Isolation and Spasmolytic Evaluation of New Alkaloids from Dichrostachys cinerea ( L . ) Wight et Arn . ( Fabaceae )

Dichrostachys cinerea (L.) Wight et Arn. (Fabaceae) root bark is used in Ivorian Traditional Medicine to treat asthma, which is a respiratory disorder characterized by inflammation and the restriction of tracheal muscles obstructing the air circulation. The tracheal relaxant effect of a crude aqueous-alcoholic extract of the plant root bark was previously shown. For the present study, alkaloids were isolated from the same extract and investigated ex vivo in C57Bl/6j mice isolated trachea contracted with carbachol 1 μM, in comparison with a reference bronchodilatator, i.e. salbutamol. Two extraction procedures allowed isolating 2 Alkaloids that monodimensional and bi-dimensional nuclear magnetic resonance (NMR) and mass specters allowed identifying a pyrolidine structure type nucleus with a long bi-hydroxyled alkyl chain. Alkaloid 1, carrier of a sugar, is a glycoside of Alkaloid 2. Both alkaloids induced similar spasmolytic effects, but Alkaloid 1 was more effective than Alkaloid 2 at 9 × 10 M (p ˂ 0.01), 3 × 10 M, and 9 × 10 M (p ˂ 0.001). Salbutamol induced its spasmolytic effect in a different way, and its maximal effect Emax (less than 30%) was obtained at 9 × 10 M, while Emax of both alkaloids (100%) was obtained at 3 × 10 M.


Introduction
Dichrostachys cinerea (L.) Wight et Arn.(Fabaceae), among others numerous plants, is commonly used, alone or in association with other plants, in African Traditional Medicine.The ethnobotanic inquiries state that the roots of this plant are astringent and used in rheumatism, urinary calculi and renal troubles [1]; in Togo, the root decoction is administered by oral route in abscesses [2].In Ivory Coast, whereas the root decoction is used as mouthwash in case of tooth decays by people of the North, people of the South use it to treat asthma [3].As asthma is a respiratory disorder which is essentially characterized by the restriction of tracheal muscles obstructing the air circulation [4], the tracheal relaxant effect of a crude aqueous-alcoholic extract of the plant root bark was previously shown [5].Besides, bisnordihydrotoxiferine, a tertiary indole alkaloid isolated from the root of Strychnos divaricans was shown to antagonize ace-tylcholine-induced contractions in rat uterus and in guineapig ileum [6]; other alkaloids were also shown to have spasmolytic effects on guinea-pig isolated trachea contracted by carbachol, histamine, or KCl [7].For the present study, alkaloids were isolated from the root bark extract and investigated ex vivo in mice isolated trachea in comparison with a reference bronchodilatator, i.e. salbutamol.

Plant Material
The roots of D. cinerea were collected in September 2009 in the South-East of Ivory Coast in bushes near Grand-Bassam.The plant was authenticated by Professor Aké-Assi Laurent, a taxonomist at the Centre National de Floristique (Ivory Coast), in comparison with identified specimens and vouchers were deposited there.The barks were removed from the roots, washed in distilled water, air-dried at air-conditioning temperature (18˚C) for two weeks, and pulverized.Dehydration yield was 55.2%.

Extraction Procedure
The research for alkaloids in the crude extract was made by reactions of characterization in tube with the reactive of Valsen-Mayer, then on Thin Layer Chromatography plates revealed by the reactive of Dragendorff.
After that, two ways were used to isolate alkaloids from the crude aqueous-alcoholic extract of the plant root bark: direct division and division after acidification then alkalinisation (Figure 1).

Pharmacological Tests
Alkaloids isolated from D. cinerea root bark were performed on mice isolated trachea pre-contracted with carbachol 1 µM in the aim to evaluate their spasmolytic effect.

