neuropharmacology activity of alkaloids from south american medicinal plants.pdf

Curr. Med. Chem. – Central Nervous System Agents, 2002, 2, 1-15 1

1568-0150/02 $35.00+.00 © 2002 Bentham Science Publishers Ltd.

Neuropharmacology Activity of Alkaloids from South American MedicinalPlants

A. Capasso*, R. Aquino, N. De Tommasi, S. Piacente, L. Rastrelli and C. Pizza

Dipartimento di Scienze Farmaceutiche, Università di Salerno, Via Ponte Don Melillo, 84084 Fisciano, Salerno, Italy

Abstract: Higher plants, which have served humankind as sources of biologically active molecules since its earliestbeginnings, continue to play a key role in the world health. Compounds from higher plants are of great potentialvalue as medicinal agents, as "leads" or model compounds for synthetic or semisynthetic structure modificationsand optimization, as biochemical and/or pharmacological probes.

As a consequence of the renewed interest in the search of new substances from natural sources as potentialcandidates in the drug development, since 1980 our research group has been involved in investigation of higherplants employed in Italian, Chinese, African and South-American traditional medicine. Our primary objectives are:

- to isolate as many secondary metabolites as possible for the phytochemical knowledge of the plants studied;

- to identify active principles in plants with claimed biological activity;

- to evaluate pharmacological effects of plant extracts, fractions and pure compounds in relationship to the parentplant material;

- to subject the isolated compounds to biological screenings on the basis of their structural relationship withknown drugs.

One of our approach to the study of medicinal plants is the preliminary pharmacological screening of the plantextracts, followed by a bioassay-guided fractionation of the extracts leading to the isolation of the pure activeconstituents. Such a strategy has been used in the isolation of a number of antispasmodic alkaloids from theextracts of South-American medicinal plants which showed a pronounced inhibitory activity on the electricalinduced contractions of isolated guinea-pig ileum (E.C.I.) and on morphine withdrawal.

The alkaloids represent the group of natural products that has had the major impact throughout history on theeconomic, medical, political, and social affairs of humans. Many of these agents have potent physiological effectson mammalian systems as well as other organisms, and as a consequence, some constitute important therapeuticagents. In the plant kingdom, the alkaloids appear to have a restricted distribution in certain families and genera;particularly Apocynaceae, Papaveraceae, Ranunculaceae, Rubiaceae, Solanaceae, and Berberidaceae are out-standingfor alkaloid-yielding plants.

Alkaloids are usually classified according to the nature of the aminoacids or their derivatives from which they arebiosynthetized.

Our interest has been centered on alkaloids derived from the aromatic aminoacids and in particular on isoquinolinealkaloids (biologically derived from phenylalanine) from Argemone mexicana (Papaveraceae), Aristolochiaconstricta (Aristolochiaceae) and on alkaloids with an indole nucleus (biologically derived from tryptophane)from Sickingia williamsii and Sickingia tinctoria (Rubiaceae).

ARGEMONE MEXICANA

Argemone mexicana (Papaveraceae) is a spiny herbaceousannual plant which grows prolifically across India and inSouth America where it is commonly known as "cardosanto". The aerial parts of A. mexicana, in the form of aninfusion, are widely used in folk medicine for their analgesicproperties (1).

Previous chemical investigations of various parts of thisplant revealed the occurrence of phenolic compounds (2, 3)

*Address correspondence to this author at the Dipartimento di ScienzeFarmaceutiche, Università di Salerno, Via Ponte Don Melillo, 84084Fisciano, Salerno Italia; Phone/Fax: 089/964357; E-mail: [email protected]

and alkaloids characterized by the presence of an isoquinolinenucleus (4,5). Since the isoquinoline-type alkaloids arereported to possess anticholinergic and antihistaminicproperties (6), in our continuing study on the actions of thealkaloids on intestinal activity, we evaluated the effects ofthe extracts (petroleum ether, CHCl3, CHCl3-MeOH 9:1,MeOH), partially purified fractions and pure compoundsfrom Argemone mexicana on E.C.I. of isolated guinea pigileum (7).

Fig. (1a) shows that both CHCl3/MeOH (9:1) and MeOHextracts at the concentrations used (400-200-100 µg/ml)dose-dependently reduced the E.C.I. The inhibition began 2-4 min after the administration of the extracts, it wasenhanced with time and lasted for the whole recording period

2 Curr. Med. Chem. – Central Nervous System Agents, 2002, Vol. 2, No. 1 Capasso et al.

(15 min). On the other hand, both CHCl3 and petroleumextracts from 400 up to 1000 µg/ml did not inducesignificant alterations of the E.C.I (data not shown). As theMeOH extract was more active in inhibiting the ileumcontractions, it was submitted to a further purification bySephadex LH-20 column and six main fractions were

collected and tested for the effects on the E.C.I. The partiallypurified fractions II-V (200-100-50 µg/ml) were also able toreduce significantly the E.C.I. (Fig. 1b), whereas fractions Iand VI were inactive (data not shown). Also in this case, theinhibition appeared 2-4 min after the administration, it wasenhanced with time and lasted for all the recording period(15 min).

