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BOLETIN LATINOAMERICANO Y DEL CARIBE DE

PLANTAS MEDICINALES Y AROMATICAS Desde el Río Grande a la Patagonia

Incluyendo el Caribe de habla española, inglesa y francesa

AUSPICIADO POR SOCIEDAD LATINOAMERICANA DE FITOQUÍMICA

Periodicidad cada dos meses - Editado en Santiago de Chile

Volumen 3 Nº 5 Páginas 81 – 95 Septiembre de 2004 Editado sólo para distribución por vía electrónica ISSN 0717 7917 – Registrado en LATINDEX

Editores

José L. Martínez (Santiago, Chile) Jorge Rodríguez (La Habana, Cuba)

Supervisora de Edición Patricia Landázuri (Armenia, Colombia)

Co-editores Arnaldo Bandoni (Buenos Aires, Argentina)

Maria Engracia Medina (Managua, Nicaragua) Francisco Morón (La Habana, Cuba)

Patrick Moyna (Montevideo, Uruguay) Consejo Editorial

Christian Agyare (Ghana) – Jorge Alonso (Argentina) – Elsa Anselmi (España) Pastor Arenas (Argentina) – Elizabeth Barrera (Chile) – Henry Yesid Bernal (Colombia)

Armando Cáceres (Guatemala) – Bruce Cassels (Chile) – Geoffrey Cordell (USA) Marco Dehesa (Ecuador) – Carla Delporte (Chile) – Luis Doreste (Venezuela)

Angela Duque Villegas (Colombia) – Norman R. Farnsworth (USA) – Mildred García (Costa Rica) Gabino Garrido (Cuba) – Martha Gatusso (Argentina) – Mahabir Gupta (Panamá) Alberto Hernández (Cuba) – Peter Houghton (Inglaterra) – Ana Ladio (Argentina)

Ingrid Loayza (Bolivia) – Olga Lock (Perú) – Vicente Martínez (Guatemala) Ernesto Medina (Nicaragua) – Pedro Melillo de Magalhaes (Brasil) – Jordi Molgó (Francia)

Miguel Morales (Chile) – John Ojewole (Sudáfrica) – Mahendra Rai (India) Rosalía Ramírez (México) – Elsa Rengifo (Perú) – Alicia Rodríguez (Cuba)

Carles Roersch (Rep. Dom.) – Marcela Samarotto (Chile) – Aurelio San Martin (Chile) José San Martin (Chile) – Nikolai Sharapin (Brasil) – Marco Schwartz (Chile)

Djaja D. Soejarto (USA) – Mauricio Venegas (Chile) – Carlos Vicente (Argentina) Roger Villalobos (Costa Rica) – Rita Zeichen (Argentina) – Gustavo Zúñiga (Chile)

Presidente de la Sociedad Latinoamericana de Fitoquímica

(2002 – 2005) Dra. Virginia Martino (Argentina)

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INDICE

Página Objetivos del Boletín 82 Notas del Editor 83 Editorial 83 Artículos Dr. W.D. Ratnasoorita et al., – Sri Lanka 84 Dr. J.O.A. Ojewole et al., – Sudafrica 88 Correspondencia 93

OBJETIVOS DEL BOLETÍN

Estimular a los grupos de trabajo existentes en Latinoamérica, sean investigadores, productores, funcionarios o simplemente interesados en las

plantas medicinales y aromáticas, poniendo a su disposición este Boletín para la difusión y la divulgación de sus investigaciones y de las actividades que en

general desarrollen en torno a plantas. Ser una herramienta de difusión para la Sociedad Latinoamericana de Fitoquímica

principalmente y de otras sociedades y agrupaciones que se sientan representados por este Boletín.

Constituir un nexo entre los profesionales de habla hispana, francesa, portuguesa

e inglesa de la región, relacionados con el tema central del Boletín.

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NOTAS DEL EDITOR Estamos otra vez con Uds. Ahora con varias novedades. Por un lado creo que ya lo he hecho de dar las gracias a los nuevos miembros del Comité Editorial: Peter Houghton (Inglaterra), Vicente Martínez (Guatemala), Ingrid Loayza (Bolivia), Rosalía Ramírez (México), John Ojewole (Sudáfrica), Olga Lock (Perú) y Djaja Doel Soejarto (USA). Desde el 31 de agosto BLACPMA ha sido incluido en LATINDEX. Lo cual nos llena de orgullo. También desde días recientes he sido invitado a formar parte del Comité Editorial de: AFRICAN JOURNAL OF TRADITIONAL, COMPLEMENTARY AND ALTERNATIVE MEDICINES Deseo agradecer también al Dr. Guy Mazars Presidente de la Sociedad Europea de Etnofarmacología y al Dr. José Maria Tavares de Andrade de la misma Sociedad en Francia, por haber incorporado a BLACPMA en la página oficial de esta sociedad. Para acceder: http://ethnopharma.free.fr/index.html, Luego vayan a "links", luego a "journals" y han llegado. Como se abran dado cuenta hemos iniciado la salida de un suplemento en los meses en que no sale el Boletín, ese suplemento llevara mas que nada los avisos de Curso y Congresos, el listado de quienes reciben el Boletín, los enlaces con paginas de interés. En el BLACPMA solo irán las notas del editor, Editorial, Comentarios que se reciben de los lectores y los artículos. La idea es alivianar este Volumen. Le evaluación de artículos científicos siempre es dado a dos referee que de acuerdo a la temática pueden resultar ser cercanos conocedores del tema, pero en esta oportunidad en la editorial abordaremos la evaluación en una primera parte de varias que irán saliendo a lo largo de los diferentes números de BLACPMA.

EDITORIAL1

ESPECIES NATIVAS DE CHILE

Hoy se usan masivamente un conjunto de productos derivados de las especies medicinales, que aprovechan las propiedades terapéuticas que poseen estas especies. Para dar a conocer las propiedades de cada una de ellas, organismos 1 Preparado por Marcela Samarotto, Ingeniero Agrónomo, encargada de Plantas Medicinales de la Fundación para la Innovación Agraria (FIA), E-mail: msamarot@fia.gob.cl

especializados (de cada país o internacionales) elaboran monografías, que son fichas descriptivas de las especies de plantas medicinales, las cuales establecen estándares botánicos, de composición y de calidad para las distintas especies de uso medicinal y sirven de referencia para orientar su utilización. Dada la importancia de las regulaciones establecidas para el uso de los productos medicinales y su repercusión en la comercialización de los mismos, se plantea la necesidad de desarrollar monografías de plantas medicinales nativas de Chile, con el objeto de uniformar y estandarizar las características de cada especie. Esto significará que estas plantas cuenten con el respaldo de investigaciones serias, con estudios sistemáticos de sus propiedades farmacéuticas, farmacológicas, toxicológicas y clínicas, con el fin de determinar su calidad y su eficacia, así como la inocuidad de su uso. Las monografías constituyen una herramienta para potenciar la comercialización de estas especies y su inserción en distintos mercados dependerá de ellas, pues los compradores exigen información sobre la base de estas pautas referenciales. Por esta razón, se necesita de un trabajo planificado y que involucre a equipos multidisciplinarios, el cual permitirá intensificar la elaboración de monografías e impulsar así una mayor comercialización de productos originarios de Chile. Es bajo este marco, que la Fundación para la Innovación Agraria (FIA) perteneciente al Ministerio de Agricultura de Chile, en su esfuerzo por generar espacios de discusión tendientes a favorecer la articulación y coordinación de los sectores públicos, académicos y privados en materia de innovación agraria, abordó el tema, y realizó el Primer Encuentro de Investigadores de Especies Medicinales Nativas de Chile. El objetivo de éste fue intercambiar información y conocimientos sobre las Especies Medicinales Nativas dando a conocer ejemplos de investigaciones realizadas en torno a ellas y donde se planteó realizar monografías de especies medicinales nativas. En esta línea, se analizaron en conjunto con los investigadores asistentes las especies nativas posibles de monografiar junto a los encargados de cada especie. Toda esta actividad fue realizada con la colaboración de RIPROFITO-Chile y CYTED. El modelo a usar es el de monografías referenciales, usado por el proyecto PROMFI del CYTED, que aportan información sobre calidad, seguridad y eficacia de las plantas seleccionadas, y hoy se trabaja en ello.

