Journal o f Chemical Ecology, Iiol. 12, No. 10, 1986
V A R I A T I O N OF S O M E S E C O N D A R Y M E T A B O L I T E S IN J U V E N I L E S T A G E S OF T H R E E P L A N T SPECIES F R O M T R O P I C A L RAIN F O R E S T 1
R. S I L V I A D E L A M O , 2 JOSI~ G. R A M I R E Z , 2 and O F E L I A E S P E J O 3 21nstituto Nacional de lnvestigaciones sobre Recursos Bioticos, Retorno de Cerro Tuera 21 Oxtopulco Universidad, Coyoacan 04310, Mexico, D,F. 3Divisi6n de Estudios de Postgrado, Facultad de Qu[mica. Universidad Nacional Aut6noma de Mdxico
(Received July 23, 1985; accepted December 10, 1985) Abstract--Results from qualitative and quantitative evaluation of some chemical constituents, particularly phenols and volatile terpenes, from juvenile stages of three primary species belonging to the perennial rain forest are presented. The degree of infection and the time of the year were taken into account. TLC, GC, and simple chemical methods were used to estimate differences among components. Biological assays were conducted to evaluate fungicide potential of the extracts from studied plants. In general terms, the results show significant differences in the chemical composition of the species studied (Nectandra ambigens, Omphalea oleifera, and Licaria alata) related to the time of the year and the degree of infection. These differences can be related as well to their intrinsic resistance to infection and ability to grow to the adult stage. Key Words--Secondary metabolites, juvenile stages,, phenols, volatile terpenes, degree of infection, Nectandra ambigens, Omphalea oleifera, Licaria alata.
INTRODUCTION Seedlings and j u v e n i l e stages o f p r i m a r y species from p e r e n n i a l rain forests are kept in latency d u r i n g long periods o f time due primarily to light conditions prevailing in the l o w e r layers o f the forest (Richards, 1959; W h i t m o r e , 1975; del A m o , 1978). D u r i n g this period o f growth suppression, plants are subject to c o n s t a n t pressures o f m a n y types, a n d these are a c o n s e q u e n c e o f the inter~This investigation was sponsored by Project 001077 PCECBNA, Consejo Nacional de Ciencia y Tecnolog/a. 2021 (1098-0331/86/1000-2021505.00/0 9 1986 Plenum Publishing Corporation
2022
DEL A M O ET AL.
action between biotic and abiotic factors. One of the main factors of stress during the first stages of growth is the attack of pathogenic microorganisms. The importance of chemical mechanisms of defense developed by some plants against pathogenic microorganisms, insects, and other herbivores, through formation and accumulation of secondary metabolites, has been noted by Levin (1976), Whittaker and Feeny (1971), and Seigler (1978), among others. Of the metabolites, those most frequently related to pathogen resistance have been the phenolic compounds and the terpenes (Cobb et al., 1968; Hunter et al., 1978). As for the terpenes, Cobb et al. have suggested that some monoterpenes such as myrcene, phellandrene, limonene, pinene, and carene, produced by Pinus ponderosa, play an important part in its resistance to infection by several microorganisms; they also mentioned that their activity can be related to that of phenolic compounds. Monoterpenes and sesquiterpenes are found in the socalled essential oils. The contents and production of essential oils may vary according to climate, soil conditions, stage of development of the plants, and genetic factors (Swain, 1963; Rice et al. 1978). The main goal of the present work was to evaluate the qualitative and quantitative variation of chemical components, mainly terpenes and phenolic compounds, in juvenile stages of three primary species from the perennial rain forest, taking into account the degree of infection and the time of the year. At the same time, extracts of each plant were evaluated against phytopathogenic fungi isolated from the same species studied in order to evaluate their fungicidal potential. METHODS
AND MATERIALS
Juvenile stages of plants of three primary species from Ebitrolotu (station for tropical biology at Los Tuxtlas, Veracruz, Mexico, belonging to the National University of Mexico) were used: Nectandra ambigens, Licaria alata (Lauraceae), and Omphalea oleifera (Euphorbiaceae). These species are common in the Tuxtla region, and their seedlings and young plants form a dense mass in the understory of this tropical rain forest (del Amo, 1978; Cordova, 1980). Samples were collected and classified as "damaged" and "healthy" plants, according to the degree of infection. There were no completely undamaged or healthy plants in the case of Omphalea oleifera specimens. Even so, the three species were classified in the same manner. It is important to note that the term "damaged" was given to plants with evident signs of infection or insect depredation. Samples were collected twice during the year: during the dry season in March and the rainy season in July, with the purpose of detecting variations in secondary metabolites according to environmental conditions, specifically plu-
2023
RAIN FOREST PLANT METABOLITES
vial precipitation. The samples were processed as soon as possible in order to avoid metabolic transformation due to action of plant enzymes or microorganisms. Laboratory Methods. Solvents were purified by distillation. Thin-layer chromatography (TLC) was done using silica gel GF 254-60 Merck and microcrystalline cellulose on flexible plates, "Bakerflex," and were made visible with iodine and/or anisaldehyde-H2SO 4 followed by UV light. Gas-liquid chromatography (GLC) was carried out in a Varian 3700 apparatus, using a 10% FFAP on Chromosovo WHP column. As an external standard, a mixture of pure terpenes selected according to chemotaxonomic criteria was used (Figure 1). Phenols were measured using a Varian 364 spectrophotometer. Fresh material, 300 g, was processed according to the Scheme 1. Fresh material 2g 1. Preliminary chemical test in the collection site
23 g 2. Quantitation of phenols
125 g 3. Ethanolic extractions (70% EtOH)
