**1. Introduction**

518 Pharmacology

Zheng, Y.Y.; Yang, J.; Chen, D.H. & Sun, L. (2007). Effects of the stilbene extracts from

pp.562-565.

Cajanus cajan L. on ovariectomy-induced bone loss in rats. *Yao Xue Xue Bao*, vol. 42,

Natural products have been the most successful source of drugs ever (Tulp & Bohlin, 2005). Historically, the most important natural sources have been plants. Research progressed along two mayor lines: ethnopharmacology and toxicology. These strategies have produced many valuable drugs and are likely to continue to produce hit-lead compounds. However, actually exist numerous unconventional natural sources, ecologically sustainable, of potentially medicinal compounds without research.

The development of pharmaceutical and fine chemistry in Cuba and the synthetic or natural product-oriented generation of new pharmacological and molecular entities, in the II decade of XXI century, it´s sustained in several basic conceptual and methodological principles:


Unconventional Raw Natural Sustainable Sources for Obtaining Pharmacological

**2.3 Structural elucidation** 

**at 230** 

**C)** 

**2.5 Animals** 

were used.

compounds.

**2.5.1 Pharmacological studies** 

**2.5.1.1 Acute toxicity study** 

**2.4 Synthesis of dehydroabietic acid (DHAA)** 

Principles Potentially Active on CNS Through Catalytic, Ecologically Clean, Processes 521

The structural elucidation was based on FTIR spectroscopy (spectrophotometer FT-IR Jasco, FT/IR-460 Plus, Japan, in a range of 280-7200 cm-1 with a sensibility of 0,1 cm-1) and NMR spectrometry (1H y 13C) at room temperature, using a spectrometer Brucker AC-250 MHz, at

Diazepam (DZP, Quimefa, Cuba), pentylenetetrazole (PTZ, Sigma, USA, CAS), picrotoxin (PTX, Sigma, USA, CAS), haloperidol (Esteve S.A, España), amphetamine-sulphate (Sigma, USA, CAS), were used in this study. Solvents were analytical grade and were purified by distillation before used. All drugs and their solutions were prepared immediately before use.

**2.4.1 Catalytic disproportion of colophony using piritic ash as catalyst (0,5 % (m/m)** 

mixture of 75 % ethanol/water v/v. and dried 3 h. Yield 39,3 % (overall 98,2 %)

Male albino mice (Swiss, 18–22 g) in anticonvulsant activity, elevated plus-maze, amphetamine and thiopental-induced sleep, open field activity and aggressive behaviour test and male rats (Wistar, 150–200 g) in amphetamine-induced behavioural stereotypy test

Six female rats (Wistar, 150–200 g) were used for evaluating the acute toxicity of test

All animals (Laboratory of Biological Control. CIDEM, Havana, Cuba) were housed in groups of five under standard laboratory conditions of temperature, humidity and lighting (12:12-h light/dark). Animals had free access to food and water, except during experiment.

500 grams of previously hydrothermally treated colophony is heated during 30 min (130 0C) in a glass pyrex reactor (750 mL) equipped with thermometer and stirrer. 1,0 gram of pyritic ash is added and the reaction temperature increased to 230 0C. The reaction mixture is maintained at this temperature during 3 h under intense stirring (1500 rpm). (The residual concentration of abietic acid is 0,5–0,8 %). 100 grams of disproportionated colophony in 100 mL of alcohol is filtered through SiO2-Al2O3. The solution is heated to 70 0C and then added 18 grams of 2-aminoethanol, and 250 mL of hot water (60-900C). The resulting solution is kept at 70 0C during 10 min., under gentle stirring, then the reaction mixture is extracted with iso-octane, toluene or a mixture of heptane/ciclohexanone 7/3 v/v ( 3v x 75 mL). The selective crystallization of the quaternary ammonium salt dehydroabietic acid-2 aminoethanol starts at 50 0C. The solution is cooled to 4 0C, collecting crystals which are soluble in cool 50 % ethanol (250 mL) Yield: 51,0 grams with 89,5 % of purity related to dehydroabietic acid (98,5 % overall purity). The obtained salt is dissolved 160 mL of hot ethanol and acidified with aqueous HCl (12 %, pH 4-5) and allowed to stand during 8h at room temperature. The pure dehydroabietic acid is collected, washed, re-crystallized from a

28 0C in DMSO-d6 ,using as internal reference TMS, given all the signals in ppm (δ).

