**3. Results and discussion**

The Cuban pine oleoresin, was milked and collected by incision of the bark from mature trees *Pinus caribbaea*. After distillation the main components and mixture of colophony (resinic acids), turpentine oils and neutral fraction were separated and hydrothermally treated to minimize the amount of fatty lineal and branched acids. Colophony was analyzed, treated with stecheometrical amount of NaOH, to obtain the desired sodium resinate and submitted directly, after crystallization from a mixture water: ethanol (30:70 v/v), to neuropharmacological evaluations.

The oleoresin collected from *Pinus caribbaea* is hydrothermally treated and purified through redox-acid/base protocols described in (Cuban patents CU 20060144 & CU20060252), generating a practically pure mixture of rosin-colophony as a mixture of resinic acids (RA, FTIR- cm-1: 3426(O-H), 2931(Csp3-H y Csp2-H), 2869 (s CH2) y 2929 (as CH2), 1694(C=O), 1385-1366(δs-CH3), 1450(δas-CH3), 1150-1180 isopropyl system, zone 950-970 olefinic fragments, 830-770 trisubstituted olefins; NMR,,ppm: 6 zones observed: *0.5-0,8* methyl groups, *1.0-1.2* methyl groups, *1.3-2.0* methylenic groups, *5.0-6.0* olefinic zone exo- and endocyclic bonds, *6.8-7.3* aromatic protons, *11.9-12.5* COOH) that was used directly in the evaluation of its neuropharmacological profile. This mixture (50-52 % of abietic acid),

Unconventional Raw Natural Sustainable Sources for Obtaining Pharmacological

Principles Potentially Active on CNS Through Catalytic, Ecologically Clean, Processes 525

The fundamentals (conceptual and methodological) for the analytical technique described here rely on both the optimal combination of wavelengths for detection and the chromatographic resolution of the peaks. Abietanes have distinctive spectra that we used here, together with chromatographic separation, to distinguish and quantify the resin acids by HPLC. The spectra (220-540 nm) of the individual chromatographically separated components (Fig.1), show λ max values of 255 nm (levopimaric), 251 nm (palustric), 266 nm (abietic), and 269, 278 nm (dehydroabietic). The information related is shown in Fig. 2.

Fig. 2. Spectral chromatogram of analyzed mixture of resin acids present in Cuban colophony

The developed HPLC analysis revealed the main components of the Cuban colophony, a

HO O

CH3

H

H

H3C

HO O

H3C

CH3

H

CH3

CH3

CH3

CH3

CH3

CH3

CH3

Palustric acid Levopimaric acid

Fig. 3. Main resin acids present in the Cuban colophony. (abietic acid 40 %; dehydroabietic

CH3

Abietic acid Dehydroabietic acid

starting raw material for preparing sodium resinate (Fig. 3).

CH3

H

H

HO O

CH3

H

acid 22 %; palustric acid 18 %; levopimaric acid 18-20 %).

H3C

HO O

H3C

without further purification, was used for obtaining dehydroabietic acid (DHAA) by heterogeneous catalytic disproportionation-aromatization treatment of colophony with pyritic ash (Fe2O3/FeS/Ba2+/SiO2, 2300C, stirring, air, 95%) and the selective precipitation of the 2-aminoethanol salt of DHAA (molar ratio 1:1, 500C, 89 %) in aqueous ethanol solution. The pure DHAA was obtained after acidification (pH 4-5; 98%, TLC: Silicagel G60-254, eluent: n-hexane/ethyl acetate 7:3 v/v, 2 drops of isopropanol; chromophoric agent vainilline/H2SO4, mp. 171.5-172.30C and applied column chromatography; NMR,ppm, 1H-13C selected signals: 9,78-OH/184,32-COOH; 7,15 C-11H/C-11 124,90; 6,95 C-12H/C-12 124,20; 6,88 C-14H/C-14 127,0).

The most widely protocol used for resin acid analysis and their derivatives is gas chromatography (GC) of the methyl esters (Zinkel & Engler, 1977) with detection by flame ionization (FID) or mass spectrometry (MS). However, this method has disadvantages for us, including instability of the derivatized samples (Latorre et al., 2003), hazards of methylating reagents (potentially explosive and carcinogenic), and tedious work-up of raw biological material required.

Taking this in consideration, in our laboratory, have been developed a simple analytical methodology for resin acid analysis by high-performance liquid chromatography (HPLC) in gradient conditions. We report a simple protocol for the analysis of abietanes derivatives by reversed-phase HPLC with several advantages including: (1) no sample derivatization is required; (2) extraction and chromatographic conditions are mild, (3) all components of the HPLC mobile (acetonitrile and water) phase are volatile and therefore recovery of compounds from fractionated sample is simplified. These benefits are particularly advantageous in biological studies that require rapid analysis of abietane acid mixtures and screenings for neuroprotective bioactivity.

