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

doi:10.4236/pp.2013.49095

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Pharmacology & Pharmacy, 2013, 4, 684-688

Published Online December 2013 (http://www.scirp.org/journal/pp)

http://dx.doi.org/10.4236/pp.2013.49095

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Isolation and Spasmolytic Evaluation of New Alkaloids

from Dichrostachys cinerea (L.) Wight et Arn. (Fabaceae)

Amenan Geneviève N’guessan-Irié1,2, Joël Dade1, Pierre Champy1,3, N’doua Gisèle Siransy-Kouakou2,

Véronique Leblais3,4

1Chimie des Substances Naturelles, CNRS UMR 8076 BioCIS, Châtenay-Malabry, France; 2Laboratoire de Pharmacologie et Physiologie,

UFR Sciences Pharmaceutiques et Biologiques, Université Félix Houphouët-Boigny, Abidjan, République de Côte d’Ivoire; 3Faculté

de Pharmacie, Université Paris-Sud 11, Châtenay-Malabry, France; 4INSERM UMR-S 769, Châtenay-Malabry, France.

Email: [email protected]

Received September 23rd, 2013; revised October 28th, 2013; accepted November 15th, 2013

Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is

properly cited.

ABSTRACT

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 reso-

nance (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 × 10−6 M (p ˂ 0.01), 3 × 10−5 M, and 9 × 10−5 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 × 10−6 M, while Emax of both alkaloids (100%) was obtained at 3 × 10−4 M.

Keywords: Alkaloids Isolated; Plant; Spasmolytic; Asthma

1. 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, uri-

nary calculi and renal troubles [1]; in Togo, the root de-

coction 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 ob-

structing the air circulation [4], the tracheal relaxant ef-

fect of a crude aqueous-alcoholic extract of the plant root

bark was previously shown [5]. Besides, bisnordihydro-

toxiferine, a tertiary indole alkaloid isolated from the root

of Strychnos divaricans was shown to antagonize ace-

tylcholine-induced contractions in rat uterus and in guinea-

pig ileum [6]; other alkaloids were also shown to have

spasmolytic effects on guinea-pig isolated trachea con-

tracted by carbachol, histamine, or KCl [7]. For the pre-

sent 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. sal-

butamol.

2. Materials and Methods

2.1. 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

Isolation and Spasmolytic Evaluation of New Alkaloids from Dichrostachys cinerea (L.) Wight et Arn. (Fabaceae) 685

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%.

2.2. 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).

2.3. Pharmacological Tests

Alkaloids isolated from D. cinerea root bark were per-

formed on mice isolated trachea pre-contracted with car-

bachol 1 µM in the aim to evaluate their spasmolytic effect.

Extraction : ether petroleum, 500 mL, 2 × 48 h

ether petroleum Filtrat

Negative in reactive of Dragendorff *

Marc

Filtra t

Positive in reactive of Dragendorff

Marc

1. Alca linisa tion NH

OH (pH 9),

2. Extraction CH

(500 mL) , 2 × 48 h

1. Alca linisa tion NH

OH (pH 9),

2. Extraction MeOH (500 mL) , 2 × 48 h

MeOH Filtrat

Negative in reactive of Dragendorff

Marc

Extract A

6.2 g

«Residue II »

3.1 g

«Residue I »

3.1 g

Separation of Ain 2

1. Flash Chromatography

on silica gel,

EtOH /CH

4:6

2. Preparative Chromatography

on silica pa tch,

MeOH/CH

3:7

3. Chromatography

on Sephadex

LH-20 gel,

MeOH

Alkaloid 2

29.7 mg

Alkaloid 1

4.2 mg

1. Acidification HCl 1 N (200 mL, pH 2)

2. liquid / liquid Extraction,

(3 ×150 mL)

Acid aqueous phase

Phase

Negative in reactive of Dragendorff *

1. Alca linisa tion NH

OH (pH 9)

2. liquid / liquid Extraction, CH

(3 × 150 mL)

Aqueous

phase

Phase

Positive in reactive of Dragendorff *

Dried residue

1.87 g

Alkaloid 2bis, identical to alkaloid 2

15.3 mg

1. Flash Chromatography on silica gel, EtOH /CH

4:6

2. Chromatography on Sephadex

LH-20 gel, MeOH

* Test in reactive of Dragendorff realized

on an against acid aqueous extract

evaporation

évaporation

Dichrostachys cinerea

Pulverized root bark (300 g)

Figure 1. Pure alkaloids extraction scheme.

