Secondary metabolites from Ballota undulata (Lamiaceae)

Biochemical Systematics and Ecology 33 (2005) 341–351 www.elsevier.com/locate/biochemsyseco

Secondary metabolites from Ballota undulata (Lamiaceae) Tiziana Sicilianoa, Ammar Baderb, Antonio Vassalloc, Alessandra Bracaa,*, Ivano Morellia, Cosimo Pizzac, Nunziatina De Tommasic a

Dipartimento di Chimica Bioorganica e Biofarmacia, Universita` di Pisa, Via Bonanno, 33, 56126 Pisa, Italy b Faculty of Pharmacy, Al-Zaytoonah Private University of Jordan, P.O. Box 130, 11733 Amman, Jordan c Dipartimento di Scienze Farmaceutiche, Universita` di Salerno, Via Ponte Don Melillo, 84084 Fisciano (SA), Italy Received 24 April 2004; accepted 16 October 2004

Abstract Four phenylpropanoids, forsythoside B 1, lysionotoside 2, verbascoside 3, betonyoside F 4, an iridoid, verminoside 5, seven flavonoids, compounds 6–12, and two betaine derivatives 13 and 14 were isolated from the aerial parts of Ballota undulata. The structures of the isolated compounds were established by high resolution NMR and ESI-MS. The antioxidant activity of all compounds was determined by measuring free radical scavenging activity by TEAC test. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Ballota undulata; Lamiaceae; Phenylpropanoids; Flavonoids; Iridoid; Betaine

* Corresponding author. Tel.: C39 050 2219688; fax: C39 050 2219660. E-mail address: [email protected] (A. Braca). 0305-1978/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2004.10.013

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1. Introduction The genus Ballota (Lamiaceae) consists of about 33 species growing mainly in the Mediterranean regions. Ballota undulata (Sieb. ex Fresen.) Bentham is a perennial herb with woody base and many single hairy stems, ovate-cordate and reticulate leaves, widely widespread in Jordan, particularly in waste places and mountains (AlEisawi, 1998). The plant is used by Bedouins as a remedy for the treatment of wounds, scorpion, bee, and wasp stings (Sathiyamoorthy et al., 1997). The aqueous extract of B. undulata was tested for antitumor and antimalarial activities (Sathiyamoorthy et al., 1999). The phytochemical investigations on the species B. undulata resulted in the isolation of luteolin 7-O-glucoside, apigenin 7-Oglucoside, and rutin (Radwan et al., 1997), and the composition of its essential oil has been reported recently (Bader et al., 2003). Previous phytochemical studies on the genus Ballota evidenced the presence of labdane diterpenoids, flavonoids, and phenylpropanoids (Tables 1 and 2). Table 1 Diterpenoids isolated from Ballota sp. Species

Compound

Reference

B. nigra L.

Ballotinone, ballonigrin, 7a-acetoxymarrubiin, ballotenol, preleosibirin, 13-hydroxyballonigrolide Ballonigrin, ballonigrinone, rupestralic acid Hispanolone, hispaninic acid, hispanonic acid, hispanonic acid methyl ester 18-Hydroxyballonigrin

Savona et al. (1976, 1977a, 1977b), Bruno et al. (1986), Seidel et al. (1996a) Savona et al. (1977a, 1977c)

13-Hydroxyballonigrolide, ballonigrin Hispanolone

Savona et al. (1978c) Savona et al. (1982)

Ballonigrin, 18-hydroxyballonigrin, marrubenol

Savona et al. (1982)

Ballotinone, balloaucherolide, 7b,9a,-dihydroxy-15,16-epoxi-labda-13(16), 14-dien-6-one, 7b-hydroxy-9a,13,15, 16-bis-epoxilabd-14-en-6-one, 6b-hydroxy-15-methoxy-9a,13,15, 16-bis-epoxilabdan-7-one, 15-epi-6b-hydroxy-15-methoxy-9a,13,15, 16-bis-epoxilabdan-7-one, 6b-hydroxy-15-ethoxy-9a,13,15, 16-biepoxi-labdan-7-one, ballonigrin, 6b-hydroxy-15,16-epoxy-labda-8,13(16), 14-trien-7-one, seco-labdane, persianone Hispanone, 18-hydroxyballonigrin

Rustaiyan et al. (1991, 1995)

B. rupestris (Biv.) Vis. B. hispanica (L.) Benth.

B. acetabulosa (L.) Benth. B. lanata L. B. andreuzziana Pampan. B. pseudodictamnus (L.) Benth. B. aucheri Boiss

B. saxatilis C. Presl

Savona et al. (1978a), Rodriguez et al. (1979), Lopez de Lerma et al. (1980) Savona et al. (1978b)

Hersel et al. (2000), Bruno et al. (2001)

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Table 2 Flavonoids and phenylpropanoids isolated from Ballota sp. Species Flavonoids B. nigra L. B. acetabulosa (L.) Benth.

