Caffeine and theobromine analysis of Paullinia yoco, a vine harvested by indigenous peoples of the upper Amazon

by Luke M. Weiss, MF 2015¹ & James K. Kearns, PhD²

Abstract

Paullinia yoco R.E. Schult. & Killip (Sapindaceae) or ‘yoco’, is a liana (a woody vine) used traditionally by indigenous peoples of the upper Amazon region as a stimulating beverage, because it contains both caffeine and theobromine. However, as the wild plants become increasingly rare and hard to find because of permanent settlement within a much-reduced territory, there is growing interest among indigenous tribes to learn how to successfully cultivate it for community consumption and potentially as a cash crop. To date, there has been very little research conducted on the stimulant chemical composition of the liana and biochemical variation within- and between plants.

This paper provides the results of chemical (high-performance liquid chromatography) analysis of caffeine and theobromine concentrations of multiple samples of phloem and leaf material from 18 Paullinia yoco lianas. Caffeine values ranged from 0.1% to 3.6% with a significantly higher amount in the stem phloem material than the leaf material, and there was a positive linear correlation between stem diameter and caffeine concentration (% dry weight). Although the highest theobromine level was found in leaf material (1.8%), overall there was no significant difference between phloem and leaf material, and no correlation between stem diameter and theobromine concentration. Finally, we found a significant amount of variance of both caffeine and theobromine between individual plants. These results are important, first because they shed light on the apparently conflicting previous reports on P. yoco caffeine and theobromine content; and second, they provide important clues about the phytochemical architecture of P. yoco which in turn has important implications for the design of a cultivation strategy for local indigenous communities to potentially produce a yet-to-be domesticated plant of substantial market potential.

Paullinia yoco R. E. Schult. & Killip (Sapindaceae) o ‘yoco’, es una liana (una enredadera leñosa) utilizado tradicionalmente por los pueblos indígenas de la región amazónica superior como una bebida estimulante, ya que contiene cafeína y teobromina. Sin embargo, como las plantas silvestres son cada vez más raros y difíciles de encontrar debido a asentamiento permanente en un territorio muy reducido, existe un creciente interés entre las tribus indígenas para aprender cómo cultivar con éxito para el consumo de la comunidad y, potencialmente, como cultivo comercial. Hasta la fecha, ha habido muy poca investigación realizada sobre la composición química estimulante de la liana y bioquímica intra-variación y entre plantas.

Este trabajo presenta los resultados del análisis química (cromatografía líquida de alta resolución) de las concentraciones de cafeína y teobromina de múltiples muestras de material floema y hoja de 18 lianas Paullinia yoco. Valores de cafeína variaron de 0,1% a 3,6% con una cantidad significativamente mayor en el material de floema vástago de que el material de hoja, y había una correlación lineal positiva entre el diámetro del tallo y la concentración de cafeína (% peso seco). Aunque el nivel teobromina más alta se encontró en el material de la hoja (1,8%), no hubo diferencia significativa entre el floema y el material de hoja, y no hay correlación entre el diámetro del tallo y la concentración de teobromina. Finalmente, se encontró una cantidad significativa de varianza de cafeína y teobromina, tanto entre los individuos. Estos resultados son importantes, primero porque arrojan luz sobre las aparentemente contradictorios informes anteriores sobre P. yoco cafeína y teobromina contenido; y segundo, que proporcionan importantes pistas sobre la arquitectura fitoquímico de P. yoco que a su vez tiene implicaciones importantes durante el diseño de una estrategia de cultivo para las comunidades indígenas locales para producir una planta todavía-a-ser domesticada del potencial de mercado importante.

