1. Introduction
Salvia triloba L. (Lamiaceae), traditionally known as Greek sage, is also known by several other names includ- ing Salvia fruticosa, Salvia libanotica, Salvia lobryana, and Salvia cypria due to a taxonomic confusion over the years. It is a perennial Mediterranean herb and has been used to enhance memory [1] , as a sedative [2] and to treat headaches [3] . Many phytochemicals are known to interact with receptors for the major inhibitory neurotransmitter GABA [4] . Such interaction may underlie some of the pharmacological effects of S. triloba. The constituents of an ethanolic extract of S. triloba have been shown to have moderate affinity to GABAA benzodiazepine receptor sites [5] . Another study demonstrated that compounds isolated from this plant increased the hypnotic effect of hexobarbital in the rat [6] [7] , suggesting a benzodiazepine like effect.
Ionotropic GABA receptors are ligand gated chloride channels that mediate much of the fast inhibitory neuro- transmission in the brain [8] . GABAA receptors are antagonised by the convulsant alkaloid bicuculline [9] and modulated by drugs such as benzodiazepines and barbiturates as well by a range of phytochemicals including flavonoids [4] [10] . The less numerous homomeric GABAC receptors are insensitive to bicuculline and most of the chemicals that modulate GABAA receptors. The receptor complex is a pentamer composed of a combination of five subunits of these subunits (α1-6, β1-3, γ1-3, δ, ε, π, θ). However, most GABAA receptors appear to be formed by 2α, 2β subunits in combination with one subunits form another class. The most common subunit combination in the human brain was found to be a1b2g2 [8] [11] .
We have studied the effects of extracts and isolated compounds from S. triloba on functional assays using two-electrode voltage clamp methods on recombinant GABAA receptors containing a1b2g2L subunits expressed in Xenopus laevis oocytes. Using bio-assay guided fractionation we found seven phytochemicals that modulated these receptors.
2. Materials and Methods
2.1. Plant Materials
Dried Salvia triloba plants were purchased from herbal markets located in Amman. The plants were identified by Prof. Dawud AL-Eisawi (Department of Biological Sciences, Faculty of Science, University of Jordan). Voucher specimens were deposited at the herbarium of the University of Jordan.
2.2. Chemicals, Materials and Instrumentation
All chemicals used were purchased from Aldrich Chemical Co. Ltd (St Louis, MO, USA) unless otherwise stated and were of highest commercially available purity. Solvents were distilled by standard techniques prior to use. Silica gel for column chromatography (CC) was performed on silica gel (Merck silica gel 60H, particle size 5 - 40 μm) and Sephadex LH-20 gel. Routine thin layer chromatography (TLC) was performed on Merck alu- minium backed plates, pre-coated with silica (0.2 mm, 60F254).
The UV-Spectra were recorded on Hitachi U-2000 double beam UV/Vis Spectrophotometer. Mass spectra were carried out on a ThermoFinnigan (Waltham, MA, USA) PolarisQ Ion Trap system using a direct exposure probe. Nuclear magnetic resonance 1H NMR and 13C NMR spectra were recorded on 400 MHz Varian Gemini spectrometer (Palo Alto CA, USA) in DMSO-d6 (Sigma-Aldrich, USA) with tetramethylsilane as internal standard. Melting points were determined using a Stuart (Stone, Staffordshire, UK) SMP10 melting point apparatus.
2.3. Preparation of the Extracts and Solvent Fractionation
The dried material (5 kg) was ground into fine powder and defatted by extraction with petroleum ether at room temperature for 7 days. After filtration, the remaining materials were then extracted extensively with ethanol for (3 times, 10 days each) at room temperature. The resulting ethanol extract was partitioned between CHCl3 and H2O (1:1 v/v). The dried chloroform extract was further partitioned between n-hexane and 10% aqueous metha- nol. The dried methanolic extract was then extracted with ethyl acetate to give the final extract.
2.4. Isolation of Constituents
The final extract (43 g) was chromatographed on a silica gel column chromatography (Φ28 × 8 cm) eluted with a gradient of MeOH/CHCl3 of increasing polarity resulting in six fractions (SI-SVI). Each fraction was further purified by a combination of column chromatography and preparative thin layer chromatography using suitable solvent systems.