B. limbata Linn. B. saxatilis C. Presl B. hirsuta Benth.

B. foetida Lam. B. undulata Fresen (Benth.) Phenylpropanoids B. nigra L.

Compound

Reference

Tangeretin, luteolin-7-lactate, luteolin-7-glucosyl-lactate Scutellarein 4#,7-dimethyl ether, apigenin-7-glucoside, acacetin-7-glucoside, chrysoeriol-7-glucoside, luteolin-7-glucoside, chrysoeriol-7-O-b-(3$-Z-p-coumaroyl)glucopyranoside, chrysoeriol-7-O-b-(3$-E-p-coumaroyl)glucopyranoside, chrysoeriol-7-O-b-glucopyranoside, apigenin-7-O-b-(3$-E-p-coumaroyl)glucopyranoside, apigenin-7-O-b-glucopyranoside, eutigoside A Eupatorin Ladanein, kaempferol 3,7,4#-tetramethyl ether, quercetin 3,7,3#,4#-tetramethyl ether Salvigenin, kumatokenin, genkwanin, ladanein, nuchensin, isokaempferide, apigenin, luteolin, apigenin-7-( p-coumaroyl)-glucoside, apigenin-7-glucoside, luteolin-7-glucoside, quercetin-3-glucoside, luteolin-7-rutinoside, vicenin 2 Apigenin-7-glucoside, vicenin 2 Luteolin-7-glucoside, apigenin-7-glucoside, rutin

Kisiel and Piozzi (1995), Bertrand et al. (2000) Mericli et al. (1988), Sahpaz et al. (2002)

Darbour et al. (1986) Radwan et al. (1997)

Verbascoside, forsitoside B, arenarioside, ballotetroside

Seidel et al. (1996b, 1997)

Parvez et al. (2001) C¸itoglu et al. (1999) Ferreres et al. (1986)

In the course of our ongoing project on detection of chemical constituents from Jordanian plants (Braca et al., 2002, 2003), we now report the isolation, structural characterization, and antioxidant activity by measuring radical scavenger activity with TEAC assay (Re et al., 1999) of four phenylpropanoids, one iridoid, seven flavonoids, and two betaine derivatives from B. undulata aerial parts. The total amount of 3,4-dihydroxyphenylalanine derivatives in the aerial parts was determined by a colorimetric method (Arnow, 1937).

2. Materials and methods 2.1. General experimental procedures UV spectra were recorded on a Perkin–Elmer-Lambda 12 spectrophotometer. A Bruker DRX-600 NMR spectrometer using the UXNMR software package was used for NMR experiments (Braca et al., 2003). ESI-MS spectra (positive and negative mode) were obtained from a Finningan LC-Q Advantage Termoquest spectrometer, equipped with a Xcalibur software. TLC was performed on precoated Kieselgel 60 F254 plates (Merck, Darmstadt, Germany); compounds were detected by