Introduction

Stimulating beverages have been developed and are consumed by virtually all human cultures (Weinberg & Bealer 2001). Of the six caffeine-containing plant genera used as stimulants (Coffea, Camellia, Theobroma, Cola, Ilex, and Paullinia), Paullinia, a genera of woody vines or lianas in the Sapindaceae family, is the least studied (Weckerle et al. 2003). Two species within this genus have been found to contain purine Alkaloids (PuA): Paullinia cupana Kunth and Paullinia yoco R.E. Schult. & Killip. The seed of Paullinia cupana, more commonly known as guaraná, has received the most attention because of its economic importance as the key ingredient in commercial beverages (Henman 1982), that include internationally marketed brands such as Red Bull^TM, and Sobe^TM, as well as many lesser known national (Brazilian) brands. Paullinia yoco, on the other hand, remains one of the most important cultural plants of the indigenous tribes of the Amazonian regions of Ecuador, Colombia, and Peru (Miño et al. 1995). The fresh phloem layer of the vine is rasped and infused in cold water (Fig. 1 and 2) and drunk in early morning for its stimulating properties, attributed to its composition of caffeine and theobromine (Schultes 1987). Richard Evans Schultes (1987) reported P. yoco to be “amongst the Indians of Colombia and Ecuador, the most important non-alimentary plant in the economy.” Paullinia yoco is used by most of the tribes and communities affiliated with the western Tukanoan linguistic family (Schultes 1942, 1951), as well as their neighboring Cofán and Ingáno tribes that represent other language families (Zuluaga 2004). All of these groups reside in the triple-border region of Ecuador, Peru, and Colombia, and all refer to this liana ubiquitously as ‘yoco’ (Belaunde & Echeverri 2001). In recent years however, a severe reduction in the extent of many indigenous territories from colonial encroachment, in tandem with other social and demographic changes, has led to a notable shortage of P. yoco in the wild. Traditional indigenous silvicultural management is no longer sufficient to maintain this plant within the current social and geographical context.

Fig. 1. A cross section of a Paullinia yoco cutting (4 cm diameter) showing the dark orange phloem layer and white resin. Credit: Luke Weiss.

As a means of addressing this imminent shortage, the Secoya indigenous nation of the province of Sucumbios, Ecuador, is interested in learning how to more successfully cultivate P. yoco to meet the demands of the local community and also potentially to create a marketable product. To assess the economic potential of P. yoco, a better understanding of the plant’s stimulant chemical concentration, the within-plant distribution of stimulant chemicals, and variance in chemistry between genetic provenances is critical. To date, very little research has been conducted on P. yoco, and worldwide only 40 herbarium specimens have been collected (Weckerle et al. 2003). The first reported chemical analysis was in 1926, where levels of caffeine were found to range between 2.0 and 2.7% (Michiels & Denis 1926). Another more recent study used previously collected herbarium specimens and found concentrations two orders of magnitude lower—median values for phloem material were 0.1% caffeine and 0.05% theobromine; and for leaf material were 0.03% caffeine and 0.03% theobromine. This contrast resulted in Weckerle et al. (2003) summarizing their findings by saying “clearly fresh stem material of various P. yoco provenances will be necessary to fully resolve the ‘yoco mystery.’”

Fig. 2. A Secoya elder, Delfin Payaguaje, during his morning ritual of rasping the phloem layer of Paullinia yoco into a gourd of cold water, which will be kneaded into a bitter beverage. Credit: Mitchell Anderson.

This research, therefore, aims to elucidate the ‘mystery’ of this Amazonian liana through the analysis of fresh stem material of 18 P. yoco provenances, with sampling from multiple stem diameters and vegetative parts, collected under the guidance of indigenous experts. Specifically, the following hypotheses will be addressed: (1) caffeine and theobromine concentrations from fresh material are higher than those found in herbarium material by Weckerle et al. (2003), (2) caffeine and theobromine concentrations are higher in phloem than in leaves and positively correlated to stem diameter, and (3) there is considerable between-plant variation in caffeine and theobromine content.

Methods and materials

Field collection

Plant material from 18 Paullinia yoco lianas was collected under the guidance of local expert indigenous guides within the indigenous territory of the Secoya Nation (SIEKOPAI) located in the north-eastern province of Sucumbios, Ecuador (Fig. 3). This region is a lowland tropical rain forest on the eastern toe slopes of the Andes mountains at approximately 300 m elevation and with an average annual rainfall of 3,555 mm and mean average temperature of 24°C (Miño et al. 1995). The sampling sites were distributed over an area of approximately 10,000 hectares of varzea flood-plain and terra firme forest types, with an average distance of 6,947 m between sampled individuals. Paullinia yoco vines were positively identified by indigenous guides. The outer layer of bark was scraped away, and sampling of the phloem layer was conducted with a small knife. Sample sizes ranged between 0.25 and 1.0 grams, and were collected at the vine’s base (approx. 50 cm from ground) and one to three other accessible regions. Liana diameter was recorded at each sampling point. Mature leaves were also collected when accessible. Plant samples were left to air dry during the course of the collection period; no preserving chemicals were applied to specimens.

