Doi:10.1016/j.jep.2005.01.037

Journal of Ethnopharmacology 98 (2005) 367–370 Alkaloids from Boophane disticha with affinity to the Mikkel Sandager , Nicolaj D. Nielsen , Gary I. Stafford , a Research Centre for Plant Growth and Development, School of Botany and Zoology, University of KwaNatal Pietermaritzburg, P/Bag X01, Scottsville 3209, South Africa b Department of Medicinal Chemistry, The Danish University of Pharmaceutical Sciences, Universitetsparken 2, DK-2100 Copenhagen Ø, Denmark Received 17 August 2004; received in revised form 21 January 2005; accepted 28 January 2005 Abstract
Bulbs and leaves of Boophane disticha are used in South African traditional medicine in the treatment of anxiety. Crude extracts of the leaves have shown affinity to the SSRI site on the serotonin transporter in a radioligand binding assay. In this study, two compounds, buphanadrineand buphanamine, were isolated by bioassay-guided fractionation on VLC and preparative TLC. The structures of the compounds weredetermined by 1H and 13C NMR. Fractions were tested for affinity to the serotonin transporter in a binding assay using [3H]-citalopram asligand. The IC50 values of buphanidrine and buphanamine were 274 ␮M (Ki = 132 ␮M) and 1799 ␮M (Ki = 868 ␮M), respectively. The twoalkaloids were also tested for affinity to the 5HT1A receptor, but only showed slight affinity.
2005 Published by Elsevier Ireland Ltd.
Keywords: Boophane disticha; Buphanamine; Buphanidrine; [3H]-Citalopram; Depression; Radioligand binding assay; Serotonin transporter; 5HT1A Plant leaves of Boophane disticha (L.f.) Herb (Amarylli- Previously isolated classes of constituents 11 alkaloids daceae) were collected in the Botanical Gardens, Pietermar- have been isolated from the bulb, with a total yield of 0.31%.
itzburg, South Africa in March. A voucher specimen (Stafford Buphanidrine constituted 19.4% and buphanamine 14.1% of 53 NU) is deposited in the Bews Herbarium, University of Uses in traditional medicine scales from the bulb of Boo- phane disticha is traditionally used for numerous of purposes, 1. Materials and methods
e.g., treatment of hysteria in young women The bulb has narcotic properties and leads to visual hallucinations in toxic doses In ascreening of plants used for anxiety and depression for affin- Hundred g fresh leaves of Boophane disticha were ex- ity to the serotonin transporter in rat brain, leaf extracts of tracted with 70% ethanol (3 × 500 ml). The extract was frac- Boophane disticha had high affinity for the SSRI site ( tionated on 150 g of Merck Silica gel 60 in a vacuum column.
Five hundred millilitres of each of following solvents wereused as eluents:hexane; hexane:ethyl acetate 50:50; 25:75;ethyl acetate; ethyl acetate:methanol 90:10; 80:20; 70:30; Abbreviations: DPAT, dipropylaminotetralin; SERT, serotonin trans- 60:40; 50:50; 40:60; 30:70; 20:80; 10:90; 2 × methanol; wa- porter; SSRI, selective serotonin reuptake inhibitor ∗ Corresponding author. Tel.: +45 3530 6339; fax: +45 3530 6041.
ter. Active fractions from VLC (ethyl acetate:methanol 90:10 E-mail address: [email protected] (A.K. J¨ager).
and 80:20) where dissolved in 100 ml 70% ethanol, adjusted 0378-8741/$ – see front matter 2005 Published by Elsevier Ireland Ltd.
doi:10.1016/j.jep.2005.01.037 M. Sandager et al. / Journal of Ethnopharmacology 98 (2005) 367–370 Fig. 1. Structures of (1) buphanidrine and (2) buphanamine.
to pH 3 with 4% acetic acid and partitioned against di- ethylether (3 × 100 ml). The pH was adjusted to 10 withNaOH and the aqueous phase was partitioned against di- All procedures were carried out at 0–4 ◦C. Whole rat ethylether (3 × 100 ml), ethylacetate (3 × 100 ml) and bu- brains, except cerebellum, were homogenised with an Ul- tanol (4 × 70 ml). The active fraction (diethylether from basic tra Turrax homogenizer in 1:10 (w/v) buffer (5 mM TRIS partition) was separated on preparative TLC using ethyl ac- base, 150 mM NaCl and 20 mM EDTA, pH 7.5). The ho- etate:methanol:water (90:20:10) as mobile phase. The TLC mogenate was centrifuged at 16,000 × g for 10 min and the plate was detected under UV 254/365 nm and a small part of tissue membranes washed with 120 ml of the same buffer.