In order to establish the principles responsible for theobserved effects, fractions II-V, containing an alkaloidmixture, were submitted to Si-gel column chromatografy,affording three main compounds 1-3. Compounds 1-3 werefound to exert different activity on the E.C.I., as reported infig. (1c). In fact, while compounds 1-2 (5 x 10-6 - 1 x 10-5 - 5x 10-5 M) induced a concentration-related and significantinhibition of the E.C.I., compound 3 (5 x 10-6 - 1 x 10-5 - 5x 10-5 M) produced the opposite effect, and significantlyincreased the E.C.I.

By NMR analysis, compounds 1-3 were respectivelyidentified as protopine, allocryptopine and berberine (8-10)(Fig. 2). Protopine-type alkaloids are widely distributedamong members of the plant families Berberidaceae, Fuma-riaceae, Ranunculaceae, Rutaceae and Papaveraceae (11).

Fig. (2). Compounds 1-3 from Argemone mexicana

Protopine and allocryptopine are reported to possessanticholinergic properties (6), whereas berberine is reportedto be an inhibitor of acetylcholine esterase (10). Therefore, inour experiments, the ability of protopine and allocryptopineto inhibit E.C.I. may be related to their anticholinergicactivity, (thus confirming their spasmolytic effects), whereasthe potentiating effect of berberine on E.C.I. may beattributed to an increase of Ach. This hypothesis may besupported by the results of experiments on the Ach-inducedcontractions of guinea-pig ileum (12). In fact both protopineand allocryptopine (5 x 10-6 - 1 x 10-5 - 5 x 10-5 M) were able

Fig. (1). Dose related effects of (A) CHCI3/MeOH (9:1) andMeOH extracts, (B) partially purified fractions II -V, (C) purecompounds 1-3 from Argemone mexicana on the electricallvinduced contractions of guinea-pig ileum. Results areexpressed as mean ± SEM. *p <0.05, **p <0.01.

O

O NO

CH3

R1

R2

1 protopine OCH2O

2 allocryptopine OCH3

R1 R2

OCH3

O

O N+

OCH3

OCH33 berberine

Neuropharmacology Activity of Alkaloids Curr. Med. Chem. – Central Nervous System Agents, 2002, Vol. 2, No. 1 3

to reduce significantly the Ach-induced contractions ofguinea-pig ileum (IC50 = 1.3 x 10-6 M for protopine; IC50 =2.3 x 10-6 M for allocryptopine). By contrast berberine at thesame concentrations significantly increased the Ach-inducedcontractions (EC50 = 5.7 x 10-5 M) of guinea-pig ileum.

The prevalent inhibitory effect of both the CHCl3-MeOH(9:1) and MeOH extracts, and partially purified fractionsfrom the above extract could be due to the higherconcentration of protopine and allocryptopine or to thepresence of other active components, acting synergistically oras vehicles enhancing the inhibitory activity on the E.C.I.

ARISTOLOCHIA CONSTRICTA

Aristolochia constricta (Mutis ex H.B.K.) is a medicinalplant found in Ecuador and widely distributed in SouthAmerica. It belongs to the Aristolochiaceae in whicharistolochic acids and aristolactams are the known maincomponents (13,14). Although the aerial parts of A.constricta are empirically used in folk medicine asantispasmodic (15), emmenagogue and against the snakebites (16), there were no data in the literature on the possiblepharmacological effects exerted by the extracts, fractions andpure compounds from this plant.

Taking into account the antispasmodic propertiesattributed to A. constricta, each extract [light petroleumether, CHCl3, CHCl3-MeOH (9:1) and MeOH] of the aerialparts of this plant was tested on the electrically inducedcontractions of guinea-pig ileum (17). Both CHCl3-MeOH(9:1) and MeOH extracts at the concentrations used (125,250 and 500 µg/ml) dose-dependently reduced the ECI (Fig.3a) while both petroleum and CHCl3 extracts from 300 to1000 µg/ml did not induce significant alterations of theE.C.I. (data not shown).