The addition of ethyl acetate to the methanolic extracts of S. triloba yielded an impure solid that was then washed with methanol for further purification and afforded ursolic acid (3-hydroxy-urs-12-en-28-oic acid). Add- ing chloroform to fraction SI lead to the precipitation of impure solid which was then recrystallize by methanol to give carnosol as a white crystals. The TLC of the mother liquor of fraction SI showed a major UV active spot resulting in precipitation of an impure solid which give upon further purification by chloroform salvigenin (5-hydroxy-6,7,4’-trimethoxyflavone). Fraction SII was subjected to column chromatography (Φ31 × 4 cm) and was eluted with hexane/CHCl3 mixtures of increasing polarity until pure chloroform was used providing three subfractions (SII-1- SII-3). Fraction SII-2 afforded a pure white solid that was then identified as oleanolic acid (3-hydroxy-olean-12-en-28-oic acid). The addition of methanol to fraction SIV yielded an impure solid that produced two major UV active spots on TLC with Rf values of 0.4 and 0.3 (2% MeOH/CHCl3 as the solvent system). This solid was separated by column chromatography on a Sephadex LH-20 gel (Φ21 × 4 cm) to yield rosmanol (Rf = 0.4) and cirsimaritin (Rf = 0.3) (5,4’-dihydroxy-6,7-dimethoxyflavone). An impure solid precipi- tated upon treating fraction SV with methanol. Further washing with hot acetonitrile of this precipitate gave his- pidulin (5,7,4’-trihydroxy-6-methoxyflavone) as pure yellow solid.
2.5. Physical and Spectroscopic Data of the Isolated Compounds
Ursolic acid (1): light white powder, m.p. 209˚C - 220˚C; ESMS m/z (%): 479.4 (100), 457 (5), 439 (15), 425 (44), 413 (12). 1H-NMR (DMSO-d6) δ ppm: 5.11 (H, t, J = 3.4 Hz, H-12), 2.99 (H, m, H-3), 2.08 (H, d, J = 11.6 Hz, H-18), 1.02 (3H, s, Me-27), 0.89 (3H, s, Me-23), 0.87 (3H, s, Me-26) 0.84 (H, d, J = 6 Hz, H-30), 0.79 (H, d, J = 6 Hz, H-29), 0.73 (3H, s, Me-24), 0.65 (3H, s, Me-25); 13C-NMR (DMSO-d6) δ ppm: 178.7 (C-28), 138.6 (C-13), 125.0 (C-12), 77.3 (C-3), 55.2 (C-5), 52.8 (C-18), 47.5 (C-9), 47.3 (C-17), 42.1 (C-14), 39.5 (C-8), 38.9 (C-4), 38.9 (C-19), 38.8 (C-20), 38.7 (C-1), 37.0 (C-10), 36.8 (C-22), 33.2 (C-7), 30.6 (C-21), 28.7 (C-15), 28.0 (C-23), 27.4 (C-2), 24.3 (C-16), 23.7 (C-27), 23.3 (C-11), 21.5 (C-30), 18.4 (C-6), 17.5 (C-29), 17.4 (C-26), 16.5 (C-24), 15.7 (C-25).
Carnosol (2): white crystals, m.p. 209˚C - 220˚C; UV lmax (MeOH) nm: 209 and 285; ESMS m/z (%): 683.4 (100), 331.0 (19); 1H-NMR (DMSO-d6) δ ppm: 6.68 (H, s, H-14), 5.44 (1H, dd, J = 3.8, 1.4 Hz, H-7), 3.21 (1H, sept, J = 6.8, H-15), 2.64 (1H, dd, J = 14, 1.6 Hz, H-1a), 2.43 (1H, ddd, J = 14, 4.6 Hz, H-1β), 2.01 (1H, ddd, H-6a), 1.6 (1H, m, H-6β), 1.59 (1H, dd, J = 10.6, 5.4 Hz, H-5), 1.50 (1H, m, H-2β), 1.42 (1H, dd, J = 13, 1.4 Hz, H-3a), 1.21 (1H, ddd, J = 13.4, 3.2 Hz, H-3β), 1.10 (6H, d, J = 6.8, H-17, H-16), 0.79 (3H, s, H-18), 0.77 (3H, s, H-19); 13C-NMR (DMSO-d6) δ ppm: 175.9 (C-20), 143.7 (C-11), 143.5 (C-12), 134.7 (C-13), 132.0 (C-8), 122.3 (C-9), 111.7 (C-14), 77.4 (C-7), 48.3 (C-10), 45.4 (C-5), 41.0 (C-3), 34.6 (C-4), 31.8 (C-18), 29.7 (C-6), 29.2 (C-1), 26.6 (C-15), 23.2 (C-17), 23.1 (C-16), 19.8 (C-19), 19.0 (C-2).