the TLC plate was sprayed with Dragendorrf reagent to detect The supernatant was discarded and the pellet was suspended alkaloids. Five bands were scraped off the TLC, eluted with in buffer (5 mM TRIS base and 5 mM EDTA, pH 7.5), left methanol and tested for activity. The isolated compounds to react for 20 min and then centrifuged at 16,000 × g for were dissolved in CDCl3 and the structures elucidated by 10 min. The supernatant was discarded and the pellet was washed with 120 ml buffer (50 mM TRIS base, 120 mM NaCl Table 1NMR data for buphanamine (100 MHz for 13C, 600 MHz for 1H, CDCl3) 6.05 dddd (3J2,3 = 10.0, 3J2,1 = 5.6, 3J2,4␤ = 2.8, 3J2,4␣ = 1.9) 5.86 ddd (3J3,2 = 10.0, 3J3,4␣ = 4.6, 3J3,4␤ = 2.9) ␣: 2.83 dddd (2J4␣,4␤ = 19.6, 3J4␣,4a = 5.6, 3J4␣,3 = 4.6, ␤: 2.21 ddt (2J4␤,4␣ = 19.6, 3J4␤,4a = 8.2, 3J4␤,3 = 4J4␤,2 = 2.8) 3.79 t (3J4a,4␣ = 3J4a,4␤ = 8.2) endo: 1.99 dddd (2J11endo,11exo = 12.3, 3J11endo,12endo = 8.7, 3J11endo,12exo = 3.0, 4J11endo,4a = 1.3)exo: 2.12 ddd (2J11exo,11endo = 12.3, 3J11exo,12exo = 10.9, endo: 2.99 ddd (2J12endo,12exo = 13.2, 3J12endo,1endo = 8.7, M. Sandager et al. / Journal of Ethnopharmacology 98 (2005) 367–370 and 5 mM KCl, pH 7.5) and then centrifuged at 16,000 × gfor 10 min. The supernatant was discarded and the pellet fi-nally suspended in 120 ml buffer (50 mM TRIS base, 120 mMNaCl and 5 mM KCl, pH 7.5). This homogenate was kept at−70 ◦C until use.
1.3. [3H]-Citalopram binding assay with modifications Two hundred mi-crolitres of test solution were mixed with 50 ␮l of [3H]-citalopram (4 nM) and 50 ␮l of tissue suspension, respec-tively. Paroxetin (1.5 ␮M) was used for the determinationof unspecific binding. The total binding of [3H]-citalopramwas determined with a buffer blank. All samples were incu-bated for 2 h at 25 ◦C and then filtered under vacuum usingglass fibre filters. After 24 h the radioactivity on the filterswas determined by liquid scintillation. Specific binding wascalculated as total binding minus unspecific binding. All ex-periments were done in duplicate.
1.4. 8-Hydroxy[3H]DPAT binding assay Two hundred microlitres of test solution were mixed with 50 ␮l of 8-hydroxy[3H]DPAT and 50 ␮l of tissue suspension.
Buspirone (1 ␮M) was used to determine unspecific binding.
Subsequent procedures were as for the [3H]-citalopram bind-ing assay.
Fig. 2. IC50 determinations of buphanidrine and buphanamine in the SERTand 5HT1A assays.
1.5. Estimation of IC50 values and Ki values 50 values were calculated using GraFit 5 from i values of the two compounds were calculated to be Erithacus Softwarethe utilising a full four-parameter equa- 132 ␮M for buphanidrine and 868 ␮M for buphanamine. For i values, which is independent of ligand con- 50 value was 1.3 nM, giving a Ki value of centration and thus more useful for inter-lab comparison, 0.6 nM. Both buphanidrine and buphanamine bound to the i = IC50/(1 + [L]/Kd), where [L] is the 1A receptor with low affinity, the IC50 values were 1203 ligand concentration (here 0.75 nM) and K For the SSRIs, and most other types of antidepressant drugs, it takes 2–4 weeks for the therapeutic action to de-velop. A leading hypothesis of this delayed pharmacologi- 2. Results
cal action is desensitation of somatodendritic serotonin 1Aautoreceptors (5HT1A) in the midbrain raphe, which act Two compounds, buphanidrine (7.6 mg) and buphanamine (5.6 mg) (with activity on the SERT were isolated.
A major goal of antidepressant development is to im- Buphanidrine was identified by comparison with NMR data prove preceding drug classes with more rapid onset of an- tidepressant action and fewer unwanted side effects. Ther- apeutic agents acting both by inhibition of serotonin reup- Structurally, buphanamine and buphanadrine have the take and by inhibiting the action of 5HT1A autoreceptors benzo-1,3-dioxole moiety in common with the clinically used might result in a more rapid onset of antidepressant action.
SSRI paroxetin, which could explain their affinity to the Buphanidrine and buphanamine had only slight affinity to SERT. The hallucinogenic effects obtained after accidental or 5HT1A, eliminating the possibility of a dual-action system.
purposeful overdosing with Boophane disticha extracts Although the activities of buphanidrine and buphanamine on the SERT were lower than the activity of the clinically used SSRI citalopram, the activity supports the traditional use of Boophane disticha as a remedy for depression and buphanamine were 274 and 1799 ␮M, respectively M. Sandager et al. / Journal of Ethnopharmacology 98 (2005) 367–370 Acknowledgements
Holmes, A., Yang, R.J., Murphy, D.L., Crawley, J.N., 2002. Evaluation of antidepressant-related behavioral responses in mice lacking the sero- The Danish Medical Research Council and The National tonin transporter. Neuropsychopharmacology 27, 914–923.
Nielsen, N.D., Sandager, M., Stafford, G.I., J¨ager, A.K., van Staden, J., Research Foundation, Pretoria are thanked for financial sup- 2004. Screening of indigenous plants from South Africa for affinity port. NMR equipment used in this work was purchased with to the serotonin reuptake transport protein. Journal of Ethnopharma- grants from Apotekerfonden af 1991, Copenhagen, and The Danish University of Pharmaceutical Sciences. Dan Stærk is Plenge, P., Mellerup, E.T., Nielsen, M., 1990. Inhibitory and regulatory thanked for performing the NMR experiments.
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