**3.3 Thiopental-induced sleep**

526 Pharmacology

The colophony was administered in 3 dosis levels (100, 200 y 400 mg/Kg) in all experiments, except in the case of bioassay of labyrinth in cross (50, 100 y 150 mg/Kg). The DHAA dosis levels were 50, 100 and 200 mg/Kg. The injection volume of administration in mice was 0.4 ml/20 g and 1mL/100 g in rats. The colophony (sodium salt-SR) was dissolved in distillated

SR (100, 200 and 400 mg/kg, po.) reduced locomotor activity and rearing in a dosedependent manner during the observation period. The observations are given in Table 1. Doses of 400 mg/Kg of SR showed similar behaviour to diazepam (DZP) 1 mg/Kg

The present study demonstrated that SR prepared from natural and pre-treated (hydrothermally and by acid-basic reaction) resin extracted from Cuban *Pinus* reduced spontaneous locomotor activity in mice. Usually the rodents show an exploratory behaviour when they are collocated in a novel place. However, if the animals are pre-treated with depressant central nervous system drugs, the locomotor activity is decreased. This result is

> Tested groups mean±S.E.M Distilled water 24,5 ± 4,20a SR 100 mg/Kg. 20,2 ± 3,38 b SR 200 mg/Kg. 19,6 ± 2,22 b SR 400 mg/Kg. 12,9 ± 4,48 c DZP 1 mg/Kg. 10,5 ± 3,39 c

Table 1. Effects of SR (100, 200 and 400 mg/kg, po.) on spontaneous locomotor activity.

Social isolation induces aggressive behavior in several strains of mice. The isolation-induced aggression is proposed to be useful as an animal model for assessing inhibitory activity on central nervous system. Different neurotransmitters such as serotonin, noradrenaline, dopamine and gamma-aminobutyric acid (GABA) are considered to be involved in mediating aggressive behaviour; there are conflicting results on brain neurotransmitter metabolism (Matsuda et al., 2001; Sakaue et al., 2001). Table 2. shows the effects of SR on aggressive behaviour in isolated mice. Test compound reduces an aggressive behaviour in a dose-dependent manner. A similar result to open field test in between 400 mg/Kg of SR and diazepam 1 mg/Kg doses was obtained. This behaviour was reported by Valzelli in 1973 as a classic pattern for central nervous system depressor (Valzelli, 1973). Our results show an anti-aggressive behaviour in orally SR-treated mice. This result can be mediated by inhibitory effects on brain biogenic amines action or excitatory neurotransmitter release and

Groups with unequal letters differ to each other for p 0.05.

suggests the inhibitory effect of SR on the central nervous system.

water and administered orally.

**3.1 Open field activity** 

(standard anxiolytic drugs).

typical for sedative drugs.

**3.2 Aggressive behavior** 

SR as well as diazepam, a standard reference drug, increased the number of sleeping animals and prolonged the thiopental-induced sleeping time in mice.

All SR doses increase the number of sleeping animals (Table 3) compared with the control, doses of 200 and 400 mg/Kg caused sleep in all animals.

SR (400 mg/Kg) and diazepam (1 mg/Kg) prolonged thiopental induced sleep in the similar manner (Table 4).


Table 3. Effect of SR on percentage of sleeping animal.


Table 4. Effect of SR on sleeping time (min). Groups with unequal letters differ to each other for p0.05.

#### **3.4 Drug-induced convulsion**

Numerous excitatory drugs such as PTZ and PTX, can induce convulsion *via* GABA receptor antagonism, due to, the anxiolytic-like drugs (ex. diazepam) might be inhibit the druginduced convulsion via inhibition of GABA-ergic inter-neurons.

Unconventional Raw Natural Sustainable Sources for Obtaining Pharmacological

Time spent in close arms

> 198.9 ± 44.70 a

112.56 ± 30.25 a

85.4 ± 42,77 a

93.15 ± 29.15 a

70.18 ± 7.83 b

amphetamine (5 mg/Kg)-induced sleeping and its extension in time.

**3.5 Elevated plus-maze behavior** 

in open arms at any of the doses tested.