Oleanolic Acid (3): light white powder; ESMS m/z (%): 456 (100), 431 (15), 391 (8); 1H-NMR (DMSO-d6) δ ppm: 5.14 (H, t, H-12), 2.98 (H, t, H-3), 2.78 (H, dd, J = 6.8, 4 Hz, H-18), 1.07 (3H, s, Me-27), 0.87 (3H, s, Me-25), 0.85 (6H, s, Me-30, 24), 0.70 (H, s, H-29), 0.66 (3H, s, Me-26), 0.65 (3H, s, Me-23); 13C-NMR (DMSO-d6) δ ppm: 179.0 (C-28), 144.3 (C-13), 122.0 (C-12), 77.3 (C-3), 55.2 (C-5), 47.5 (C-9), 46.1 (C-19), 45.9 (C-17), 41.8 (C-14), 41.3 (C-18), 38.8 (C-8), 38.5 (C-1), 37.0 (C-10), 33.8 (C-21), 33.3 (C-29), 32.9 (C-22), 32.5 (C-7), 30.8 (C-20), 28.7 (C-23), 27.6 (C-15), 27.4 (C-2), 26.0 (C-27), 23.8 (C-30), 23.3 (C-16), 23.1 (C-11), 18.5 (C-6), 17.3 (C-26), 16.5 (C-24), 15.5 (C-25).
Salvigenin (4): light yellow powder; UV lmax (MeOH) nm: 332 (Band I), 277 (Band II); +NaOMe, 327, (Band I), 278 (Band II); +AlCl3, 349 (Band I), 300 (Band II); +HCl, 349 (Band I), 299 (Band II); ESMS m/z (%): 350.9 (100), 328.9 (4); 1H-NMR (DMSO-d6) δ ppm: 12.86 (1H, s, 5-OH), 8.06 (2H, d, J = 8.8 Hz, H-2’, H-6’), 7.11 (2H, d, J = 8.8 Hz, H-3’, H-5’), 6.95 (1H, s, H-8), 6.93 (1H, s, H-3), 3.92 (3H, s, 7-OMe), 3.85 (3H, s, 4’-OMe), 3.72 (3H, s, 6-OMe); 13C-NMR (DMSO-d6) δ ppm: 182.7 (C-4), 164.1 (C-2), 162.9 (C-4’), 159.2 (C-7), 153.1 (C-5), 152.5 (C-9), 132.4 (C-6), 128.8 (C-2’, C-6’), 123.2 (C-1’), 115.1 (C-3’, C-5’), 105.6 (C-10), 103.8 (C-3), 92.1 (C-8), 60.5 (6-OMe), 56.9 (7-OMe), 50.1 (4’-OMe).
Rosmanol (5): light white powder; ESMS m/z (%): 314.9 [M-CH3]+ (100), 369.0 (93), 301.0 (28), 347 (8); 1H-NMR (DMSO-d6) δ ppm: 6.68 (H, s, H-14), 5.08 (1H, d, J = 4.4 Hz, H-7), 4.13 (1H, t, J = 4 Hz, H-6), 3.21 (1H, sept, J = 1.6 Hz, H-15), 2.59 (1H, d, J = 14.4 Hz, H-1a), 2.57 (1H, m, H-5), 2.53 (1H, ddd, J = 13.8, 4.4 Hz, H-1β), 1.60 (1H, m, H-2a), 1.19 (1H, d, J = 4 Hz, H-3a), 1.49 (1H, m, H-2β), 1.21 (1H, ddd, J = 13.4, 3 Hz, H-3β), 1.25 (3H, d, J = 1.6 Hz, H-17), 1.11 (3H, d, J = 1.6 Hz, H-16), 0.94 (3H, s, H-18), 0.79 (3H, s, H-19); 13C-NMR (DMSO-d6) δ ppm: 175.2 (C-20), 143.9 (C-11), 143.4 (C-12), 134.6 (C-13), 128.8 (C-8), 122.2 (C-9), 115.2 (C-14), 80.0 (C-6), 67.7 (C-7), 55.0 (C-5), 47.9 (C-10), 41.1 (C-3), 34.3 (C-4), 32.3 (C-18), 29.2 (C-1), 26.7 (C-15), 23.3 (C-17), 23.0 (C-16), 21.3 (C-19), 18.9 (C-2).
Cirsimaritin (6): light yellow crystals, m.p. 267˚C - 269˚C; UV lmax (MeOH) nm: 333 (Band I), 275 (Band II); +NaOMe, 387, (Band I), 272 (Band II); +AlCl3, 353 (Band I), 299 (Band II); +HCl, 353 (Band I), 299 (Band II); ESMS m/z (%): 336.8 (100), 314.9 (43), 300.9 (4); 1H-NMR (DMSO-d6) δ ppm: 12.91 (1H, s, 5-OH), 7.94 (2H, d, J = 8.8 Hz, H-2’, H-6’), 6.91 (2H, d, J = 8.8 Hz, H-3’, H-5’), 6.91 (1H, s, H-8), 6.82 (1H, s, H-3), 3.90 (3H, s, 7-OMe), 3.71 (3H, s, 6-OMe); 13C-NMR (DMSO-d6) δ ppm: 182.7(C-4), 164.5 (C-2), 161.9 (C-4’), 159.0 (C-7), 153.1 (C-5), 152.5 (C-9), 132.3 (C-6), 129.0 (C-2’, C-6’), 121.4 (C-1’), 115.4 (C-3’, C-5’), 105.5 (C-10), 103.1 (C-3), 92.0 (C-8), 60.5 (6-OMe), 56.9 (7-OMe).
