Cocaine and Cinnamoylcocaine Content of Erythroxylum Species

The dried leaves of 31 species and two varieties of the genusErythroxylum were quantitatively analysed for cocaine and bothcis- and trans-cinnamoylocaine, using a stable-isotope dilutionmethod and gas chromatography-mass spectrometry selected-ionmonitoring. Both cocaine and cinnamoylcocaine were detectedin all four varieties of cultivated coca (E. coca var. coca,E. coca var. ipadu, E. novogranatense var. novogranatense andE. novogranatense var.truxillense). Cinnamoylcocaines (cis andtrans) were found in much higher concentrations in both varietiesof E. novogranatense than in either variety of E. coca. Amazoniancoca (E. coca var. ipadu) contained consistently lower cocainelevels than the montane variety (E. coca var. coca). Twenty-ninewild species of Erythroxylum, selected to represent morphological,ecological and taxonomic diversity, were analysed for the samealkaloids; cocaine was detected in 13 neotropical species, representingfive sections of the genus. No cocaine was detected in the OldWorld species. Two wild species from Venezuela, E. recurrensand E. steyermarkii, contained cocaine levels comparable tothose found in the commercially cultivated species. Erythroxylaceae, Erythroxylum, coca, cocaine, cinnamoylcocaine, alkaloids, chemotaxonomy     

Determination of cocaine in some south american species of Erythroxylum using mass fragmentography

Thirteen South American species of Erythroxylum have been analyzed for their cocaine content. Cocaine was found only in E. coca Lam., E. novogranatense (Morris) Hieron. and E. novogranatense var. truxillense (Rusby) Machado. The amount of cocaine was determined by mass fragmentography using deuterium labelled cocaine as internal standard     

Content and Distribution of Erythroxylum coca Leaf Alkaloids

Fully expanded leaves of Erythroxylum coca var. coca Lam. (Erythroxylaceae)35-70-d-old were harvested from 21-month-old plants grown undergreenhouse conditions. Each harvested leaf was placed on a plexiglasssilhouette according to its dimensions and divided into fourprimary sections (petiole, base, mid and anterior) and threesubsections (lamina periphery, false mid-rib, and true mid-rib)to determine the distribution and content of hygrine, cuscohygrine,trans-cinnamoylcocaine, cis -cinnamoylcocaine, tropacocaine,tropinone, methyl ecgonine and cocaine. The leaf sub-sectionsand petiole were extracted and analysed for alkaloid content(%) by HPLC (cocaine alkaloid only) gas chromatography and GC/MS.Cocaine, methyl ecgonine and hygrine were highest in the laminaperiphery with a content of 0·48, 0·46 and 0·32%,respectively. Trans-cinnamoylcocaine was pre-eminent of thecinnamoylcocaines and was most abundant in the petiole at acontent of 0·24%. Cis-cinnamoylcocaine, tropinone, cuscohygrineand tropacocaine were ubiquitously distributed throughout theleaf where the average contents were 0·14, 0·004,0·16 and 0·04%, respectively.Copyright 1995, 1999Academic Press Alkaloids, Erythroxylum coca var. coca, E. coca, leaf alkaloids, petiole alkaloids, cocaine, cuscohygrine, methyl ecgonine, hygrine, tropinone, tropacocaine, trans-cinnamoylcocaine, cis-cinnamoylcocaine     

Variation of Alkaloid Content in Erythroxylum coca Leaves from Leaf Bud to Leaf Drop