Further purification of the MeOH extract by SephadexLH-20 column with MeOH as eluent afforded seven mainfractions which were tested on the E.C.I. at concentrations of50, 100 and 200 µg/ml. Fractions I - V and VII did notshow inhibitory activity on the E.C.I. (data not show),whereas fraction VI was able to reduce significantly theE.C.I. only at the higher concentration used (200 µg/ml)(Fig. 3b). By means of reversed phase HPLC, compounds 4-9 were isolated from fraction VI.

These pure compounds, tested at concentrations of 1 x10-5, 5 x 10 -5, 2.5 x 10 -5 M exerted a significant differentactivity on the E.C.I. The relative order of potency was:compound 9 inactive; 4 = 8 = 7 < 5 and 6 (Fig. 3c). Finallythe above pure compounds were also able to reducesignificantly both Ach- and Histamine induced contractionsof isolated guinea-pig ileum (Fig. 4a, 4b).

The structural elucidation of the new compounds 4-9proceded as follows.

The molecular formulae of compounds 4-9 (C19H17O3Nfor 4, C19H19O3N for 5, C17H15O3N for 6, C17H13O3N for 7,C18H15O3N for 8, C 26H27O4N for 9) were determined fromEIMS, 13C NMR (Table 1), and DEPT-13C NMR.

The 500 MHz 1H NMR spectrum of 4 (Table 2) exhibitedthe presence of 17 protons, each of which was identified withthe help of 1H-1H COSY spectrum. Two signals (3H, s) for -OMe protons resonated at δ 3.85 and 3.87. Signals at δ 7.28(1H, d, J = 8.60 Hz, H-1), 6.90 (1H, dd, J = 8.60 and 2.45Hz, H-2), 8.05 (1H, d, J = 2.45 Hz, H-4) and at δ 7.16 (1H,d, J = 2.50 Hz, H-9), 6.86 (1H, dd, J = 8.70 and 2.50 Hz,H-11), 7.15 (1H, d, J = 8.70 Hz, H-12) suggested twoaromatic rings 1, 3, 4 trisubstituted. An uncoupled proton atδ 8.77 (1H, s) could be assigned to the C-8 proton, whereastwo coupled olefinic protons at δ 8.04 and 8.30 (both d, J =5.12 Hz) were assigned to the C-5 and C-6 protonsrespectively. HETCOR experiment correlated each hydrogensignal to the corresponding carbon signal and allowed theassignment of all the resonances (Table 1). Protopinealkaloids were usually reported as C-2 substituted also onthe basis of biogenetic considerations (8). The inusualabsence of C-2 substituent in 4 required further spectroscopicevidences such as those derived from NOESY and COLOCexperiments which allowed to establish clearly thesubstitution pattern and to locate the methoxyl groups at C-3and C-10. On the basis of the foregoing data the structurereported in Fig. (5) was assigned to compound 4, a newnatural product named 3,5-di-O-methylconstrictosine.

The EIMS of 5 gave a molecular ion at m/z 309 whichwas two mass units higher than that of 4. From the 1H-1HCOSY data, a spin system was assigned to a segment CH2-CH2- (C-5, C-6), which was connected with H-8 through thenitrogen atom (N-7) by the 1H-13C long-range couplingsobserved in HMBC spectrum of 5 (cross peaks: H-8/C-6, H2-6/C-8). Furthermore, the HMBC data provided evidence forthe position of the substituents (HMBC cross peaks:OMe/C-3, OMe/C-10). From these data it clearly appearedthat compound 5 differed from 4 for the absence of the ∆5

double bond; thus 5 is the new compound 5,6-diidro-3,5-di-O-methylconstrictosine.

Compounds 6 and 7 (Fig. 5) differed from 5 and 4,respectively, only in the substitution of the methoxy groupsat C-3 and C-10 with phenolic functions, as suggested bythe absence of the signals at δ 3.85 and 3.87 for 6 and at δ3.85 and 3.88 for 7 in the 1H NMR spectra and at δ 57.10and 57.00 for 6 and at δ 57.15 and 57.05 for 7 in the 13CNMR spectra and by some small ∆δ differences in thecarbon resonances of ring A and D. Thus 6 and 7 wererespectively named 5,6-dihydroconstrictosine and cons-trictosine.

The NMR data of compound 8 (Fig. 3) were very similarto those of 4, the main differences being the absence of thesignal ascribable to one methoxyl group and the chemicalshifts of one aromatic ring. NOE experiments allowed tolocate the methoxyl group at C-3 and to establish for 8 thestructure 3-O-methylconstrictosine (Fig. 5).