Hispidulin (7): light yellow powder, m.p. 290˚C - 292˚C; UV lmax (MeOH) nm: 334 (Band I), 274 (Band II); +NaOMe, 392, (Band I), 276 (Band II); +AlCl3, 353 (Band I), 300 (Band II); +HCl, 351 (Band I), 299 (Band II); ESMS m/z (%): 322.9 (100), 300.9 (98), 271.0 (13), 238.9 (8), 197.7 (7); 1H-NMR (DMSO-d6) δ ppm: 13.06 (1H, s, 5-OH), 7.90 (2H, d, J = 8.8 Hz, H-2’, H-6’), 6.90 (2H, d, J = 8.8 Hz, H-3’, H-5’), 6.76 (1H, s, H-3), 6.57 (1H, s, H-8), 3.73 (3H, s, 6-OMe); 13C-NMR (DMSO-d6) δ ppm: 182.6 (C-4), 164.2 (C-2), 161.6 (C-4’), 157.7 (C-7), 153.2 (C-5), 152.8 (C-9), 131.8 (C-6), 128.9 (C-2’, C-6’), 121.7 (C-1’), 116.4 (C-3’, C-5’), 104.5 (C-10), 102.8 (C-3), 94.7 (C-8), 60.4 (6-OMe).
2.6. Pharmacological Analysis
Pharmacological evaluation of the final extract and the isolated compounds from Salvia triloba were carried out on functional assays using two-electrode voltage clamp methods on recombinant GABA receptors expressed in Xenopus laevis oocytes using the methods described previously [12] . Bio-assay guided fractionation led to seven compounds being isolated from S. triloba; these are: ursolic acid, carnosol, oleanolic acid, salvigenin, rosmanol, cirsimaritin and hispidulin.
3. Results
The final extract of Salvia triloba tested at 200 mg/mL did not show any effect at uninjected oocytes. The ex- tract of S. triloba activated the receptor by 53.2% ± 0.2% when applied alone at a1b2g2L GABA receptors and in- hibited currents due to 300 μM GABA by 54.1% ± 0.2%.
3.1. Ursolic Acid
Ursolic acid it did not show any effect at sham injected oocytes or at a1b2g2L GABAA receptors when adminis- tered alone. However, At 300 µM ursolic acid inhibited currents due to 100 µM GABA by 29.0% ± 0.4% (Fig- ure 1, Table 1). GABA dose response curves were carried out both with and without ursolic acid (Figure 1) at a1b2g2L GABAA receptors. Ursolic acid (100 µM) shifted the GABA dose response curves at a1b2g2L GABAA receptors to the right, increasing the mean GABA EC50 from 123.3 to 210.4 µM (95% CI: 29.4 to 1508), with a Hill slope of 0.48 ± 0.16 (compared to 0.73 ± 0.07 in the case of GABA alone). The effect of ursolic acid was greater at higher concentrations of GABA, with no effect at concentrations below 30 µM GABA.
3.2. Oleanolic Acid
Oleanolic acid (Figure 2) did not have any effect on sham-injected oocytes (n = 3, data not shown). Also, olea- nolic acid demonstrated no activity at a1b2g2L GABA receptors when administered alone but inhibited currents due to 3000 µM GABA by 31.0% ± 1.6% (Table 1).
3.3. Carnosol
Carnosol (Figure 3) did not have any effect at sham-injected oocytes (n = 3, data not shown) or at a1b2g2L GABAA receptors when administered alone, but inhibited currents due to 100 µM GABA with an IC50 of 80.11 µM (95% CI: 69.6 to 92.3) and a Hill coefficient of 6.0 ± 1.5 (Figure 3(a)). GABA dose response curves were
Table 1. Percentage inhibition and enhancement values for the isolated compounds.
aPercentage inhibition of maximal GABA response by 100 µM compound, bPercentage enhancement of GABA EC5response by 100 µM compound.
