Erythroxylum Novogranatense Var Research Articles

Plantas medicinales: Efecto antibacteriano in vitro de Plantago major L, Erythroxylum novogranatense, Plowman var truxillense y Camellia sinensis sobre bacterias de importancia estomatológica

El objetivo de la investigación fue comparar la actividad antibacteriana in vitro de los extractos hidroalcohólicos de tres plantas medicinales: Plantago major L. (llantén), Erythroxylum novograntense var truxillense (coca trujillo) y Camellia sinensis (té verde) mediante el método de difusión en agar con discos, sobre cinco cepas patrones de bacterias orales: Streptococcus mutans ATCC 25175, Lactobacillus acidophilus. ATCC 314, Actinomyces viscosus ATCC 15987, Prevotella melaninogenicus ATCC 25845 y Fusobacterium nucleatum ATCC 25586. Se obtuvieron extractos hidroalcohólicos de principios activos totales procedentes de las hojas secas de cada una de las plantas, mediante maceración alcohólica con alcohol etílico al 70 % y posterior evaporación del solvente con el empleo del rotavapor. Cada extracto se diluyó en alcohol etílico al 70 % en las concentraciones de 25 μg/mL y 50 μg/mL. Estas soluciones fueron comparadas con PerioAid® (clorhexidina 0,012 %) como control positivo y con alcohol etílico al 70 %, como control negativo. Al realizar las pruebas de sensibilidad in vitro, se obtuvieron los siguientes resultados: los tres extractos hidroalcohólicos en ambas concentraciones presentaron actividad antibacteriana mayor al alcohol etílico (5,8 mm) y menor que el PerioAid® (22,0 mm) sobre las cinco cepas bacterianas en estudio. La mayor actividad presentó el extracto hidroalcohólico de Camellia sinensis a 50 μg/mL, la menor actividad presento Plantago major a 25 μg/mL. Se concluye que los tres extractos hidroalcohólicos en las diluciones de 25 y 50 μg/mL presentaron actividad antibacteriana sobre Streptococcus mutans, Lactobacillus acidophilus, Actinomyces viscosus, Prevotella melaninogenica y Fusobacterium nucleatum. El efecto antibacteriano aumentó con la concentración en P. melaninogenica, que fue la cepa más sensible y A. viscosus la menos sensible.

Chemosystematic identification of fifteen new cocaine-bearing Erythroxylum cultigens grown in Colombia for illicit cocaine production

Colombian coca farmers have historically cultivated three varieties of coca for cocaine production (Erythroxylum novogranatense var. novogranatense, Erythroxylum novogranatense var. truxillense, and Erythroxylum coca var. ipadu). Within the past 13 years, 15 new cultigens of cocaine-bearing Erythroxylum have been propagated by Colombian coca farmers; each with differing physical characteristics, yet producing cocaine alkaloids at similar levels found in the historical and native varieties. Fifteen new cultigens were collected from throughout Colombia and propagated along with the three historical varieties within an experimental field in Colombia. Five plants/cultigen were randomly selected and examined for alkaloid content to determine their varietal characteristics when compared to the three known varieties. Ten cultigens gave classic Erythroxylum coca var. ipadu alkaloid profiles, four cultigens produced alkaloid profiles consistent with a hybridization of Erythroxylum novogranatense and Erythroxylum coca var. ipadu, while one cultigen gave heterogeneous alkaloid profiles that could not be characterized.