 Integration of *in silico* screen to natural products or their mixtures with minimal complexity

Taking this into account, in Cuba (2008-2011) it has been attempted, starting from natural products of the forest industry and raw renewable materials ecologically sustainable, such as rosin, colophony and resinic acids isolated from endemic botanic species belonging to gen. *Pinus* (Pinaceae) the development and application of new heterogeneous catalytic procedures, optimization of design and synthesis of new pharmacological agents structurally based on sodium resinate and dehydroabietic acid (DHAA), with a great added value, as therapeutics for medical treatment of pathologies of CNS, including GABA agonist-antagonists, cannabinoid analogues and sedative molecular systems.

Cuban resin acids, an ecological sustainable natural product, derived form Cuban forestry industry, have been shown to have broad and highly active biological properties, potent microbiocidal and fungicidal actions and potential neuroprotective effects on central nervous system (CNS). In this communication, we studied this novel ecologically sustainable source of potentially therapeutic compounds using, as starting raw material, resins from endemic Cuban *Pinus* specie and show their effect on central nervous system in rodents.

## **2. Materials and methods**

#### **2.1 Chemicals and drugs**

#### **2.1.1 Oleoresin, colophony and starting resin acids**

The Cuban oleoresin, was milked and collected by incision of the bark from mature trees (*Pinus caribbaea* 7-13 years) planted in Viñales Forestry Station, 168 Km West of Havana, serpentine soil) located in the Western zone of the Cuban archipelago. It was submitted to distillation as reported previously (Franich & Gadgil, 1983). The main components, a mixture of colophony (resin acids), turpentine oils and neutral fraction were separated. The acid fraction was submitted to identification of abietane acids (mixture of abietic, dehydroabietic, levopimaric & pimaric acid) and directly used in pharmacological tests.

For the preparation of extracts, approximately 500 mg of colophony were grinding in an agate mortar into 2-mm pieces, extracted with 4 ml methanol, filtered through glass wool, and the extracts stored at −10°C. Extracts were filtered through 0.45-μm Teflon syringe filters prior to HPLC and used in the next steep for obtaining the sodium salt.

The sodium salt (sodium resinate-SR) was prepared as reported in: CU/2006-0144 patent. Crystallized from a mixture of water: ethanol (8: 2 v/v)

#### **2.2 Chromatography conditions**

Analytical HPLC-PDA (Kanuer Smart Line-2005): Column: LichroCART 250 x 0.44 cm RP-18, 5µm particle size (Lichropher 100); mobile phase: acetonitrile-water in gradient conditions (40% to 100%) during 45 min, held at 100% for further 2 min; temperature: 25 0C; flow rate: 1mL/min; sample injection 10 µL; detection: 200 nm to 505 nm. Data were analyzed with ChromGate 3.1 (Germany) for LC 3D software.

#### **2.3 Structural elucidation**

520 Pharmacology

Integration of *in silico* screen to natural products or their mixtures with minimal

Taking this into account, in Cuba (2008-2011) it has been attempted, starting from natural products of the forest industry and raw renewable materials ecologically sustainable, such as rosin, colophony and resinic acids isolated from endemic botanic species belonging to gen. *Pinus* (Pinaceae) the development and application of new heterogeneous catalytic procedures, optimization of design and synthesis of new pharmacological agents structurally based on sodium resinate and dehydroabietic acid (DHAA), with a great added value, as therapeutics for medical treatment of pathologies of CNS, including GABA