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ARTICULOS

DIURETIC ACTIVITY OF HOT FLOWER INFUSION OF NYCTANTHES ARBO-TRISTIS IN RATS

W. D. RATNASOORIYA2

AND J. R. A. C. JAYAKODY3

RECEIVED. 1 OCTOBER 2003

ACCEPTED: 6 FEBRUARY 2004 Summary: The objective of this study was to evaluate the diuretic potential of Nyctanthus arbo-tristis Linn. (Oleaceae) flowers using a hot water infusion in rats. Different concentrations of the infusion (3.7, 7.5, 12.5, or 18.7 mg/ kg ), or vehicle, or furosemide (the reference drug at 13 mg/kg), was orally administered to hydrated rats. Their cumulative urine output was monitored at hourly intervals for 6 hours. The flower infusion induced a significant (P<0.05) diuresis in a dose-related manner. The onset of this diuretic action was extremely rapid (within 1 hr) and lasted throughout the study period. Further, the flower infusion caused a significant impairment (P<0.05) of the urinary Na+ and K+ levels and caused slight but significant (P<0.05) alkalisation. However, it had no significant (P>0.05) effect on urine specific gravity or Na+/K+ ratio of urine. Collectively, these data suggest that the infusion induces diuresis by basically acting as a potassium sparing diuretic. It is concluded that these flowers have a promising potential as a safe, effective and cheap diuretic. Key words: Nyctanthes arbo-tristis, diuresis, potassium sparing diuretic, urine output RESUMEN4: El objetivo de este estudio fue evaluar el potencial diuretico de las flores de Nyctanthus arbo-tristis Linn. (Oleaceae), usando una infusión de agua caliente en ratas. Diferentes concentraciones de la infusión (3.7, 7.5, 12.5, or 18.7 mg/ kg) o de vehículo, o furosemida (la droga de referencia 13 mg/kg) fueron administradas oralmente a ratas hidratadas. Se monitoreó el volumen de orina en intervalos de 6 horas. La infusión de flores indujo una diuresis estadisticamente significativa (P< 0.05) y en forma dosis dependiente. El inicio de la acción diurética fue extremadamente rápido (dentro de 1 hora) y permaneció a lo largo de todo el periodo de estudio. Aun más, la infusión de flores provocó una significativa alteración (P<0.05) de los niveles 2 Corresponding author: Prof. W.D.Ratnasooriya, E-mail address: wdrratna@webmail.cmb.ac.lk 3Department of Zoology, University of Colombo, Colombo 03, Sri Lanka. 4 Abtract traducing to Dr. Miguel Morales, University of Chile, E-mail: mmorales@uchile.cl

urinarios de Na+ y K+ y causó una significativa (P<0.05) alcalinización. Sin embargo, no tuvo un efecto significativo en la cantidad de orina o en la relación Na+/K+ de ésta. Analizados de modo conjunto, estos datos sugieren que la infusión induce una diurésis actuando básicamente como un diurético del tipo ahorrador de potasio. Se concluye que estas flores tienen un promisorio potencial como diurético efectivo, seguro y barato. Palabras claves: Nyctanthes arbo-tristis, diuresis, diurético ahorrador de potasio, volumen urinario.

INTRODUCTION Nyctanthes arbo-tristis Linn. (Family Oleacea), night flowering jasmine in English, Sepalika in Sinhala, is a small tree which is introduced to Sri Lanka (1), but which is now commonly found in Buddhist temples and home gardens almost throughout the country. The tree flowers at night all year round and the flowers start to fall in large numbers from mid night on. These flowers can be collected, if required, with no payment. In the traditional and folk medicine of Sri Lanka the flowers are claimed to have a diuretic action (2), which has not been so far tested by scientifically controlled investigations. Phytochemically, N. arbo-tristis flowers are reported to contain appreciable amounts of flavonoids (Ratnasooriya et al, unpublished data) and an essential oil similar to that of jasmine (3). The presence of these classes of compounds suggested that this flower may have diuretic potential (4). Further, it is now known that many of the medicinal claims for plants are indeed valid (5). This prompted us to investigate the diuretic potential of N. arbo-tristis flowers. If the claimed diuretic potential is established, then it could be used as an effective and cheap herbal diuretic in Sri Lanka, as in Sri Lanka, about 35% of the population are mainly dependent on Ayurvedic and traditional systems for their health care (6).

MATERIALS AND METHODS Animals: Healthy adult cross bred male albino rats (weighing 200-250 g ) from our colony were used. They were housed in standard environmental conditions (temperature: 28-31°C; photoperiod: approximately 12 hrs natural light per day; relative humidity: 50-55%) with free access to pelleted food (Master Feed Ltd, Colombo, Sri Lanka) and water. Collection of flowers: Fresh flowers from a mature N. arbo-tristis tree which were fallen on the ground, were collected in the early morning (5.00-6.00 h) from a home garden at Hunupitiya, Colombo 02, Sri Lanka, between September and December 2002. The identification and authentication was done by Prof. R.N. De Fonseka, Department of Botany,

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University of Colombo. A voucher specimen (WDR/Sepalika 1) is deposited at the museum of the Department of Zoology, University of Colombo. Preparation of infusion: The fresh flowers were oven dried (60°C) for 24 h and powdered. 4.5 g of this material was soaked in 24 mL boiling distilled water (DW) for 30 minutes. The dark brown infusion (I) was then filtered through a muslin cloth (yield: 25.7% w/v), and used directly for experimental work at doses of 3.7, 7.5, 12.5 or 18.7 mg / kg in 1 mL. These doses are identical to what we have used previously in the evaluation of sedative potential of the flower infusion (Ratnasooriya et al, unpublished data, submitted to Pharmaceutical Biol.). Evaluation of diuretic activity: The evaluation was done as described previously (7). 43 rats were deprived of water but not food for 18 hrs. Their bladders were emptied by gentle compression of the pelvic area and by pulling their tails. Each rat was then orally administered with 15 mL of isotonic saline (NaCl 0.9% w/v) to impose a uniform liquid load. The rats were assigned to 6 groups and treated orally in the following manner: 1(n=8) 1 mL of DW, 2(n= 8) 3.7 mg/ kg of I, 3 (n=8) 7.5 mg/ kg of I, 4 (n= 8) 12.5 mg/ kg of I, 5 (n=5) 18.7 mg/ kg of I and 6 ( n= 6) 13 mg /kg of furosemide, the reference drug. Each of the rats was individually placed in a metabolic cage and the cumulative urine output determined at hourly intervals, for 6 hrs. The colour of urine was noted. The pH of urine was determined using a pHmeter (Toa Electronics Ltd., Tokyo, Japan). The specific gravity, presence or absence of glucose and proteins were determined using Combistix® reagent strips (Bayer Diagnostics Manufacturing Ltd., Bridgenl, UK). Urinary Na+ and K+ levels were determined flame photometrically (Compact Atomic Absorption Spectrometer, GFC Scientific Equipment Pvt. Ltd., Sydney, Australia). Statistical Analysis: Data are given as mean ±SEM. Statistical comparisons were made using Mann-Whitney U- test. Significance was set at P ≤ 0.05.

