Antifungal Diterpenoids and Flavonoids from Ballota inaequidens.

ABSTRACT

Two diterpenoids (hispanolone 1, ballonigrine 2) and six flavonoids (5-hydroxy-3,7,4′-trimethoxyflavone 3, retusin 4, 5-hydroxy-7,4′-dimethoxyflavone 5, pachypodol 6, 5-hydroxy-3,6,7,4′-tetramethoxyflavone 7, 5-hydroxy-7,3′,4′-trimethoxyflavone 8) have been isolated from the aerial parts of Ballota inaequidens. Hub.-Mor. & Patzak. Among them, 7 has not been reported previously in the genus Ballota.. The antibacterial and antifungal activities of 1–4, 6, and 8 were tested against Bacillus subtilis., Staphylococcus aureus., Escherichia coli., Pseudomonas aeruginosa., Candida albicans., and Candida crusei..

Keywords:

• Antifungal activity
• Ballota inaequidens
• ballonigrine
• hispanolone
• pachypodol
• retusin
• 5-hydroxy- 3,7,4′-trimethoxyflavone
• 5-hydroxy-7,4′-dimethoxyflavone
• 5-hydroxy-3,6,7,4′-tetramethoxyflavone
• 5-hydroxy-7,3,4′-trimethoxyflavone

Introduction

Ballota. L. is represented by 16 species in Turkey (Davis, 1982). Ballota. species have been used in Turkish folk medicine as antiulcer, antispasmodic, and sedative agents (Çitoğlu et al., 1998). In our previous studies, three diterpenoids (hispanolone, ballonigrine, dehydrohispanolone) and ten flavonoids (kumatakenin, pakipodol, 5-hydroxy-7,3′,4′-trimethoxyflavone, velutin, corymbosin, 5-hydroxy-3,7,4′-trimethoxyflavone, retusin, 5-hydroxy-7,4′-trimethoxyflavone, 5-hydroxy-3,6,7,4′-tetramethoxyflavone, ladanein) were isolated, chemically characterized, and analyzed by HPLC in different species of Ballota. (Çitoğlu et al., 199819992003; Sever, 2000). Although Ballota inaequidens. Hub.-Mor. & Patzak is also widespread in South Anatolia, studies on its flavonoid constituents have not been reported to date. As a continuation of our research on the Ballota. species, we isolated the main constituents of Ballota inaequidens. and investigated their antimicrobial properties.

Materials and Methods

General experimental procedures

UV spectra (in MeOH) were recorded using a UV-1601 Shimadzu instrument. ¹H NMR spectra in CDCl₃ were recorded at 400 MHz on a Varian XLAA 400 spectrometer. Melting points were recorded on a Büchi SMP-20 instrument.

Plant material

Ballota inaequidens., in flower, was collected and identified in 1998 near Antalya (Turkey) by B. Sever and F. Tezcan. Voucher specimens are kept in the Herbarium of Ankara University, Faculty of Pharmacy (AEF no. 19901).

Isolation of flavonoids

Air-dried and powdered aerial parts of the plant (500 g) were extracted with Me₂CO (5 l) at room temperature for 3 days. After evaporation, the residue was extracted with EtOAc and the extract washed with H₂O and dried. The extract was concentrated to dryness in vacuo. to afford a syrupy residue (34.45 g) that was purified on SiO₂ of 70–230 mesh (300 g) with light petroleum ether and increasing amounts of EtOAc until 80:20 (v/v) (Savona et al., 19781982; Rodriguez et al., 1979). Fractions of 50 ml were collected, and similar fractions were purified on a PTLC (Kieselgel 60 PF₂₅₄) using CHCl₃:MeOH (10:0,05) to give: hispanolone 1 (76 mg), ballonigrine 2 (85 mg), 5-hydroxy 3,7,4′-trimethoxyflavone 3 (116 mg), retusin 4 (120 mg), 5-hydroxy-7,4′-dimethoxyflavone 5 (20 mg), pachypodol 6 (44 mg), 5-hydroxy-3,6,7,4′-tetramethoxyflavone 7 (58 mg), and 5-hydroxy-7,3′,4′-trimethoxyflavone 8 (53 mg) (Fig. 1). The structures of these compounds were elucidated on the basis of their UV and NMR spectral data.

