Ilex guayusa: A systematic review of its Traditional Uses, Chemical Constituents, Biological Activities and Biotrade Opportunities

Abstract

Ilex guayusa is an emblematic tree of the Ecuadorian Amazon region, widely used in folk medicine, ritual uses and household and industrial beverages. Despite the daily consumption only a few number of studies have been carried out and the species deserves a deepener bioprospecting activity, also in order to define a new Biotrade strategy for the Ecuadorian amazon region. This review summarizes the ethno pharmacological data and the researches concerning I.guayusa. Promising biological activities have been detected, especially as new source of antioxidant agents due to the presence of phenolic compounds. Also a preliminary study as antidiabetic natural product accounts for new researches.

Full text

Mol2Net, 2016, 2, Section M, doi: 10.3390/MOL2NET-02-M??? 1
http://sciforum.net/conference/mol2net-02
Mol2Net
Ilex guayusa: A systematic review of its Traditional Uses,
Chemical Constituents, Biological Activities and Biotrade
Opportunities
Matteo Radice 1,*, Neyfe Cossio 1 and Laura Scalvenzi1
1 Universidad Estatal Amazónica (Km 2 ½ Via Napo (paso lateral), Puyo, Pastaza, Ecuador); E-Mail:
mradice@uea.edu.ec; nsablon@uea.edu.ec; lscalvenzi@uea.edu.ec
* Corresponding author. E-mail address: mradice@uea.edu.ec. Tel: +593 032-888-118 / 032-889-118
Received: / Accepted: / Published:
Abstract:
Ilex guayusa is an emblematic tree of the Ecuadorian Amazon Region (EAR), widely used in folk
medicine, ritual uses and household and industrial beverages. Despite the daily consumption only a
few number of studies have been carried out and the species deserves a deepener bioprospecting
activity, also in order to define a new Biotrade strategy for the EAR. This review summarizes the
ethno pharmacological data and the researches concerning I. guayusa. Promising biological activities
have been detected, especially as new source of antioxidant agents due to the presence of phenolic
compounds. Also a preliminary study as antidiabetic natural product accounts for new researches
Keywords: Ilex guayusa, Ecuadorian Amazon Region, folk medicine, antioxidant, antidiabetic,
Biotrade
1. Introduction
Ilex guayusa is an emblematic tree of the
Amazonian region, widely present in the
Amazonian region of Ecuador, Colombia, Peru
and Bolivia. As reported by many authors (1 – 8)
I. guayusa was mentioned in several researches
regarding pre-Columbian archaeological
collections, old historical manuals and
ethnobotanical studies, legitimizing the fame of
this specie as the most important plant in the
daily life of Kichwa Amazonian communities (5)
and local farmer from “colono” and “mestizos”
communities. Despite the wide presence of
ethnobotanical studies regarding several uses in
folk medicine (6), there are only few researches
about the phytochemistry and biological
activities of I. guayusa, and this lack
compromises a complete understanding about the
concurrence between folk medicine and
pharmaceutical applications. Moreover, deepener
studies may propose new promising applications
as nutraceutical or cosmetic ingredient. We
aimed to compile an up to date and
comprehensive review of I. guayusa that mainly
covers the phytochemistry and pharmacology
SciForum

Mol2Net, 2016, 2, Section M, doi: 10.3390/MOL2NET-02-M??? 2
http://sciforum.net/conference/mol2net-02
information, in order to suggest new researches
and to offer a complementary paper to the
ethnobotanical research.
2. Results and Discussion
2.1 Botanical description, historical
information and folk medicine
Ilex guayusa is an evergreen tree belonging to
Aquifoliaceae family, native to the Amazon. The
plant is dioecious and reaches between 6 to 10
meters tall. The leaves are simple, pinnate,
glabrous, oblong, elliptic with serrate margin;
they are 7-20 cm long and 2.5-7 cm wide (4). I.
guayusa is distributed from 200 to 2000 m above
sea level along the Andes and contiguous
Amazonian piedmont (8). Historical information
about I. guayusa was mentioned by Schultes (9).