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2.3.1. Preparation of Solutions

Alkaloids isolated, as well as the reference product (sal-

butamol), were extemporaneously dissolved and serially

diluted in distilled water (3 × 10−5, 3 × 10−4, 3 × 10−3 and

3 × 10−2 M).

2.3.2. Chemicals

Salbutamol and carbachol were purchased from Sigma-

Aldrich Chemicals, France; all salts (NaCl, KCl, KH2PO4,

MgSO4, CaCl2 and NaHCO3) and d-glucose from Pro-

labo.

2.3.3. Animals

All experiments were performed in accordance with the

European Community guiding principles in the care and

use of animals (86/609/CEE, CE Off. J. no. L358, 18th

December 1986), the European Ethics Committee

(CREEA Ile-de-France Sud) guidelines, and the French

Decree no. 87 - 848 (J Off République Française, 20 Oc-

tober 1987: pp 12245-12248). Experiments were per-

formed on male C57BL/6J mice at 7 - 8 weeks of age (25

- 30 g; Elevage Janvier, Le Genest Saint Isle, France).

2.3.4. Preparation of Trachea Rings and ex Vivo

Procedure

Mice were anesthetized with pentobarbital (60 mg/kg

i.p.). The upper respiratory tract and associated alimen-

tary tissue were rapidly excised and placed in ice-cold

Krebs bicarbonate solution containing: NaCl 117 mM,

KCl 5.36 mM, NaHCO3 25 mM, KH2PO4 1.03 mM,

MgSO4 0.57 mM, CaCl2 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 sus-

pended isometrically between 2 stainless steel hooks in

organ chambers containing 5 mL Krebs bicarbonate so-

lution 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 trans-

ducer connected to the PowerLab® data acquisition sys-

tem 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.

2.3.5. Data Analysis and Statistics

Chemically, isolated components’ structure has been ana-

lyzed using monodimensional NMR (1H, 13C), bi-dimen-

sional NMR (DEPT, COSY, HMBC, HSQC, NOESY),

and mass (ESI, APCI, high resolution) specters.

Biologically, the relaxant response of the tracheal

rings was expressed as percentage of the precontractile

tone induced by carbachol. The effect of vehicle (dis-

tilled water) was systematically subtracted from the ef-

fect of the products.

Results are expressed as mean ± standard errors of the

mean (S.E.M) of 6 experiments. Emax is the maximal re-

laxation obtained and CEX 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 sta-

tistical significance at p < 0.05.

3. Results and Discussion

The analysis of the root bark extract highlighted the

presence of an alkaloid fraction (tests in reactive of Val-

sen-Mayer and in reactive of Dragendorff were positive)

which purification allowed obtaining 49.2 mg of total

alkaloids, purification yield being 0.0164%.

The division allowed isolating 2 majority pure prod-

ucts. Their monodimensional and bi-dimensional nuclear

magnetic resonance specters (NMR) allowed identifying

a new structure in alkaloids’ serial: pyrolidin with a

bi-hydroxyled long alkyl chain (Figures 2 and 4) con-

firmed by mass spectrometry. These compounds are

called Alkaloid 1 (4.2 mg; y = 0.0014%), Alkaloid 2

(29.7 mg; y = 0.0099%) and Alkaloid 2 bis (15.3 mg; y =

0.0051%), alkaloids 2 and 2 bis being identical com-

pounds isolated by two different ways.