The 13C and 13C DEPT NMR spectra of 9 revealed 26carbons constisting of 8 sp3 and 18 sp2 ones, the lattercorresponding to three aromatic rings. The 1H NMRspectrum of 9 (Table 3) showed signals due to nine aromaticprotons, two methoxyl groups, two sp3 methines and foursp3 methylenes, identified with the help of 1H-1H COSY


Fig. (3). Dose-related effects of (A) CHCl3/MeOH (9:1) and MeOH extracts, (B) partially purified fraction VI, (C) pure compounds 4-8from Aristolochia constricta on the electrically induced contractions of guinea-pig ileurn. Results are expressed as mean ± SEM. *p<0.05, **p <0.01.


Fig. (4). Dose-response relations of compounds 4-8 from Aristolochia constricta on (A) Ach-induced contractions of guinea pigileum, (B) Hist-induced contractions of guinea pig ileum. Results are expressed as mean ± SEM. *p <0.05, **p <0.01.


Table 1. 13C NMR Data for Compounds 4-8 (CD3OD) a.

Carbon 4 5 6 7 8

C-1 111.50 113.80 113.50 113.45 111.65

C-2 112.00 111.80 113.50 113.90 112.20

C-3 158.75 158.00 154.75 154.15 159.00

C-4 106.65 106.10 107.86 107.86 106.80

C-4a 132.75 132.75 132.75 132.38 132.85

C-5 116.85 21.00 19.95 118.60 116.90

C-6 137.80 50.10 50.00 137.95 137.85

C-8 140.00 139.10 139.10 139.40 140.10

C-8a 126.60 126.59 126.59 115.70 115.75

C-9 102.90 102.90 103.70 106.90 107.00

C-10 156.10 156.10 152.07 154.15 154.30

C-11 113.00 113.00 114.00 113.89 113.90

C-12 114.55 114.50 116.50 119.85 120.00

C-12a 122.75 128.73 128.73 122.45 122.55

C-13 50.00 50.00 50.00 50.00 49.00

C-14 185.05 185.00 184.44 189.30 189.30

C-14a 129.70 128.73 128.73 129.55 129.70

OCH3 57.10 57.15 ------ ------ 57.20

OCH3 57.00 57.05 ------ ------ ------

a Assignments were confirmed by 1H-1H COSY and 1H-13 C HETCOR experiments.

Fig. (5). Compounds 4-9 from Aristolochia constricta

N

OR1

RO

A

D

1

2

3

4 56

8

9

1011

12

13

4a

14a

O

12a

8a

N

OR1

RO

O

4 R = CH3, R1 = CH37 R = H, R1 = H8 R = CH3, R1 = H

5 R = CH3, R1 = CH36 R = H, R1 = H

N

OR1

CH3O

A

D

1

2

34 5

6

8

9

1011

12

13

4a

14a

12a

8a

CH3OH

14

14

OHE1'

2' 3'

4'

5'6'

15

H

9


Table 2. 1H NMR Data for Compounds 4-8 (CD3OD)a.

Proton 4 5 6 7 8

H-1 7.28 d 7.22 d 7.21 d 7.25 d 7.28 d

(8.60) (8.70) (8.75) (8.60) (8.60)

H-2 6.90 dd 6.88 dd 6.73 dd 6.67 dd 6.90 dd

(8.60, 2.45) (8.70, 2.45) (8.75, 2.44) (8.60, 2.40) (8.60, 2.45)

H-4 8.05 d 7.91 7.75 d 7.93 d 8.05 d

(2.45) (2.45) (2.44) (2.40) (2.45)

H-5 8.04 d 2.86 t 2.86 t 8.06 d 8.01 d

(5.12) (8.38) (8.40) (5.18) (5.14)

H-6 8.30 d 3.95 t 3.90 t 8.80 d 8.28 d

(5.12) (8.38) (8.40) (5.18) (5.14)

H-8 8.77 s 8.10 s 8.06 s 8.78 s 8.73 s

H-9 7.16 d 7.12 d 6.94 d 7.45 d 7.45 d

(2.50) (2.50) (2.50) (2.40) (2.45)

H-11 6.86 dd 6.83 dd 6.75 dd 7.44 dd 7.42 dd

(2.50, 8.70) (2.50, 8.70) (2.50, 8.70) (2.40, 8.75) (2.45, 8.70)

H-12 7.15 d 7.15 d 7.17 d 7.48 7.48 d

(8.70) (8.70) (8.70) (8.75) (8.70)

OCH3 3.85 s 3.85 ------ ------ 3.83 s

OCH3 3.87 s 3.88 ------ ------ ------

a J values are in parentheses and reported in Hz; chemical shifts are given in δ units.

spectrum. The assignments of all protonated carbons wereenabled by interpretation of the HETCOR spectrum (Table3). Particularly, the 1H-1H COSY spectrum of 9 revealedspin systems due to three segments [one AA'BB' (C-5-C-6)and two ABX patterns (C-8-C-15 and C-13-C-14)]. Thesethree segments were connected one another through thenitrogen atom (N-7) by the 1H-13C long range couplingsobserved in HMBC spectrum (H-8/C-6, H-14/C-6, H2-6/C-8,H2-6/C-14 and H-8/C-9). HMBC data also showed key-correlations among these segments and three aromatic rings,allowing to deduce for 9 a benzyltetrahydroprotoberberinering system characterized by two hydroxyl and two methoxylgroups. HMBC provided evidences to discriminate methoxybearing carbons from hydroxy bearing carbons (Table 3).