Figure 1. Dose response curves of GABA (●) and GABA in the presence of 100 μM ursolic acid (◊) at a1b2g2L GABAA receptors expressed in Xenopus oo- cytes. Data are the mean ± SEM (n = 3 - 6 oocytes). Da- ta are the mean ± SEM (n = 3 - 6 oocytes). Inset: repre- sentative current traces from individual oocytes for GABA (100 mM) alone and in the presence of 100 μM ursolic acid at a1b2g2L GABAA receptors expressed in Xenopus oocytes.
Figure 2. Representative current traces from indi- vidual oocytes for GABA (3000 mM) alone and in the presence of 100 mM oleanolic acid at a1b2g2L GABAA receptors expressed in Xenopus oocytes.
Figure 3. (a) Effect of carnosol in the presence of GABA (100µM) at a1b2g2L GABAA re- ceptors expressed in Xenopus oocytes. (b) Dose response curves of GABA (●) and GABA in the presence of 100 (Δ), 300 (○) and 1000 μM (◊) carnosol at a1b2g2L GABAA receptors ex- pressed in Xenopus oocytes. All data are the mean ± SEM (n = 3 - 6 oocytes).
in the presence and absence of carnosol (100, 300 and 1000 µM) at a1b2g2L GABA receptors. Carnosol had no significant effect on EC50 of GABA. However, 100, 300 and 1000 µM of carnosol inhibited currents due to 3000 µM GABA by 17.5% ± 4.0%, 40.7% ± 3.3% and 58.5% ± 3.5%, respectively (Figure 3(b)). Carnosol appears to have a slight positive modulation of GABA at lower does of GABA.
3.4. Salvigenin
Salvigenin (Figure 4) did not have any effect on sham-injected oocytes and had no activity at a1b2g2L or a1b2 GABAA receptors when administered alone. Salvigenin exhibited a biphasic effect at a1b2g2L GABAA receptors. At high concentrations of GABA (100 µM), salvigenin (100 µM) inhibited currents due to GABA by 40.2% ± 0.7% and this action was unaffected by the addition of 10 µM flumazenil, while at low concentrations of GABA (10 µM) salvigenin positively modulated the current by 89.9% ± 1.5% (Table 1). This enhancement was re- duced to 21.8% ± 0.2% by flumazenil (10 μM). At a1b2 GABAA receptors (data not shown), no potentiation of current due to low doses of GABA was observed, however salvigenin reduced the current due to 10 µM GABA by 49.2% ± 0.3% at 100 µM.
3.5. Rosmanol
Rosmanol (Figure 5) did not have any effect on sham-injected oocytes (n = 3, data not shown) and had no activ- ity at a1b2g2L or a1b2 GABAA receptors when administered alone. Rosmanol appears to have two modes of ac- tion at a1b2g2L GABAA receptors. At high concentrations of GABA (3000 µM), rosmanol (100 µM) inhibited currents due to GABA by 26.7% ± 2.1% and this inhibition was not affected by the addition of flumazenil (10 µM), while at low concentrations of GABA (10 µM) rosmanol positively modulated the current by 99.7% ± 0.5% (Table 1) and this enhancement was reduced to 50.8% ± 0.3% by flumazenil (10 μM). At a1b2 GABAA recep- tors, no enhancement of the GABA response was observed, however, rosmanol inhibited currents due to 10 µM GABA by 29.2% ± 0.2% at 100 µM (data not shown), similar to its effect at a1b2g2L GABAA receptors. Ros- manol (100 µM) decreased the maximal GABA response by approximately 50% and shifted the GABA EC50 and decreased the EC50 of GABA from 123.3 (95% CI: 91.5 to 166.2) to 19.51 µM (95% CI: 9.5 to 40.1), with a Hill slope of 0.88 ± 0.15 (Figure 5).
3.6. Cirsimaritin
Cirsimaritin (Figure 6) exhibited a biphasic effect at a1b2g2L receptors, inhibiting currents due to 100 µM GABA by 23.0% ± 0.5% at 100 µM and positively modulating currents due to 10 µM GABA by 89.9% ± 1.5%. The inhibitory effect of cirsimaritin at high concentrations of GABA showed further inhibition in the presence of 10 µM flumazenil, however, this is most likely due to receptor desensitization. Flumazenil had no effect on the enhancement of the GABA response at low concentrations of GABA. Cirsimaritin was inactive at a1b2 GABAA receptors (data not shown). At a1b2g2L GABAA receptors cirsimaritin (60 µM) shifted the GABA dose response curve to the right at low GABA concentrations (<60 µM) and to the left at higher GABA concentra- tions.
Figure 4. Representative current traces from individual oo- cytes showing that the effect of a low dose of GABA (10 mM) is potentiated by 100 mM salvigenin and that this potentiation is inhibited by 10 mM flumazenil; and that a high dose of GABA (100 mM) is inhibited in the presence of 100 mM sal- vigenin and that this inhibition in is unaffected by 10 mM flumazenil at a1b2g2L GABAA receptors.