Figure 1. Compounds isolated from aerial parts of Ballota inaequidens..

Hispanolone (1)

White needles (76 mg), m.p. 144–145°C (lit. 147–148°C) (Rodriguez et al., 1979; Savona et al., 1982; Davies-Coleman & Rivett, 1990; Çitoğlu et al., 1998). MS m/z. (rel. int.): 318 (M⁺, 15), 219 [(300-C₂H₅O)⁺, 16], 195 (13), 177 (44), 165 (41), 135 (25), 107 (30), 79 (28). ¹H NMR (CDCl₃) δ 7.37 [1H, d (J. = 8.8 Hz) H-15], 7.25 [1H, s, H-16], 6.29 [1H, d (J. = 8.8 Hz) H-14], 2.76 [1H, q (J. = 6.6 Hz) H-8], 2.49 (2H, m, H-12), 2.31 (2H, t, H-6), 2.04 [1H, dd (J. = 3.0–4.1 Hz) H-5], 1.95 (2H, m, H-11), 1.60 (2H, m, H-3), 1.47 (2H, t, H-1), 1.25 [2H, d (J. = 4.2) H-2], 1.20 (3H, s, H-20), 1.14 [3H, d (J. = 6.4 Hz) H-17], 0.92 (3H, s, H-19), 0.90 (3H, s, H-18); ¹³C NMR (CDCl₃): 31.2 (C-1), 17.8 (C-2), 40.6 (C-3), 32.9 (C-4), 45.7 (C-5), 38.5 (C-6), 210.0 (C-7), 50.2 (C-8), 81.0 (C-9), 42.6 (C-10), 34.0 (C-11), 20.8 (C-12), 124.1 (C-13), 109.9 (C-14), 142.3 (C-15), 137.8 (C-16), 7.5 (C-17), 32.3 (C-18), 20.7 (C-19), 15.5 (C-20).

Ballonigrine (2)

White needles (853. mg), m.p. 178–180°C (lit. 212°C) (Savona et al., 197619771978; Çitoğlu et al., 1998). MS m/z. (rel. int.): 328 (5.2), 313 (5.7), 300.2 (6.3), 285.2 (6.9), 203.1 (25.9), 109.1 (8.4), 95.1 (10.4), 81.0 (100). ¹H NMR (CDCl₃) δ 7.99 (1H, s, H-14), 7.39 (1H, brs, H-16), 6.31 (1H, brs, H-15), 4.76 [1H, d (J. = 6.0 Hz) H-6], 2.20 [1H, d (J. = 5.75 Hz) H-5], 1.82 (3H, s, H-17), 1.57 (2H, m, H-3), 1.35 (2H, m, H-8), 1.1 (3H, s, H-20). ¹³C NMR (CDCl₃): 31.15 (C-1), 18.65 (C-2), 28.56 (C-3), 42.70 (C-4), 50.46 (C-5), 76.23 (C-6), 193.78 (C-7), 131.95 (C-8), 163.32 (C-9), 37.39 (C-10), 24.88 (C-11), 30.81 (C-12), 124.39 (C-13), 111.28 (C-14), 143.94 (C-15), 139.55 (C-16), 12.73 (C-17), 25.32 (C-18), 180.72 (C-19), 28.76 (C-20).

5-Hydroxy-3,7,4′-trimethoxyflavone (3)