It is reported that the tetrahydroprotoberberine alkaloids,if rings B and C assume a half-chair conformation exist inthe equilibrium of one B/C trans-quinolizidine (I trans) andtwo B/C cis-quinolizidine systems (II cis and III cis) (8,18)(Fig. 6) The three systems are called conformers although thenitrogen configuration in trans-quinolizidine is opposite tothat of cis-quinolizidine. Previous 1H NMR literature datafor cis and trans junction showed that for B,C-trans

junction H-14 and H-8 resonated in CD3OD both at ~ 3.6 ±0.2 ppm, whereas for the cis junction a ~ 0.7 ppm downfieldshift was recorded (~ 4.3 ± 0.2 ppm). Moreover, as reportedby R. Suau et al. (18), in the 13C NMR spectrum the trans-quinolizidine conformation is associated with a low field C-14 in contrast with the analogous signal of its conformer (∆δ~ 9 ppm). Resonances at δ 4.22 and 4.46 respectively for H-8 and H-14 in the 1H NMR spectrum and at δ 51.45 for C-14 in the 13C NMR spectrum of 9 indicated a B/C cisconformation (Table 4). The chemical shift, the multiplicityand the coupling constant values of H-14 (1H, dd, J = 11and 5 Hz) were consistent for an axial proton. Irradiation ofthis proton yielded a strong NOE with H-8 as expected for aII cis conformation of 9 (Fig. 4). These observation alsoindicated the relative configuration at C-8 with an equatorialbenzyl group.

Therefore to compound 9 was attributed the structure (-)-8b-[4'-hydroxybenzyl]-2,3-dimethoxyberbin-10-ol. 8-benzyl-berberine alkaloids have been isolated previously from othernatural sources. The novelty of compound 9 resides in thedifferent substitution pattern of rings A (methoxy groups atC-2 and C-3) and D (hydroxy group at C-10) never reported,as far as we know, in the literature.


SICKINGIA WILLIAMSII AND SICKINGIATINCTORIA

The bark of Sickingia williamsii and Sickingia tinctoria,typical South-American small trees belonging to the familyRubiaceae, are empirically used for their analgesic andantinflammatory activity for the treatment of a variety ofinflammatory diseases in Peruvian folk medicine (19). In thecourse of our work on biological active Rubiaceae, we haveisolated five glucoindole alkaloids (10-14) from themethanol extract and a β-carboline alkaloid (harmine, 17) aswell as scopoletin 18, and oleanolic acid 19 from thechloroform extract of the two species (20). All the alkaloidsisolated from S. williamsii and S. tinctoria werecharacterized by the presence of an indole nucleus. Alkaloidswith the β-carboline structure are present in plants known to

affect the central nervous system (CNS) including e.g.Banisteriopsis species, Alianthus altissima , Passifloraincarnata; the pharmacological activity of these plants isrelated to their indole alkaloid content (21). Administrationof crude alkaloid extract of various Banisteriopsis has, forexample, resulted in in vivo various CNS effects includingsedation, passivity of movements, prolongation ofhexobarbital sleeping time, antagonism of reserpine-inducedhypothermia, analgesia in mice and an in vitro inhibitoryaction of the spontaneous contractions of rabbit ileal strips.Given the above literature the purpose of our study were asfollows:

- the confirmation or invalidation of the traditional effectsascribed to the extracts of S. williamsii and the study of theinvolvment of its metabolites in these properties;

Table 3. 1H and 13C NMR Data for Compound 9 (CD3OD) a.