Time spent in open arms

8.02 a

7.07 a

14,35 a

8.23 a

5.56 b

unequal letters differ to each other for p0.05.

**3.6 Amphetamine-induced behavioral stereotypy** 

(Pellow, 1985).

Tested groups

Distilled water 8.2 ±

SR 50 mg/Kg 9.12 ±

SR 100 mg/Kg 9,6 ±

SR 150 mg/Kg 10.08 ±

DZP 0.5 mg/Kg 55.23 ±

Principles Potentially Active on CNS Through Catalytic, Ecologically Clean, Processes 529

Rodents usually avoid open arms and prefer enclosed arms in an elevated plus-maze. Time spent in open arms and numbers of entries into open arms are indexes of neophobic anxiety in animals. Standard anxiolytic drugs such as diazepam increase open-arm exploration, as reflected by increases in the time spent and the number of entries into the open arms

Diazepam exhibited the conventional profile of anxiolytics in the elevated plus-maze test; it increased the percentage of open arm entries and time spent in open arms (Table 7). However, SR did not significantly modify the percentage of either time spent or arm entries

> Open arm entries (%)

> > 14.81 ± 12.45 a

> > 16.23 ± 14.36 a

> > 18,68 ± 23,70 a

> > 17.59 ± 9.23 a

> > 65,17 ± 12.18 b

Table 7. Effect of SR on the elevated plus-maze test in mice ( see Table 6). Groups with

Psycho stimulants (such as, amphetamine a simpatic-mimetic amine) administration in rats increase dopamine levels in different brain zones, induce stereotyped behaviours, characterized by repetitive sniffing, biting, grooming, and head movements. The dopamine receptor antagonist or sedative drugs can be reducing its behaviour (Ralph et al. 2001). Table 8 shows behavioral stereotypy after subcutaneously administration of amphetamine, 1.5 mg/Kg (p.sc). Rats treated with SR reduced its behaviour in a dose-dependent manner compared with the water-treated group and amphetamine. The observed results reveal that colophony decreases the stimulant effect either for an antagonism of dopaminergic transmission, inhibition of dopamine releasing, blocking of its post-synaptic receptor or by an activation of some inhibitory transmission with the decrease of excitation caused by the dosis of employed amphetamine. The data suggest that the colophony has not a characteristic profile of active antidepressants. It was confirmed by the evaluation of

Open close entries (%)

> 85.18 ± 12.45 a

> 87.26 ± 10.25 a

> 81,31 ± 23,70 a

> 83.26 ± 15.15 a

> 34,83 ± 6.65 b

Number of open arm entries (counts)

> 1.80± 1.62 a

> 1.93± 0.98 a

> 1.50± 2.01 a

> 1.75± 1.29 a

19.05± 2.15 b

Number of closed arm entries (counts)

> 8.9± 2.33 a

> 8.03± 2.15 a

> 7.6± 3.60 a

> 7.98± 2.23 a

10.18± 1.98 a

To further investigate the inhibitory effect of SR on the central nervous system, drugs that can excite or block excitation in the central nervous system were used. Diazepam (4 mg/kg, po.) was highly effective in delaying the occurrence of clonic convulsion (Table 5 and 6) even in protecting animals against convulsion induced by PTX and PTZ. However the different doses of SR tested were unable to protect animals against convulsion and death, and unable to increase the latency of the clonic convulsion induced by pro-convulsivant drugs. Our findings show an ineffective action of SR to avoid the PTZ and PTX-induced convulsion. These results suggest that the sedative effects of test compound might not be mediated via GABA or glycine systems.


Table 5. Effect of SR on clonic convulsion induced by PTX. Groups with unequal letters differ to each other for p0.05.


Table 6. Effect of SR on clonic convulsion induced by PTZ. Groups with unequal letters differ to each other for p0.05.