Cuban resin acids, an ecological sustainable natural product, derived form Cuban forestry industry, have been shown to have broad and highly active biological properties, potent microbiocidal and fungicidal actions and potential neuroprotective effects on central nervous system (CNS). In this communication, we studied this novel ecologically sustainable source of potentially therapeutic compounds using, as starting raw material, resins from endemic Cuban *Pinus* specie and show their effect on central nervous system in

The Cuban oleoresin, was milked and collected by incision of the bark from mature trees (*Pinus caribbaea* 7-13 years) planted in Viñales Forestry Station, 168 Km West of Havana, serpentine soil) located in the Western zone of the Cuban archipelago. It was submitted to distillation as reported previously (Franich & Gadgil, 1983). The main components, a mixture of colophony (resin acids), turpentine oils and neutral fraction were separated. The acid fraction was submitted to identification of abietane acids (mixture of abietic, dehydroabietic, levopimaric & pimaric acid) and directly used in pharmacological tests.

For the preparation of extracts, approximately 500 mg of colophony were grinding in an agate mortar into 2-mm pieces, extracted with 4 ml methanol, filtered through glass wool, and the extracts stored at −10°C. Extracts were filtered through 0.45-μm Teflon syringe

The sodium salt (sodium resinate-SR) was prepared as reported in: CU/2006-0144 patent.

Analytical HPLC-PDA (Kanuer Smart Line-2005): Column: LichroCART 250 x 0.44 cm RP-18, 5µm particle size (Lichropher 100); mobile phase: acetonitrile-water in gradient conditions (40% to 100%) during 45 min, held at 100% for further 2 min; temperature: 25 0C; flow rate: 1mL/min; sample injection 10 µL; detection: 200 nm to 505 nm. Data were

filters prior to HPLC and used in the next steep for obtaining the sodium salt.

agonist-antagonists, cannabinoid analogues and sedative molecular systems.

complexity

rodents.

**2. Materials and methods 2.1 Chemicals and drugs** 

**2.1.1 Oleoresin, colophony and starting resin acids** 

Crystallized from a mixture of water: ethanol (8: 2 v/v)

analyzed with ChromGate 3.1 (Germany) for LC 3D software.

**2.2 Chromatography conditions** 

The structural elucidation was based on FTIR spectroscopy (spectrophotometer FT-IR Jasco, FT/IR-460 Plus, Japan, in a range of 280-7200 cm-1 with a sensibility of 0,1 cm-1) and NMR spectrometry (1H y 13C) at room temperature, using a spectrometer Brucker AC-250 MHz, at 28 0C in DMSO-d6 ,using as internal reference TMS, given all the signals in ppm (δ).

Diazepam (DZP, Quimefa, Cuba), pentylenetetrazole (PTZ, Sigma, USA, CAS), picrotoxin (PTX, Sigma, USA, CAS), haloperidol (Esteve S.A, España), amphetamine-sulphate (Sigma, USA, CAS), were used in this study. Solvents were analytical grade and were purified by distillation before used. All drugs and their solutions were prepared immediately before use.

#### **2.4 Synthesis of dehydroabietic acid (DHAA)**

#### **2.4.1 Catalytic disproportion of colophony using piritic ash as catalyst (0,5 % (m/m) at 230 C)**

500 grams of previously hydrothermally treated colophony is heated during 30 min (130 0C) in a glass pyrex reactor (750 mL) equipped with thermometer and stirrer. 1,0 gram of pyritic ash is added and the reaction temperature increased to 230 0C. The reaction mixture is maintained at this temperature during 3 h under intense stirring (1500 rpm). (The residual concentration of abietic acid is 0,5–0,8 %). 100 grams of disproportionated colophony in 100 mL of alcohol is filtered through SiO2-Al2O3. The solution is heated to 70 0C and then added 18 grams of 2-aminoethanol, and 250 mL of hot water (60-900C). The resulting solution is kept at 70 0C during 10 min., under gentle stirring, then the reaction mixture is extracted with iso-octane, toluene or a mixture of heptane/ciclohexanone 7/3 v/v ( 3v x 75 mL). The selective crystallization of the quaternary ammonium salt dehydroabietic acid-2 aminoethanol starts at 50 0C. The solution is cooled to 4 0C, collecting crystals which are soluble in cool 50 % ethanol (250 mL) Yield: 51,0 grams with 89,5 % of purity related to dehydroabietic acid (98,5 % overall purity). The obtained salt is dissolved 160 mL of hot ethanol and acidified with aqueous HCl (12 %, pH 4-5) and allowed to stand during 8h at room temperature. The pure dehydroabietic acid is collected, washed, re-crystallized from a mixture of 75 % ethanol/water v/v. and dried 3 h. Yield 39,3 % (overall 98,2 %)