Describing an archeological finding from a
shaman excavated tomb in Bolivia (Tihuanacoid
culture), the author described the presence of
dried and pressed leaves, a mortar and pestle.
The finding probably describes the use of the
species as snuff during ritual activities and it is
feasible that the species has been used for at least
1,500 years. Since the XVI century until today,
many authors described folk medicine and
commercial activities related to I. guayusa. Even
for Jesuit missionary in Ecuador the species was
an important source of income (1,3) and,
currently, a few companies from the EAR are
selling beverages and infusions obtained from I.
guayusa.
Regarding the folk medicine information, the
Table 1 summarized several traditional uses
which include ritual and magical application.
According to the “cosmovisión” concept of the
Amazonian ethnic groups, I. guayusa can be used
for multiple porpoises, from human health
remedy to the custom of cleansing the stomach
daily as a ritual purification. I. guayusa tea is
considered a “magical drink” and is also given to
the hunting dogs, before a hunting expedition, in
order to improve their abilities and skills. For
indigenous people, the infusion can also
provokes a soft hypnotic effect in which “little
dreams” can inspire or dissuade in advance a
hunting expedition (1).
2.2 Phytochemistry
Leaves contain caffeine, theobromine, phenolic
compounds and flavonoids as the main
components (10-13). Also guanidine was
mentioned as an important component of I.
guayusa leaves extracts (14,15). Another
research performed by Ruiz and Roque (16),
mentioned a phytochemical preliminary assay on
ethanolic, methanolic and hydroalcoholic
extracts of I. guayusa, the study revealed the
presence of tannins, alkaloids, flavonoids,
glycosides, phenolic compounds and quinones.
A study performed by liquid chromatography
with tandem mass spectrometry (17) of a I.
guayusa leaves extract detected several amino
acids (Table 2), which provide an interesting
information about nutraceutical profile and taste.
From the same author (18), another research on I.
guayusa leaves extracts, using gas and liquid
chromatography and mass spectrometry, revealed
the presence of two pentacyclic triterpenoid,
oleanolic (1,18 mg/g) and ursolic acid (18,22
mg/g) respectively.
2.3 Biological activity
Stimulant and protective effect of caffeine and
theobromine are extremely reported in literature
(19-23). As reported by Jara et al. (13), dried
leaves of I. guayusa were extracted with ethanol
(EtOH) and ethyl acetate (EtOAc). The total
phenolic content was determined
spectrophotometrically according to Folin–
Ciocalteu’s phenol method and calculated as
gallic acid equivalent (GAE). The total
flavonoids content (TFC) was determined
spectrophotometrically, the antioxidant activity
was determined using free radical DPPH (2,2-
diphenyl-1-picrylhydryzyl) scavenging method
and the β-Carotene bleaching. Results are
reported in Table 3.
Anyway, the presence of phenolic compounds
and flavonoids may indicate a protection against
cellular damage induced by free radical oxidative
injury or reactive oxygen species. These
antioxidant properties are associated with the
presence of phenolic compounds and flavonoids.
Although guanidine was reported but wasn’t
quantified, its presence explains the preliminary
hypoglycemic effect of I. guayusa leaves extracts
in animal model. Guayusa may reduce
hyperglycemia without affect the parameter of
glucose homeostasis in non-diabetic mice (14).

Mol2Net, 2016, 2, Section M, doi: 10.3390/MOL2NET-02-M??? 3
http://sciforum.net/conference/mol2net-02
Oleanolic and ursolic acid are recognized as
antiviral and anti-inflammatory bioactive
compounds and also were reported the in vitro
inhibition activity of these molecules against
various cancer cell type (18). Finally, estrogenic
activity of ethanolic extracts from leaves of I.
guayusa was tested in an in vivo model (female
albino rats) shoving a remarkable increase of
serum estradiol levels and ovaries and uteri
weights. This finding is a preliminary but
promising data in order to confirm the traditional
use of I. guayusa against female infertility (24).
In another study, it was observed that methanolic
and hydroalcoholic extracts from I. guayusa have
fungicidal action against Candida albicans, the
hydroalcoholic extracts was effective also against
Microsporum canis (16).