AFLP Phylogeny of 36 Erythroxylum Species

Four taxa of the plant genus Erythroxylum; Erythroxylum coca var. coca (Ecc), Erythroxylum coca var. ipadu (Eci), Erythroxylum novogranatense var. novogranatense (Enn) and Erythroxylum novogranatense var. truxillense (Ent) are cultivated primarily for the illicit extraction and processing of cocaine. Despite their economic and medical importance, the evolutionary history of these species remains unknown in a modern phylogenetic framework. The aims of this study were to: (a) investigate the relationship among the cultivated and a select number of non-cultivated taxa, and (b) test Plowman’s (Journal of Psychodelic Drugs 11:103–117, 1979b) linear progression hypothesis of the cultivated Erythroxylum taxa versus Johnson’s et al. (Annals of Botany 95:601–608, 2005) hypothesis that Ec and En are sister species. AFLP phylogeny was used to compare the relationships among 36 Erythroxylum species (133 accessions) spanning the geographic distribution of the genus. A Maximum Parsimony tree revealed both geographic and taxonomic partitioning into clades representing species from Africa, Asia-Pacific and the New World (Tropical Americas). Ec and En formed distinct clades, indicating they are sister species and a cluster of non-cultivated species were the most closely related to the cultivated species. Multivariate ordination analysis was used to evaluate the relationship between cultivated and non-cultivated Erythroxylum taxa from the Tropical Americas. Our results support the hypothesis that the cultivated species are more closely related to each other than to any other species of Erythroxylum, but refute the hypothesis that Ent (and Enn) descended from Ecc. Instead our data suggest an independent, non-linear evolutionary relationship between Ec and En. Finally, the AFLP analyses identified significantly different genetic groups within Erythroxylum suggesting that the current intrageneric classification of this genus be revised.

The Effects of Ambient Solar UV Radiation on Alkaloid Production by Erythroxylum novogranatense var. novogranatense†

Truxillines are alkaloids produced by Erythroxylum species and are thought to be derived from the UV-driven dimerization of cinnamoylcocaines. This study was conducted to determine the effects of ambient UV radiation on the production of truxillines in Erythroxylum novogranatense var. novogranatense. Field plants were grown under shelters covered with plastic filters that were transparent to UV radiation, filtered UV-B, or both filtered UV-B and UV-A radiation. The treatments had no significant effect on plant biomass or specific leaf weight. Absorption values in the UV-C and UV-A region of acidified-methanol leaf extracts were higher for plants exposed to UV radiation compared to the no UV radiation treatment. There was a trend in decreasing levels of trans-cinnamoylcocaine and a statistically significant decrease in levels of cis-cinnamoylcocaine in the leaves of plants exposed to UV radiation compared to the no UV radiation treatment. Truxilline levels increased in leaves from plants exposed to UV radiation compared to the no UV radiation treatment. Most significantly, the ratio of truxillines to total cinnamoylcocaines in the leaves was affected by UV, increasing with increased UV exposure. The results support the hypothesis that UV radiation is involved in the formation of truxillines from cinnamoylcocaines.

Apparent Effects of Glyphosate on Alkaloid Production in Coca Plants Grown in Colombia

During the routine analysis of coca leaf material from South America, alkaloids in Erythroxylum coca var. ipadu (ECVI) leaf samples from fields suspected of being treated with glyphosate were compared with those from non-treated E. coca var. ipadu and Erythroxylum novogranatense var. novogranatense (ENVN) plants. Cocaine levels in leaf tissue from non-treated ECVI and ENVN were 0.53+/-0.08% and 0.64+/-0.08% (w/w), respectively, whereas leaves from treated plants were nearly devoid of cocaine. Further analysis demonstrated the presence of several previously undescribed N-nortropane alkaloids, several of which were tentatively identified. The results suggest that applications of glyphosate to coca plants can have dramatic effects on the quantity and quality of alkaloids produced by surviving or subsequent leaves. The analytical data presented will be of value to forensic chemists who encounter illicit cocaine preparations containing alkaloids produced from coca plants treated with glyphosate.

Calystegines in wild and cultivated Erythroxylum species

Calystegines were identified in the genus Erythroxylum for the first time. Erythroxylum novogranatense var. novogranatense, a species cultivated for cocaine production, contained 0.2% total calystegines in dry leaves. Forty six Erythroxylum herbarium species consisting mostly of leaf tissue were analysed for calystegines, and 38 were found positive. Calystegines were compared qualitatively and quantitatively between individual Erythroxylum species. Calystegines A 3 and B 2 were the major calystegines in most species. Total calystegine content reached up to 0.32% dry mass. The simultaneous occurrence of calystegines, cocaine, other alkaloids of a 3α-hydroxy- or 3β-hydroxytropane structure together with nicotine supports the concept of common biosynthetic steps of these alkaloids in Erythroxylum. The present results are the basis for further investigations of the phylogenetic origin of tropane alkaloid biosynthesis in the taxonomically remote families Solanaceae and Erythroxylaceae.

