Popular medicinal uses of Calea uniflora Less . ( Asteraceae ) and its contribution to the study of Brazilian medicinal plants

Calea uniflora Less. is widely used in southern Santa Catarina (Brazil), but there are no scientific studies which support its use. Then, this study was proposed to determine of the percentage use of C. uniflora in a city of southern Brazil and documentation of the knowledge that the population has about this species. The survey was conducted with semi-structured interviews using a questionnaire applied to 372 participants. In analyzing the results, it was observed that of the 94.1% who recognized C. uniflora, 74.3% utilize it as a medicinal plant and 65.4% of such knowledge originates in childhood, mainly through the family (84.6%). 93% reported using inflorescences macerated in alcohol or rum; this extract is generally used topically for wound healing and muscle pain. Furthermore, some reported using small quantities of this extract orally to treat cold and flu. Regarding effectiveness and safety, 97% stated an improvement in symptoms with the use of the plant, while 98.5% stated that it has no toxicity. In light of these results, future phytochemical, pharmacological and toxicological analyses should be designed in order to ensure rational and safe use of this species.

intRoduCtion Saslis-Lagoudakis et al. (2014) claim that an important point for change in history is the ability of humans to learn from others and transmit this knowledge to those who live around them, thus ensuring the transmission and conservation of information.In the context of medicinal plant use this is no different, as traditional knowledge is usually transmitted orally and associated with families, communities or ethnic groups (Hamilton 2004, Abbet et al. 2014).However, lifestyle change associated with lack of ethnobotanical surveys and recording of information, may result in the loss of LUAN S. RAMOS et al. traditional knowledge for future generations (Dovie et al. 2008, Simbo 2010, Maroyi 2011, Wambugu et al. 2011).
Such observations under in the importance of ethnobotany, as studies in this discipline are intended to investigate for what purposes plants are used, while recording and ensuring the preservation of information (Sargin et al. 2013, Liu et al. 2014).In addition, these studies may also support the evolution of phytochemical and pharmacological analyses, since plant extracts present a promising resource for the discovery of substances with pharmacological activity (Nascimento andOliveira 2004, Heinrich et al. 2014).
Among the diverse plant kingdom is the family Asteraceae, with approximately 1.535 genera and 23.000 species distributed in cosmopolitan fashion, but occurring predominantly in tropical and subtropical regions (Maia et al. 2011, Nascimento andOliveira 2004).Within this family is found the Calea genus of plants, comprising around 110 species, including the medicinal species Calea uniflora Less., a perennial herb with yellow inflorescences, popularly known as arnica-da-praia (Nascimento et al. 2002(Nascimento et al. , 2004)).As described by (Mondin et al. 2015), C. uniflora is heliophytic and develops in a subtropical climate.This species is native to Paraguay, Argentina, Uruguay and Brazil.In Brazil, is found in the center-south of the country (Mato Grosso do Sul, Minas Gerais, São Paulo, Paraná, Santa Catarina and Rio Grande do Sul (Mondin et al. 2015).In particular, the plant is well distributed in the south of Santa Catarina in the coastal area in well-drained soil regions, appearing spontaneously in Balneário Rincão city, where usually is collected by the local population and neighboring municipalities.Some scientific studies have been developed to determine the phytochemical and pharmacological activities.Phytochemical studies reported to date with C. uniflora have identified a range of secondary metabolites including rel-2-methyl-2,3,4-trihydroxybutanoic acid-1,4-lactone, 5-deoxyflavone glycoside, 3',4',7-trihydroxyflavone-7-O-β-glucopyranoside, 2',4-dihydroxy-3-methoxychalcone-4'-Oβ-glucopyranoside and quercetin-3-O-βgalactopyranoside (Nascimento and Oliveira 2004).Also, Lima et al. (2016) describes the presence of nine phenolic compounds in leaves of C. uniflora, identified as noreugenin, ethyl caffeate, a mixture of butein + orobol, α-hydroxybutein, caffeic acid, butein 4›-O-glucopyranosyl, quercetin 3-O-glucopyranosyl and 3,5-di-Ocaffeoylquinic acid.The same authors demonstrate the trypanocidal effect of ethyl caffeate, a mixture of butein + orobol obtained by this plant.Furthermore, Nascimento et al. (2007) demonstrated that chromanones, uniflorol-A and uniflorol-B, isolated from C. uniflora inhibited the growth of promastigote forms of Leishmania major.In another study, both a dichloromethane extract of the roots and two isolated compounds derived from p-hydroxyacetophenone also showed trypanocidal properties.All compounds tested were also active against dermatophyte fungi (Trichophyton rubrum and T. mentagrophytes), but only three against Candida albicans and C. glabrata (Nascimento et al. 2004).The dichloromethane extracts of both xylopodia and aerial parts showed in vitro trypanocidal activity against trypomastigotes of Trypanosoma cruzi (Nascimento et al. 2002).
According to local knowledge transmitted orally, this plant is widely used as a medicinal plant in southern Brazil, but to date scientific studies to support this use are lacking, an important factor in motivation of this research.Fundamentally, this research project aimed to determine the percentage use of C. uniflora in a city of Santa Catarina state in southern Brazil and establish the extent of knowledge that the population has about this species.Based on this information, we glimpse subsidize studies on phytochemistry and the pharmacology of this plant.The holding of interviews began after the participants gave informed consent, pursuant to Initially, socio-demographic information on the participants was collected, and then the participants were asked whether they know arnica, and them an exemplar of plant was shown to each participant in order to confirm their recognition of the species, mentioning its popular name (arnica-da-praia) in situations where there was difficulty in recognition.All completed and signed interviews were collated, including those in which the participants reported not knowing or not using the plant.