RESULTS As shown in Table1, 3.7 (by 61%), 7.5 (by 57%) and 12.5 (by 66%) mg/kg doses of I significantly (P<0.05) and markedly increased the cumulative urine output. Furosemide, the reference diuretic, also increased the total urine output significantly (P<0.05) (by 46%). The highest dose of I failed to show significant (P>0.05) diuretic effects. The diuretic effects of the infusion exhibited a curvilinear dose-response relationship (r2=0.91, P<0.05). EC50 value for the diuretic effect was 1.14 mg/kg. The onset of the I-induced diuresis (Figure 1) was extremely rapid (in the first hour). The peak diuresis was also evident in

the first hour, as with furosemide. However, at the peak diuresis furosemide increased diuresis by 262%, while the 12.5 mg/kg dose of the I increased diuresis by 441%. The diuretic effects of the I persisted up to the termination of the experiment.

0

1

2

3

4

5

6

7

8

1 2 3 4 5

Time (Hours)

Urin

e ou

tput

(ml)

Control

3.7mg/kg

7.5mg/kg

12.5mg/kg

18.7mg/kg

Furosemide

Figure: Time course of diuresis in rats treatment with (3.7, 7.5, 12.5 or 18.7 mg/kg) different doses of infusion of Nyctanthes arbo-tristis flowers, vehicle and reference drug furosemide. Each point represents the mean of 6-8 rats and vertical bars indicates SEM. *P<0.05

Dose Total urine output

(mL)

Control

3.7 mg/ kg

7.5 mg/ kg

12.5mg/kg

18.7mg/ kg

Furosemide

5.6 ±0.45

9.0* ±1.4

8.8* ±1.1

9.3* ±1.3

6.6 ±0.8

8.2* ±0.9

Table 1: Cumulative urine output in rats over 5 hour period following oral administration of infusion of Nyctanthes arbo-tristis flowers. (mean ± SEM). *P<0.05 as compared with control (Mann-Whitney, U- test)

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As shown in Table 2, 12.5 mg/kg dose of I significantly (P<0.05) decreased the urinary output of both Na+ (by 64%) and K+ (by 60%). However, Na+/K+ ratio remained unaltered. The pH of the urine was slightly (by 17%) but significantly (P<0.05) increased by I, but not its specific gravity. Further, the colour of the urine of the treated rats appeared to be similar to that of control and had no glucose or proteins at detectable level in it.

DISCUSSION The results show, for the first time that, N. arbo-tristis flowers can function as an orally active moderately strong diuretic agent. The onset of the diuretic action was extremely rapid, almost similar to that of strong loop diuretics (within 1h), indicating quick absorption from the gastrointestinal tract. Interestingly, the peak activity was also evident at the first hour and the diuretic action lasted throughout the study period (6h), suggesting a slow clearance. The diuretic action of N. arbo-tristis showed a curvilinear dose-response relationship. The existence of a dose-related activity suggest that the observed effect was intrinsic and causal, and may not have been result from a non-specific action, such as high salt content of the flower, as reported for some Thai indigenous medicinal plants (8). N. arbo-tristis flowers have extremely low concentrations of electrolytes (3). Some herbal drugs induce diuresis by stimulating thirst and thereby enhancing fluid intake (9). Such a mode of action is unlikely here as rats had no access to water after the administration of I. The N. arbo-tristis flower-induced urinary output was not accompanied by a corresponding increase in Na+, K+, Na+/K+ ratio or the specific gravity. Collectively, these observations suggest that the I is not acting as an osmotic diuretic (10). Osmotic diuretics are usually given intravenously and are pharmacologically inert (11,12). In contrast, this flower is orally active and contains flavonoids, an essential oil, anthocyanines and modified diterpenoids, which have other biological effects (3). These evidences provide additional support in favour of the previous notion. ADH plays a vital role in the regulation of urinary output (11,12). A possibility exists that the flower I may stimulate diuresis by inhibiting ADH release, or its action on the uriniferous tubules. However, such a mechanism seems unlikely to be operative, as there was no change in the specific gravity of urine. Inhibition of ADH action causes polyurea with low osmolality (12). N. arbo-tristis flower I could produce diuresis by stimulating the release of endogenous natriuretic peptides, which promote sodium and water secretion (11,12). Such a mode of action is also unlikely, as there was no increase in urinary Na+ level although the urinary volume was raised. The flower I did not promote an increase in natriuresis and kaleuresis with its diuretic action. Therefore the

flower is not acting as a loop diuretic (11,12). Loop diuretics are the most powerful of all diuretics and they inhibit the Na+/K+/2Cl- co-transporter system of the ascending limb of Henle' s loop (11,12). The flower I is not acting as thiazides and related diuretics, as these act by inhibiting the Na+/Cl- co-transporter in the distal convoluted tubule and increase urinary Na+ and K+ loss (11,12). However, in this study there was profound reduction in urinary Na+

and K+ levels. PARAMETER CONTROL

GROUP TREATED GROUP

pH 7.06 ± 0.31

7.18* ± 0.2

Specific gravity

1.013 ± 0.0 1.011*± 0.0

Na+ (ppm)

10336.3 ± 1539.7 3697.5*± 258.7

K+ (ppm)

4490.6 ± 844.6

1737.7*± 232.2

Na+/K+ ratio

2.5 ± 0.3

2.33 ± 0.2

Glucose (mg/dL)

negative

negative

Protein (mg/dL)

negative negative

Table2: Effects of orally administrated infusion of Nyctanthes arbo-tristis flowers. 12.5 mg/kg on some urine parameters (up to 5h) of rats (mean±SEM). *P<0.05 compared to control (Mann Whitney, U-test, T-test). On the other hand, the reduction of urinary K+ level accompanied with an increased urinary output strongly suggest that the flower I is acting as a potassium-sparing diuretic. Clinically used potassium-sparing diuretics on their own are weak diuretics and act in the collecting tubules by blocking the Na+

channels (11,12). They also impair Na+/H+ exchange mechanisms, resulting in slight alkalization of urine (12). Although an alkalization of urine was evident in this study, supporting a potassium-sparing diuretic mechanism of the flower I, there was no increase in Na+ excretion. All the therapeutically used diuretics are known to increase Na+ excretion (11,12). In this aspect it is noteworthy that recently we showed another plant claimed as a diuretic in traditional medicine in Sri Lanka, Anisomeles indicia (Lumiacae), has a powerful diuretic action which was mediated by an identical mechanism (13). This K+ retention ability of N. arbo-tristis flowers is an important feature as it indicates the potential of this extract as an antihypertensive, for a negative correlation exists between total body potassium and blood pressure in hypertensive patients (14). N.

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arbo-tristis flowers are shown to have an encouraging safety profile even with sub-chronic administration (in terms of overt clinical signs of toxicity, serum GOT, GPT, alkaline phosphatase, creatinine, urea, total protein levels). These flowers have no effects on blood tryglycerides and glucose levels or on male libido and fertility (Ratnasooriya et al., unpublished data). On the other hand, an elevated tryglyceride concentration in blood, hyperglycemia, and impotence are common side effects of several clinically used diuretics (11). In conclusion, this study provides the first scientific evidence in support of the claimed diuretic potential of N. arbo-tristis flowers. It also shows that the diuresis is mediated via a potassium-sparing mechanism.

REFERENCES 1. Jayaweera DHA. (1981) Medicinal plants. National

Science Council of Sri Lanka, Colombo, Sri Lanka 151 pp.

2. Dahanayaka DDS. (1994) Osuturu Visituru Ed. Weragoda PB. Department of Ayurveda, Colombo, Sri Lanka. 297-300 pp.

3. Sastvi SBN. (1962) The wealth of India: A Dictionary of Indian Raw Materials and Industrial Products. Council of Scientific and Industrial Research. New Delhi, India. 69 pp.