Additionally, at each sampling site, the support tree identity (to genus level) and diameter at breast height (1.3m, DBH), forest type (varzea flood plain or terra firme) and GPS coordinates were recorded.

Fig. 3. Location of research site: Secoya Indigenous territory in the Province of Sucumbios, Ecuador.

Plant sample preparation

Upon return to Yale University, plant samples were freeze-dried to constant mass and homogenized into a powder-sized particulate using a freezer mill. Plant sample analysis closely followed the methodology of Weckerle et al. (2003). Extraction of the samples was carried out with 5 ml 0.1N HCl in a snap-cap Falcon tube vial for 30 minutes at 50 degrees Celsius. An aliquot of 500 ml of the extract was applied onto a Merck Extrelut NT 1-100 column for initial solid phase separation. After 5 minutes, elution was carried out with 5 ml methylene chloride (CH₂Cl₂). The organic solvent (CH₂Cl₂) was removed by a stream of N₂ gas, and the residue dissolved in 500 ml 8% MeOH. Aliquots of 50 ml were analyzed by high performance liquid chromatography (HPLC).

HPLC analysis

Separation was performed on a Capcell Type UG 120 5μm column; size: 4.6mm x 150mm, and no pre-column, with elluents H2O [A] and MeOH [B], both with 1% THF, at a total flow rate of 1 ml min^-1 and by the following gradient (%B over A): 0–10 min (8–25). Peaks were identified by using a UV detector set at the λmax = 272 nm. The retention times were determined by running separate pure standards of caffeine and theobromine and were as follows (min): theobromine (4.0), caffeine (9.0). The detection limit was determined by preparing authentic standards at decreasing concentrations until no significantly observable peak could be identified.

Calibration curves were established by preparing mixed standards of equal concentrations (by mass) of caffeine-theobromine at various concentrations ranging between 0.2 ppm and 200 ppm. Individual P. yoco samples (% dry weight) of caffeine and theobromine were calculated by multiplying the peak area of the plant sample chromatograms by the calculated coefficient derived from the calibration curves.

Results

Calibration curves

The calibration curves were as follows (Fig. 4). For caffeine (R2 = 0.998):

y = 0. 0025x

and theobromine (R2 = 0.9996):

y = 0. 0031x

Fig. 4. A) Calibration curve for caffeine derived from prepared samples of caffeine (Sigma 99.9%) dissolved in 8% MeOH. Prepared standard concentrations were (ppm): 0.02, 0.05, 0.1, 0.2, 2.0, 10.0, 20.0, 25.0, 35.0, 50.0, 75.0, 100.0, 150.0, 200.0. B) Calibration curve for theobromine derived from prepared samples of theobromine (Sigma 99.9%) dissolved in 8% MeOH. Prepared standard concentrations were (ppm): 0.05, 0.02, 0.2, 0.1, 1.0, 2.0, 10.0, 20.0, 25.0, 35.0, 50.0, 75.0, 100.0.

Chemical analysis of phloem and leaf material

A total of 36 samples of phloem material and 11 samples of leaf material representing 18 individual P. yoco lianas were collected (Table 1). Mean caffeine content (% dry weight) was 1.17±0.01% in phloem material (range = 0.01–3.68%) and 0.20±0.003% (range = 0.00–0.75%) for leaf material. Mean theobromine content was 0.18±0.002% (range = 0.01–0.74%) for phloem and 0.54±0.01% (range = 0.01–1.63%) for leaf material.

Table 1. Summary of caffeine and theobromine mean and range values of 36 phloem samples and 11 leaf samples.