Moreover, toxicological study was conducted on
ethanolic and water extracts using a Brine-
Shrimp assay (25), in both cases the test shown
respectively low (LC50 500–1000 μg/ml) and
median toxicity (LC50 250–499 μg/ml), in
according with safe traditional use, especially for
the aqueous extract. Another research performed
by Ames test and a chromosome aberration study
in human lymphocytes demonstrated a no
harmful effects (26).
2.4 Biotrade opportunities
In the EAR, ritual infusion, beverages and tea
bags obtained from I. guayusa leaves are widely
present in local market, restaurants and houses.
Furthermore, there are some experiences about
the development of a local and international Fair
Trade market, based on social sustainability
approaches and eco-friendly criteria. Actually, in
order to valorize the Ecuadorian Amazonian
biodiversity, the I. guayusa derivatives may be a
sustainable alternative to design natural products,
relevant for local economies, such as: tea (27),
phytopharmaceuticals (12). In order to maintain
national and international markets, it is necessary
to design a bi-commercial business strategy that
enhances the balance between conservation
politics and entrepreneurs needs.
A recent study performed by Sidali and Garrido
Pérez (8), focuses on a food tourism model,
based on guayusa case, as a strategy of
sustainable development for Kichwas
communities in Napo (Ecuador). The qualitative
research confirms as food tourism may be a
viable strategy and a future trend for EAR.
Moreover, the research identifies four principles
of Kichwa communities’ cosmovision
(worldview) which are compatible with Western-
based theory on niche tourism, respectively:
mutual learning, empowerment, regulated access
to intellectual property and community
legislation.
.
.
.
.
.
.
.
Table 1. Traditional, magical and ritual uses of Ilex guayusa.
Traditional, magical and ritual uses
Country
Ethnic group
Ref.
Glycemic effect
n.r.
Amaguajes
14, 15
Ritual uses
Brazil (outskirts of
Manaus)
n.r.
11
Diabetes, venereal diseases, improving digestion and appetite,
strengthening the body and treat pain, increase fertility, daily
purging. Promoting conviviality, stimulant, stomach tonic,
diuretic, and flu remedy. Increase fertility and libido. Can
help to avoid insect and snakebites. Guayusa helps people to
dream.
Ecuador, Colombia,
Perú
Amazonian Kichwa,
Shuar, Achuar, Cofán,
Secoya, Awajún
Mestizo and white
people
6

Mol2Net, 2015, 1(Section A, B, C, etc.), 1- x, type of paper, doi: xxx-xxxx 4
Daily morning drink, can help to avoid insect and snakebites,
improves hunting and fishing ability.
Ecuador
Amazonian Kichwa
5
Ritual uses, scatological purification, ailment, emetic,
narcotic, hypnotic, stimulant or tonic, diaphoretic and
diuretic, purgative. Increase woman fertility and helps people
to dream for knowing in advance.
Ecuador
Several Amazonian
communities
1
Health tonic, emetic, venereal diseases, improved the
digestion and appetite, can cure dysentery and amenorrhea.
Ecuador, Peru
Amazonian
communities
3
Use before and after drinking ayahuasca. Stomach trouble
aphrodisiac.
Colombia, Ecuador,
Peru
Amazonian
communities
4
Emetic and stimulant tea
Ecuador
n.r.
9
Gastritis, relaxant, helping woman fertility.
Ecuador
Saraguros, Shuar
28
Energizing and stomach pain.
Ecuador
Kichwa, mestizo
7
Additives for hallucinogenic rituals and ritual snuff.
Ecuador, Peru
Shuar
29, 30
Table 2. Amino acids in Ilex guayusa extract (mg/g).
