coupled. The 13C spectrum revealed 20 carbon signals: one carbonyl, 10 aromatic and 9 nonolefinic, of which one was oxygen bearing (δ 69.6). In the DEPT-13C NMR experiment, the quaternary carbons were not shown as expected whilst the CH and CH3 carbons were shown as positive peaks and the CH2 carbons as negative peaks, and hence allowing the establishment of all carbon functional groups in the molecule. Direct 13 C and 1H correlations (1J) were further established through HMQC experiments. The establishment of the structure as shown in 1 was based on extensive HMBC studies. The major 2J and 3 J connectivities are shown in Table 2. The two gemdimethyls at C-4 were good reference points for the correlation studies and showed all the expected 2J and 3 J correlations (Table 2). In addition to 3J coupling of the gem-dimethyl groups to each other and expected 2 J coupling to the C-5 quaternary carbon, a distinctive correlation to the methylene (C-3) and another quaternary carbon at δ 45.0 was evident. The lack of correlation of the gem-dimethyls with a methine functional group as expected in labdane/abietane skeleton and the further correlation of the δ 45 carbon with a methyl singlet at δ 1.26 supports the assignment of the C-5 methyl diterpene skeleton as shown in 1. The two methylene protons (C-3) appearing as doublets with no other coupling in the 1H NMR spectrum and their 2J coupling with the carbonyl carbon and 3J coupling with one aromatic (CH) carbon and C-5 further firmly established the assignment of the A-ring. Further HMBC correlation from the olefinic H-1 and H3-20 protons as shown in Table 1 was clearly indicative of the A-ring and C-6-C-7 double bond assignment. In addition to the expected coupling with H/C-16 and H/C-17 groups, H-15 showed 2J and 3J correlations with the aromatic carbons. This allowed the assignment of the aromatic functional groups in ring C. In the HMBC study, the only carbon that did not show correlation was C-11 due to its distance being more than three bonds from neighbouring protons. The ESI-MS analysis using the accurate mass capable Waters Synapt G2 TOF mass spectrometer in positive ion mode assigned the molecular formula of 1 as C20H24O4. The MS spectrum further showed the classical [M Na]+, [2 M + H]+ and [2 M + Na]+ ion peaks (Fig. 2). This observation ruled out the possible alternative structure 2. The IR spectrum showing bands for the carbonyl at low frequency (1628 cm 1); the 13C NMR data for shielded C-1 and deshielded C-10 carbons as well as the relatively downfield position of C-6 as compared with C-7 in the 13C NMR spectrum were all evidence for the α,β-unsaturated ketone system and the extended aromatic structure. The aromatic C-11

Table 1. Cytotoxicity and antioxidant effects of test compounds. IC50 values obtained from a minimum of three determinations in the cytotoxicity and five determinations for antioxidant study are shown Cytotoxic Activity in μg/mL [μM] Test sample Crude RB extract 1 Etoposide Caffeic acid

SHSY-5Y

B16

31.7 ± 12.6 1.5 ± 0.3 [ 4.7 ± 1.0]* 0.66 ± 0.1 [1.1 ± 0.1]* —

109 ± 4.9 4.7 ± 0.9 [ 14.4 ± 2.7]** 0.9 ± 0.04 [1.5 ± 0.07]** —

Antioxidant Effect in μg/mL [μM] DPPH Radical Scavenging — 6.7 ± 0.4 [20.4 ± 1.3]*** — 2.6 ± 0.3 [14.4 ± 1.6]***

RB, root bark. Symbols *, **, *** indicate that data in the same column are significant from each other. Copyright © 2014 John Wiley & Sons, Ltd.

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Table 2. 1H, 13C and 2J and 3J HMBC NMR data for compound 1

δH

Position 1 2 3a 3b 4 5 6 7 8 9 10 11 12 13 14 15

7.21 s

16a 16b 17 18 19 20

4.02 br d (9.2) 3.74 br t (8.6) 1.32 d (7.1) 0.95 s 1.18 s 1.26 s

— 2.73 d (17.0) 2.15 d (17.0) — — 5.95 d (9.8) 6.35 d (9.8) — — — — — — 6.43 s 3.23 m

Key HMBC Correlations

δC 125.6 208.8 48.5 38.9 45.0 131.1 125.3 127.4 114.5 160.4 146.7 142.4 133.4 117.1 37.2 69.6 15.0 26.2 24.3 22.2

C-3, C-5, C-9 — C-2, C-4 C-1, C-2, C-4, C-5 — — C-8, C-10 C-8, C-9, C-14

C-7, C-9, C-12, C-13 C-12, C-13, C-14, C-16, C-17 C-13 C-13 C-13, C-15, C-16 C-3, C-4, C-5, C-19 C-3, C-4, C-5, C-18 C-4, C-5, C-6, C-10,

All experiments were run in CDCl3-δ values in ppm and coupling constant (J) in parenthesis are shown.