Material	Caffeine (% DW)	Theobromine (% DW)
Phloem (n = 36)	mean = 1.17 (range = 0.11 – 3.68)	0.18 (0.01–0.74)
Leaf (n = 11)	0.17 (0.00–0.75)	0.38 (0.01–1.63)

Within-plant variation in purine alkaloids

For all plant individuals pooled, there was significantly more caffeine in phloem than leaf material (Paired t-test, t = 3.99, p = 0.0026; Fig. 5A). There was no significant difference in theobromine concentration between phloem and leaf material (Paired t-test, t =-1.599, p = 0.141; Fig. 5B).

Fig. 5. Caffeine (A) and theobromine (B) content of phloem and leaf material from Paullinia yoco lianas from Amazonian Ecuador (n = 18). Significantly greater caffeine content was found in the phloem than leaf, but there was no difference in theobromine content.

Alkaloids versus stem diameter

As predicted, there was a significant positive relationship between phloem caffeine content and stem diameter, with around a 1% dry weight increase in caffeine for every 4cm increase in stem diameter (linear regression, y = -0.18 + 0.34x, t = 7.541, p < 0.001; Fig. 6A). However, the slight negative trend between phloem theobromine content and stem diameter was not significantly different from zero (linear regression, t = -0.698, p = 0.490; Fig. 6B), and there was no significant trade-off between percent dry weight caffeine and theobromine (linear regression, t = -0.939, p = 0.353).

Fig. 6. Caffeine content (A) of Paullinia yoco lianas increased with increasing stem diameter, but theobromine content did not (B), for 18 lianas sampled from Amazonian Ecuador.

Comparison with Weckerle et al. (2003)

A comparison of these results to the values found in the analysis of herbarium samples by Weckerle et al. (2003), revealed a significantly higher amount of both caffeine and theobromine values in the phloem material (Welch two sample t-test: Caffeine in phloem, t = -6.111, p < 0.001; Theobromine in phloem, t = -2.7107, p = 0.0096). However, our results did not differ significantly from those reported by Weckerle for caffeine or theobromine values in the leaf material (Welch two sample t-test: Caffeine in leaf, t = 0.989, p = 0.336; Theobromine in leaf, t = -1.227, p = 0.245).

Between-plant variation in purine alkaloids

Caffeine and theobromine content varied significantly between individuals, even controling for stem diameter (ANOVA, caffeine: F = 47.31, df = 1, p < 0.001; theobromine: F = 48.55 , df = 1 , p < 0.001; Fig. 7A and 7B).

Fig. 7. There was significant variation in caffeine (A) and theobromine (B) content among individual Paullinia yoco lianas sampled from Amazonian Ecuador.

Support trees

The support trees of the P. yoco lianas included trees of 10 families and 15 genera (Table 3) in a wide range of successional guilds: from fast-growing pioneer species (Cecropia and Solanum), to long-lived pioneers (Virola and Ficus spp.), as well as late-successional species (Nectandra and Ocotea). Lauraceae was the most represented family with four individuals. The support trees ranged in diameter from 12 to 80 cm (mean = 37.2, sd = 18.9).

Table 2. List of support trees identified at each Paullinia yoco sampling site.

Specimen	Family	Genus/species	DBH (cm)
yoco 1	Moraceae	Helicostylis herrerensis	26.0
yoco 2	Solanaceae	Solanum sp.	75.0
yoco 3	Mimosaceae	Inga alata	36.0
yoco 4	Melastomataceae	Blakea cf. rosea	24.0
yoco 5	Moraceae	Ficus sp.	80.0
yoco 6	Bombacaceae	Matisia sp.	48.0
yoco 7	Lauraceae	Lauraceae sp. 1	30.3
yoco 8	Mimosaceae	Inga sp.	31.8
yoco 9	Malvaceae	Theobroma cacao	12.0
yoco 10	Cecropiaceae	Cecropia sciadophylla	45.0
yoco 11	Unknown	Unknown sp. 1	21.0
yoco 12	Caesalpiniaceae	Brownea grandiceps	30.0
yoco 13	Myristicaceae	Virola divergens	47.0
yoco 14	Malvaceae	Theobroma cacao	14.0
yoco 15	Lauraceae	Lauraceae sp. 2	45.0
yoco 16	Lauraceae	Nectandra paucinervia	40.1
yoco 17	Lauraceae	Ocotea sp.	49.0
yoco 18	Unknown	Unknown sp. 2	15.0

Discussion

New data on the ‘yoco mystery’