A sequential study describing the content (%) of alkaloids inleaves of Erythroxylum coca var. coca Lam. from leaf bud developmentto leaf drop has not previously been conducted. The length oftime the leaf resides on the plant and its concurrent metabolicactivity also has not been defined. In the present study, cocaine,methyl ecgonine, hygrine, tropinone, trans -cinnamoylcocaine,cis-cinnamoylcocaine, tropacocaine and cuscohygrine were monitoredto determine: (1) their content and patterns of accumulationfrom leaf bud to leaf drop; (2) the time leaves resided on theplant; and (3) whether leaves were metabolically active untilleaf drop. E. coca plants were grown in a controlled environmentfor 37 months. Leaves similar in chronological age were extractedand analysed for alkaloid content by gas chromatography (GC)and gas chromatography/mass spectrometry (GC/MS). Cocaine washighest in 7 d old rolled leaves (0·75%) and declinedto 0·39% at leaf drop. Cocaine, methyl ecgonine, hygrine,tropinone, trans -cinnamoylcocaine, cis-cinnamoylcocaine, cuscohygrineand tropacocaine content in 35 d old (mature) leaves was 0·61,0·59, 0·68, 0·08, 0·31, 0·55,0·52, and 0·05%. respectively. Cocaine, methylecgonine, hygrine, cis -cinnamoylcocaine, and cuscohygrine displayeda gradual decline from week 2 to week 36 of leaf duration. Tropinoneand tropacocaine were the least abundant of the alkaloids monitored.Cis-cinnamoylcocaine content exceeded cocaine at week 12, 16,and weeks 19 to 23 of leaf duration. Trans -cinnamoylcocainewas highest in rolled leaves (week 1) and in expanded leavesafter week 30. The monitored alkaloids appeared to be part ofthe metabolically active pool of the leaf. Leaves remained onthe E. coca plants for 36 weeks.Copyright 1994, 1999 AcademicPress Cocaine, methyl ecgonine, hygrine, tropinone, trans-cinnamoylcocaine, cis-cinnamoylcocaine, cusco-hygrine, tropacocaine, leaf bud, rolled leaves, expanded leaves, alkaloids, patterns, fluctuation, Erythroxylum coca var. coca, E, coca     

Inter- and intra-specific variation among five Erythroxylum taxa assessed by AFLP

BACKGROUND: and Aims The four cultivated Erythroxylum taxa (E. coca var. coca, E. novogranatense var. novogranatense, E. coca var. ipadu and E. novogranatense var. truxillense) are indigenous to the Andean region of South America and have been cultivated for folk-medicine and, within the last century, for illicit cocaine production. The objective of this research was to assess the structure of genetic diversity within and among the four cultivated alkaloid-bearing taxa of Erythroxylum in the living collection at Beltsville Agricultural Research Center. METHODS: Amplified fragment length polymorphism (AFLP) fingerprinting was performed in 86 Erythroxylum accessions using a capillary genotyping system. Cluster analysis, multidimensional scaling (MDS) and analysis of molecular variance (AMOVA) were used to assess the pattern and level of genetic variation among and within the taxa. KEY RESULTS: A clear distinction was revealed between E. coca and E. novogranatense. At the intra-specific level, significant differentiation was observed between E. c. var. coca and E. c. var. ipadu, but the differentiation between E. n. var. novogranatense and E. n. var. truxillense was negligible. Erythroxylum c. var. ipadu had a significantly lower amount of diversity than the E. c. var. coca and is genetically different from the E. c. var. ipadu currently under cultivation in Colombia, South America. CONCLUSIONS: There is a heterogeneous genetic structure among the cultivated Erythroxylum taxa where E. coca and E. novogranatense are two independent species. Erythroxylum coca var. coca is most likely the ancestral taxon of E. c. var. ipadu and a founder effect may have occurred as E. c. var. ipadu moved from the eastern Andes in Peru and Bolivia into the lowland Amazonian basin. There is an indication of artificial hybridization in coca grown in Colombia.     

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?C117, 1979b) linear progression hypothesis of the cultivated Erythroxylum taxa versus Johnson??s et al. (Annals of Botany 95:601?C608, 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.     

Content and De Novo Synthesis of Cocaine in Embryos and Endosperms from Fruit of Erythroxylum coca Lam

There is controversy as to whether the mature fruit of Erythroxylumcoca var. coca Lam. contains the cocaine alkaloid (benzoylmethylecgonine).In the present study, cocaine was monitored to determine ifit was present in embryos and endosperms of mature fruit ofE. coca var. coca Lam., and if present, the time required forde novo synthesis in imbibing seed. Seeds from mature fruitof E. coca were dissected to separate the embryos from the endosperms.The separated embryos and endosperms were analysed for cocaine.Subsequently, endosperms and embryos from seed imbibed. undera light and dark treatment were separated on days 3, 6, 9, 12and 15 and analysed for cocaine. Cocaine was present in embryos(0.005% of d. wt) and endosperms (0–001% of d. wt) ofmature fruit of E. coca. De novo synthesis of cocaine occurredonly in embryos of seed imbibed under light after day 9 of imbibition. Erythroxylum coca, alkaloid, benzoylmethylecgonine, cocaine, embryo, endosperm, seed imbibition     