Figure 5. Dose response curves of GABA (●) and GABA in the presence of 100 μM (◊) rosmanol at a1b2g2L GABAA receptors expressed in Xenopus oocytes. Data are the mean ± SEM (n = 3 - 6 oocytes). Inset: Representative current traces from individual oocytes showing that the effect of GABA (3000 mM) is inhibited by 100 mM rosmanol and this inhibition is unaffected by 10 mM flumazenil; and thatthe effect ofGABA (10 mM) is enhanced by 100 mM rosmanol and that this enhance- ment is inhibited by 10 mM flumazenil at a1b2g2L GABAA receptors.
3.7. Hispidulin
Hispidulin (Figure 7) produced no effect on sham-injected oocytes (n = 3, data not shown) or at a1b2g2L and a1b2GABAA receptors when administered alone but inhibited currents due to 100 µM GABA with an IC50 of 81.7 µM (95% CI: 45.24 to 147.8) and a Hill coefficient of 1.6 ± 0.64. At a1b2g2L GABAA receptorshispidulin (100 µM) shifted the GABA dose response curve to the right at GABA concentrations less than 300 µM and to the left at higher GABA concentrations. This resulted in a change to the GABA EC50 from 123 µM (95% CI: 91.5 to 166.2) to 60 µM (95% CI: 19.5 to 185.5). Hispidulin acts as a positive modulator when applied at low concentrations of GABA but at high concentrations it acts as a negative modulator. This resulted in a change to the GABA EC50 from 123 µM (95% CI: 91.5 to 166.2) to 60 µM (95% CI: 19.5 to 185.5). Hispidulin acts as a
Figure 6. Dose response curves of GABA (●) and GABA in the presence of 60 μM (◊) cirsimaritin at human a1b2g2L GABAA receptors expressed in Xe- nopus oocytes. Data are the mean ± SEM (n = 3 - 6 oocytes). Inset: Representative current traces from in- dividual oocytes expressing a1b2g2L GABAA recep- tors showing that the effect of GABA (100 mM) is in- hibited by 100 mM cirsimaritin and that this inhibi- tion is unaffected by 10 mM flumazenil; the effect of GABA 10 mM is enhanced by the presence of 100 mM cirsimaritin and this enhancement is unaffected by 10 mM flumazenil.
Figure 7. (a) Effect of hispidulin in the presence of GABA (100 µM) at a1b2g2L GABAA re- ceptors expressed in Xenopus oocytes. Data are the mean ± SEM (n = 3 - 6 oocytes) Inset: Representative current traces from individual oocytes showing that the effect of a high dose of GABA (100 mM) is inhibited by 100 mM hispidulin and that this inhibition of the GABA re- sponse is unaffected by 10 mM flumazenil (b) Dose response curves of GABA (●) and GABA in the presence of 100 μM (◊) hispidulin at human a1b2g2LGABAA receptors expressed in Xenopus oocytes. Data are the mean ± SEM (n = 3 - 6 oocytes) Inset: Representative current traces from individual oocytes showing that the effect of GABA 10 mM is enhanced by the presence of 100 mM hispidulin and this enhancement is inhibitedby 10 mM flumazenil at a1b2g2L GABAA receptors.
positive modulator when applied at low concentrations of GABA but at high concentrations it acts as a negative modulator. The effect of hispidulin on low concentrations of GABA was blocked by 10 µM flumazenil. Howev- er, the effect of hispidulin on high concentrations of GABA was not affected by 10 µM flumazenil. At a1b2 GABAA receptors, hispidulin inhibited currents due to 100 µM GABA by 52.2% ± 0.5% at 100 µM (Table 1). The maximum concentration of hispidulin applied was 300 µM due to the limits of its solubility.
4. Discussion
Ursolic acid is a pentacyclic triterpenoid that exists widely in many medicinal plants used in traditional medicine, and it appears that ursolic acid and carnosol are partly responsible for the antitumorigenic activity of rosemary [13] . Although ursolic acid has been reported to inhibit GABA transaminase (GABA-T) [14] , to date, no stu- dies have investigated the action of ursolic acid at GABAA receptors. In the current study ursolic acid inhibited currents due to 100 µM GABA with an IC50 of 98.7 µM and shifted the GABA dose response curve at a1b2g2L GABAA receptors to the right increasing the GABA EC50approximately two-fold to 210.4 µM. Interestingly, this result is in contrast to the in vivo results reported in a recent study by Taviano et al. in which ursolic acid showed significant CNS depressant properties in mice when administrated orally. In this study ursolic acid pro- duced a potentiation of pentobarbital-induced sleeping time and a protective action against pentylenetetrazol (PTZ) induced convulsion [15] . As GABA is an inhibitory neurotransmitter, inhibition of the GABA response would be expected to have an overall excitatory response, and in fact most GABAA antagonists are convulsants. This suggests that ursolic acid is exerting its CNS depressant actions either through a different mechanism or a different GABAA receptor sub-type.