Our results support the suggestion that fresh material of Paullinia yoco has much higher purine alkaloid content than herbarium material such as that reported by Weckerle et al. (2003). Specifically, Weckerle et al. (2003) analyzed 23 phloem samples of P. yoco and found a median caffeine content of only 0.1% (range 0.00–0.525%) and median theobromine content of only 0.048% (range 0.005–0.185%). Our study reported much higher levels of both alkaloids in phloem, with mean caffeine levels of 1.18% and as high as 3.68%, and mean theobromine levels of 0.18% and as high as 0.74%, approximately four times higher than Weckerle et al. (2003).

However, this difference in alkaloid content between fresh and herbarium specimens was not apparent for leaf material. Weckerle et al. (2003) found a caffeine content range in leaf material of 0.00–1.3%, and theobromine of 0.00–0.438%, while our research found a caffeine range of 0.00–0.75% and theobromine range of 0.01–1.63%. In both studies, wide ranges of values were obtained, with a higher maximum value of caffeine found in Weckerle et al. (2003), but a higher theobromine maximum found in our study. Evidently, further studies are required to better understand the purine alkaloid distribution within the leaves.

One notable distinction between this research and the previous Weckerle et al. (2003) study is that we used a homogenized sample of 2-3 leaves from each liana, rather than analyzing specific regions of individual leaves. Perhaps closer attention to the particular region of the leaf and stage of leaf development during collection would further help to elucidate leaf phytochemical architecture. For example, caffeine content of tea leaves (Camellia sinensis) have been found to have higher caffeine levels along the edges and in younger leaves postulated as a defense against insects (Lin et al. 2003). Furthermore, the relative amount of shade and sun exposure may also contribute to varying amounts of purine alkaloid accumulation. In one study, shade-grown tea (C. sinensis) leaves were found to contain higher levels of caffeine than those grown in full sun (Hirai et al. 2008).

Within-plant variation in purine alkaloids

As predicted, there was significant within-plant variation in purine alkaloid content. Overall, phloem had a greater concentration of caffeine than leaves, but leaf material did not have a significantly higher amount of either purine alkaloid.

Furthermore, the results of this research revealed an important correlation between stem diameter and caffeine concentration (Fig. 6A), suggesting that larger and older plants accumulate caffeine as they grow. It may be this phytochemical architecture of P. yoco that can explain the relatively low caffeine content found by Weckerle et al. (2003). In their research, although stem diameters are not given, most of the phloem samples were removed from herbarium samples, which are traditionally taken from the upper, small branches where leaf and flower parts are present, and that according to our data will likely have much lower caffeine content than larger basal stems. Interestingly, this phytochemical architecture does not hold true for theobromine (Fig. 6B), and further research is certainly warranted to better understand the distribution of theobromine in Paullinia. In cocoa trees (Theobroma cacao) for example, the accumulation of theobromine is over 18 times higher in bean (33.20 mg g-1) than in leaf (1.80 mg g-1) material (Hammerstone et al. 1994).

Between-plant variation in purine alkaloids

Finally, the data from phloem samples highlight the striking differences in caffeine content between individual Paullinia yoco lianas. Not surprisingly, this variation between lianas is also recognized by the local people, where it is common practice to comment on the relative strength of a newly harvested ‘yoco’ liana (pers. comm., July 2014). Without much more extensive sampling, it is hard to say whether this variance is most likely due to genetic differences or site differences. However, the caffeine content of Paullinia cupana also varies significantly between seeds of different provenance, ranging between 2.7 and 5.8% (Henman 1982).

Conclusion

Clearly, the popularity of Paullinia yoco as the preferred stimulant beverage among many indigenous tribes of the upper Amazon region is a testament to the liana’s high caffeine and theobromine content. Although much remains to be understood in terms of its theobromine distribution, the within-plant distribution of caffeine is correlated to stem diameter, as well as considerable variance between individual ‘yoco’ lianas.