Identification of Erythroxylum taxa by AFLP DNA analysis

Erythroxylum coca, indigenous to the Andean region of South America, is grown historically as a source of homeopathic medicine. However, in the last century, cultivation of E. coca and several closely-related species for the production of illicit cocaine has become a major global problem. Two subspecies, E. coca var. coca and E. coca var. ipadu, are almost indistinguishable phenotypically; a related cocaine-bearing species also has two subspecies (E. novogranatense var. novogranatense and E. novogranatense var. truxillense) that are phenotypically similar, but morphologically distinguishable. The purpose of this research was to discover unique AFLP DNA patterns ("genetic fingerprinting") that characterize the four taxa and then, if successful, to evaluate this approach for positive identification of the various species of coca. Of seven different AFLP primer pairs tested, a combination of five proved optimal in differentiating the four taxa as well as a non-cocaine-bearing species, E. aerolatum. This method of DNA fragment separation was selective, and faster, for coca identification, compared with analyses based on flavonoid chemotaxonomy. Using the 5-primer AFLP approach, 132 known and unknown coca leaf accessions were evaluated. Of these, 38 were collected in 1997-2001 from illicit coca fields in Colombia, and all were genetically differentiated from coca originating in Peru and Bolivia. Based on the DNA profiling, we believe that the Colombian coca now represents a hybridization of E. coca var. ipadu. Geographical profiling within Colombia also seems feasible as new coca production areas are developed or new types of coca are introduced within traditional growing areas.     

Flavonoids as chemotaxonomic markers for Erythroxylum australe

Methanolic leaf extracts of Erythroxylum australe F. Muell. produced eight O-conjugated flavonoids. Six of the flavonoid aglycones were dihydroisoflavones (all dihydro-orobol derivatives), one a flavanone, eriodictyol, and one a flavonol, quercetin. The major glycosides of the flavonoids included mono-glucosyl-rhamnosyls and dirhamnosyl-glucosides with either 3, 5, 7 or 3', 4' linkage or a combination thereof The array of flavonoids present in E. australe suggests kinship to E. ulei and linkage to the four cultivated alkaloid-bearing Erythroxylum, especially the ancestral E. coca var. coca. Because of the uniqueness of the flavonoids present in leaf tissue of E. australe they are unambiguously useful as chemotaxonomic markers for the taxon.     

Flavonoid glycosides from needles of Pinus massoniana

Seven flavonoids have been isolated from Pinus massoniana needles and identified as taxifolin and its 3′-O-β-D-glucopyranoside, (+)-catechin, naringenin-7-O-β-D-glucopyranoside and three new flavonoid glycosides, 6-C-methylaromadendrin 7-O-β-D-glucopyranoside, taxifolin 3′-O-β-D-(6″-O-phenylacetyl)-glucopyranoside and eriodictyol 3′-O-β-D-glucopyranoside.     

Chemophenetics of Azorean Leontodon taxa (Cichorieae,Asteraceae)

The genera Leontodon s.str. and Hedypnois are so far the only known sources of hydroxyhypocretenolides, a rare subclass of guaianolide type sesquiterpene lactones. In this study the three endemic species from the Azorean Archipelago, L. filii, L. hochstetteri, and L. rigens, were analyzed together with L. hispidus and L. × grassiorum, a hybrid originating from L. hispidus and L. hochstetteri. Flowering heads were analyzed by UHPLC-DAD-MS with regards to their phenolics' profiles, establishing qualitatively identical profiles for all taxa. The following phenolics were detected in flowering heads of all investigated taxa: caffeoyltartaric acid, cichoric acid, chlorogenic acid, 3,5-di-O-caffeoylquinic acid, luteolin, luteolin 7-O-β-d-glucopyranoside, luteolin 4′-O-β-d-glucopyranoside, and luteolin 7-O-β-d-glucuronide.In UHPLC-DAD-MS analyses of the rhizomes, no flavonoids were detected. In rhizomes, caffeoyltartaric acid was only detected in L. hispidus. However, in addition to caffeoylquinic acid derivatives already found in the flowering heads, 1,5-di-O-caffeoylquinic acid, 3,4-di-O-caffeoylquinic acid, and 4,5-di-O-caffeoylquinic acid were detected in rhizomes of all investigated taxa.The chemophenetically most interesting group of hydroxyhypocretenolides was detected in rhizomes of all investigated taxa. 11,13β-Dihydro-14-dihydroxyhypocretenolide was detected in L. filii and L. hochstetteri, while 11,13β-dihydro-14-hydroxyhypocretenolide-β-d-glucopyranoside was present in all Azorean taxa. 1,10-Epoxy-14-hydroxyhypocretenolide-β-d-glucopyranoside and 1,10-epoxy-14-hydroxyhypocretenolide-β-d-glucopyranoside-6′-O-p-hydroxyphenylacetic acid ester were restricted to the Azorean taxa and the hybrid L. × grassiorum, while the dimeric sesquiterpenoid 14-hydroxyhypocretenolide-β-d-glucopyranoside-4′,14″-hydroxyhypocretenoate ester was restricted to L. hispidus and L. × grassiorum.     