Yellow needles (116 mg), m.p. 140–141°C (lit. 146–148°C) (Vidari et al., 1971; Çitoğlu et al., 1999). UV λ_max (MeOH) 347, 267; (+NaOMe) 366, 283; (+AlCl₃) 394, 276, 351, 304; (+AlCl₃/HCl) 395, 344, 302, 277; (+NaOAc) 346, 267; (+NaOAc/H₃BO₃) 347, 267; ¹H NMR (CDCl₃ + DMSO) δ 4.10 (3H, s, OCH₃), 4.11 (3H, s, OCH₃), 4.14 (3H, s, OCH₃), 6.58 [1H, d (J. = 2 Hz) H-6], 6.68 [1H, d (J. = 2 Hz) H-8], 7.27 [2H, dd (J. = 2.5 and 8.5 Hz), H-3′ and H-5′], 8.32 [2H, d (J. = 2.5 and 8.5 Hz), H-2′ and H-6′], 12.89 (1H, brs, 5-OH); ¹³C NMR (CDCl₃): 156.3 (C-2), 139.2 (C-3), 179.1 (C-4), 157.1 (C-5), 98.2 (C-6), 139.2 (C-7), 92.5 (C-8), 162.0 (C-9), 106.4 (C-10), 123.2 (C-1′), 130.5 (C-2′), 114.4 (C-3′), 162.4 (C-4′), 114.4 (C-5′), 130.5 (C-6′), 60.3 (OCH₃), 56.1 (OCH₃), 55.8 (OCH₃).

Retusin (5-hydroxy-3,7,3′,4′-tetramethoxyflavone) (4)

Yellow needles (120 mg), m.p. 147–148°C (lit. 160–162°C) (Vidari et al., 1971; Sakakibara et al., 1976; Arisawa et al., 1991; Çitoğlu et al., 1999). UV λ_max (MeOH) 350, 268; (+NaOMe) 370, 281; (+AlCl₃) 405, 268; (+AlCl₃/HCl) 405, 269, 352; (+NaOAc) 348, 268; (+NaOAc/H₃BO₃) 347, 268, 254; ¹H NMR (CDCl₃) δ 3.79 (3H, s, OCH₃), 3.80 (3H, s, OCH₃), 3.89 (3H, s, OCH₃), 3.90 (3H, s, OCH₃), 6.28 [2H, d (J. = 2.5 Hz) H-6], 6.37 [2H, (d, J. = 2.5 Hz) H-8], 6.91 [2H, d (J. = 9 Hz), H-5′], 7.65 (2H, m, H-2′ and H-6′], 12.57 (1H, brs, 5-OH); ¹³C NMR (CDCl₃): 149.1 (C-2), 139.3 (C-3), 179.1 (C-4), 162.4 (C-5), 98.2 (C-6), 165.8 (C-7), 92.6 (C-8), 156.2 (C-9), 106.4 (C-10), 123.3 (C-1′), 111.2 (C-2′), 149.1 (C-3′), 151.7 (C-4′), 111.2 (C-5′), 122.5 (C-6′), 60.6 (OCH₃), 56.4 (OCH₃), 56.4 (OCH₃), 56.2 (OCH₃).

5-Hydroxy-7,4′-dimethoxyflavone (5)

Yellow needles (20 mg), m.p. 177–179°C (lit. 168°C) (Silva et al., 1971). UV λ_max (MeOH) 327, 268; (+NaOMe) 352, 285; (+AlCl₃) 345, 276; (+AlCl₃/HCl) 339, 277; (+NaOAc) 327, 268; (+NaOAc/H₃BO₃) 328, 268; ¹H NMR (CDCl₃) δ 3.81 (3H, s, OCH₃), 3.82 (3H, s, OCH₃), 6.29 [2H, d (J. = 2.5 Hz) H-6], 6.40 [2H, d (J. = 2 Hz) H-8], 6.50 [1H, s, H-3], 6.96 [2H, dd (J. = 2.5 and 8.5 Hz), H-3′ and H-5′], 7.75 [2H, dd (J. = 2.5 and 8.5 Hz), H-2′ and H-6′], 12.66 (1H, brs, 5-OH).

Pachypodol (5,4′-dihydroxy-3,7,3′-trimethoxyflavone) (6)