Carnosol is primarily responsible for the high antioxidant activity of Rosmarinus officinalis [13] [16] [17] . Carnosol significantly increased tyrosine hydroxylase activity suggesting that it may be a potential for the treat- ment for Parkinson’s disease (PD) [18] . In a study by Rutherford and colleagues, carnosol inhibited the binding of t-butylphosphorothionate (TBPS) to the chloride channel of the GABA/benzodiazepine receptor but it had no effect on the binding of muscimol, diazepam or flunitrazepam, suggesting that the site of action of carnosol to be directly on the chloride channel [19] .
The current study investigates the effects of carnosol at human recombinant a1b2g2L receptors expressed in Xenopus laevis oocytes. Carnosol had no activity at a1b2g2L GABA receptors when administered alone but inhi- bited currents due to 100 µM GABA with an IC50 of 80.11 µM. Carnosol was found to non-competitively block GABA, being more effective at high dose of GABA. These features are characteristic of a channel blocker.
Oleanolic acid is an isomer of ursolic acid, differing only on the location of one methyl group. In a study by Ha et al., oleanolic acid was tested for its ability to modulate binding to GABAA benzodiazepine receptors in vi- tro; in this study oleanolic acid did not affect the binding of [3H] Ro15-1788 or [3H] flunitrazepam in the pres- ence of GABA [20] . In another study oleanolic acid was found to inhibit GABA transaminase (GABA-T) by 20% at 10 μM/mL [14] . In the current study oleanolic acid inhibited currents due to 3000 µM GABA by 30% most probably by acting as a channel blocker with weaker activity than carnosol.
To date, no studies have investigated the action of salvigenin at GABAA receptors. In the current study salvi- genin inhibited currents due to 100 µM GABA by 40% at 100 µM and positively modulated currents due to 10 µM GABA by 90%. This compound appears to have two modes of action, with positive modulation occurring via the high-affinity flumazenil benzodiazepine site and the inhibitory action occurring via a site that is inde- pendent of the presence of the g-subunit.
Rosmanol is one of the compounds that gives sage free radical scavenging activity [21] [22] . 7-Methoxyros- manol and galdosol are two derivatives of rosmanol that have been shown to competitively inhibit3H-flumazenil bindingto the benzodiazepine receptor with IC50 values of 7.2 and 0.8 µM, respectively [23] . In the current stu- dyrosmanol at 100 µM inhibited currents due to 3000 µM GABA by 25%. GABA dose response curves were carried out both without and with rosmanol (100 µM) at a1b2g2L GABA receptors shifting the GABA dose re- sponse curves to the right with a GABA EC50 19.51 µM. Rosmanol has a biphasic mode of action, positively modulating the effect of GABA at low concentrations of GABA and inhibiting the response to high concentra- tions of GABA. This suggests that rosmanol may have two sites of action on the GABA receptor complex.
The two phases of rosmanol at a1b2g2 GABAA receptors are thought to be mediated via two distinct mechan- isms. The positive modulation of the GABA response by rosmanol at a1b2g2L GABAA receptors was sensitive to antagonism by flumazenil only at low concentrations of GABA indicating the involvement the “high affinity” benzodiazepine binding site which requires the g subunit. The inhibitory second phase action of rosmanol is not affected by flumazenil and is observed at receptor combinations both with and without a g subunit, indicating that the presence of the g subunit is not a requirement. Therefore the inhibitory phase is not mediated via the high-affinity flumazenil sensitive benzodiazepine site. This inhibitory phase may occur via the “low-affinity” benzodiazepine binding site, which is known to be present on ab combinations or at a novel site independent of benzodiazepine activity.
Competitive inhibition of 3H-flumazenil binding was detected for cirsimaritin when tested for its affinity to the benzodiazepine receptor in a membrane preparation from human frontal cortex with an IC50 of 350 mM [23] . Cirsimaritin inhibited the binding of [methyl-3H] diazepam to rat brain benzodiazepine receptors with an IC50 of 23 µM and induced a small increase in [35S]TBPS binding [24] . In the current study, the positive modulation by cirsimaritin of low concentrations of GABA at a1b2g2L GABAA receptors was found to be insensitive to anta- gonism by flumazenil indicating that the high affinity benzodiazepine binding site is not involved which sug- gests the involvement of the “low-affinity” binding site, or an alternative novel biding site. At high concentra- tions of GABA, the inhibitory effect of cirsimaritin appeared to be slightly increased by the presence of fluma- zenil, however this is most likely due to desensitization of the GABA receptors. Interestingly, although cirsima- ritin was insensitive to flumazenil, it showed no activity at a1b2 GABAA receptors, indicating that the presence of the g subunit is essential for activity, suggesting that cirsimaritin may modulate the response to GABA via a novel binding site.