#### **3.5 Elevated plus-maze behavior**

528 Pharmacology

To further investigate the inhibitory effect of SR on the central nervous system, drugs that can excite or block excitation in the central nervous system were used. Diazepam (4 mg/kg, po.) was highly effective in delaying the occurrence of clonic convulsion (Table 5 and 6) even in protecting animals against convulsion induced by PTX and PTZ. However the different doses of SR tested were unable to protect animals against convulsion and death, and unable to increase the latency of the clonic convulsion induced by pro-convulsivant drugs. Our findings show an ineffective action of SR to avoid the PTZ and PTX-induced convulsion. These results suggest that the sedative effects of test compound might not be

> Latency of first clonic convulsion

Distilled water 12.0 ± 1.94a 20.66 ± 4.55a 22.66 ± 5.03a 80 30

SR 100 mg/Kg. 12.0 ± 6.00a 17.0 ± 4.36a 23.0 ± 8.48a 70 20

SR 200 mg/Kg. 15.90 ± 8.60a 20.2 ± 4.76a 26.0 ± 1.41a 80 20

SR 400 mg/Kg. 16.8 ± 6.37a 21.62 ± 5.70a 24.0 ± 2.91a 80 20

DZP 4 mg/Kg 23.0 ± 3.21b 26.0 3.80b 28.1 ± 2.12b 20 0

Latency of first clonic convulsion

Distilled water 7.30 ± 1.66 a 11.66 ± 3.07 a 14.80 ± 3.83 a 60 50

SR 100 mg/Kg 6.00 ± 2.45 a 11.50 ± 1.52 a 13.17 ± 3.97 a 60 60

SR 200 mg/Kg 7.27 ± 1.35 a 13.16 ± 2.23 a 13.0 ± 3.16 a 60 40

SR 400 mg/Kg 7.90 ± 2.28 a 11.50 ± 3.45 a 13.33 ± 1.86 a 60 40

DZP4 mg/Kg 25.0 ± 2.18 b 27.1 3.26 b 29.0 ± 1.18 b 20 0

Table 6. Effect of SR on clonic convulsion induced by PTZ. Groups with unequal letters

Table 5. Effect of SR on clonic convulsion induced by PTX. Groups with unequal letters

Time of death

Time of death

Percentage of animals with clonic convulsion

Percentage of animals with clonic convulsion Percentage of death

Percentage of death

mediated via GABA or glycine systems.

Latency of first convulsion

Latency of first convulsion

Tested groups

PTX 5 mg/Kg +

PTX 5 mg/Kg +

PTX 5 mg/Kg +

PTX 5 mg/Kg +

PTX 5 mg/Kg +

Tested groups

PTZ 85 mg/Kg +

PTZ 85 mg/Kg +

PTZ 85 mg/Kg +

PTZ 85 mg/Kg +

PTZ 85 mg/Kg +

differ to each other for p0.05.

differ to each other for p0.05.

Rodents usually avoid open arms and prefer enclosed arms in an elevated plus-maze. Time spent in open arms and numbers of entries into open arms are indexes of neophobic anxiety in animals. Standard anxiolytic drugs such as diazepam increase open-arm exploration, as reflected by increases in the time spent and the number of entries into the open arms (Pellow, 1985).

Diazepam exhibited the conventional profile of anxiolytics in the elevated plus-maze test; it increased the percentage of open arm entries and time spent in open arms (Table 7). However, SR did not significantly modify the percentage of either time spent or arm entries in open arms at any of the doses tested.


Table 7. Effect of SR on the elevated plus-maze test in mice ( see Table 6). Groups with unequal letters differ to each other for p0.05.

## **3.6 Amphetamine-induced behavioral stereotypy**

Psycho stimulants (such as, amphetamine a simpatic-mimetic amine) administration in rats increase dopamine levels in different brain zones, induce stereotyped behaviours, characterized by repetitive sniffing, biting, grooming, and head movements. The dopamine receptor antagonist or sedative drugs can be reducing its behaviour (Ralph et al. 2001). Table 8 shows behavioral stereotypy after subcutaneously administration of amphetamine, 1.5 mg/Kg (p.sc). Rats treated with SR reduced its behaviour in a dose-dependent manner compared with the water-treated group and amphetamine. The observed results reveal that colophony decreases the stimulant effect either for an antagonism of dopaminergic transmission, inhibition of dopamine releasing, blocking of its post-synaptic receptor or by an activation of some inhibitory transmission with the decrease of excitation caused by the dosis of employed amphetamine. The data suggest that the colophony has not a characteristic profile of active antidepressants. It was confirmed by the evaluation of amphetamine (5 mg/Kg)-induced sleeping and its extension in time.