Preparation of Trachea Rings and ex Vivo
Procedure Mice were anesthetized with pentobarbital (60 mg/kg i.p.).The upper respiratory tract and associated alimentary tissue were rapidly excised and placed in ice-cold Krebs bicarbonate solution containing: NaCl 117 mM, KCl 5.36 mM, NaHCO 3 25 mM, KH 2 PO 4 1.03 mM, MgSO 4 0.57 mM, CaCl 2 2.5 mM, D-glucose 11.1 mM.The tracheas were dissected free from surrounding tissue and cut into 2-mm length segments, which were suspended isometrically between 2 stainless steel hooks in organ chambers containing 5 mL Krebs bicarbonate solution at 37˚C and continuously gassed with a mixture of 95% oxygen and 5% carbon dioxide.Isometric tension was recorded in real time by a force-displacement transducer connected to the PowerLab ® data acquisition system controlled by the Chart ® version 5 data analysis software (AD Instruments, Bella Vista, Australia).The rings were stretched in a stepwise manner to a resting value of 0.6 g for at least 1 hour.Tracheal rings were then challenged with 10 −5 M carbachol (Sigma-Aldrich Chemicals, France) to evaluate their functional integrity.After a 45-minutes washout period, tracheal preparations were precontracted with a submaximal concentration of carbachol (10 −6 M).After stabilization of the contraction, cumulative additions of the different products and of distilled water as control vehicle were performed.
Biologically, the relaxant response of the tracheal rings was expressed as percentage of the precontractile tone induced by carbachol.The effect of vehicle (distilled water) was systematically subtracted from the effect of the products.
Results are expressed as mean ± standard errors of the mean (S.E.M) of 6 experiments.E max is the maximal relaxation obtained and CE X is the concentration of product which induces X% relaxation.Data were analyzed with Sigmaplot ® software by an unpaired Student's t-test or by one way analysis of variance (ANOVA) followed by Holm-Sidak or Bonferroni-test, with criterion set for statistical significance at p < 0.05.

Results and Discussion
The analysis of the root bark extract highlighted the presence of an alkaloid fraction (tests in reactive of Valsen-Mayer and in reactive of Dragendorff were positive) which purification allowed obtaining 49.2 mg of total alkaloids, purification yield being 0.0164%.
These data allow proposing the fact that Alkaloid 1, carrier of a sugar, is a glycoside of Alkaloid 2 (baptized JEMIGRACINE by our care).
Not aromatic, these isolated alkaloids probably arise from lysine metabolism [8].
Both alkaloids 1 and 2 induced almost similar regular spasmolytic effects (E max value in Table 1).However, Alkaloid 1 was more effective than Alkaloid 2 at 9 × 10 −6 M (p ˂ 0.01), 3 × 10 −5 M and 9 × 10 −5 M (p ˂ 0.001) just like indicated on Figure 3 and in Table 1     like the numerous hydroxyls of aminosides (amino-glycosides) that modulate the penetration of the antibiotic in the bacterium and so play on the specter of action by widening it.E max for salbutamol (less than 30%) was obtained at 9 × 10 −6 M while the one of both alkaloids, corresponding to a total deletion of contractions, was obtained at 3 × 10 −4 M.
Salbutamol induced its spasmolytic effect in a different way [from the lowest concentrations its relaxant effect appeared and reached the maximal level at 9 × 10 −6 M, then decreased for the highest concentrations (Figure 3)], due to a difference in the mechanisms of action.Indeed, the spasmolytic effect of the crude extract from which alkaloids were isolated was not inhibited by β 2 -blockers, but was sustained by hyperpolarization [5]; however salbutamol is known to have a β 2 -mimetic action [9].Besides, the increase of salbutamol's spasmolytic effect, for its lowest concentrations, testimonies β 2 -receptors sensitation.The decrease of this effect, for its highest concentrations, might be due to β 2 -receptors saturation, with desensitation of the subsequent signaling cascade [10].Nevertheless this probable saturation might not do prejudice to the molecule activity, as salbutamol is well known for its neutralizing action of bronchitis spasm during asthma crisis.Concerning the isolated alkaloids, the steadiness of relaxation going until to a total inhibition of the carbachol-induced pre-contractions is-it in relation with a potent spasmolytic effect?In this connection, it is important to note that authors defend the thesis that herbal in vitro data are questionable in absence of in vivo observations [11].As a matter of fact, factors like absorption or metabolism of those substances are liable to great differences between their in vitro and in vivo activities [12].
The E max value is the percentage of relaxation obtained at the maximal tested concentration.The EC x value is defined as the concentration of extract that induces x% relaxation.n.d is not determined value.

Conclusion
This study allowed isolating new structures of alkaloids from plants.Those new components exerted a good ex vivo activity on contracted trachea.Nevertheless, further investigations will be required to completely identify these components, and to confirm their real potency with in vivo bronchodilatation tests.