These results have important implications in the design of a cultivation strategy for this liana. First, it is crucial to cultivate the liana in such a manner to maximize its diameter, as well as the thickness of the phloem layer. For example, a single, larger vine would be preferable to more numerous but smaller, branching vines. Second, a closer examination of the canopy architecture of support trees is warranted to identify the most appropriate support-tree candidate for maximizing the liana’s leaf canopy index, which has been shown to positively influence phloem thickness in other tree species (Shrimpton & Thomson 1985). Third, the results reveal the potential importance of further genetic studies of various P. yoco provenances, perhaps over a much larger geographic range than the 10,000 hectares of this research, and to encompass provenances over the entire tri-country region. This genetic research would be needed in order to better understand the pathways of caffeine and theobromine production, and in selecting and cross-breeding to achieve a seed source with the greatest potential of producing the desired stimulant chemicals. Finally, it is worth adding that beyond the specific data collected in this research, this project illustrates the broader importance of combining indigenous knowledge and Western science during the creation of development projects for local communities that has both deep cultural implications and high economic potential.

Acknowledgments

We are grateful to the Tropical Resources Institute and Career Development Office at the Yale School of Forestry & Environmental Studies, the Center for Latin American Studies, and the Carpenter-Sperry Research fund for their generous financial support. We would also like to thank the many individuals who assisted us with advice, guidance, and technical support during this research, especially Delfin Payaguaje, Helmut Ernstberger, Chad Oliver, Mark Ashton, Simon Queenborough and Florencia Montagnini.

References

Belaunde, L.E. & Echeverri, J.A. 2008. El yoco del cielo es cultivado: Perspectivas sobre Paullinia yoco en el chamanismo airo-pai (Secoya-Tucano occidental). Anthropologica 26, 87–112.

Hammerstone, J.F., Romanczyk, L.J. & Aitken, W.M. 1994. Purine alkaloid distribution with Herrania and Theobroma. Phytochemistry 35, 1237–1240.

Henman, A.R. 1982. Guaraná (Paullinia cupana var. sorbilis): Ecological and social perspectives on an economic plant of the central Amazon basin. Journal of Ethnopharmacology 6, 311–338.

Hirai, M., Yoshikoshi, H., Kitano, M., Wakimizu, K., Sakaida, T., Yoshioka, T., Nitabaru, J., Nakazono, K., Hayashi, M. & Maki, T. 2008. Production of value-added crop of green tea in summer under the shade screen net: Canopy microenvironments. ISHS Acta Horticulturae 797: International Workshop on Greenhouse Environmental Control and Crop Production in Semi-Arid Regions 411–417.

Michiels, L. & Denis, P. 1926. Sur la liane Yocco et sur la presence de Cafeine dans les Paullinias. J. Pharm. Belg. 43, 795–797.

Miño, G.P.Y., Balslev, H. & Valencia, R. 1995. Useful lianas of the Siona-Secoya indians from Amazonian Ecuador. Economic Botany 49, 269–275.

Schultes, R.E. 1942. Plantae Columbianae II. Yoco, a stimulant of Southern Colombia. Botanical Museum Leaflets, Harvard University. 10, 301–324.

Schultes, R.E. 1951. Plantae Austro-Americanae VII: De festo seculari Ricardi Sprucei America australi adventu commemoratio atque de plantis principaliter vallis Amazonicis diversae observationes. Botanical Museum Leaflets, Harvard University 15, 29–78.

Schultes, R.E. 1987. A caffeine drink prepared from bark. Economic Botany41, 526–527.

Shrimpton, D.M. & Thomson, A.J. 1985. Relationship between phloem thickness and lodgepole pine growth characteristics. Canadian Journal of Forest Research 15, 1004–1008.

Weckerle, C.S., Stutz, M.A. & Baumann, T.W. 2003. Purine alkaloids in Paullinia. Phytochemistry 64, 735–742.

Weinberg, B.A. & Bealer, B.K. 2001. The World of Caffeine: The science and culture of the world’s most popular drug. Routledge, New York, NY, USA.

Lin, Y.S., Tsai, Y.J., Tsay, J.S. & Lin, J.K. 2003. Factors affecting the levels of tea polyphenols and caffeine in tea leaves. Journal of Agriculture and Food Chemistry 51, 1864–1873.

Zuluaga Ramirez, G. 2004. El Yoco (Paullinia yoco): La savia de la selva. Centro Editorial Universidad del Rosario, Bogotá, Colombia.