4. Stanic G, Samarzija I. (1993) Diuretic activity of Satureja montana. Sub sp: Montana extracts and oil in rats. Phytotherapy Res. 7: 363-366.

5. Farnsworth NR. (1999) Screening plants for new medicines. pp. 83-97. Biodiversity Ed. Wilson EO. National Academy Press, Washington USA

6. Mahindapala R.(2000) Sri Lanka,s medicinal plants. Loris 22: 32-36.

7. Ediriweera ERHSS, Ratnasooriya WD. (2002) Antidiuretic effects of Scoparia dulcis in rats. J. Trop. Med.Plants 3: 55-58.

8. Sripanidkulchai B, Wongpanich V, Laupattarakasem P, Suwansaksri J, Jirakulsomchok D. (2001) Diuretic effects of sedative Thai indigenous medicinal plants in rats. J.Ethnopharmacol. 75: 185-190.

9. Neuman M.(2002) Metabolic effects and drug interactions provoked by certain vegetables: grapefruit, St. John,s wort and garlic. Press Med. 31: 1416-1422.

10. Tanira MOM. Shah AH, Mohsin A, Ageel AM, Qureshi S. (1996) Pharmacological and toxicological investigations on Foeniculum vulgare dried fruit extract in experimental animals. Phytotherapy Res. 10: 33-36.

11. BNF (2001) British National Formulary, British Medical Association and Royal Pharmaceutical Society of Great Britain, London 63-69 pp.

12. Rang HP, Dale MM, Ritter JM.(1995) Pharmacology. Churchill Livingstone, London. 367-384 pp.

13. Dharmasiri MG, Ratnasooriya WD. Thabrew MI. (2003) Diuretic activity of leaf and stem decoctions of Anisomeles indica. J.Trop. Med. Plants (in press).

14. Siana A, Strazzullo P, Russo L, Guglielmi S, Iacoviello L, Ferrara LA, Mancini M.(1987) Controlled trial of long term oral potassium supplements in patients with mild hypertension. Br. Med. J. 294: 1453-1456.

FRASES

No te afanes en las cosas que el mundo se afana.

Afánate en las cosas en que el mundo no se afana.

Confucio

Descifrar los signos del mundo quiere decir siempre luchar contra cierta inocencia de los

objetos Roland Bartles

La fortuna vende muy caro lo que parece regalar

Vincent Voiture Para obrar, el que da debe olvidar pronto, y el que

recibe, nunca Séneca

Jamás en la vida encontraréis ternura mejor, más profunda, más desinteresada ni verdadera que la

de vuestra madre. Honoré de Balzac

Nunca prediques porque tienes que decir algo,

sino porque tienes algo que decir Richard Whately

Desde el lugar más reducido del mundo,

cualquier hombre puede contemplar la inmensa grandeza del firmamento

Vicente Risco

Así como el hierro se oxida por falta de uso, también la inactividad destruye el intelecto

Leonardo da Vinci

El hombre honrado es el que mide su derecho por su deber

Henri Lacordaire

Un padre sirve para dejar que te equivoques y ayudarte después

Daniel Múgica

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MOLLUSCICIDAL, CERCARIACIDAL, LARVICIDAL AND ANTIPLASMODIAL PROPERTIES OF

BARRINGTONIA RACEMOSA FRUIT AND SEED EXTRACTS

JOHN A.O. OJEWOLE56, NIRASHA NUNDKUMAR7

AND CLEMENT O. ADEWUNMI8

RECEIVED. 10 MAY 2003 WITH CORRECTION 10 SEPTEMBER 2003

ACEPTED: 11 FEBRUARY 2004 Abstract: Aqueous extracts of Barringtonia racemosa (Linn) Roxb. [Family: Lecythidaceae] fruit (pericarp) and seed have been evaluated for molluscicidal, cercariacidal, mosquito larvicidal and antiplasmodial activities. The plant extracts produced approximately equipotent molluscicidal, cercariacidal, mosquito larvicidal and antiplasmodial properties in the experimental models used. It is speculated that the observed biological effects of the plant aqueous extracts may largely be due to the triterpenoid saponins, especially barringtogenol and barringtogenic acid, present in the fruit and seed of the plant. Key Words: Barringtonia racemosa, fruit and seed extracts, triterpenoid saponins, molluscicidal, cercariacidal, antiplasmodial, mosquito larvicidal activities. Resumen9: El extracto acuoso de los frutos (pericarpio) y semillas de Barringtonia racemosa (Linn) Roxb. [Familia: Lecythidaceae ), han sido evaluados para determinar su actividad molusquicida, cercariacida, larvicida (mosquitos) y antiplasmodica. El extracto de la planta produce 5 Department of Pharmacology, Faculty of Health Sciences, University of Durban-Westville, Private Bag X54001, Durban 4000, South Africa. 6 Correspondence to Dr. JOHN A. O. OJEWOLE, Department of Pharmacology, Faculty of Health Sciences, University of Durban-Westville, Private Bag X54001, Durban 4000, South Africa. Actually in: Senior Professor of Pharmacology & Dean, Faculty of Health Sciences (Westville Campus), University of KwaZulu-Natal, Private Bag X5400, Durban 4000 SOUTH AFRICA. E-mail: ojewolej@ukzn.ac.za Tel: +27-31-260-7356/7767/8019 Fax: +27-31-260-7907 7 Department of Biochemistry, Faculty of Science, University of Durban-Westville, Private Bag X54001, Durban 4000, South Africa 8 Drug Research & Production Unit, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Nigeria. 9 Traduction of Dr. Rita Zeichen, ANMAT, Buenos Aires, Argentina. E-mail: rzeichen@anmat.gov.ar

aproximadamente equipotente actividad en los modelos experimentales usados. Se especula que los efectos bioogicos observados del extracto acuoso, pueden ser debidos a los saponinas triterpenoides, especialmente barringtogenol y el ácido barringtogenico, presente tanto en fruto y semilla de la planta. Palabras clave: Barringtonia racemosa, extracto de fruto y semilla, saponinas triterpenoides,actividad molusquicida, cercariacida, larvicida (mosquitos) y antiplasmodica

INTRODUCTION Malaria and schistosomiasis are the two leading parasitic diseases of tropical and subtropical countries of the world in terms of their overall prevalence, morbidity, socio-economic and public health burden, and human impact. Effective control and management of these infectious diseases require holistic approaches which not only include curative, chemotherapeutic and chemoprophylactic measures, but also preventive methods which include control measures that interfere with the life-cycles of the parasites. In developing, poor third-world countries, availability of cheap, effective, safe and environmentally-friendly indigenous alternatives to the existing expensive, toxic, synthetic, imported drugs used in the control and/or management of the parasitic diseases are certainly warranted. In our search for local, effective, safe alternatives for the control and/or management of malaria and schistosomiasis in South Africa, we have screened several species of higher plants from various families and diverse genera. One of the few such medicinal plants that have shown some promise as potential candidates for malaria and schistosomiasis control and/or management is Barringtonia racemosa. Barringtonia racemosa (Linn) Roxb. [Family: Lecythidaceae] occurs as a small to medium-sized tree (4-10 metres) in KwaZulu-Natal Province of South Africa. Common names of this tree include ‘brackwater mangrove’, ‘horse chestnut’ and ‘powder puff tree’. The Zulus of South Africa refer to this tree as iboqo, iliboqo, umlukula or umululuka (Pooley, 1993). The fruit of B. racemosa is said to make a good emetic solution against malaria (Hutchings et al, 1996; Palgrave, 1977). The stem-bark is reported to have insecticidal effects (Palgrave, 1977), and extracts of the fruit and stem-bark are claimed to be toxic to mosquitoes, snails and brine-shrimp (Hutchings et al, 1996). In India, the root- and stem-barks of the tree are used for stomachache and skin diseases, while the fruit sap is applied to eczema (Watt and Breyer-Brandwijk, 1962). The stem-bark is often used in decoctions dabbed on bodies of sick people, and is also used in non-venereal stricture in Australia. The fruit is used to poison wild pigs in the

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Phillipines, and is also used as a fish poison in Africa (Hutchings et al, 1996).