Yellow needles (44 mg), m.p. 154–156°C (lit.168–170°C) (Valesi et al., 1971; Fraser & Lewis, 1973; Sakakibara et al., 1976; Miyazawa et al., 2000; Orabi et al., 2000). UV λ_max (MeOH) 356, 268; (+NaOMe) 403, 356, 264; (+AlCl₃) 397, 361, 267; (+AlCl₃/HCl) 394, 360, 267; (+NaOAc) 360, 254; (+NaOAc/H₃BO₃) 356, 253; ¹H NMR (CDCl₃); δ 3.78 (3H, s, OCH₃), 3.80 (3H, s, OCH₃), 3.91 (3H, s, OCH₃), 6.28 [1H, d (J. = 2 Hz) H-6], 6.37 [1H, d (J. = 2 Hz) H-8], 6.96 [1H, d, (J. = 8.5 Hz) H-5′], 7.59 [2H, dd (J. = 2.5 and 9 Hz) H-6′], 7.61 [2H, dd (J. = 2.0 and 8.5), H-2′], 12.56 (1H, brs, 5-OH); ¹³C NMR (CDCl₃): 148.0 (C-2), 139.0 (C-3), 179.1 (C-4), 164.0 (C-5), 98.2 (C-6), 165.8 (C-7), 92.5 (C-8), 157.1 (C-9), 106.4 (C-10), 123.0 (C-1′), 114.9 (C-2′), 146.7 (C-3′), 149.0 (C-4′), 114.9 (C-5′), 122.8 (C-6′), 60.5 (OCH₃), 56.5 (OCH₃), 56.2 (OCH₃).

5-Hydroxy-3,6,7,4′-tetramethoxyflavone (7)

Yellow needles (58 mg), m.p. 159–160°C (Southwick et al., 1972) UV λ_max (MeOH) 336, 272; (+NaOMe) 375, 290; (+AlCl₃) 363, 279; (+AlCl₃/HCl) 356, 282; (+NaOAc) 335, 221; (+NaOAc/H₃BO₃) 336, 271; ¹H NMR (CDCl₃) δ 3.79 (3H, s, OCH₃), 3.83 (3H, s, OCH₃), 3.85 (3H, s, OCH₃), 3.88 (3H, s, OCH₃), 6.43 (1H, s, H-8), 6.94 [1H, dd (J. = 2.5 and 8.5 Hz), H-3′ and H-5′], 7.99 [1H, dd (J. = 2.5 and 8.5 Hz), H-2′and H-6′], 12.55 (1H, brs, 5-OH); ¹³C NMR (CDCl₃): 153.2 (C-2), 139.4 (C-3), 179.0 (C-4), 158.4 (C-5), 132.8 (C-6), 162.1 (C-7), 90.7 (C-8), 152.7 (C-9), 107.0 (C-10), 123.2 (C-1′), 130.5 (C-2′), 114.4 (C-3′), 159.4 (C-4′), 114.4 (C-5′), 130.5 (C-6′), 55.8(OCH₃), 56.7 (OCH₃), 60.5 (OCH₃), 61.2 (OCH₃).

5-Hydroxy-7,3′,4′-trimethoxyflavone (8)

Yellow needles (53 mg), m.p. 156–158°C (lit. 163–165°C) (Nakanishi et al., 1985) UV λ_max (MeOH) 338, 269; (+NaOMe) 370, 283; (+AlCl₃) 355, 274; (+AlCl₃/HCl) 349, 276; (+NaOAc) 338, 269; (+NaOAc/H₃BO₃) 339, 269; ¹H NMR (CDCl₃) δ 3.81 (3H, s, OCH₃), 3.89 (3H, s, OCH₃), 3.91 (3H, s, OCH₃), 6.30 [1H, d (J. = 2 Hz) H-6], 6.42 [1H, d (J. = 2 Hz) H-8], 6.51 [1H, s, H-3], 7.26 [1H, d (J. = 8.5 Hz), H-5′], 7.44 [1H, d (J. = 2.5), H-2′], 7.46 [1H, dd (J. = 2 and 8 Hz), H-6′], 12.72 (1H, brs, 5-OH); ¹³C NMR (CDCl₃): 148.2 (C-2), 104.5 (C-3), 182.7 (C-4), 162.6 (C-5), 98.4 (C-6), 165.8 (C-7), 98.1 (C-8), 158.1 (C-9), 105.1 (C-10), 128.3 (C-1′), 114.9 (C-2′), 149.7 (C-3′), 152.7 (C-4′), 120.5 (C-5′), 124.1 (C-6′), 56.5 (OCH₃), 56.2 (OCH₃), 55.9 (OCH₃).