Gly
Asn
Ser
Asp
Gln
Thr
Ala
Glu
Pro
Lys
0.0100
0.2795
0.0107
0.0533
0.0502
0.0136
0.1069
0.0501
0.0253
0.0092
Val
His
Met
Arg
Tyr
Ile
Leu
Phe
Trp
Total
0.0174
0.0129
0.0052
0.0429
0.0129
0.0132
0.0125
0.0110
0.0794
0.8161
Table 3. Phenolic content, flavonoid content and antioxidant activity in Ilex guayusa extract
Sample
Total Phenolics
(GAE mg/g)
Total flavonoids
(RE mg/g)
DPPH IC50
(μg/mL)
β-carotene IC50
(μg/mL)
Guayusa EtOH
54.0±3.8
46.0±2.0
17.5±1.4
55.6±1.6
Guayusa EtOAc
36.0±2.2
20.0±1.8
52.7±4.3
85.7±3.7
3. Materials and Methods
The present systematic review was achieved
adopting the following electronic databases:
SciFinder, PubMed, Google Scholar, SciElo,
Taylor & Francis and Scopus. Data were
independently extracted from three reviewers and
the final paper selections were completed
avoiding duplication of data. The following
keywords were selected: Ilex guayusa, guayusa.
The reviewers selected articles were in English
and Spanish language and were excluded data
from patents. The above mentioned criteria
allowed selecting 20 eligible articles; we also
considered some additional key papers for
introduction, discussion and result chapters.
Anyway, it is deserved to remember that many
authors mentioned remarkable letters and
historical sources from XVI until XIX century
.
4. Conclusions
Despite the widespread presence of beverages
and commercial products obtained from I.
guayusa, mainly ethnobotanical research has
been realized in the last decades. All
phytochemical researches known until today
were developed exclusively on leaves extracts,
without a deepener studies on other parts of the
plant. A recent review about Ilex genus (31)
reported wide information about active
constituents and their biological activities, but

Mol2Net, 2015, 1(Section A, B, C, etc.), 1- x, type of paper, doi: xxx-xxxx 5
present basic information on I. guayusa
regarding the presence of caffeine. For many
others Ilex species were been identified many
molecules as triterpenoids, saponins, flavonoids,
alkaloids, anthocyanins and other phenolic
compounds which can explain the mentioned
biological activities. The lack of a deepener
phytochemical research about I. guayusa is
undeniable and the future trend may be to
increase the researches about antidiabetic and
estrogenic activity above-mentioned.
Furthermore, others studies about plants which
contain caffeine (32,33) reported the effect of
leaf age effects on the quantitative contents of
caffeine, theobromine, methylxanthines and total
phenolic compounds, essentially showing a
decrease amount of mentioned active compounds
in old leaves. Moreover was observed that
caffeine presence seems to be cultivar-specific,
tissue-specific, and season-dependent. In order to
optimize nutraceutical and cosmetic formulations
based on I. guayusa extracts, all these findings
suggest a deepener research about caffeine
presence and phenolic compounds focusing
different parts of the plant, different plant ages
and harvest seasons. Finally, I. guayusa
represents a promising bio-active compound
source and an alternative income wellspring for
local farmers from the Ecuadorian Amazonian
Region.
Acknowledgments
The authors gratefully acknowledge the financial support of the Amazonian State University of the
Republic of Ecuador.
Conflicts of Interest
State any potential conflicts of interest here or “The authors declare no conflict of interest”.
References and Notes
1. Patiño VM. Guayusa, a Neglected Stimulant from the Eastern Andean Foothills. Economic
Botany. 1968, Vol. 22, No. 4 (Oct. - Dec., 1968), pp. 310-316.
2. Bruhn JG, Holmstedt B, Lindgren JE, Henry Wassen B. The tobacco from niño korin:
identificaction of nicotine in a bolivian archaeologic collection. Goteborgs etnografiska museum
Ârstryck. 1976, pp. 45-48.
3. Shultes ES. Discovery of an ancient guayusa plantation in Colombia. Botanicai Mrsprm Leaflets.
1979, May Jun Vol. 27, No. 56.
4. Shemluck M. The flowers of Ilex guayusa. Botanical Museum Leaflets, Harvard University. 1979,
Vol. 27, No. 5/6 (May-June), pp.155-160.