MATERIALS AND METHODS Plant Material: Mature fruits of Barringtonia racemosa were collected from the University of Durban-Westville’s Campus in Durban, South Africa, between March and August, 2001. The fruits were identified by the Taxonomist/Curator of the Department of Botany, University of Durban-Westville, as those of Barringtonia racemosa (Linn) Roxb. [Family: Lecythidaceae]. Extraction Procedure: One kilogramme (1000 g) each of Barringtonia racemosa’s dry fruit (pericarp) and seeds were finely ground in an industrial mill. The ground materials were separately extracted twice, on each occasion with 5 litres of distilled water, at room temperature for 24 hours, with shaking. The aqueous solubles obtained were filtered and concentrated to dryness under reduced pressure at 30±10C. The crude, aqueous extracts thus obtained were freeze-dried, finally yielding 1.5% and 6.1% powdery crude aqueous fruit and seed extract residues respectively. Aliquot portions of the crude fruit and seed extracts were weighed out and dissolved in distilled water on each day of our experiment. Molluscicidal Evaluation: The World health Organization’s method (WHO, 1965) for screening molluscicides was used. The intermediate host of Schistosoma mansoni in Nigeria, Biomphalaria pfeifferi snails, collected from a stream in Edun-Abon near Ile-Ife in Osun State of Nigeria, were separately exposed to graded concentrations of B. racemosa fruit and seed aqueous extracts. Niclosamide (Bayluscide), a synthetic, chemical molluscide, was used as the reference, control molluscicide for comparison. Ten snails were used per extract or niclosamide dose, with three replicates per dose of the extract or control. The snails were exposed to each extract dose for 24 hours, after which the snails were recovered and mortality recorded. Cercariacidal Testing: Infected Biophalaria pfeifferi snails were placed in petri-dishes and then exposed to artificial light (100 Watt bulbs) for 30 minutes. The emerging cercariae were pooled and counted with the aid of a microscope. Laboratory bred, male albino mice (weighing 20-25 g) were inoculated intraperitoneally, with 0.1 ml of the suspension containing 200 cercariae (pre-determined infective dose) per mouse. The method used was adapted from that described by Ogboli and co-workers (2000). Mosquito Larvicidal Screening: 0.01%, 0.1% and 1.0% (w/v) solutions of each extract were made-up in

distilled water. Third (3rd) instars of Anopheles arabiensis mosquito larvae were obtained from the Medical Research Council (MRC) of South Africa, Durban Office. The larvicidal assay method used in this study was adapted from the one described in detail earlier by Marston and co-workers (1993). The experimental room temperature was set at 28-300C, with a relative humidity of 70-80%. 20-ml of each of the test solutions (0.01%, 0.1% and 1.0%w/v) were placed in 100-ml beakers. 20-ml distilled water (placed in 100-ml beakers) was used as controls for the aqueous extracts. ‘Test’ and ‘control’ beakers were set-up in triplicates. Into each of the ‘test’ and ‘control’ beakers were placed 20 third instar Anopheles arabiensis larvae. Mortality count was taken 1, 3, 6, 12 and 24 hours after exposing the larvae to the plant extract solutions. This was done by gently swirling and agitating the contents of the beakers, and then allowing them to settle for 5 minutes, before counting the number of dead and living larvae. Whereas dead larvae sank to, and remained motionless at the bottom of beakers, those that were alive were able to swim back to the surface of the solutions in the beakers. Antiplasmodial examination: This assay part of the study was carried out in the Department of Pharmacology of the University of Cape Town, Cape Town, South Africa. Chloroquine-sensitive Plasmodium falciparum D10 strain was used for this assay. Stock solutions of the plant extracts (2 mg/ml) were made up in water. 100 µl of each of the above stock solutions was added to 900 µl of the assay medium to give a final concentration of 200 µg /ml. Red blood cells with 2% parasitaemia as well as a stock of unparasitized red blood cells were also used in this assay. The experiments were carried out in 96-well microtiter plates (see Figure 1). The assay procedure was carried out under sterile conditions, within a laminar flow hood. Firstly, 100 µl medium was placed into all the wells except those in column 3. Into each of the wells in column 3 were placed 200µl of the plant extracts (with concentration of 200µg/ml). This was done in duplicate with the same extract being tested in rows A and B. In this way, four plant extract concentrations were tested on each plate. The plant extract solutions were then serially diluted across the plate from column 3 to column 12, with the final concentration of plant extract in each row being 100 µg/ml, 50 µg/ml, 25 µg/ml, 12.5 µg/ml, 6.25 µg/ml, 3.13 µg/ml, 1,56 µg/ml, 0.78 µg/ml, 0.39 µg/ml, and 0.20 µg/ml. A volume of 100 µl of unparasitized red blood cells was placed in all the wells of column 1 and 100 µl of 2% parasitized red blood cells were placed into all the wells of columns 2 to 12. The plate was covered and placed into an incubation chamber. The chamber was then gassed

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for 5 minutes with filtered gas comprising 3% oxygen, 4% carbon di oxide and 93% nitrogen. The chamber was thereafter placed into a 370 incubator for 48 hours. After 48 hours incubation, the plates were removed from the incubator. The red blood cells in each well were carefully re-suspended. A volume of 100 µl Malstat and 25 µl NBT/PES was placed into all the wells of a new 96 well microtiter plate. Next, 15 µl of the re-suspended red blood cells from the original microtiter plates were placed into the corresponding wells of the plate containing the Malstat and NBT/PES, and the plate was immediately placed into a microplate reader (Cambridge Technology, Inc. – Microplate Reader Series 7500 VER-3.00), set at a wavelength of 620 nm. After 5 minutes (the time within which the tetrazolium would have been reduced to the blue formazan salt), readings were taken and recorded.

Molluscicide LD50 values

(in ppm)

Barringtonia racemosa

fruit (pericarp) aqueous

extract

seed aqueous extract

iclosamide

(Bayluscide)

521.48

(490.53-564.58)

556.23

(540.36-581.76)

0.112

(0.075-0.210)

Table 1: Comparative toxicity of Barringtonia racemosa fruit (pericarp) and seed aqueous extracts on Biomphalaria pfeifferi snails.

RESULTS

Molluscicidal Activity: The LD50 values for Barringtonia racemosa extracts and niclosamide (Bayluscide) on Biomphalaria pfeifferi snails are shown in Table 1. The fruit (pericarp) extract was found to possess slightly stronger molluscicidal activity than the seed extract of the plant. Cercariacidal Property: The non-infected, non-treated (negative control) mice had normal organs with no gross lesions, and with a mean liver weight of 1.16 g (Table 2). The infected but non-treated (positive control) mice had a mean liver weight of 2.37 g, and a mean worm load of 5.65/g of intestinal tissue. Pathologically, the mice in this positive control group had enlarged, fatty and friable liver, enlarged

and congested spleen and kidney, and were anaemic. Infected mice treated with aqueous extracts of Barringtonia racemosa fruit (pericarp) and seed extracts showed a mean liver weight of 1.65 g and 1.71 g respectively, and a mean worm load of 1.61/g and 1.68/g of intestinal tissue respectively. Compared with the positive control mice, the latter values represent 71.50% and 70.27% reductions in the parasite load caused by the Barringtonia racemosa fruit- (pericarp) and seed-extract treatments respectively. Pathologically, all the Barringtonia reacemosa fruit- and seed-extract treated mice showed haemorrhagic and friable dark liver, enlarged spleen and kidney, and anaemic carcass. These observations are in tandem with the findings of Ogboli et al (2000).