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Ce(SO4)2/H2SO4 (Sigma–Aldrich, Milano, Italy). Column chromatography was performed over Sephadex LH-20 (Pharmacia); HPLC separations were performed on a Waters 515 series pumping system equipped with a Waters R401 refractive index detector and with a Waters m-Bondapak C18 column and U6K injector. 2.2. Plant material Aerial parts of B. undulata Fresen (Benth.) were collected during the flowering period in Ain Ghazal, Amman, Jordan, on May 2001. The plant was identified by Dr. Ammar Bader, Al-Zaytoonah Private University of Jordan, Amman. A voucher specimen (number 7279/4) is deposited at the Herbarium Horti Pisani, Pisa, Italy. 2.3. Extraction, isolation and identification of compounds Extracts were obtained from the air-dried and powdered aerial parts of B. undulata. A representative sample of the air-dried plant (200 g) was defatted with n-hexane and successively extracted with CHCl3, CHCl3–MeOH (9:1, V:V), and MeOH, by exhaustive maceration, to give 5.8, 2.7, 5.2, and 6.5 g of the respective residues. The methanol extract was partitioned between n-BuOH and H2O, to afford an n-BuOHsoluble portion. The n-BuOH residue (3.0 g) was submitted to a Sephadex LH-20 (200 g), using MeOH as eluent; fractions of 8 mL were collected and grouped into four major fractions (A, B, C, and D) by TLC results on silica 60 F254 gel-coated glass sheets with n-BuOH–AcOH–H2O 60:15:25 and CHCl3–MeOH–H2O 40:9:1, together with pure compounds 6 (15 mg) and 8 (20 mg). Fractions A (120 mg), B (120 mg), C (30 mg), and D (30 mg) were separately purified by RP-HPLC on a C18 m-Bondapak column (30 cm ! 7.8 mm, 125 A˚ 10 mm, flow rate 2.0 mL minÿ1) with MeOH–H2O (3:7, V:V) for fraction A, MeOH–H2O (3.5:6.5, V:V) for fraction B, MeOH–H2O (1:1, V:V), for fraction C, MeOH–H2O (3:2, V:V), for fraction D to afford pure compounds 1 (4.8 mg, tR Z 40 min) and 2 (7.0 mg, tR Z 50 min) from fraction A, compound 3 (5.5 mg, tR Z 22 min) from fraction B, compounds 4 (7.0 mg, tR Z 15 min) and 5 (10 mg, tR Z 25 min) from fraction C, compound 7 (5.8 mg, tR Z 14 min) from fraction D. The chloroform–methanol residue (5 g) was chromatographed on Sephadex LH-20 (265 g), using MeOH as eluent. The fractions obtained (each tube was of 8 mL) were collected like reported above for n-butanolic portion and the fraction A obtained was purified by RP-HPLC on a C18 m-Bondapak column (30 cm ! 7.8 mm, 125 A˚ 10 mm, flow rate 2.0 mL minÿ1) with MeOH–H2O (1:9, V:V) to yield pure compounds 13 (35 mg, tR Z 2 min) and 14 (20 mg, tR Z 3 min). Finally the chloroform extract (2.5 g) was chromatographed on a silica gel column (4 ! 20 cm, 63–200 mm, 50 g of silica gel for 1 g of crude extract), eluting with chloroform followed by increasing concentrations of MeOH (between 1% and 50%) in CHCl3. Fractions of 35 mL were collected, analysed by TLC (silica gel plates, in CHCl3 or mixtures CHCl3– MeOH 99:1, 98:2, 97:3, 9:1, 4:1, V:V), and grouped into five big fractions (A, B, C, D, and E). The fractions A and B were subjected to RP-HPLC on a C18 m-Bondapak column (30 cm ! 7.8 mm, 125 A˚ 10 mm, flow rate 2.0 mL minÿ1) with MeOH–H2O (4:1, V:V) for fraction A and MeOH–H2O (7:3, V:V) for fraction B to yield compounds

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9 (5 mg, tR Z 9 min) and 10 (3.5 mg, tR Z 12 min) from fraction A and compounds 11 (2 mg, tR Z 9 min), and 12 (3 mg, tR Z 15 min) from fraction B. The structures of the isolated compounds were established by different spectroscopic methods (UV; ESIMS; 1H, 13C, 13C DEPT, 1D-TOCSY, and 2D-NMR experiments, DQF-COSY, HSQC, HMBC) and confirmed by comparison with those reported in literature (Endo et al., 1982; Miyase et al., 1996; Andary et al., 1982; Liu et al., 1998; Tomas and Salmeron, 1976; Tomas-Barberan et al., 1986; Biswas et al., 1978; Kumari et al., 1986; Das et al., 1970; Sticher and Afifi-Yazar, 1979; Wood et al., 2002). OR2

O

O

O R1O

HO

O

OH

OR OH

HO

R

R1

H

1

R2

H3C HO

O OH

O

HO OH H3C HO

H

2

OH

HO

O

H

HO O O OH OH

HO

H

3

H3C HO

H

O HO OH

4

H3C HO

O OH

O

H

HO HO

OH

OH

HO

+

COO

N H3C

CH3

13

-

+

COO

N H3C

CH3

14

-

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T. Siciliano et al. / Biochemical Systematics and Ecology 33 (2005) 341–351 R2 R1 R