Leaf flavonoids from Croton urucurana and C. floribundus (Euphorbiaceae)

Flavonoids were isolated by PVPP column chromatography of leaf extracts of Croton floribundus Spreng and C. urucurana Baill. and identified by NMR and co-chromatography with standards. The two species revealed highly distinct flavonoid profiles. C. urucurana, belonging to the phylogenetically basal section Cyclostigma, yielded the flavone C-glycosides vitexin and orientin, quercetin and the O-glycosides quercetin 7-O-rhamnoside, rhamnitrin and rutin, in addition to tiliroside. Instead, C. floribundus, from the more derived section Lasiogyne, yielded no C-glycosides, but a high diversity of classes of flavonols, including kaempferol, three flavonol O-methyl ethers, isoquercitrin, three tri-O-galactosides, in addition to tiliroside and an isorhamnetin-coumaroyl-O-glycoside. The present work is the first report for Croton of two rhamnosides (isolated from C. urucurana). It is also the first report for Euphorbiaceae of two tri-O-glycosides obtained from C. floribundus. The distribution of flavonoids in the two species as determined by HPLC-DAD of extracts of small leaf samples of herbarium specimens is highly similar with the profiles resulting from isolation of compounds from bulky leaf samples. Differences among specimens of the same species were restricted to relative proportions of individual constituents. The results indicate that flavonoid profiles are effective to characterize and distinguish the two species. The present results, combined with literature data, supports the condition of tiliroside as a marker of Croton and the hypothesis of an evolutionary trend in the genus toward the loss of C-glycosides and a progressive complexity of flavonoid profiles.     

Fate of cocaine in the lymantriid Eloria noyesi, a predator of Erythroxylum coca

Larvae of the lymantriid moth Eloria noyesi, which are obligate feeders on Erythroxylum coca, excrete most of the ingested cocaine as unchanged base. Cocaine, analysed by mass fragmentography, is readily, detectable in the blood of larvae and is presumably sequestered during larval feeding, since it is present in the bodies of adult moths that do not feed on E. coca. Cocaine is an effective feeding deterrent for the ant Monomorium pharaonis when present at a concentration below that found in the leaves of E. coca.     

Antitermitic and antioxidant activities of heartwood extracts and main flavonoids of Hymenaea stigonocarpa Mart.

Hymenaea stigonocarpa, is an endemic tree from the Brazilian cerrado (savannah) and popularly known as jatobá-do-cerrado. The wood of this species is resistant to biodegradation and is used in naval and civil construction. Cuttings from heartwood of this species showed resistance to fungi and termites. In this paper, we report the antitermitic and antioxidant activities of H. stigonocarpa heartwood ethyl acetate extract and its main flavonoids constituents, hultenin (1), taxifolin (2), quercetin (3) and 7-methoxycathequin (4). The structure elucidation of the isolated flavonoids was performed by spectroscopic methods. The ethyl acetate extract possesses highest antitermitic and antioxidant activity when compared with isolated flavonoids.     

Leaf flavonoids of eupomatiaceae

The leaf flavonoids of Eupomatia bennettii and E. laurina were examined and five flavonoid compounds were detected. The most distinctive of these compounds were two methylated flavones: 7-O-methylapigenin and 7,3′-di-O-methylluteolin (velutin). The flavonoids of Eupomatiaceae are most similar to those of the Winteraceae and this similarity may be indicative of a phylogenetic relationship between the two families.     