Mice group

Treatment Mean liver weight (g)

Mean worm

load (per g of

intestinal tissue)

% Parasite

reduction

A(1)

B. racemosa fruit (pericarp) aqueous extract

1.65±±0.018

1.61±±0.75

71.50

B(2)

B. racemosa seed aqueous extract

1.71±±0.25

1.68±±0.81

70.27

C(3)

Positive control (Infected but not treated)

2.37±±0.17

5.65±±1.30

Nil

D(4)

Negative control (Non-infected and Non-treated)

1.16±±0.05

Nil

Nil

Pathological signs (1) Enlarged, haemorrhagic, dark liver, spleen

and kidney; anaemic carcass. (2) Enlarged liver, spleen and kidney with severe

lesions; anaemic carcass (3) Enlarged, fatty and friable liver, spleen and

kidney; anaemic carcass (4) Normal organs, no gross lesions

Table 2: Effects of Barringtonia racemosa fruit (pericarp) and seed aqueous extracts on Schistosoma mansoni infected mice. Mosquito Larvicidal Property: 1% (w/v) solutions of the fruit (pericarp) and seed extracts of Barringtonia racemosa produced far more pronounced larvicidal activities than the 0.1% and 0.01% (w/v) solutions of the extracts. Consequently, our discussion section of this paper will refer to the results obtained with the 1 % (w/v) solutions.

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The aqueous extracts of the fruit and seed of B. racemosa produced approximately equal larvicidal activity. A 50% mortality of the mosquito larvae were observed within 3 hours of exposing the larvae to 1% solutions of the extracts. 100% mortality of the larvae was obtained within 12 hours of incubating the larvae in 1% solutions of the extracts (see Figure 2). The distilled water control used in this set of experiments produced a maximum of 5% mortality (figure not shown). This observation strongly suggests that the plant extracts were actually responsible for the observed larvicidal activities.

Figure 2: Percentage mortality of Anopheles arabiensis larvae in 1% solutions of aqueous extracts of B. racemosa fruit (pericarp) and seed. Antiplasmodial Activity: Barringtonia racemosa fruit and seed aqueous extracts displayed similar antiplasmodial activities (see Figure 3). They both gave IC50 values of approximately 30 µg/ml. Also worthy of note is the fact that the extracts caused 100% parasite death at 100 µg/ml. It is, therefore, speculated that the same chemical compound/s might be responsible for the anti-plasmodial activities of the two extracts. Although chemical samples with IC50 values of <10 µg/ml are usually considered active, it must be noted that the extracts tested in this study are very crude. Thus, in the present circumstances, the IC50 values of approximately 30 µg/ml may be considered significant in the sense that these crude extracts may contain antiplasmodial compounds but at low concentrations. The efficacy of the plant crude extracts may, therefore, only become obvious after their purification.

DISCUSSION AND CONCLUSION: The results of this experimental study indicate that Barringtonia racemosa fruit and seed extracts possess molluscicidal, cercariacidal, antiplasmodial and mosquito larvicidal properties. These observations are in consonance with some of our preliminary studies on the plant’s extracts (Nundkumar and Ojewole, 2002). The molluscicidal and cercariacidal properties of the plant extracts observed in this study, coupled with their

antiplasmodial and mosquito larvicidal effects, make B. racemosa fruit and seed ideal candidates for scientific scrutiny in the search for new molluscidal and antimalarial agents among South African flowering plants. Indeed, Barringtonia racemosa fruits and seeds could assist mankind in the battle against schistomiasis and malaria by providing chemical compounds to control the vectors, and also for the chemotherapy of the two debilitating and deadly parasitic diseases.

Our preliminary chemical analysis of B. racemosa fruit (pericarp) and seed aqueous extracts indicated the presence of flavonoids, terpenoids, fatty acids and saponins. Triterpenoids have been generally credited with mosquito larvicidal (Gbolade, 2000), molluscicidal (Hostettmann et al, 1982; Hostettmann, 1984) and cercariacidal (Ogboli, 2000) activities. Although it is premature at this stage, to attribute the biological activities of the plant extracts observed in this study to a specific group of chemical compounds, we speculate that the triterpenoid saponins, especially barringtogenol and barringtogenic acid, present in the fruit and seed of B. racemosa (Hutchings et al, 1996) might have contributed largely to the observed biological effects of the extracts. Further studies are in progress to determine the chemical constituent/s of the plant that is/are responsible for the biological activities obtained in this study, and to shed more light on the value of B. racemosa fruit and seed as potential plant molluscicides and larvicides.

Figure 3: Percentage parasite survival after treatment with aqueous extracts of Barringtonia racemosa fruit (pericarp) and seed. Chloroquine was used as the reference antiplasmodial drug for comparison.

ACKNOWLEDGEMENTS

This study was partly supported by a research grant from SIDA/NRF (for which the authors are grateful). One of us (JAO) will like to thank the council of the University of Durban-Westville for the provision of research grant R642 to support this study.

0

20

40

60

80

100

0 3 6 9 12 15 18 21 24

TIME (HOURS)

% M

OR

TALI

TY

FRUIT

SEED

0

10

20

30

40

50

60

70

80

90

100

110

120

0.1 1 10 100 1000

Concentration (microgram/ml)

% P

aras

ite s

urvi

val

fruitseedchloroquine

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REFERENCES

1. Gbolade, A.A. (2000). Plant derived insecticides in the control of malaria vector. In: “Phytomedicines in Malaria and Sexually-transmitted Diseases: Challenges for the New Millenium”, edited by Adewunmi, C.O. and Adesina, S.K., Drug Research and Production Unit, Faculty of Pharmacy, Obafemi, Awolowo University, Ile-Ife, Nigeria, pp. 48-50.

2. Hostettmann, K. (1984). On the use of plants and plant-derived compounds for the control of schistosomiasis. Natur wissenschaften, 71: 247 – 251.

3. Hostettmann, K.; Kizu, H. and Tomimon, T. (1982). Molluscicidal properties of various saponins. Planta medica, 44: 34 – 35.

4. Hutchings, A., Scott, A.H., Lewis, G and Cunningham, A. (1996). Zulu Medicinal Plants - An inventory. University of Natal Press, Pietermaritzburg, South Africa, p. 212.

5. Marston, A., Maillard, M. and Hostettman, K. (1993). Search for antifungal, molluscicidal and larvicidal compounds from African Medicinal Plants. Journal of Ethnopharmacology, 38: 215 – 223.

6. Nundkumar, N. and Ojewole, J.A.O. (2002). Studies on antiplasmodial properties of some South African medicinal plants used as antimalarial remedies in Zulu folk medicine. Methods and Findings in Experimental and Clinical Pharmacology, 24(7): 397 – 401.

7. Ogboli, A.U., Nock, I.H., Abdurahman, E.M. and Ibrahim, N.D.G. (2000). Medicinal application of Vernonia amygdalina Del. leaf extracts in the treatment of schistosomiasis in mice. Nig. J. Nat. Prod. And Med., 4: 73 – 75.

8. Palgrave, K.C. (1981). Trees of Southern Africa. Struik, Cape Town, South Africa.

9. Pooley, E. (1993). The complete field guide to trees of Natal, Zululand and Transkei. Natal Flora Publications, Natal Herbarium, South Africa.

10. Watt, J. M. and Breyer-Brandwijk, M. G. (1962). The Medicinal and Poisonous Plants of Southern and Eastern Africa. 2nd Edition, E&S Livingstone, Edinburgh and London, p. 534.

11. WHO (1965). Molluscide screening and evaluation. Memoranda, Bulletin of The World Health Organization 33: 567 – 581.

THE END

Figure 1: Diagrammatic representation of the ‘microtitre plate’ used for the antiplasmodial assay.