Figure 2. The ESI-MS spectrum of compound 1.

position that did not show HMBC correlation appeared down-field in the 13C NMR and hence could be easily assigned to the hydroxyl-bearing carbon. The stereochemistry of the isolated compound depicted from the NOESY studies is shown in 1. Even though, the close chemical shift values of the C-18 and C-20 protons did not permit to establish NOESY correlations, information from other correlations Copyright © 2014 John Wiley & Sons, Ltd.

is in firm support of the placement of C-20 methyl α-orientation. From the available NOESY information, it is not possible to establish the stereochemistry at C-15 position. In the biosynthesis of C-5 methyl diterpenes, such as the formation of clerodane skeleton of Premna species from their geranylgeranyl pyrophosphate precursor, one of the C-4 methyl groups migrates to C-5 whilst the C-10 methyl moves to C-9 position (Habtemariam et al., 1990, 1992). In compound 1, however, the gemdimethyl C-4 group is intact as with Premna labdanes, abietanes, icetexanes and pimaranes (Habtemariam et al., 1991, 1992; Salae et al., 2012; Suresh, et al., 2011a; Yadav et al., 2010) whilst the C-10 methyl moves to the unexpected biogenetically unique position of C-5 to form a novel diterpene skeleton. This unique biosynthesis pathway that allowed the migration of the C-20 methyl group from C-10 position made it possible for the extended aromatisation in 1 through the three rings of the diterpene skeleton. It remains to be seen if such unique structure is associated with novel biological activities, but the present study already established a potent cytotoxicity activity against cancer cell lines (Fig. 1). As shown in Table 1, the activity of compound 1 against the neuroblastoma and melanoma cells were 21 and 23 times better than the crude RB extract. In contrast to the standard anticancer compound etoposide, however, the new compound was 5.2 and 9.6 times, respectively, less potent against SHSY-5Y and B16 cells. The observed in vitro cytotoxicity data against two representative cancer cell lines is a further evidence of Premna species as a rich source of novel potential anticancer agents (Habtemariam, 1995; Suresh et al., 2011a, 2011b) The further advantage of the extended α,β-unsaturated carbonyl unsaturated functional group together with the aromatic diorthohydroxyl moieties of compound 1 was to offer antioxidant activity. The standard antioxidant compound, caffeic acid that has similar structural moiety with compound 1, was also included in our study as a positive control. As shown in Table 1, both compounds were potent radical scavengers with caffeic acid slightly more potent than compound 1 (p < 0.05). Because reactive oxygen species are involved in the development of various pathologies including inflammatory and liver diseases (Habtemariam and Jackson, 2007; Nabavi et al., 2014), the observed antioxidant effect of the crude extract and 1 partly explains the reported traditional uses of P. serratifolia. Premna serratifolia L. is a highly polymorphic species with a number of other species known in the literature such as P. corymbosa (Burm. f.) Merr., P. integrifolia L. and P. obtusifolia R. Br. are now all considered synonyms (Munir, 1984). In agreement with Munir (1984) observation, we have noted varieties of P. serratifolia growing in Kerala (India) region as trees, shrubs and Phytother. Res. 29: 80–85 (2015)

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miniature under-shrubs far below a metre long. Whilst some are gowning inland at various altitudes, we noted some salt-resistant varieties growing on sea salt-infested river beds of Kerala. As different morphotypes with respect to leaf sizes and appearance are also available, the various distinct chemical and pharmacological reports published for the species under different synonyms may be due to the species polymorphism. For example, 11 novel and 15 known diterpenes belonging to various structural classes such as pimaranes, rosanes, abietanes, icetexanes and rearranged icetexanes have been isolated from the roots and aerial parts of P. obtusifolia of Thailand origin (Salae et al., 2012), whereas similar work on P. injtegrifolia of Indian origin yielded three novel pimaranes and abietanes diterpenes (Yadav et al., 2010). Novel dimeric icetexane diterpenoids have also been recently isolated from P. obtusifolia (Salae and Boonnak, 2013). In conclusion, the present study revealed a novel compound belonging to a structurally new diterpene skeleton.

The observed biological activity including potential anticancer coupled with antioxidant lead compound means that this class of diterpenes need to be further explored as a potential lead for various disease conditions. It should also be noted that P. serratifolia is a highly polymorphic species and the present finding is related to P. serratifolia of serrate leaves morphotypes. It is therefore imperative that the number of published phytochemical and pharmacological work on this plant should be correlated to the various morphotypes of the species. Acknowledgements The technical assistance of staff at the Mass and NMR Laboratories, University of Greenwich is greatly appreciated.

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

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Phytother. Res. 29: 80–85 (2015)

A novel diterpene skeleton: identification of a highly aromatic, cytotoxic and antioxidant 5-methyl-10-demethyl-abietane-type diterpene from Premna serratifolia.

Premna serratifolia Linn. (syn: . P. corymbosa (Burm. f.) Merr., P. integrifolia L. and P. obtusifolia R. Br.) is a member of the Verbenaceae family t...
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