O

R3 OH

O

R HOH2C HO HO

6

R1

R2

R3

OH

OH

H

OH

OCH3

H

OCH3

OH

H

O O OH O O

7 HO

8

O

HO HO

O OH

HOH2C HO HO

O O OH

9

OCH3

OCH3 OCH3 OCH3

10

OCH3

OCH3

11

OCH3

OH

OCH3 OCH3

12

OCH3

OH

OCH3

OH

OH

O O H O O HOH2C HO HO

H CH2OH O

O

OH

5

H

OCH3

H

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2.4. Antioxidant activity assay Pure compounds were tested by using the TEAC assay. The TEAC value is based on the ability of the antioxidant to scavenge the radical cation 2,2#-azino-bis-(3ethylbenzothiazoline-6-sulfonate) (ABTScC) with spectrophotometric analysis (Re et al., 1999). The ABTScC cation radical was produced by the reaction between 7 mM ABTS in H2O and 2.45 mM potassium persulfate, stored in the dark at room temperature for 12 h. The ABTScC solution was then diluted with phosphate buffered saline (pH Z 7.4) to an absorbance of 0.70 at 734 nm and equilibrated at 30  C. Samples were diluted with methanol to produce solutions of 0.3 mM, 0.5 mM, 1 mM, 1.5 mM, and 2 mM concentration. The reaction was initiated by the addition of 1 mL of diluted ABTS to 10 mL of each sample solution. Determinations were repeated three times for each sample solution. The percentage inhibition of absorbance at 734 nm was calculated for each concentration relative to a blank absorbance (methanol) and was plotted as a function of concentration compound or standard 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox, Aldrich Chemical Co., Gillingham, Dorset, UK). The antioxidant activities of compounds 1– 13 are expressed as Trolox Equivalent Antioxidant Capacity (TEAC) values in comparison with TEAC activity of reported reference compound quercetin (Re et al., 1999). Compounds are considered active when their TEAC is near to that of quercetin. The TEAC value is defined as the concentration of standard Trolox with the same antioxidant capacity as a 1 mM concentration of the antioxidant compound under investigation. 2.5. 3,4-Dihydroxyphenylalanine derivatives content Total 3,4-dihydroxyphenylalanine derivatives of B. undulata aerial parts were determined by colorimetric methods based on o-dihydroxycinnamic derivatives estimation (Arnow, 1937). An aliquot (1 mL) of hydroalcoholic solution was added to 2 mL of 0.5 N HCl, 2 mL of 10% (w/v) aqueous solution of sodium molybdate (Arnow reagent), and 2 mL of 1 N NaOH. The solution was adjusted to 10 mL with water. The adsorption was measured at 525 nm. Result is expressed in g/100 g of dry plant material with respect to verbascoside 3.

3. Results and discussion Four phenylpropanoids 1–4, one iridoid 5, seven flavonoids 6–12, and two betaine derivatives 13 and 14 were isolated from the aerial part of B. undulata. The 1H and 13C NMR spectral data and ESI-MS analysis for compounds 1–4 were in accordance with those reported in literature for forsythoside B 1, betonyoside F 2, verbascoside 3, lysionotoside 4 (Endo et al., 1982; Miyase et al., 1996; Andary et al., 1982; Liu et al., 1998). Also the results of NMR and ESI-MS experiments for the flavonoids 6–12 were confirmed with those reported in the literature for luteolin 7-O-b-D-glucopyranoside 6, luteolin 3#-methyl ether-7-(6$-p-coumaroyl)-b-D-glucopyranoside 7, diosmetin