Unconventional Raw Natural Sustainable Sources for Obtaining Pharmacological

of the GHS or not classified (mortality> 2000 mg/kg).

25

Fig. 4. Effect of SR 2000 mg/Kg on acute oral toxicity test.

not detected any macroscopic or anatomic effect on organs.

28

31

34

weigh gain (g)

37

40

43

Principles Potentially Active on CNS Through Catalytic, Ecologically Clean, Processes 531

disappeared within 4 h. With this exception, no outward behavioral abnormalities were noted during the 2-week post-treatment period. The body weight gains were observed in the similar manner in both groups (Figure 4). Macroscopic alterations were not observed in selected organs and tissues removed (stomach, liver, kidney, brain, spleen and lungs). The administration of SR didn't cause significant toxic symptoms for what classifies in 5 category

Acute oral toxicity

Taking into consideration the structural similarity, *grosso modo,*(molecular modeling are underway and are unpublished results yet) between secondary metabolites, DHAA, related derivatives and cannabinoids, the observed pharmacological effects could be attribute to a potential antagonism with glutamate receptors and/or inhibition of noradrenalin-dopamine releasing. The acute toxicity study revealed that no toxic effects were observed, and any symptom (increase of cleaning-up behavior, exploration, decreasing of frequency of movement, etc) disappeared after 4 hrs. In the autopsy were

In the case of dehydroabietic acid (DHAA) generated by disproportionationaromatization of resinic acids the results reveal a dosis depending effect on decreasing of

SR 2000mg/Kg

Vehicle (destilled

water)


Table 8. Effect of SR on behavioural stereotypy induced by amphetamine. Groups with unequal letters differ to each other for p0.05.

#### **3.7 Amphetamine -induced sleep in mice**

Mice treated with different doses of SR exhibited similar behaviour against amphetamine induced sleep in mice compare with water-treated group (Table 9).


Table 9. Effect of SR on sleep induced by amphetamine in mice.

Although SR showed a sedative effect in evaluated tests (thiopental-induced sleep, open field activity, aggressive behaviours and amphetamine-induced behavioral stereotypy). The interaction of SR with convulsant drugs, confirms that the sedative effect of test compound might not be related via the GABA or glycine systems.

In this context, recently our group found that abietic and dehydroabietic acids might be responsible of the sedative effects of the SR (Unpublished result).

#### **3.8 Acute oral toxicity study**

The death of any animal was not observed during the study. The major adverse effects were related with CNS depression (motor impairment and sedation), but these symptoms

SR 100 mg/Kg. + amphetamine 1,5 mg/Kg. 22,71 ± 4.90b

SR 200 mg/Kg. + amphetamine 1,5 mg/Kg. 23,92 ± 5,75b

SR 400 mg/Kg. + amphetamine 1,5 mg/Kg. 20,70 ± 5,85b

Distilled water + amphetamine 1,5 mg/Kg. 31,64 ± 5,98c

Haloperidol 5 mg/Kg. + amphetamine 1,5 mg/Kg. 12,28 ± 2,56a

Mice treated with different doses of SR exhibited similar behaviour against amphetamine -

Distilled water + amphetamine 5 mg/Kg 189.90 ± 9.65

SR 100 mg/Kg. + amphetamine 5 mg/Kg 190.00 ± 11.51

SR 200 mg/Kg. + amphetamine 5 mg/Kg 195.86 ± 5.05

SR 400 mg/Kg. + amphetamine 5 mg/Kg 190.22 ± 7.07

Although SR showed a sedative effect in evaluated tests (thiopental-induced sleep, open field activity, aggressive behaviours and amphetamine-induced behavioral stereotypy). The interaction of SR with convulsant drugs, confirms that the sedative effect of test compound

In this context, recently our group found that abietic and dehydroabietic acids might be

The death of any animal was not observed during the study. The major adverse effects were related with CNS depression (motor impairment and sedation), but these symptoms

Tested groups Media ± D.S

Table 8. Effect of SR on behavioural stereotypy induced by amphetamine. Groups with

unequal letters differ to each other for p0.05.