The fact that cirsimaritin displaces benzodiazepine binding, yet is flumazenil insensitive in functional assays is not without precedent. For example 6-methylflavone has been reported to competitively displace [3H]-Ro 15-1788 binding in assays on rat brain membranes in vitro and human recombinant GABAA/BZD receptors ex- pressed in Sf-9 insect cells [25] . However, 6-methylflavone has been shown to be a flumazenil insensitive posi- tive modulator at a1b2g2L GABAA receptors expressed in Xenopus oocytes [26] . Similarly, amentoflavone has been shown to be on of the most potent non-nitrogen containing ligands for the benzodiazepine site in binding assays with Ki = 6 nM [27] . However, at a1b2g2L GABAA receptors expressed in Xenopus oocytes, amentofla- vone has been shown to be a flumazenil insensitive negative modulator [28] [29] .
Hispidulinwas tested for its affinity to the benzodiazepine receptor and was found to competitively inhibit the binding of 3H-flumazenil with an IC50’s of 1.3 mM [23] and 8 µM [30] . Synthetic hispidulin was investigated at recombinant GABAA receptors expressed in Xenopus laevis oocytes and found to act as a positive allosteric modulator at GABAA receptor subtypes (a1-3,5,6b2g2) being more potent at a1,2,5b2g2 subtypes than at a3,6b2g2 [31] . In the same study, hispidulin was shown also to have an anticonvulsant action in seizure prone mongolian ger- bils and to cross the blood brain barrier. Unlike diazepam, hispidulin was found to act as a positive modulator at a6b2g2L GABAA receptors at which 10 mM of hispidulin enhanced the action of GABA at these receptors by 65%, with the enhanced response being reduced to 37% by 1 μM flumazenil [31] .
Hispidulin also appears to have a biphasic mode of action at a1b2g2L GABAA receptors: acting as a positive modulator when applied with low concentrations of GABA but at high concentrations of GABA it acts as a neg- ative modulator. The two phases of hispidulin at a1b2g2 GABAA appear to be mediated via two distinct mechan- isms. The positive modulation of low concentrations of GABA by hispidulin at a1b2g2L GABAA receptors was sensitive to antagonism by flumazenil indicating the involvement the “high affinity” benzodiazepine binding site that requires the g subunit. The second phase or inhibitory action of hispidulin is not affected by flumazenil and is observed at receptor combinations both with and without a g subunit, indicating that it is not acting at the high-affinity benzodiazepine binding site.
5. Conclusions
Although previous studies have investigated some of the chemical constituents of sage at the GABAA/benzo- diazepine complex and cholinergic receptors, few studies have investigated the constituents of Salvia at GABAA receptors using a functional electrophysiological assay.
GABAA receptors are known to be implicated in memory and cognition with enhancers of GABA function such as benzodiazepines having a well-documented detrimental effect on cognitive function. However, many GABA inhibitors or antagonists are not sub-type selective, acting at GABA receptors throughout the central nervous system and usually have the significant activity as convulsants, making them unsuitable for use as cognitive enhancers. In this study, the majority of compounds demonstrated some inhibitory activity at a1b2g2L GABAA receptors, which supports the concept that this herb may have cognition enhancing properties. Interestingly, many of the compounds demonstrated a biphasic activity at a1b2g2L GABAA receptors, enhancing the activity of GABA at lower concentrations of GABA and showing inhibition at higher GABA concentrations. In most cases these activities are likely to be occurring via different modulatory sites on the GABAA receptor complex. It may be that the combination of these activities permits cognition enhancement whilst offering protection from convulsant activity.
The action of salvigenin, rosmanol, and hispidulin at low concentrations of GABA were found to be flumaze- nil sensitive, these compounds also demonstrated flumazenil insensitive actions at high concentrations of GABA. Particularly interesting is the action of cirsimaritin, which was found to be flumazenil insensitive, but requires the presence of the g-subunit, suggesting that cirsimaritin may act at a novel modulatory site on the GABAA re- ceptor complex. Further studies to determine the properties of this site may provide another target for therapeu- tic modulation of GABAA function.
Acknowledgements
A. A. gratefully acknowledges the financial support of the Government of Australia for an Endeavour Interna- tional Postgraduate Research Scholarship (EIPRS).
NOTES
*Corresponding author.