A B C D E F G H

1 2 3 4 5 6 7 8 9 10 11 12

Unparasitized red blood cells

Parasitized red blood cells only

Serially diluted plant extracts (100 µg/ml – 0.2 µg/ml) with 2% parasitized red blood cells

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CORRESPONDENCIA

Desde Argentina: Estimado José Luis: Recibi el nuevo volumen del BLACPMA.Quiero felicitarte por la tarea que estás llevando a cabo. Realmente se destacan la calidad y la actualidad de los trabajos presentados, entre ellos el referente a los microorganismos endofíticos y sus perspectivas de Pedro Acciol y colaboradores. Creo que el boletín está cumpliendo ampliamente con el objetivo de ser una herramienta de difusión muy útil para todos aquellos involucrados en la investigación de las plantas medicinales bajo sus diversos aspectos. Un cordial saludo, Virginia Martino, Presidente Sociedad Latinoamericana de Fitoquímica y Profesora Universidad de Buenos Aires. Desde Nigeria: Dear Prof. Jose Luis Martinez, Thanks for you mail and attached files: BLACPMA Vol. 3 Nº 4 in pdf, once again, thank you very much for the kind consideration. Pius I. Odigie, Lagos, Nigeria. Desde Perú: Hola Jose Luis, Gracias por la ultima versión del BLACPMA de Julio. Voy a insistir con algunos Doctores peruanos a ver si se animan a aportar estudios/articulos al Boletín. Un abrazo, Mark Hein, Peruvian Heritage S.A.C. / Repex S.A.C., Av. Benavides 4562, Santiago de Surco, Lima, Perú. Desde Chile: ¡Muchas gracias José Luis! Interesante como siempre... y me sorprende tu colección inagotable de citas, Bruce Cassels, Universidad de Chile. Desde Bolivia: Estimado José Luis: Vengo recibiendo regularmente los números que envías de BLACPMA, quiero felicitarte por el esfuerzo que realizas al hacer posible esta distribución. Quiero decirte que gracias al Boletín me he enterado de muchos eventos que de otra forma no lo haría, sigue adelante. Ingrid Loayza, Centro de Tecnología Agroindustrial, Universidad Mayor de San Simon, Cochabamba, Bolivia. Desde México: Estimado José Luis, Mi agradecimiento y felicitaciones por el nuevo Boletín. BLACMA es una contribución para el medio interesado en el conocimiento sobre las potencialidades del recurso herbolario medicinal, un gran esfuerzo que valoramos. Un saludo muy cordial. Mariana Meckes. Unidad de Investigación Médica en Farmacología de Productos Naturales. CMN Siglo XXI, IMSS. Desde Chile: Buen día, cuso recibo de tan importante revista.gracias por mantenernos en su base de datos. Andrés Carmona, Administrador del Medio Ambiente y de los Recursos Naturales Desde Bolivia: Estimado José Luis: Una vez más, muchas gracias por la gentileza que tienes con nosotros, como siempre la revista será difundida entre los integrantes del Centro donde trabajo. Un cordial saludo. Gloria Saavedra, Centro de

Tecnología Agroindustríal, Facultad de Ciencias y Tecnología, Universidad Mayor de San Simón, Cochabamba, Bolivia. Desde Argentina: José Luis, Gracias por el envío (lo recibí duplicado). Adhiero con varios de las personas que se comunicaron en este número en que el BLACPMA es no sólo un forma de difundir nuestro trabajo y conocernos, sino también de estar al tanto de los eventos que son de nuestro interés. Gracias otra vez!, Dra. Silvia B. González, Univ. de la Patagonia sede Esquel Desde Chile: Estimado José Luis: Por el momento te informo que recibí la revista de Julio, Atte, Prof. Fernando López, Pontificia Universidad Católica de Chile. Desde Brasil: Prezado señor, Recebi o BLACPMA de julho e agradeço, Atenciosamente, Elfriede Marianne Bacchi, Universidad de Sao Paulo. Desde Argentina: Estimado José Luis, Acabo de recibir tu último envío como siempre muy interesante. Agradezco tu trabajo y el de tus colaboradores por mantenernos informados y actualizados. Recibe los más atentos saludos de Cristina Résico, Programa de Productos Forestales No Madereros, Dirección de Bosques, Secretaría de Ambiente y Desarrollo Sustentable Desde Chile: Estimado José Luis: Muchas gracias por los envíos de los diferentes números del Boletín BLACPMA. Todos son muy interesantes y muestran un gran esfuerzo de tu parte. Cordialmente, René Torres, Universidad de Santiago de Chile. Desde Brasil: Prezado José Luiz, Acuso o recebimento do Boletim, que aprecio muito. Considero-o muito importante como forma de união entre todos nós, pesquisadores latinoamericanos. Grata, Atenciosamente, Profa. M. Graças Lins Brandão, Universidade Federal de Minas Gerais. Desde México: Recibí vol. 3, No 4. como siempre interensatísimo, gracias por enviarmelo, me es de mucha utilidad la información que contiene. Saludos, Delia Cruz, Investigador Asociado Centro de Investigación Biomédica del Noreste, IMSS., 2 de Abril y San Luis Potosí, Col. Independencia, Monterrey, N.L. Desde Chile: De mi consideración. Agradezco a usted el envió del boletín BLACMA, a nosotros los estudiantes de pregrado que nos interesa el estudio de plantas medicinales nos es de mucha ayuda saber sobre las actividades realizadas en el resto de América latina y el Caribe. Reitero mis agradecimientos y se despide atentamente de usted. José Gigoux, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile. Desde Brasil: Olá, José Luis, Estou muito agradecida pelo Boletim. Ele tem sido muito útil para os alunos, pesquisadores e para mim também. Um grande abraço Maria de Fátima Barbosa Coelho. Desde Chile: Muchas gracias por el envío del ejemplar de julio, agradecería se me incluyera en el

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futuro en su distribución en forma permanente. Atentamente. Leonora Fuentes, Centro de Información Silvoagropecuaria (CIS), Oficina de Estudios y Políticas Agrarias (ODEPA). Desde Argentina: Estimado José Luis: Te agradezco el nuevo número del Boletín el cual es de sumo interés para todos aquellos que nos vinculamos a la investigación en plantas medicinales y aromáticas. Muchos saludos. Diego Estomba, San Martín de los Andes, Neuquen, Argentina. Desde Brasil: Prezado Martinez, Confirmo recebimento do BLACPMA vol 3, No 4, 2004. Obrigado, José María Barbosa. Desde España: Hola José Luis: Agradecido por la gentileza de enviarme el número de Julio. La revista continua creciendo en interés y calidad. Saludos. Dr. Lázaro Vidal. Madrid. España. Desde Francia: Dear Dr. Martinez, I acknowledge with many thanks the receipt of the BLACPMA vol.3 N° 4. I am pleased to let you know that a links to your interesting BOLETIN was put in the LINKS section of the Website of the European Society of Ethnopharmacology at: http://ethnopharma.free.fr/links3.htm#journals I hope you will reciprocate, by placing a links to our Society in the section “Sitios de Interes” of your own Website. However, it seems that this section is not available. Please check your Website ! With all best , wishes, Dr. Guy MAZARS, President, European Society of Ethnopharmacology, Institut d’Histoire des Sciences, Université Louis Pasteur. Desde Chile: Estimado José Luis, deseo expresa mis más sinceros agradecimientos por integrarme en esta red de conocimientos y volver a expresar las felicitaciones pertinentes por la emisión de este tan importante boletín. La Edición está bastante simple para el pronto entendimiento de los temas. Los temas nos interesan de sobremanera, en especial si apareciera algo sobre el uso del cultivo de Aloe vera. Nuevamente Felicitaciones. Renato Mella Pavez. Escuela de Agronomía. Universidad Iberoamericana de Ciencias y Tecnología (UNICIT), Santiago, Chile. Desde Argentina: Estimado José Luis: Acuso recibo del último Boletín. Estoy sumamente feliz del éxito que esta teniendo entre nuestros colegas. Su contenido no solo científico, sino humano, es de gran valor y nos da fuerzas para continuar. Muchas gracias. Dra Rita Zeichen, Jefa Servicio de Fitoterapia, Departamento de Farmacología, ANMAT-INAME, ARGENTINA. Desde Brasil: Estimado José Luis: Agradeço-lhe o envio do vol. 3 N. 4 do BLACPMA, sempre interessante e informativo. Abraços, Nikolai Sharapin Desde Perú: Acusamos recepción y muchas gracias por la información es muy útil para nuestro trabajo. Atte, Amarilda Luque, Proyecto GTZ/IICA, Coordinación Nacional FOMRENA PERU.