#### **2.5 Animals**

#### **2.5.1 Pharmacological studies**

Male albino mice (Swiss, 18–22 g) in anticonvulsant activity, elevated plus-maze, amphetamine and thiopental-induced sleep, open field activity and aggressive behaviour test and male rats (Wistar, 150–200 g) in amphetamine-induced behavioural stereotypy test were used.

#### **2.5.1.1 Acute toxicity study**

Six female rats (Wistar, 150–200 g) were used for evaluating the acute toxicity of test compounds.

All animals (Laboratory of Biological Control. CIDEM, Havana, Cuba) were housed in groups of five under standard laboratory conditions of temperature, humidity and lighting (12:12-h light/dark). Animals had free access to food and water, except during experiment.

Unconventional Raw Natural Sustainable Sources for Obtaining Pharmacological

percentage of arm entries in open arms (Sukma et al., 2002).

**2.5.2.6 Amphetamine-induced behavioral stereotypy** 

**2.5.2.7 Amphetamine -induced sleep in mice** 

of righting reflex were recorded. **2.5.2.8 Acute oral toxicity study** 

**3. Results and discussion** 

v/v), to neuropharmacological evaluations.

**2.5.2.9 Statistics** 

Principles Potentially Active on CNS Through Catalytic, Ecologically Clean, Processes 523

at the centre of the maze with its head facing an open arm and allowed to explore the maze for 5 min. Entry into an arm was defined as placement of all four paws into an arm and were recorded: number of entries into each type of arm, the percentage of time spent and the

Amphetamine (1.5 mg/Kg) was injected subcutaneously 30 min after administration of vehicle or test compound in rats and the animals were collocated in individually cage to

Mice were divided in four groups of ten each. The animals were pre-treated with SR 30 min before the administration of amphetamine 5 mg/Kg (p sc). An animal was placed on its back on a warmed (35 ºC) pad and the number of sleeping animals and the duration of loss

A single dose of SR (2000 mg/kg) or distilled water was administered orally (10 mL/Kg) in equal number female (n=3) animals; and rats were returned to an *ad libitum* diet immediately after dosing. All animals were monitored continuously for 12 h after dosing for signs of toxicosis and daily for changes additional behavioural or clinical signs. The animal weights were recorded weekly. Rats were euthanized on day 14 by ether inhalation, and selected organs removed and examined macroscopically for toxicant-induced changes (OECD, 2001).

Drug effects were assessed by single factor analysis of variance followed by the Student-

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

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),

/Newman-/Keuls post-hoc test. The level of significance was set at p<0.05.

recorder the behavioral stereotypy each 5 min during 1 h. (Kuczenski et al., 1999).

They were deprived of food but not water 6 h before the drug administration and each group consisted of ten animals. All experiments were carried out between 8:00 am and 11:00 am in accordance with the Institutional Animal Ethical Committee approved the study and animal care was in conformity with Canadian Council for Animal Care guidelines

SR was administrated in three doses levels (100, 200 y 400 mg/Kg) in all experiment except the elevated plus-maze behaviour test (50, 100 y 150 mg/Kg). The volume of injection in mouse was 0.4 ml/20 g and in rat was 1 ml/100 g. The SR was dissolved in distilled water and administered orally.