**3.7 Amphetamine -induced sleep in mice** 

induced sleep in mice compare with water-treated group (Table 9).

Table 9. Effect of SR on sleep induced by amphetamine in mice.

responsible of the sedative effects of the SR (Unpublished result).

might not be related via the GABA or glycine systems.

**3.8 Acute oral toxicity study** 

Tested groups mean±S.E.M

Distilled water 10.45 ± 3.70a

disappeared within 4 h. With this exception, no outward behavioral abnormalities were noted during the 2-week post-treatment period. The body weight gains were observed in the similar manner in both groups (Figure 4). Macroscopic alterations were not observed in selected organs and tissues removed (stomach, liver, kidney, brain, spleen and lungs). The administration of SR didn't cause significant toxic symptoms for what classifies in 5 category of the GHS or not classified (mortality> 2000 mg/kg).

Fig. 4. Effect of SR 2000 mg/Kg on acute oral toxicity test.

Taking into consideration the structural similarity, *grosso modo,*(molecular modeling are underway and are unpublished results yet) between secondary metabolites, DHAA, related derivatives and cannabinoids, the observed pharmacological effects could be attribute to a potential antagonism with glutamate receptors and/or inhibition of noradrenalin-dopamine releasing. The acute toxicity study revealed that no toxic effects were observed, and any symptom (increase of cleaning-up behavior, exploration, decreasing of frequency of movement, etc) disappeared after 4 hrs. In the autopsy were not detected any macroscopic or anatomic effect on organs.

In the case of dehydroabietic acid (DHAA) generated by disproportionationaromatization of resinic acids the results reveal a dosis depending effect on decreasing of

Unconventional Raw Natural Sustainable Sources for Obtaining Pharmacological

*Biochem Psychopharmacol,* Raven Press, pp. 14.

pharmacological action on CNS.

mice. *J-Neural-transm*. 105(6-7), 587-99.

*Neuropsychopharmacol.* 21, 71-76.

*Neuroscience*. 21(1), 305-313.

*Ethnopharmacology.* 83, 87:94.

432, 163-166.

Method" Nº 423 Adopted 20 December 2001.

*Bioorganic & Medicinal Chemistry*. 13 5274–5282

piritic ash and obtention of dehydroabietic acid.

**5. References** 

17, 37-64.

23:183-195.

699-709.

A 991:205-215.

Principles Potentially Active on CNS Through Catalytic, Ecologically Clean, Processes 533

Carlini, E.A., Contar, J.D.P., Silva, A.R., Silveira- Filho, N.G., Frochtengarten, M.L., Bueno,

Costa, E., Greengard, P. (1975). Mechanism of action of benzodiazepines. New York: *Adv* 

*CU Patent 20060144*, (2006). Tacoronte J.E et al. Disproportionation of Cuban colophony with

*CU Patent 20060252*, (2006). Tacoronte J.E et al. Natural cannabinoid precursors with

Franich R A, Gadgil, P. D. (1983). Fungistatic effects of *Pinus radiata* needle

Fischer, W., Vander, H. (1998). Effect of clobenpropit, a centrally acting histamine H3-

Kuczenski, R., Segal, D.S. (1999). Sensitization of amphetamine-induced

Latorre A, Rigol A, Lacorte S, Barcelo D. (2003). Comparison of gas chromatography-mass

Matsuda, T., Sakaue, M., Ago, Y., Sakamoto, Y., Koyama, Y., Baba, A. (2001). Functional

OECD Guideline For Testing Of Chemical "Acute Oral Toxicity – Acute Toxic Class

Pellow, S. (1985). Validation of open: closed arm entries in an elevated plus-maze as a measure of anxiety in the rat. *Journal of Neuroscience* Method 14, 149-167. Ralph, R.J., Paulus, M.P., Fumagalli, F., Caron, M.G., Geyer, M.A. (2001). Prepulse Inhibition

Sakaue, M., Ago, Y., Murakami, C., Sowa, C., Sakamoto, Y., Koyama, Y., Baba, A., Matsuda,

Sukma, M., Chaichantipyuth, C.H., Murakami, Y., Tohda, M., Matsumoto, K., Watanabe, H.