Position 13 C 1H HMBC correlation 1H

1 112.50 6.65 s H-14

2 145.80 OMe, H-4

3 147.00 OMe, H-1

4 112.60 6.60 s H-5

4a 130.05 H-6

5 30.15 2.70 t (8.0)

6 49.00 3.38 t (8.0) H-8, H-14

8 69.05 4.22 br t (7.0) H-6

8a 127.10 H2-13

9 108.10 7.50 d (2.0) H-8

10 154.20

11 114.35 7.40 dd (2.0, 7.5)

12 119.90 7.42 d (7.5) H2-13

12a 133.05 H-8

13 (α) 35.05 2.60 dd, (12.5, 11.0)

13 (β) 2.55 dd (12.5, 5.0)

14 51.45 4.46 dd (11.0, 5.0) H2-6

14a 125.70 H-5, H 2-13

15 41.50 3.78 m

1’ 132.00 H-8

2’ 131.10 6.90 br d (8.5) H-15

3’ 116.10 6.70 br d (8.5)

4’ 158.05

5’ 116.10 6.70 br d (8.5)

6’ 131.10 6.90 br d (8.5) H-15

OCH3 57.00 3.80 s

a Assignments were confirmed by 1H-1H COSY and 1H-13 C HETCOR experiments. J values are in parentheses and reported in Hz; chemical shifts are given in δ units.


- the examination of possible analgesic and antispasmodiceffects often associated with the reported antinflammatoryeffects of extracts, and partially purified fractions and thestudy of the involvment of indole alkaloids and iridoid.

Therefore, we proceeded to bioassay-directed extractionand fractionation of S. williamsii as reported for the previous

plants (22). This approach has led to the isolation andcharacterization of glucoindole alkaloids 10, 11, 13, and 14as the active antispasmodic principles besides some iridoids(loganin 15, secoxyloganin 20, sweroside 21, loganic acid22), and a quinic acid derivative (4,5-dicaffeoylquinic acid16) (Fig. 7).

Fig. (6). Possible isomeric forms of compound 9 from Aristolochia constricta

Fig. (7). Glucoindole alkaloids and iridoids from Sickingia williamsi and Sickingia tinctoria.

N

OH

HHCH3O

CH3O

BC

R

14

I trans

N

OH

HH

CR14

CH3O

CH3O8

8

II cis

OH

CH3O

CH3O

N

HR

14

III cis

NN+

H

H

OR2

H

H

OR120 O OH

OH

OH

HO

12 R1 = H R2 = COO-

13 R1 = COO- R2 = H

NN

COOH

O

HH

HO

O OHOH

OH

HO

14

NN

O

HH

HO

O OHOH

OH

HO

10

H

COOH

O

OO OH

OH

OH

HO

OO

COOMe

O

OO OH

OH

OH

HO

21

HO

CH3

H

H

15

NN

COOCH3

O

HH

HO

O OHOH

OH

HO

11

H

COOH

H2

678


The plant extracts (petroleum ether, chloroform,CHCl3/MeOH (9:1), and MeOH) were tested at concentrationof 300, 150 and 30 µg/ml organ bath, dissolved in DMSO,with a contact period of 15 min. Between the tested extracts,the CHCl3/MeOH (9:1) was more active (IC50 = 116.1 µg)than the CHCl3 (IC50 = 400.9 µg) (Fig. 8a), whereas thepetroleum ether and the MeOH extracts did not induce anysignificant modifications on the ileum contractions. For thisreason, CHCl3/MeOH (9:1) extract was submitted to afurther purification by Sephadex LH-20 column to yield fivemain fractions (I-V) which were tested under the sameexperimental conditions at concentrations of 500, 250 and100 µg/ml organ bath, dissolved in distilled water, with acontact period of 15 min. All the fractions tested showed adifferent potency in inhibiting ECI; the relative order ofpotency (Fig. 8b) was : fraction V inactive, fraction I <fraction II< fraction III and IV (most active, IC50 = 356.2µg). In order to identify the molecules responsible for theactivity of the above extract and fractions and how structuraldifferences may influence the activity, the fractions weresubmitted to a further purification. Thus, by means of RP-HPLC, compounds 12 (ophiorine A) and 13 (ophiorine B)were isolated from fraction I; compounds 10 (sickingine), 12and 13 from fraction II; compounds 11 (5α-carboxystrictosidine) and 14 (lyalosidic acid) from fractionIII; compounds 15 (loganin), 16 (4,5-dicaffeoylquinic acid),20 (secoxyloganin), 21 (sweroside), and 22 (loganic acid),from fraction IV; compounds 17 (harmine), 18 (scopoletin),and 19 (oleanolic acid) from fraction V. The experimentsperformed with the pure compounds indicated (Fig. 8c) thatall the tested glucoindole alkaloids sickingine 10 (IC50 =1x10-4 M), 5α-carboxystrictosidine 11 (IC50 = 1.3x10-4 M),ophiorine B 13 (IC50 = 5.3x10-5 M), and lyalosidic acid 14(IC50 = 1.3x10-4 M), appeared to be more or less potentinhibitors of E.C.I., except for ophiorine A 12. Among thetested iridoids loganin 15 (IC50 = 1x10-4 M) and sweroside21 (IC50 = 4x10 -4 M) possessed a significant inhibitory effecton E.C.I. while loganic acid 22 and secoxyloganin 20 didnot exert significant effects (data not shown). Also, thealkaloid harmine 17 and scopoletin 18 and oleanolic acid 19proved to be inactive.