The results are shown in Fig. 1

Fig. 1. Abietanes (resin acids present in the Cuban colophony) HPLC analysis of methanol extract obtained from distilled oleoresin. 1= Levopimaric, 2= Palustric, 3= Abietic, 4= Dehydroabietic

without further purification, was used for obtaining dehydroabietic acid (DHAA) by heterogeneous catalytic disproportionation-aromatization treatment of colophony with pyritic ash (Fe2O3/FeS/Ba2+/SiO2, 2300C, stirring, air, 95%) and the selective precipitation of the 2-aminoethanol salt of DHAA (molar ratio 1:1, 500C, 89 %) in aqueous ethanol solution. The pure DHAA was obtained after acidification (pH 4-5; 98%, TLC: Silicagel G60-254, eluent: n-hexane/ethyl acetate 7:3 v/v, 2 drops of isopropanol; chromophoric agent vainilline/H2SO4, mp. 171.5-172.30C and applied column chromatography; NMR,ppm, 1H-13C selected signals: 9,78-OH/184,32-COOH; 7,15 C-11H/C-11 124,90; 6,95 C-12H/C-12

The most widely protocol used for resin acid analysis and their derivatives is gas chromatography (GC) of the methyl esters (Zinkel & Engler, 1977) with detection by flame ionization (FID) or mass spectrometry (MS). However, this method has disadvantages for us, including instability of the derivatized samples (Latorre et al., 2003), hazards of methylating reagents (potentially explosive and carcinogenic), and tedious work-up of raw

Taking this in consideration, in our laboratory, have been developed a simple analytical methodology for resin acid analysis by high-performance liquid chromatography (HPLC) in gradient conditions. We report a simple protocol for the analysis of abietanes derivatives by reversed-phase HPLC with several advantages including: (1) no sample derivatization is required; (2) extraction and chromatographic conditions are mild, (3) all components of the HPLC mobile (acetonitrile and water) phase are volatile and therefore recovery of compounds from fractionated sample is simplified. These benefits are particularly advantageous in biological studies that require rapid analysis of abietane acid mixtures and

Fig. 1. Abietanes (resin acids present in the Cuban colophony) HPLC analysis of methanol extract obtained from distilled oleoresin. 1= Levopimaric, 2= Palustric, 3= Abietic, 4=

124,20; 6,88 C-14H/C-14 127,0).

biological material required.

screenings for neuroprotective bioactivity.

The results are shown in Fig. 1

Dehydroabietic

The fundamentals (conceptual and methodological) for the analytical technique described here rely on both the optimal combination of wavelengths for detection and the chromatographic resolution of the peaks. Abietanes have distinctive spectra that we used here, together with chromatographic separation, to distinguish and quantify the resin acids by HPLC. The spectra (220-540 nm) of the individual chromatographically separated components (Fig.1), show λ max values of 255 nm (levopimaric), 251 nm (palustric), 266 nm (abietic), and 269, 278 nm (dehydroabietic). The information related is shown in Fig. 2.

Fig. 2. Spectral chromatogram of analyzed mixture of resin acids present in Cuban colophony

The developed HPLC analysis revealed the main components of the Cuban colophony, a starting raw material for preparing sodium resinate (Fig. 3).

Fig. 3. Main resin acids present in the Cuban colophony. (abietic acid 40 %; dehydroabietic acid 22 %; palustric acid 18 %; levopimaric acid 18-20 %).

Unconventional Raw Natural Sustainable Sources for Obtaining Pharmacological

animals and prolonged the thiopental-induced sleeping time in mice.

doses of 200 and 400 mg/Kg caused sleep in all animals.

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

induced convulsion via inhibition of GABA-ergic inter-neurons.

**3.3 Thiopental-induced sleep** 

manner (Table 4).

for p0.05.

**3.4 Drug-induced convulsion** 

Principles Potentially Active on CNS Through Catalytic, Ecologically Clean, Processes 527

Tested groups mean±S.E.M Distilled water 24.90 ± 4.15 a SR 100 mg/Kg. 17.60 ± 3.38 b SR 200 mg/Kg. 16.75 ± 2.94 b SR 400 mg/Kg. 12.90 ± 4.48 c DZP 1 mg/Kg. 10.50 ± 3.39 c Table 2. Effects of SR (100, 200 and 400 mg/kg, po.) on aggressive behaviour (biting attacks and wrestling) in isolated mice Groups with unequal letters differ to each other for p0.05.

SR as well as diazepam, a standard reference drug, increased the number of sleeping

All SR doses increase the number of sleeping animals (Table 3) compared with the control,

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

Tested groups mean±S.E.M Distilled water 2,25 ± 6.16 a SR 100 mg/Kg. 8,25 ± 8.65 b SR 200 mg/Kg. 33,75 ± 8.83 c SR 400 mg/Kg. 40,00 ± 0 d DZP 1 mg/Kg. 40,00 ± 0 d Table 4. Effect of SR on sleeping time (min). Groups with unequal letters differ to each other

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 drug-

Distilled water 12.50 SR 100 mg/Kg. 56.25 SR 200 mg/Kg. 100 SR 400 mg/Kg. 100 DZP 1 mg/Kg. 100

Tested groups Percentage of sleeping animal

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 water and administered orally.

#### **3.1 Open field activity**

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 (standard anxiolytic drugs).

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 typical for sedative drugs.


Table 1. Effects of SR (100, 200 and 400 mg/kg, po.) on spontaneous locomotor activity. Groups with unequal letters differ to each other for p 0.05.

#### **3.2 Aggressive behavior**

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


Table 2. Effects of SR (100, 200 and 400 mg/kg, po.) on aggressive behaviour (biting attacks and wrestling) in isolated mice Groups with unequal letters differ to each other for p0.05.