Coca chewing for exercise: hormonal and metabolic responses of nonhabitual chewers

Favier, Roland, Esperanza Caceres, Laurent Guillon, BrigitteSempore, Michel Sauvain, Harry Koubi, and Hilde Spielvogel. Cocachewing for exercise: hormonal and metabolic responses of nonhabitualchewers. J. Appl. Physiol. 81(5):1901-1907, 1996.To determine the effects of acute coca use onthe hormonal and metabolic responses to exercise, 12 healthynonhabitual coca users were submitted twice to steady-state exercise(~75% maximal O₂ uptake). Onone occasion, they were asked to chew 15 g of coca leaves 1 h beforeexercise, whereas on the other occasion, exercise was performed after 1 h of chewing a sugar-free chewing gum. Plasma epinephrine,norepinephrine, insulin, glucagon, and metabolites (glucose, lactate,glycerol, and free fatty acids) were determined at rest before andafter coca chewing and during the 5th, 15th, 30th, and 60th min ofexercise. Simultaneously to these determinations, cardiorespiratoryvariables (heart rate, mean arterial blood pressure, oxygen uptake, andrespiratory gas exchange ratio) were also measured. At rest, cocachewing had no effect on plasma hormonal and metabolic levels exceptfor a significantly reduced insulin concentration. During exercise, theoxygen uptake, heart rate, and respiratory gas exchange ratio weresignificantly increased in the coca-chewing trial compared with thecontrol (gum-chewing) test. The exercise-induced drop in plasma glucoseand insulin was prevented by prior coca chewing. These results contrastwith previous data obtained in chronic coca users who display duringprolonged submaximal exercise an exaggerated plasma sympatheticresponse, an enhanced availability and utilization of fat (R. Favier,E. Caceres, H. Koubi, B. Sempore, M. Sauvain, and H. Spielvogel.J. Appl. Physiol. 80: 650-655, 1996). We conclude that, whereas coca chewing might affect glucose homeostasis during exercise, none of the physiological data provided bythis study would suggest that acute coca chewing in nonhabitual userscould enhance tolerance to exercise.

     

Archaeological evidence of coca (Erythroxylum coca,erythroxylaceae) in the upper mantaro valley,Peru

Prehistoric remains of coca (Erythroxylum spp.) are rarely uncovered by archaeologists or positively identified by botanists because of their fragile nature and the lack of rigorous archaeological collection techniques. This absence of plant evidence has made evolutionary studies of diffusion and use of coca difficult. From special depositional conditions in the Mantaro area of central Peru, one coca leaf and two endocarps have been uncovered and identified as Erythroxylum coca var. coca. These three specimens came from elite-status contexts dating to the Late Intermediate and the Late Horizon-Early Colonial Periods. These remains provide the first highland evidence for access to coca-producing, ceja de montaña farms, which lie more than 50 km away on the eastern slope of the Andes.     

Degradation of Quercetin and Luteolin by Eubacterium ramulus

The degradation of the flavonol quercetin and the flavone luteolin by Eubacterium ramulus, a strict anaerobe of the human intestinal tract, was studied. Resting cells converted these flavonoids to 3,4-dihydroxyphenylacetic acid and 3-(3,4-dihydroxyphenyl)propionic acid, respectively. The conversion of quercetin was accompanied by the transient formation of two intermediates, one of which was identified as taxifolin based on its specific retention time and UV and mass spectra. The structure of the second intermediate, alphitonin, was additionally elucidated by ¹H and ¹³C nuclear magnetic resonance analysis. In resting-cell experiments, taxifolin in turn was converted via alphitonin to 3,4-dihydroxyphenylacetic acid. Alphitonin, which was prepared by enzymatic conversion of taxifolin and subsequent purification, was also transformed to 3,4-dihydroxyphenylacetic acid. The coenzyme-independent isomerization of taxifolin to alphitonin was catalyzed by cell extract or a partially purified enzyme preparation of E. ramulus. The degradation of luteolin by resting cells of E. ramulus resulted in the formation of the intermediate eriodictyol, which was identified by high-performance liquid chromatography and mass spectrometry analysis. The observed intermediates of quercetin and luteolin conversion suggest that the degradation pathways in E. ramulus start with an analogous reduction step followed by different enzymatic reactions depending on the additional 3-hydroxyl group present in the flavonol structure.