The two classes (glucoindole alkaloids and iridoids) ofnaturally occurring substances isolated from S. williamsii arebiogenetically related because iridoids like loganin andsecologanin are the key monoterpenoid intermediate in thebiosynthetic pathway leading to strictosidine which is theprecursor of all the indole alkaloids. Therefore, the iridoidmoiety is included into the glucoindole alkaloid structutes.Considering the molecular structures reported in Fig. (7), wenote that the active glucoindole alkaloids 10, 11, 13, and 14possess both an indole and iridoid nucleus, whereas the otheractive compounds 15 and 21 possess only an iridoid nucleusthus suggesting that the base cycle of the iridoids affects theinhibitory activity on the ECI. Also we can note that thealkaloid harmine 17, having only an indole nucleus andlacking the iridoid moiety in its molecular structure showedno inhibitory effect on ECI. Taken together, these resultssupport the hypothesis that a combination of an indolenucleus with an iridoid moiety seems responsible for theappearance of the activity. Furthermore, stereochemicalinfluences and interactions between the various substituentson the molecules appear to affect the inhibitory activity. For

example when all the other characters in the structure werethe same as in alkaloid 12, the molecule was inactivewhereas its C-16 stereoisomer 13 appeared to be one of themost active principles of S. williamsii in this assay.

Regarding the possible pharmacological mechanismunderlying the observed inhibition, we suggest that thecompounds were not anticholinergic but their effects werepresynaptically mediated. This may be supported by datashowing that the active compounds were not able to reduceacetylcholine-induced contractions of guinea-pig ileum. Onthe other hand, the question of whether this effect is relatedto an interaction with specific receptors or neurotransmitterson smooth muscle control is a subject for further study.

EFFECTS OF THE ALKALOIDS ON MORPHINE-WITHDRAWAL IN VITRO

Protopine-type alkaloids 1-2 from A. mexicana and 4-7from A. constricta resulted able to reduce Ach-inducedcontractions of isolated guinea-pig ileum (23,24). Thereforewe studied the anticholinergic activity on morphinewithdrawal of compounds 1-2 and 4-7 since acetylcholinehas been reported to be one of the neurotransmitter mainlyinvolved in opioid withdrawal (25,26).

Furthermore, alkaloids with an indole nucleus are knownto decrease drug craving in the addicted user and to reduceself-administration of both morphine and cocaine (27-29).On this basis also alkaloids 11-14 from S. williamsii wereinvestigated for their potential in treating drug abuse (30).

Opiate withdrawal syndrome by opioids is a well knownphenomenon and its cellular mechanisms have also beenstudied (31). Although several methods may be used toinduce opiate dependence both in vivo and in vitro (31),recently, the similarities of the enteric nervous system withthe central nervous system makes the first a mostinvestigated tissue to study cellular biology of neurons (32).Therefore, as the responses obtained from the guinea-pigisolated ileum share many features in common with thoseobserved in the central nervous system, it has been thoughtthat this tissue may provide a new and simple model for thestudy not only of the acute effects of opioids, but also of thelong-term effects of tolerance and dependence (33).Significant advances in understanding the dependencephenomena have been obtained as it has been demonstratedthat a strong naloxone-induced contracture could be obtainednot only from the ilea of opiate-treated animals but also fromuntreated animals after a brief in vitro exposure to opioids(34). This indicated that the cellular mechanisms ofdependence may occurr very rapidly following occupation ofreceptors and that these mechanisms are operating within themyenteric plexus. The characteristics of dependencedevelopment and its precipitation with naloxone in theguinea-pig ileum are very similar to those of acutedependence in experimental animals and man (35).

A reproducible acute opiate dependence was obtainedperforming the following experimental procedure. A typicaltracing of contracture responses of the ileum to repeated


Fig. (8). Dose related effects of (A) CHCl3/MeOH (9:1) and CHCI3 extracts, (B) partially purified fractions I-IV, (C) pure compounds10 -17 , 21 from Sickingia willamsii on the electrically induced contractions of guinea-pig ileum. Results are expressed as mean± SEM.*p<0.05. *~p <0.01.