5. Innerhofer S. and Bernhardt KG. Ethnobotanic garden design in the Ecuadorian Amazon.
Biodivers Conserv. 2011, 20:429–439 DOI 10.1007/s10531-010-9984-9.
6. Dueñas JF, Jarrett C, Cummins I, Logan-Hines E. Amazonian Guayusa (Ilex guayusa Loes.): A
Historical and Ethnobotanical Overview. Economic Botany. 2016, XX(X), pp. 1–7.
7. Abril Saltos RV, Ruiz Vásquez TE, Lazo JA, Viáfara Banguera D, Ríos Guayasamín PD, Aginda
Vargas JK, Vega Peña I. The use of medicinal plants by rural populations of the Pastaza province
in the Ecuadorian Amazon. Acta Amazónica. 2016, VOL. 46(4): 355 – 366
http://dx.doi.org/10.1590/1809-4392201600305.
8. Sidali KL, Morocho PY, Garrido-Pérez EI. Food Tourism in Indigenous Settings as a Strategy of
Sustainable Development: The Case of Ilex guayusa Loes. in the Ecuadorian Amazon.
Sustainability. 2016, 8, 967; doi:10.3390/su8100967.
9. Schultes RE. Fifteen years of study of psychoactive snuffs of South America: 1967-1982 - A
Review. Journal of Ethnopharmacology. 1984, 11: 17-32.
10. Lewis WH, Kennelly EJ, Bass GN, Wedner HJ, Elvin-Lewis MP, Fast D. Ritualistic use of the
holly Ilex guayusa by Amazonian Jívaro Indians. J. Ethnopharmacol. 1991, Sep;34(2-3):293.
11. Callaway JC, McKenna DJ, Grob CS, Brito GS, Raymon LP, Poland RE, Andrade EN, Andrade
EO, Mash DC. Pharmacokinetics of Hoasca alkaloids in healthy humans. Journal of
Ethnopharmacology. 1999, 65 243–256.

Mol2Net, 2015, 1(Section A, B, C, etc.), 1- x, type of paper, doi: xxx-xxxx 6
12. Radice M and Vidari G. Caracterización fitoquímica de la especie Ilex guayusa Loes. y
elaboración de un prototipo de fitofármaco de interés comercial. La Granja. 2007, Vol. 6, Núm. 2,
3-11.
13. Jara A, Rodriguez Y, Cornejo J. Cazar ME, Gutierrez M, Astudillo L. Antioxidant activity and
total phenolics of plants used in traditional medicine in Ecuador. The 17th International
Electronic Conference on Synthetic Organic Chemistry. 2013, doi:10.3390/ecsoc-17-b001.
14. Swanston-Flatt SK, Day C, Flatt PR, Gould BJ, Bailey CJ. Glycemic effects of traditional
European plant treatments for diabetes: Studies in normal and streptozotocin diabetic mice.
Diabetes Research. 1989, 10, 69-83.
15. Bailey C and Day C. Traditional Plant Medicines as Treatments for Diabetes. Diabets Care.
1989, Vol. 12, No. 8, September.
16. Ruiz JR and Roque MR. Antimicrobial activity of four plants from Peruvian north-east. Ciencia e
Investigación. 2009, 12(1): 41-47.
17. Moldoveanu CS, Zhu J, Qian N. Free amino acids analysis by liquid chromatography with tandem
mass spectrometry in several botanicals with antioxidant character. J. Sep. Sci. 2015, 38, 2208–
2222.
18. Moldoveanu CS and Scott WA. Analysis of four pentacyclic triterpenoid acids in several
bioactive botanicals with gas and liquid chromatography and mass spectrometry detection. J. Sep.
Sci. 2016, 39, 324–332.
19. Ali A, O’Donnell J, Foskett A, Rutherfurd-Markwick K. The influence of caffeine ingestion on
strength and power performance in female team-sport players. Journal of the International Society
of Sports Nutrition. 2016, 13:46. DOI 10.1186/s12970-016-0157-4.