150 g 4. Water steam extraction
Extract
Extract
l
Partition CHC13-H20 Aqueous
Chloroformic phase (C1) ~
phase (AqlL ~ ~
I AcOEt Extraction Essential oil
5. Biological assay SCHEME ] .
Preliminary Chemical Test (at Site of Collection). One gram of fresh material was triturated in a mortar, extracted with ca. 10 ml of EtOH and filtered; aliquots of this filtrate were used to search for phenols (reaction with ferric chloride), saponins (foam formation), alkaloids (reaction with Mayer, Dragendorff, Wagner, and phosphotungstic acid reagents); and essential oils (characteristic odor). Evaluation of Phenols. Phenols were quantified by the Price and Butler (1977) method, but using 6 g of fresh material instead of the dry material used by these authors. The material was extracted with 60 ml of distilled water and the extracts filtered. An aliquot of 5 ml of filtrate was diluted to 50 ml with
2024
DEL
COMPOUN o
{rN MIN)
MYRCENE 1 8,ClNEOLE p CYMENE 8ENZALOEHYDE LINALOL BORNEOL TERPINEOL CUMINALDEHYDE GERAN]OL 9 NEROL
3 I 4
19 25 34 86 94 125 134 I 56 162
SAFROLE
180
METHYLEUGENOL CINNAMALDEHyDE
21 4 22 2
CRESOL
22 9
EUGENOL BENZYL BENZOATE
25 I 346
I---
zI
uum ouJ
V A R I A T I O N OF S O M E S E C O N D A R Y M E T A B O L I T E S IN J U V E N I L E S T A G E S OF T H R E E P L A N T SPECIES F R O M T R O P I C A L RAIN F O R E S T 1
R. S I L V I A D E L A M O , 2 JOSI~ G. R A M I R E Z , 2 and O F E L I A E S P E J O 3 21nstituto Nacional de lnvestigaciones sobre Recursos Bioticos, Retorno de Cerro Tuera 21 Oxtopulco Universidad, Coyoacan 04310, Mexico, D,F. 3Divisi6n de Estudios de Postgrado, Facultad de Qu[mica. Universidad Nacional Aut6noma de Mdxico
(Received July 23, 1985; accepted December 10, 1985) Abstract--Results from qualitative and quantitative evaluation of some chemical constituents, particularly phenols and volatile terpenes, from juvenile stages of three primary species belonging to the perennial rain forest are presented. The degree of infection and the time of the year were taken into account. TLC, GC, and simple chemical methods were used to estimate differences among components. Biological assays were conducted to evaluate fungicide potential of the extracts from studied plants. In general terms, the results show significant differences in the chemical composition of the species studied (Nectandra ambigens, Omphalea oleifera, and Licaria alata) related to the time of the year and the degree of infection. These differences can be related as well to their intrinsic resistance to infection and ability to grow to the adult stage. Key Words--Secondary metabolites, juvenile stages,, phenols, volatile terpenes, degree of infection, Nectandra ambigens, Omphalea oleifera, Licaria alata.
INTRODUCTION Seedlings and j u v e n i l e stages o f p r i m a r y species from p e r e n n i a l rain forests are kept in latency d u r i n g long periods o f time due primarily to light conditions prevailing in the l o w e r layers o f the forest (Richards, 1959; W h i t m o r e , 1975; del A m o , 1978). D u r i n g this period o f growth suppression, plants are subject to c o n s t a n t pressures o f m a n y types, a n d these are a c o n s e q u e n c e o f the inter~This investigation was sponsored by Project 001077 PCECBNA, Consejo Nacional de Ciencia y Tecnolog/a. 2021 (1098-0331/86/1000-2021505.00/0 9 1986 Plenum Publishing Corporation
2022
DEL A M O ET AL.