Alkaloid Content in Two Erythroxylum Taxa During Juvenile Growth and Development

ABSTRACT Erythroxylum coca var. coca Lam. And Erythroxylum novogranatense var. novogranatense (Morris) Hieron (E.n. var. novogranatense) were grown in a controlled environment for 52 weeks to monitor the content of hygrine, tropinone, tropacocaine, methyl ecgonine, cocaine and cis- and trans-cinnamoylcocaine in seeds, plant parts, and organs during juvenile growth. Embryos and endosperms of var. coca contained cocaine while only embryos of var. novogranatense contained cocaine. Tropacocaine was present in hypocotyls, stems and roots of var. novogranatense, but not in var. coca. Trans-cinnamoylcocaine was the most abundant alkaloid in cotyledons (12 weeks after seeding) and leaves (24 weeks after seeding) in both var. coca and var. novogranatense. After 52 weeks of seeding growth, cocaine was the preeminent alkaloid in leaves of both taxons with a cocaine content of 0.66 percent in var. coca and 1.04 percent in var. novogranatense.

Flavonoids as markers for Erythroxylum Taxa

Leaf extracts of Erythroxylum coca var. ipadu Plowman ( E. c . var. ipadu ) produced five O -conjugates of taxifolin and one O -conjugate of quercetin flavonoids, while the equivalent extract from Erythroxylum novogranatense var. truxillense (Rusby) Plowman ( E. n . var. truxillense ) afforded four flavonoids: (a) the first, an O -conjugate of quercetin; (b) the second, an O -conjugate of fisetin; and (c) the third and fourth O -conjugates of kaempferol. The flavonols of E. n . var. truxillense contained 4′-OEt whereas in E. c . var. ipadu none was found. E. n . var. truxillense contained the flavonol fisetin which was not found in E. c . var. ipadu and has not been previously reported for the taxa. The major sugars in leaf extracts were acylated rhamnosides with either 7, 3 or 4′ linkage or combinations thereof. Flavonoids of the four cultivated Erythroxylum taxa, all of which contain the cocaine alkaloid, differ, and may be used as chemotaxonomic markers for the taxa.

Histochemical and Immunocytochemical Localization of Tropane Alkaloids in Erythroxylum coca var. coca and E. novogranatense var. novogranatense

The tropane alkaloids of Erythroxylum coca var. coca and Erythroxylum novogranatense var. novogranatense were localized using histochemical, cytochemical, and immunocytochemical probes. At the tissue level, these alkaloids were localized in leaves, stems, and fruits with Dragendorff's reagent. Alkaloids were found in the mesophyll, including palisade, spongy, and vascular parenchyma cell layers, and in some cells of the collenchyma. No alkaloids were detected in the epidermis of either Erythroxylum species. Alkaloids were also detected in the endosperm and embryos of orange and mature red fruits but not in tissues of immature green fruits. Quantitative HPLC reveals that embryos had 2.5-5 times more cocaine (w/w%) than endosperms, but 40 times less than leaves. In contrast to leaves, cocaine was a minor alkaloid present in fruits. Dragendorff's reagent was used to develop thin-layer chromatography plates and specifically stained the alkaloids extracted from leaves of both species. Complexing of alkaloids with tannins resulted in aggregates in the vacuole of E. coca leaf cells as visualized by transmission electron microscopy. Immunocytochemical localization, using anticocaine polyclonal antibodies on leaf tissues, proved that these compounds are localized in vacuoles of both photosynthetic and vascular parenchyma, as demonstrated by the use of Dragendorff's reagent. The alkaloids were associated with intravacuolar globules and appear to be aggregates with a core formed by phenolic compounds and a periphery enriched in alkaloids. The vacuolar localization of the cocaine alkaloids indicate that they may be complexed with phenols in vivo, thereby rendering them relatively immobile. The implications of the cellular and tissue localization of tropane alkaloids in Erythroxylum are discussed.