Fig. (9). Typical tracing of morphine withdrawal on guinea-pig ileum. (A) Three similar acetylcholine responses (A), electricalstimulation, injection of the opioid agonist (O.A.) followed after 4 min of contact period by naloxone (N) which induces contraction(first opioid withdrawal). After washout, another A response was performed. (B) After a 30 min resting period under electricalstimulation, a further 4 min exposure of the ileum to the O.A. and naloxone elicited a reproducible response (second opioidwithdrawal). (C) After another 30 min resting period under electrical stimulation, the ileum responded again to the O.A. and naloxonewith the same intensity (third opioid withdrawal).

challenges with opiate and naloxone is shown in Fig. (9a-9c). After three similar Ach responses, the preparation waselectrically stimulated for 10-20 min, 0.5 msec pulse

delivered transmurally, at a frequency of 10 sec atsupramaximal voltage, 25 V. Before the addition of themorphine to the bath, the electrical stimulation was switched


Fig. (10). Effects of the compounds (A) 1-2 from A. mexicana, (B) 4-7 from A. constricta,(C) 11 -14 from S. williamsii on morphinewithdrawal. Compounds were iniected 10 mim before the opioid agonist. Results are expressed as mean ± SEM. *p <0.05, **p <001.


off. Under these conditions, the first contact with the opioidagonist followed after a 4 min exposure by naloxone induceda strong contracture (about 60 %) of the Ach maximum.However, after washout, another Ach response was performed(to verify whether the ileum responsiveness was modifiedafter withdrawal contracture) (Fig. 9a) and, after 30 minresting period under stimulation, a further 4 min exposure ofthe ileum (without electrical stimulation) to the opiate andnaloxone elicited reproducible response. Following washout,Ach response (Fig. 9b) and another 30 min resting periodunder stimulation, the ileum responded again to themorphine and naloxone with the same intensity (Fig. 9c). Inour experiments, to avoid a possible tolerance for repeatedmorphine injection, each preparation was submitted only tothree challenges with morphine and naloxone. Naloxone perse did not produce effects on "naive" preparations or thosewashed after opiate contact.

Compounds to be tested were injected 10 min beforemorphine followed by naloxone (second opioid withdrawal).

Four parameters were evaluated:

(1) Naloxone contraction: the size of the contractionproduced by naloxone challenge was expressed as a fractionof the maximum contraction obtained with the subsequentaddition of Ach in the same piece of tissue according to amodification of the method of Collier et al (36):

response to naloxone x 100 = Tension ratio

maximum response to Ach

(2) Ach responses before and after treatment: reduction orincrease of the Ach responses in the post-drug was expressedas a percentage of Ach response in the pre-drug.

(3) Electrically stimulated contraction before and aftertreatment: reduction or increase of the electrical stimulationcontraction in the post-drug was expressed as a percentage ofthe electrical stimulation contraction in the pre-drug.

(4) Naloxone contraction before and after treatment:reduction or increase of the naloxone contraction in the post-drug was expressed as a percentage of the naloxonecontraction in the pre-drug.

In our experiments, compounds 1,2,4,5,6,7,11,12,13,14were administered 10 min before the injection of morphine.Therefore, as during compound treatments the contact periodof morphine was 10 min when compared with the pre-drugperiod, to avoid a possible influence of the contact period,we performed a series of preliminary experiments to verifywhether a contact period longer than 4 min may effectnaloxone contraction. No differences were observed when thecontact period exposure of morphine was 4 to 10 min (datanot shown).

Tested compounds, although at different concentrations,were able to reduce dose-dependently the morphinewithdrawal (Fig. 10a-10c). After washout, both Achresponse, electrical stimulation and the final opiatewithdrawal resulted still reduced.

While there are no data in literature on the effects exertedby protopine-type alkaloids on morphine withdrawal, severaldata are reported for Tabernanthe iboga, a medicinal plantknown to contain indole alkaloids which interrupt bothphysiological and psychological aspects of opiate withdrawalin man (27-29).

Our experiments performed with indole alkaloids 11-14from S. williamsi strongly confirm the importantinvolvment of the above alkaloids in the control of opiatewithdrawal.

As far as concern the involvment of protopine-typealkaloids 1-2 from A. mexicana and 4-7 from A. constricta,We can only support some hypothesis underlying theiractivity in opiate withdrawal. The ability of protopinealkaloids to reduce morphine withdrawal may be related totheir anticholinergic properties. Our results strongly supportthe above hypothesis because both acetylcholine andelectrical stimulation resulted still reduced after washout ofalkaloids, thus confirming a direct action both on post- andpresynaptic acetylcholine receptors (5,14).

In the light of the above results our experiments providean evidence that alkaloids from A. constricta, A. mexicanaand S. williamsi possess a good antiadditive activity.Further in vivo experiments are necessary to support thishypothesis.

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