action between biotic and abiotic factors. One of the main factors of stress during the first stages of growth is the attack of pathogenic microorganisms. The importance of chemical mechanisms of defense developed by some plants against pathogenic microorganisms, insects, and other herbivores, through formation and accumulation of secondary metabolites, has been noted by Levin (1976), Whittaker and Feeny (1971), and Seigler (1978), among others. Of the metabolites, those most frequently related to pathogen resistance have been the phenolic compounds and the terpenes (Cobb et al., 1968; Hunter et al., 1978). As for the terpenes, Cobb et al. have suggested that some monoterpenes such as myrcene, phellandrene, limonene, pinene, and carene, produced by Pinus ponderosa, play an important part in its resistance to infection by several microorganisms; they also mentioned that their activity can be related to that of phenolic compounds. Monoterpenes and sesquiterpenes are found in the socalled essential oils. The contents and production of essential oils may vary according to climate, soil conditions, stage of development of the plants, and genetic factors (Swain, 1963; Rice et al. 1978). The main goal of the present work was to evaluate the qualitative and quantitative variation of chemical components, mainly terpenes and phenolic compounds, in juvenile stages of three primary species from the perennial rain forest, taking into account the degree of infection and the time of the year. At the same time, extracts of each plant were evaluated against phytopathogenic fungi isolated from the same species studied in order to evaluate their fungicidal potential. METHODS
AND MATERIALS
Juvenile stages of plants of three primary species from Ebitrolotu (station for tropical biology at Los Tuxtlas, Veracruz, Mexico, belonging to the National University of Mexico) were used: Nectandra ambigens, Licaria alata (Lauraceae), and Omphalea oleifera (Euphorbiaceae). These species are common in the Tuxtla region, and their seedlings and young plants form a dense mass in the understory of this tropical rain forest (del Amo, 1978; Cordova, 1980). Samples were collected and classified as "damaged" and "healthy" plants, according to the degree of infection. There were no completely undamaged or healthy plants in the case of Omphalea oleifera specimens. Even so, the three species were classified in the same manner. It is important to note that the term "damaged" was given to plants with evident signs of infection or insect depredation. Samples were collected twice during the year: during the dry season in March and the rainy season in July, with the purpose of detecting variations in secondary metabolites according to environmental conditions, specifically plu-
2023
RAIN FOREST PLANT METABOLITES
vial precipitation. The samples were processed as soon as possible in order to avoid metabolic transformation due to action of plant enzymes or microorganisms. Laboratory Methods. Solvents were purified by distillation. Thin-layer chromatography (TLC) was done using silica gel GF 254-60 Merck and microcrystalline cellulose on flexible plates, "Bakerflex," and were made visible with iodine and/or anisaldehyde-H2SO 4 followed by UV light. Gas-liquid chromatography (GLC) was carried out in a Varian 3700 apparatus, using a 10% FFAP on Chromosovo WHP column. As an external standard, a mixture of pure terpenes selected according to chemotaxonomic criteria was used (Figure 1). Phenols were measured using a Varian 364 spectrophotometer. Fresh material, 300 g, was processed according to the Scheme 1. Fresh material 2g 1. Preliminary chemical test in the collection site
23 g 2. Quantitation of phenols
125 g 3. Ethanolic extractions (70% EtOH)
150 g 4. Water steam extraction
Extract
Extract
l
Partition CHC13-H20 Aqueous
Chloroformic phase (C1) ~
phase (AqlL ~ ~
I AcOEt Extraction Essential oil
5. Biological assay SCHEME ] .
Preliminary Chemical Test (at Site of Collection). One gram of fresh material was triturated in a mortar, extracted with ca. 10 ml of EtOH and filtered; aliquots of this filtrate were used to search for phenols (reaction with ferric chloride), saponins (foam formation), alkaloids (reaction with Mayer, Dragendorff, Wagner, and phosphotungstic acid reagents); and essential oils (characteristic odor). Evaluation of Phenols. Phenols were quantified by the Price and Butler (1977) method, but using 6 g of fresh material instead of the dry material used by these authors. The material was extracted with 60 ml of distilled water and the extracts filtered. An aliquot of 5 ml of filtrate was diluted to 50 ml with
2024
DEL
COMPOUN o
{rN MIN)
MYRCENE 1 8,ClNEOLE p CYMENE 8ENZALOEHYDE LINALOL BORNEOL TERPINEOL CUMINALDEHYDE GERAN]OL 9 NEROL
3 I 4
19 25 34 86 94 125 134 I 56 162
SAFROLE
180
METHYLEUGENOL CINNAMALDEHyDE
21 4 22 2
CRESOL
22 9
EUGENOL BENZYL BENZOATE
25 I 346
I---
zI
uum ouJ