Effect of soil pH on mineral element concentrations of two erythroxylum species

Erythroxylum coca var. coca Lam. (E. coca) and Erythroxylum novogranatense var. novogranatense (Morris) Hieron (E. n. novogranatense) are two of four Erythroxylum species grown in the tropics of South America for cultural medicines and the alkaloid benzoylmethylecgonine. In a published study of biomass production over a soil pH range of 3.5 to 7.0, E. coca grew best at a pH equal to and below 5.5, and E. n. novogranatense grew best within the pH range of 4.7 to 6.0. Erythroxylum coca was tentatively classified as more tolerant to metal toxicities [aluminum (Al) and manganese (Mn)] than E. n. novogranatense, however, concentration patterns of mineral elements for E. coca and E. n. novogranatense tissue have not been reported, nor have the mechanisms of differential acid‐soil‐tolerance been elucidated. In the current study, the effects of soil pH on concentrations of Al, calcium (Ca), copper (Cu), iron (Fe), potassium (K), magnesium (Mg), Mn, and zinc (Zn) in leaves, stems, and roots were investigated. At pH 3.5, roots of both species accumulated high concentrations of Al that decreased as soil pH increased, however, there was no pH × species interaction. The highest concentration of Ca was found in the leaves of both species, however, E. coca accumulated more Ca as soil pH increased than did E. n. novogranatense. Manganese and Zn levels were highest in roots of both species (E. coca and E. n. novogranatense); levels in all tissues decreased with increasing pH. Manganese concentration was highest in roots of E. coca and Zn concentration was highest in tissues of E. n. novogranatense. Copper, Fe, K, and Mg levels were erratic with increasing pH, indicating that sufficient amounts of these nutrients are acquired at low pH levels. Root concentrations of Fe and K in E. coca increased markedly between pH 3.5 and 4.7. At pH 3.5, E. coca demonstrated no symptoms of mineral deficiency and/or toxicity, however, chlorosis, leaf distortion and root atrophy were prevalent at pH 6.5 and 7.0. By contrast, E. n. novogranatense demonstrated diminished growth and root atrophy at soil pH 3.5, whereas at pH 6.5 and 7.0, although biomass production was reduced, no symptoms of mineral deficiency and/or toxicity were present. The species obviously behave differentially at pH extremes and E. coca appears to be most tolerant of extremely acid soils; the two species may also differ in mineral sensitivities between the species at higher pH levels. Erythroxylum coca may compete more effectively with Al for Ca binding sites within the root, and may have greater internal tolerance of Mn, compared with E. n. novogranatense.

Leaf Flavonoids as Chemotaxonomic Markers for Two Erythroxylum Taxa

Leaf extracts of Erythroxylum coca var. coca Lam. (E. c. var. coca) yielded six O-eonjugates of Eriodictyol flavonoids, while the equivalent extracts from Erythroxylum novogranatense var. novogranatense (Morris) Hieron (E. n. var. novogranatense) contained five flavonoids, two of which were O-conjugates of Luteolin and three were O-conjugates of Kaempferol. All six of E. c. var. coca methanolic extracted peaks (resolved by HPLC) were found to have a 2, 3 single bond, which in E. n. var. novogranatense is replaced by a 2- hydroxy allene. The other primary difference in the predominant flavonoids between these taxa is the chemical composition of the sugar and/or acyl O-conjugation and site(s) at which this conjugation occurred. The results suggest that the most abundant O-conjugated flavonoids of E. c. var. coca and E. n. var. novogranatense may be used as chemotaxonomic markers for the two taxa. Therefore, the O-conjugated peaks of Eriodictyol , are distinct chemotaxonomic markers for E. c. var. coca and the O-conjugated peaks Luteolin and Kaempferol for E. n. var. novogranatense. These taxa are two of the four cultivated Erythroxylum taxa that contain commercial quantities of the cocaine alkaloid in their leaves, this entity also sets apart the taxa from other members of Erythroxylum. We suggest that the biochemistry of flavonoids of other Erythroxylum taxa may also be species selective

Effects of Temperature and Light Levels on Leaf Yield and Cocaine Content in Two Erythroxylum Species