Desde Laos: El presente es para informarle que recibí el Boletín vol. 3 No. 4. Estoy en viaje en Laos y estaré de vuelto en Chicago el día 6 de Agosto. Con saludos. D. Doel Soejarto Desde Argentina: Estimado José Luis: muchas gracias por el nuevo volumen. Me encanta recibirlo y me gustaría que se incorporaran temas de legislación de los países sudamericanos respecto a las plantas medicinales y sus preparaciones. Coordino la comisión de fitoterápicos de COOPERALA y en unos días me gustaría enviarte una gacetilla respecto a un proyecto que estamos llevando a cabo, para que puedas publicarlo en el boletín. Dra. Jelena Nadinic Desde Argentina: Estimado José Luis: Quiero darte las gracias por enviarme el Boletín lo que permite mantenerme actualizada en relación a cursos, congresos como así también en relación a las publicaciones que se realizan. También estamos infinitamente agradecidos par la oportunidad brindada a Claudia Gómez de participar del Primer Curso de Producción y Control de Calidad de Fitomedicamentos del Conosur. Sin dudas por todo lo manifestado por Claudia, el Curso realizado fue un éxito. Nuevamente muchas gracias a todos. Un afectuoso saludo de Misiones, Argentina. Gabriela DE BATTISTA Desde Argentina: Estimado José Luis Martínez, Muchas gracias por enviarme el boletín BLACPMA Vol 3 N° 4. Aprovecho para enviarle mis felicitaciones y alentarlo en este desafío que aporta a la ciencia. Un saludo. Farm. Catiana Zampini, Instituto de Estudios Vegetales, Facultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán, Tucumán, Argentina. Desde España: Apreciado José Luis, acabo de recibir el nuevo boletín. Muchísimas gracias. En otro orden de cosas, no se si te comente que ya esta activa la nueva pagina web de fitoterapia: www.fitoterapia.net., Un abrazo, Bernat Vanaclocha. Desde Argentina: Estimado José Luis. gracias por el envío del Boletín; !que buenos trabajos¡, estos intelectos latino - americanos pechan lindo la cultura. Te felicito paralelamente por el esfuerzo del rastreo de citas, tan meritorio como el de trabajos, Atte. Daniel Palacios. Desde Argentina: Estimado José Luis! Muchas gracias por el re-envío de BLACPMA Vol. 3 Nº 4. Ya no tengo problemas para leerlo. Ayer, en ARGENTINA, fue el DÍA DEL AMIGO. Vaya mi saludo para vos y todos los que trabajan en la edición de BLACPMA: FELICIDADES .AMIGOS!!!!!10, Ing. Dora Vignale, Universidad Nacional de Jujuy. Desde Barbados: Dear Jose Luis: Many thanks for the bulletin. Is it published in English by any chance? Best regards, Ena Harvey.

10 La Dra. Vignale hace referencia al 20 de Julio.

BLACPMA Vol. 3 Nº 5 September 2005 In LATINDEX from 2004

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Otras cartas: Desde Argentina: Desde hace 3 años hemos puesto en marcha un microemprendimiento orgánico en San Clemente del Tuyú, provincia de Buenos Aires, Argentina. El mismo tiene como finalidad principal el cultivo y propagación de plantas aromáticas y medicinales que prosperen a orillas del mar en la franja de terrenos medanosos del corredor atlántico. Actualmente cultivamos romeros, santolinas, marcelas, poleos, ajenjo, salvias, tomillos y otras como equisetum, marrubio, etc. Disponemos de semillas y plantines de Oenothera biennis (onagra, "prímula") para realizar una siembra o transplante de entre 1 y 2 hectáreas en diferentes condiciones climáticas y edáficas. Necesitamos contactarnos con quienes estén interesados. Por otro lado también deseamos obtener información bibliográfica acerca del cultivo de Oenothera, extracción del aceite por diferentes métodos ya que lo que hemos logrado hasta ahora ha sido muy artesanal. Quisiéramos también ofrecer un intercambio o venta de nuestros plantines por otros tales como Cymbopogonum sp.(nardus, winterianus, citratus), Artemisia dracunculus, Hyssopus officinalis, Melaleuca alternifolia u otras especies. Deseosos de ponernos en contacto con Uds y a la espera de su respuesta, saludan atentamente Agrónomos Daniel Pérez y Ana Alzamendi. Desde Chile: Tenemos el agrado de comunicar que salio el Boletín de Plantas Medicinales y Aromáticas N° 11 editado por la Fundación para la Innovación Agraria. Le agradecemos compartir esta información con aquellas personas vinculadas al rubro a quienes pudiera interesarle. Le recordamos asimismo que este y otros boletines editados por FIA pueden consultarse en nuestra página web: www.fia.gob.cl. Fundación para la Innovación Agraria Siguen solicitándonos: Desde Cuba: Agradecería el recibo del Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas (BLACPMA). Dr. Víctor R. Fuentes Fiallo. Desde Colombia: Hola. Estoy interesado en recibir el BOLETIN LATINOAMERICANO Y DEL CARIBE DE PLANTAS MEDICINALES Y AROMATICA. Gracias por su atención, Alvaro Idárraga, Herbario HUA, Universidad de Antioquia (Colombia) Desde Argentina: Sr. Martínez: me interesa recibir el Boletín ya que trabajo en aromáticas hace años y en estos últimos años en medicinales también. Doy cursos y me gusta dar toda la información posible de sitios y fuentes de interés. Incluiré el sitio de referencia. Voy a manarle el aviso de mi curso en Mar del Plata para que vea mi perfil y el motivo de mi

interés11. Muchas gracias por su labor. Ing. Agr. María Cecilia Espósito, Mar del Plata.

FIN

FRASES

Solo quien tartamudea habla verdaderamente de la interpretación de los mitos

Confucio Es mejor tener la boca cerrada y parecer estúpido

que abrirla y disipar toda duda Mark Twain

Produce una inmensa tristeza pensar que la

naturaleza habla mientras el género humano no escucha

Victor Hugo

El miedo es natural en el prudente, Y el saberlo vencer es ser valiente

Alonso de Ercilla A quien más sabe es a quien más duele perder el

tiempo Dante Alighieri

Todo el mundo es ignorante, solo que en

mentiras distintas William Rogers

La mitad de nuestras equivocaciones en la vida

nacen de que cuando debemos pensar, sentimos, y cuando debemos sentir, pensamos

John Ch. Collins

La manera de jugar revela algo de la persona; la manera de perder lo revela todo

Pelé

El mundo es la suma total de nuestras posibilidades vitales

José Ortega y Gasset

Los recuerdos no pueblan nuestra sociedad, como suele decirse; antes al contrario, la hacen

más profunda Gustave Flaubert

La alegría, cuanto más se gasta más queda

Ralph Waldo Emerson

11 Nota del editor: los datos del curso que hace referencia María Cecilia en su nota no fueron incluidos pues su curso se dictaba durante el mes de Julio.