20. Bădescu SV, Tătaru CP, Kobylinska L, Georgescu EL, Zahiu DM, Zăgrean AM, Zăgrean L.
Effects of caffeine on locomotor activity in streptozotocin-induced diabetic rats. Journal of
Medicine and Life. 2016, Vol. 9, Issue 3, July-September, pp.275-279.
21. Moreira A, Diógenes MJ, De Mendonça A, Lunet N and Barros H. Chocolate Consumption is
Associated with a Lower Risk of Cognitive Decline. Journal of Alzheimer’s Disease. 2016, 53
85–93. DOI 10.3233/JAD-160142.
22. Harpaz E, Tamir S, Weinstein A and Weinstein Y. The effect of caffeine on energy balance. J
Basic Clin Physiol Pharmacol. 2017, 28(1): 1–10.
23. Yoneda M, Sugimoto N, Katakura M, Matsuzaki K, Tanigamia H, Yachie A, Ohno-Shosaku T,
Shido O. Theobromine up-regulates cerebral brain-derived neurotrophic factor and facilitates
motor learning in mice. Journal of Nutritional Biochemistry. 2017, 39 110–116.
24. Contero F, Abdo S, Vinueza D, Moreno J, Tuquinga M, Paca N. Estrogenic activity of ethanolic
extracts from leaves of Ilex guayusa Loes. And Medicago sativa in Rattus norvegicus. PhOL.
2015, vol.2, 95-99.
25. Bussmann RW, Malca G, Glenn A, Sharon D, Nilsen B, Parris B, Dubose D, Ruiz D, Saleda J,
Martinez M, Carillo L, Walker K, Kuhlman A and A. Townesmith. Toxicity of medicinal plants
used in traditional medicine in Northern Peru. J Ethnopharmacol. 2011, September 1; 137(1):
121–140. doi:10.1016/j.jep.2011.04.071.2011.
26. Kapp RW, Mendes O, Roy S, Mc Quate RS. Kraska R. General and Genetic Toxicology of
Guayusa Concentrate (Ilex guayusa). International Journal of Toxicology. 2016, Vol. 35(2) 222-
242.
27. Caranqui J and Humanante A. Estudio sobre la Taxonomía y Estado de Conservación de la
Guayusa (Ilex guayusa Loess. ) del Cantón Pastaza. 2011,
http://dspace.espoch.edu.ec/handle/123456789/767#sthash.OyXcKLgP.dpuf.
28. Tene V, Malagón O, Vita Finzi P, Vidari G, Armijos C, Zaragoza T. An ethnobotanical survey of
medicinal plants used in Loja and Zamora-Chinchipe, Ecuador. Journal of Ethnopharmacology.
2007 111 63–81.
29. Smet PA. A multidisciplinary overview of intoxicating snuff rituals in the western hemisphere. J
Ethnopharmacol. 1985, Mar; 13 (1):3-49.

Mol2Net, 2015, 1(Section A, B, C, etc.), 1- x, type of paper, doi: xxx-xxxx 7
30. Bennet BC. Hallucinogenic plants of the Shuar and related indigenous groups in Amazonian
Ecuador and Peru. Brittonia. 1992, 44(4) pp 483-493.
31. Kothiyal KS, Sati SC, Rawat MSM, Sati MD, Semwal DK, Semwal RB, Sharma A, Rawat B and
Kumar A. Chemical Constituents and Biological Significance of the Genus Ilex (Aquifoliaceae).
The Natural Products Journal. 2012, 2, 212-224.
32. Blum-Silva CH, Chaves VC, Schenkel EP, Coelho GC, Reginatto FH. The influence of leaf age
on methylxanthines, total phenolic content, and free radical scavenging capacity of Ilex
paraguariensis aqueous extracts. Revista Brasileira de Farmacognosia. 2015, 25 1–6.
33. Mohanpuria P, Kumar V, Joshi R, Gulati A, Ahuja PS, Yadav SK. Caffeine Biosynthesis and
Degradation in Tea [Camellia sinensis (L.) O. Kuntze] is under Developmental and Seasonal
Regulation. Mol Biotechnol. 2009, 43:104–111 DOI 10.1007/s12033-009-9188-2.