Published information on the response of Erythroxylumcrops to temperature and photosynthetic photon flux density (PPFD) is more descriptive than quantitative. The objective of this study was to quantify the effects of temperature and PPFD on leaf growth and cocaine content in the major cocaine-producing species. Plants of Erythroxylum cocavar. coca (Coca) and Erythroxylum novogranatensevar. novogranatense (Novo) were grown in artificially-lighted controlled environment chambers with a 12h photoperiod and at day/night temperatures of 20/16, 25/21, 30/26 or 35/31°C and at PPFDs of 155, 250 or 400μmolm −2s −1for 53d before leaves were harvested for dry weight and cocaine concentration determinations. Subsequently, chamber temperatures were altered to provide constant day/night temperatures of 19, 23 or 27°C. Plants were grown for 180d under these conditions and harvested a second time. Leaf yields in response to temperature were best expressed as quadratic functions. The optimum average daily temperature for leaf growth was near 27°C in both species. Novo was more vegetatively vigorous than Coca. Leaf mass at the first harvest was lowest in plants grown under 155μmol m −2s −1for both species. At the second harvest the only change was that there was no difference in leaf mass between 155 and 250μmol m −2s −1in Coca. Leaf cocaine concentration was not affected by PPFDs<400μmol m −2s −1but was affected by temperature. In Coca, leaf cocaine concentration was maximum at a mean daily temperature of 24°C at the first harvest and at 19°C at the second harvest. In Novo, leaf cocaine concentration was maximum at a mean daily temperature of 25°C at the first harvest but there was no effect of temperature at the second harvest. Coca leaves had higher cocaine concentration than Novo leaves at all temperatures at the first harvest but at the second harvest, there was no significant difference in leaf cocaine concentration between species except in the lowest temperature treatment when leaf cocaine concentration was higher for Coca. Cocaine production on a per plant basis was largely a function of leaf mass.

Biomass accumulation and alkaloid content in leaves of Erythroxylum coca and Erythroxylum novogranatense var. novogranatense grown in soil with varying pH

Summary Erythroxylum coca var. coca Lam. ( E. coca ) and Erythroxylum novogranatense var. novogranatense (Morris) Hieron ( E. novo, novogranatense ) were grown in greenhouse pots of Monmouth sandy loam, over a pH range of 3.5 through 7.0, to determine patterns for biomass accumulation and the content of hygrine, tropinone, methyl ecgonine, cuscohygrine, tropacocaine, cocaine, cis - and trans -cinnamoylcocaine in their leaves. Optimal growth for E. coca occurred at pH 3.5, and for E. novo. novogranatense at pH 4.7 through 6.0. The pH extreme (sub-optimal biomass accumulation, above and below optimal pH) for E. coca was ≥6.5 and for E. novo, novogranatense ≤ 3.5 and ≥ 6.5. Hence, E. coca is tentatively termed a calcifuge (Al-tolerant, Fe-inefficient) while E. novo. novogranatense more nearly resembles a calcicole (Fe-efficient, Alsensitive). Increases in leaf alkaloid content observed at the pH extremes for E. coca and E. novo, novogranatense appeared to be caused by plant stress. E. novo. novogranatense was more tolerant to the high pH extreme than E. coca . Twenty-two months after transplanting, 55% of E. coca plants grown at soil pH 7.0 died, and those that remained were chlorotic. In addition, among E. coca plants grown at soil pH 6.5, only 55 % were alive at the third harvest. Cocaine concentration in E. coca was independent of leaf biomass. Leaves of E. coca grown in soil at pH 3.5 through 6.0 had higher concentrations of cocaine than those of E. novo. novogranatense . Conversely, leaves of E. novo. novogranatense contained a higher concentration of trans - and cis -cinnamoylcocaine and methyl ecgonine than those of E. coca . The least abundant alkaloid observed in E. coca and E. novo. novogranatense was tropinone which was low or commonly not detected. Of the eight alkaloids monitored in leaves of E. novo. novogranatense grown in soils at various pH levels, methyl ecgonine was the most abundant.