Tulp M, Bohlin L. (2005) Rediscovery of known natural compounds: Nuisance or goldmine?

epicuticular fatty and resin acids on *Dothistroma pini*. *Physiol. Plant Pathol*.

receptors antagonist, on electroshock and pentilenetetrazol-induced seizures in

stereotyped behaviors during the acute response. *J-Pharmacol-Exp-Ther*. 288(2),

spectrometry and liquid chromatography-mass spectrometry for the determination of fatty and resin acids in paper mill process waters. *J. Chromatogr*.

Alteration of Brain Dopaminergic System in Isolated Aggressive Mice, *Jpn. J.* 

Deficits and Perseverative Motor Patterns in Dopamine Transporter Knock-Out Mice: Differential Effects of D1 and D2 Receptor Antagonists. *Journal of* 

T. (2001). Involvement of benzodiazepine binding sites in an antiaggressive effect by 5-HT1A receptor activation in isolated mice. *European Journal of Pharmacology*.

(2002). CNS inhibitory effects of barakol, a constituent of Cassia siamia Lamk*. J.* 

O.F.A. (1986). Pharmacology of lemongrass (Cymbopogon citratus Stapf). I. Effects of teas prepared from the leaves on laboratory animals. *J. Ethnopharmacol*.

exploratory activity, typical for sedative pharmaceutical compositions. It coadministration (50 mg/Kg) with thiopental, at experimental dosis, increase the number of sleeping animals (65%), a typical depressing action on CNS. The administration of DHAA, at all dosis evaluated, didn't protect animals for PTZ induced convulsions. The experimental data obtained in the Labyrinth in cross model didn't show any relevant results. In the Amphetamine-induced stereotipia model DHAA decreases the stimulant action produced by the subcutaneously administration of 1,5 mg / Kg of amphetamine, in the hypothetically pathways described above. The acute toxicity (DL50 > 5000 mg/Kg) study showed that DHAA had not any toxic effects at dosis concentrations used and could be classified in the GSH in the 5 category.

It is noteworthy that synthetic DHAA has a sedative action on CNS and could be employed as starting raw material for designing of new molecular and pharmacological entities potentially useful in the development of formulation for therapy of CNS pathologies where de sedative effect is needed.

Topological studies for determining any QSAR correlation between colophony and derivatives and cannabinoids are underway.

#### **4. Conclusion**

The oleoresin, a raw material isolated from Cuban Pinacea (gen. *Pinus, Pinus caribbaea*), constitutes a sustainable resource for developing potentially useful pro-drugs and pharmacologically active substances as resínic acids, sodium resinate (SR) and dehydroabietic acid. The analytical protocol developed (*all in one* wavelengths-retention time) for common abietanes present in Cuban colophony is simple, time-saving, ecofriendly, employing robust reversed-phase HPLC and offer the possibility to determine and quantify the main diterpenic acids (resínico acids) in the natural and modified mixture. The catalytic synthesis of dehydroabietic acid (DHAA) as a principal active pharmaceutical component from colophony for the potential treatment of neuropsiquiatric dysfunctions and generation of exogenic cannabinoid analogues has been developed under ecological conditions using piritic ash as re-usable catalyst (disproportionation-aromatization) at meso-scale with minimal environmental impact. The neuropharmacological profile (including acute oral toxicity) of SR in rodent behavioural tests was determined; SR reduced spontaneous locomotor activity and aggressive behaviour, increased the number of sleeping animals and prolonged the thiopental-induced sleeping time indicating a the sedative effect of test compound and it might not be related via the GABA or glycine systems. The SR is unable to protect animals against convulsion and death induced by pentylenetetrazole and picrotoxin. The SR (2000 mg/Kg p.o) didn't cause significant toxic symptoms in rats. This finding indicates that the SR can constitute a non conventional source of pharmacological molecular entities with central nervous system depressant activity. The synthetic DHAA has a sedative action on CNS and its acute toxicity (DL50 > 5000 mg/Kg) reveals that DHAA had not any toxic effects at dosis concentrations used and could be employed as starting design structural point for developing molecular entities and series leads with potentially remarkable pharmacological properties.