Antimicrobial activity

In vitro. antimicrobial studies were carried out by the macrobroth dilution assay (National Committee of Clinical Laboratory Standards, 1993) by measuring the minimal inhibition concentration (MIC) of the compounds (1–4, 6, and 8) against two Gram-positive (Staphylococcus aureus. ATCC 25923, Bacillus subtilis. ATCC 6633), two Gram-negative (Escherichia coli. ATCC 23556, Pseudomonas aeruginosa. ATCC 10145), and two fungi (Candida albicans., ATCC 10231, Candida krusei. ATCC 6258). Bacterial strains were obtained from the Refik Saydam Hygiene Center. Briefly, stock solution (3 mg/ml) of the compounds 1–4, 6, and 8 in dimethyl sulfoxide (DMSO) were two fold diluted in 0.5 ml of Mueller Hinton broth. To these broths, 0.5 ml of bacterial and fungal suspentions (10⁶ cfu/ml) were added. The bacterial and fungal suspensions had a turbidity of 0.5 MacFarland standard (ca. 1 × 10⁸ cfu/ml). The tubes were incubated at 35°C for 24 h. MIC was determinated as the lowest concentration of the compound completely inhibited the growth of the test organism. Ampicillin was used as standard antibacterial agent for bacteria, whereas fluconazole was used as a positive control for C. albicans. and C. krusei..

Results and Discussion

Isolation of two diterpenes (1, 2), two flavones (5, 8), and four flavonols (3, 4, 6, 7) has been achieved by column chromatography on silica gel using light petroleum ether and ethyl acetate as mobile phase followed by PTLC on silica gel using chloroform/methanol (10:0.05) as eluent. The structures of these compounds were elucidated on the basis of their UV and NMR spectral data. Compounds 1–8 have been found to be identical with hispanolone, ballonigrine, 5-hydroxy 3,7,4′-trimethoxyflavone, retusin, 5-hydroxy-7,4′-dimethoxyflavone, pachypodol, 5-hydroxy-3,6,7,4′-tetramethoxyflavone, and 5-hydroxy-7,3′,4′-trimethoxyflavone, respectively. The known compounds were also identified by comparision of their melting points with those reported in the literature. Among them, 5-hydroxy-3,6,7,4′-tetramethoxyflavone (7) has not yet been isolated from the genus Ballota..

The results of the screening test of the compounds 1–4, 6, and 8 against B. subtilis., S. aureus., E. coli., P. aeruginosa., C. albicans., andC. krusei. using the macrobroth dilution method are summarized in Table 1. The compounds tested have no important inhibitory activity against bacteria but showed good activities against C. albicans. and C. krusei.. We also reported three diterpenoids (hispanolone, dehydrohispanolone, ballonigrine) obtained from the aerial parts of Ballota saxatilis. subsp. saxatilis. and their effects against Gram-positive (S. aureus., S. faecalis.) and Gram-negative (P. aeruginosa., E. coli., K. pneumoniae.) microorganisms and C. albicans. (Çitoğlu et al., 1998). The results of this study confirm the previous results in this regard.

Table 1.. Minimal inhibitory concentrations (MIC) of compounds 1–4, 6, and 8 from Ballota inaequidens..

	MIC (μg/ml)
Microorganisms	1	2	3	4	6	8	amp	flu
B. subtilis.	25	25	25	25	50	25	12.5	NT
S. aureus.	25	25	25	50	50	>100	6.25	NT
E. coli.	50	50	25	25	50	25	3.1	NT
P. aeruginosa.	25	50	25	25	50	25	12.5	NT
C. albicans.	12.5	3.1	12.5	12.5	3.1	6.25	NT	12.5
C. krusei.	6.25	6.25	3.1	6.25	3.1	3.1	NT	6.25

1, hispanolone; 2, ballonigrine; 3, 5-hydroxy-3,7,4′-trimethoxyflavone; 4, retusin; 6, pachypodol; 8, 5-hydroxy-7,3′,4′-trimethoxyflavone; amp, ampicillin, flu, fluconazole; NT, not tested.

Acknowledgments

This Research was supported by the Research Foundations of Ankara University (grant no. 2000-08-03-020) and the Hungarian Nation Science Foundation (grant no. OTKA-034250).