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 (Emax 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 (lowest

EC25 and EC50 for alkaloid 1), probably because of the

sugar component which could make the cell entry easier,

CH3

OOH

Figure 2. Structure of alkaloid 1 = [5-hydroxy-12-(1-me-

thylpyrrolidin-2-yl)dodecyl hexopyranoside].

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Isolation and Spasmolytic Evaluation of New Alkaloids from Dichrostachys cinerea (L.) Wight et Arn. (Fabaceae) 687

Table 1. Emax, EC50 and EC25 of isolated alkaloids and sal-

butamol.

max (%) EC25 (×10−5 mol/L) EC50 (×10−5 mol/L)

Alkaloid 1 104.5 ± 0.9 2.22 ± 0.25 5.22 ± 0.77

Alkaloid 2 104.2 ± 1.8 4.32 ± 0.41 9.27 ± 0.8

Salbutamol 28.1 ± 3.2 6.87 ± 0.22 n.d

‐10

110

3,00E‐08 3,00E‐07 3,00E‐06 3,00E‐05 3,00E‐04

Alkaloid1

Alkaloid2

Salbutamol

%Relaxation

Log [alkaloidsandsalbutamol concentrations(mol/L)]

** ** ** ** **

Figure 3. Relaxant effects of alkaloids isolated from D. cine-

rea and salbutamol; **: statistically significance p ≤ 0.01.

NOH

CH3

Figure 4. Structure of alkaloid 2 = [12-(1-méthylpyrrolidin-

2-yl) dodecane-1,5-diol].

like the numerous hydroxyls of aminosides (amino-gly-

cosides) that modulate the penetration of the antibiotic in

the bacterium and so play on the specter of action by

widening it. Emax 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.

Differences between Alkaloids’ effects and salbuta-

mol’s ones were significant. Thus from 3 × 10−8 to 9 ×

10−6 M, salbutamol was more effective than both alka-

loids; at 3 × 10−5 M, salbutamol was more effective than

alkaloid 2 but less effective than alkaloid 1; for the two

highest concentrations tested (9 × 10−5 and 3 × 10−4 M)

salbutamol was less effective than both alkaloids (Figure

3).

Salbutamol induced its spasmolytic effect in a differ-

ent way [from the lowest concentrations its relaxant ef-

fect 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. In-

deed, the spasmolytic effect of the crude extract from

which alkaloids were isolated was not inhibited by

β2-blockers, but was sustained by hyperpolarization [5];

however salbutamol is known to have a β2-mimetic ac-

tion [9]. Besides, the increase of salbutamol’s spasmo-

lytic effect, for its lowest concentrations, testimonies

β2-receptors sensitation. The decrease of this effect, for

its highest concentrations, might be due to β2-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 inhibi-

tion of the carbachol-induced pre-contractions is-it in

relation with a potent spasmolytic effect? In this connec-

tion, 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 Emax value is the percentage of relaxation obtained

at the maximal tested concentration. The ECx value is

defined as the concentration of extract that induces x%

relaxation. n.d is not determined value.

4. 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.

5. Acknowledgements

The authors are grateful to Pr. L. Aké-Assi (Centre Na-

tional de Floristique d’Abidjan) for botanical identifica-

tion, to Dr. V. Domergue-Dupont (IFR141, Châtenay-

Malabry, France) and her staff for animal care, to Mrs. F.

Lefebvre (UMR-S 769, Châtenay-Malabry, France) for

technical assistance, to Pr. Attioua (Laboratoire de Chimie

Organique Biologique, UFR Sciences des Structures de

la Matière et Technologie, Université Félix Houphouët-

Boigny, Côte d’Ivoire) and Pr. M. Ouattara (Laboratoire

de Chimie Thérapeutique et Biomolécules, UFR Sciences

Pharmaceutiques et Biologiques, Université Félix Hou-

phouët-Boigny, Côte d’Ivoire) for their chemical help.

Financial supports were provided by the “Centre Na-

tional de la Recherche Scientifique”, the “Institut Na-

tional de la Santé et de la Recherche Médicale” and the

“Université Paris-Sud 11”.

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