1-Hydroxytropacocaine: An abundant alkaloid of Erythroxylum novogranatense var. Novogranatense and var. Truxillense

A new alkaloid, 1-hydroxytropacocaine, was isolated from leaves of greenhouse-cultivated Erythroxylum novogranatense var. novogranatense and identified. Quantitative levels of this alkaloid in dry leaf were similar to those for cocaine, i.e. 0.3–0.5% w/w. 1-Hydroxytropacocaine was also detected at 0.04–0.07% w/w (relative to dry coca leaf) in greenhouse-cultivated Erythroxylum novogranatense var. truxillense and at similar levels in Erythroxylum novogranatense var. novogranatense, grown at a tropical site other than in South America. The presence of 1-hydroxytropacocaine was <0.01% w/w (relative to dry coca leaf) in suspected Erythroxylum novogranatense var. novogranatense and Erythroxylum coca var. coca, field-cultivated in Colombia and Bolivia, respectively.

Estimating Leaf Mass from Light Interception Measurements on Isolated Plants of Erythroxylum Species

AbstractTo determine the extent of coca (Erythroxylum spp.) production in the world, some method for estimating yield (leaf mass) in a variety of habitats is required. A computer model that simulates the growth of coca plants has the versatility for making these estimates, but there must be some reliable method to validate the model. The objective of this research was to validate the use of measurements of light interception and specific leaf area (SLA, m2 kg−1) for estimating yield. Leaf area was calculated from light interception measurements made on plants grown as hedgerow or as isolated plants, then converted to leaf mass using measurements of SLA. Light interception was measured with the Decagon Sunfleck Ceptometer (SC) and the LI‐COR Plant Canopy Analyzer (PCA). Leaf disks of known area from Erythroxylum coca var. coca and Erythroxylum novogranatense var. novogranatense were cut, dried, and weighed to determine SLA values. Leaf fresh weights were measured and corrected to dry weight using the fresh to dry weight ratio obtained from a sample at harvest. Estimated leaf mass regressed on observed leaf mass demonstrated that PCA was superior to SC in estimating leaf mass on isolated plants of various sizes using the techniques described in this paper. Leaf area was consistently underestimated by the SC. The estimates of leaf mass from the PCA gave the lower mean, absolute mean, and root mean square error terms compared with SC, and the slope of the regression line of observed leaf mass vs. predicted leaf mass was closest to a value of 1.0.

Determination of Cocaine in “Mate de Coca” Herbal Tea

It came to our attention that an herbal tea, sold under the name of Health Inca Tea or “Mate de Coca”, has been available commercially in this country since 1983. This tea is produced and packed in Peru from the leaves of Erythroxylum novogranatense var. truxillense1 by “ENACO” (National Enterprise of Coca). The sellers claim that the product has been decocainized in the factory, which is not the case.1 The selling of this product is illegal under the provisions of the Control Measures of the 1961 Single Convention of the United Nations because it contains cocaine and, therefore, carries a potential for abuse. In 1986, Elsohly et al.2 found cocaine metabolites in the urine of an individual after consumption of only one cup of Health Inca Tea. The same year, Siegel et al.3 reported the availability of Health Inca Tea and “Mate de Coca” in the United States and reported cases of abuse.

Biosystematics and Evolution of Cultivated Coca (Erythroxylaceae)

ABSTRAcr. Erythroxylum coca vars. coca and ipadu and E. novogranatense vars. novogranatense and truxillense are closely related taxa cultivated in South America for their cocaine-bearing leaves. Taxonomically they have been variously treated, ranging from one to three separate species. Erythroxylum novogranatense var. truxillense is morphologically somewhat intermediate between E. c. var. coca and E. n. var. novogranatense and has been suggested to be a hybrid between the two. Evidence from artificial hybridizations, leaf flavonoid chemistry, and breeding system studies was used to clarify the taxonomic and evolutionary relationships of these plants. This evidence suggests that E. n. var. truxillense is intermediate between E. coca and E. n. novogranatense but it is not a hybrid between them. These three taxa represent a linear evolutionary series, with E. c. var. coca the ancestral taxon, E. n. var. truxillense derived from it, and E. n. var. novogranatense derived from E. n. var. truxillense. Eythroxylum c. var. ipadu was independently derived from E. c. var. coca. All of the taxa are partially isolated genetically except E. c. var. coca and E. c. var. ipadu. The taxonomic treatment that best reflects their relationships is to regard E. novogranatense var. novogranatense and E. n. var. truxillense as varieties of a species distinct from E. coca. Only limited material of E. c. var. ipadu was available for study, but it might better be regarded as a cultivar of E. coca.