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7-O-b-D-glucopyranoside 8, quercetin 3,7,3#,4#-tetramethyl ether 9, kaempferol 3,7,4#-trimethyl ether 10, quercetin 3,7,3#-trimethyl ether 11, luteolin 7,3#-dimethyl ether 12 (Tomas and Salmeron, 1976; Tomas-Barberan et al., 1986; Biswas et al., 1978; Kumari et al., 1986; Das et al., 1970). The NMR data and ESI-MS spectrum for compound 5 were also in accordance with the literature data for verminoside (Sticher and Afifi-Yazar, 1979; Chaudhuri et al., 1980). Compounds 13 and 14 are two betaine derivatives (Wood et al., 2002; Blunden et al., 1996). From a literature survey (Tables 1 and 2), diterpenes and flavonoids are the main classes of substances representative of the Ballota genus. The secondary metabolites isolated from B. undulata could be all grouped into: flavonoids, phenylpropanoids, betaine derivatives, and iridoids. As shown in Table 2, flavonoids p-coumaroyl glucosides and polymethoxylated flavonoids are commonly found in different Ballota species and they are generally considered as valuable chemotaxonomic markers of the Lamiaceae family (Tomas-Barberan et al., 1992). Flavonoids 7, 8, 11, and 12 are now reported for the first time in the genus, while 9 and 10 were isolated before from Ballota saxatilis (C¸itoglu et al., 1999). A large number of species in the Lamiaceae family contains phenylpropanoid glucosides, but exclusively 3,4disubstituted cinnamic acid derivatives (caffeic or ferulic acid) (Pedersen, 2000). The isolation of compounds 1–4 from B. undulata is completely in accordance with this chemotaxonomic evidence. Also betaines have been shown to have taxonomic significance in the Lamiaceae family: in the genus Ballota, both trans-4hydroxyprolinebetaine 13 and prolinebetaine 14, were previously isolated (Blunden et al., 1996). B. undulata seemed not to biosynthesize diterpenes, that are typical of the genus; anyway the presence of an iridoid constituent (verminoside 5) should be interesting from a chemotaxonomic point of view. Compounds 1–14 were evaluated for their antioxidant activity by measuring free radical scavenging activity by TEAC test. Polyphenols have been shown to scavenge various reactive oxygen species and have been implicated as inhibitors of lipid peroxidation. The antioxidant property of flavonoids was the first mechanism of action studied, in particular with regard to their protective effect against cardiovascular diseases. On the other hand, the antioxidant activity of phenylpropanoid glycosides was not extensively investigated. Only few works reported the activity of some phenylpropanoids as inhibitor of in vitro LDL peroxidation (Seidel et al., 2000), but this is the first report on their radical scavenger evaluation by TEAC test. The radical scavenger activity of the tested compounds was expressed as TEAC (Trolox Equivalent Antioxidant Capacity) values (Re et al., 1999). TEAC results for compounds 1–14 and quercetin, used as reference compound, are summarized in Table 3. Among phenylpropanoid glycosides, forsythoside B (1) and verbascoside (3) are the most active compounds with a TEAC value of w1.6 mM, while lysionotoside (2) and betonyoside F (4) are slightly less active (TEAC value w1.0 mM). The antioxidant activity of compounds 1–4 could be related to the presence of two orthohydroxyl groups on phenolic moieties. Luteolin derivative 6 is the most active among flavonoids (TEAC value w1.2 mM), while compounds 7, 8, and 12 yielded lower activity; the lack of activity is probably due to the presence of a methoxyl group on ring B. Flavonol derivatives 9–11 and betaines 13 and 14 are completely inactive as

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Table 3 Antioxidant activities of compounds 1–14 in the TEAC assaya Compound

TEAC assay (mM) G SDb

1 2 3 4 5 6 7 8 9 10 11 12 13 14 Quercetin

1.562 G 0.005 1.010 G 0.02 1.677 G 0.05 1.029 G 0.04 0.351 G 0.003 1.155 G 0.15 0.725 G 0.02 0.683 G 0.02 !0.05 !0.05 !0.05 !0.05 !0.05 !0.05 2.60 G 0.03

a b

For protocols used, see Section 2. n Z 3.

radical scavenger. Since the antioxidant activity of these compounds could be due to the presence of two ortho-hydroxyl groups on phenolic moieties, the total 3,4dihydroxyphenylalanine derivative content in the aerial part of B. undulata was determined colorimetrically by the Arnow method (Arnow, 1937), accounting for 8.8% of dry weight, a high amount compared with other reports from Ballota genus (Didry et al., 1999). Therefore, B. undulata aerial parts might be useful also to identify a source of antioxidant components for the development of additives with appropriate protective properties in food or herbal drugs. Acknowledgements The MIUR (Ministero dell’Istruzione, dell’Universita` e della Ricerca, Roma) is gratefully acknowledged for financial support.

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