ReSuLtS
Of the sample of 372 participants, the age profiles panned from 18 to over 70 years, with 37.1% between 30 and 50 years and 46.5% over 50 years.It was observed that women represented 74.2% of interviewees.The majority of C. uniflora users reported a monthly household income of no more than two minimum wages.Regarding time spent in formal education, it was observed that approximately 73% of C. uniflora users had completed primary education.However, over 20% also reported completion of high school or third level education (Table I).
On data relating to the study species, 50% of respondents recognized C. uniflora directly by the image, 44.1% by its popular name and only 5.9% reported not knowing it.Of the 94.1% (n = 350) familiar with it, 74.3% (n=260) use it as a medicinal plant.Of those who use it, 65.4% said they knew it or used it since childhood, with an additional 13.8% reporting knowledge acquired in adulthood but retained for20 years or longer.When asked about the origin of their knowledge, 84.6% said they learned of the plant through their family, 12.3% through friends or neighbours, and 3.1% from other sources.
Considering the sourcing of the plant for consumption, 81.5% reported its collection in the region of Balneário Rincão in fields or in the bush, and while 56.5% said that the ideal harvesting period was from December to March, 25.8% could not identify the optimal harvesting time.
In relation to its popular name, 97% knew C. uniflora only as arnica-da-praia, while the remaining 3% mentioned other names as arnica-docampo and arnica-do-mato, which may be related to plant collection sites.
In the preparation of the extracts and use of C. uniflora, multiple approaches were commonly encountered, including use of more than one part of the plant, more than one mode of preparation and more than one route of administration (Table II).A majority of users employed the plant's inflorescences, which are macerated in alcohol or rum by 93% of users.It was also observed that 75% preferred use of the fresh plant, with 19.6% drying it before use and 5.4% did not specify.
Upon analyzing the popular indications described by C. uniflora users, 39 different therapeutic uses were identified (Tables III and IV), the most cited being summarized in Figure 2.However, it is important to note that a majority of participants did not mention just one therapeutic use, but a combination of them.Apply to the affected area 1-5 times a day until disappearance of symptoms.
Aphthae and sore throat.Gargle, 2-3 times a day until the disappearance of symptoms Flowers or leaves Toothache.
Use cotton soaked with the extract or use a gargle, rinse or apply a macerated flower when in pain.

Flowers or leaves and roots
Headache.
Pass the extract on the forehead, 2 times a day until the disappearance of symptoms Leaves Wound healing, muscle pain and swelling.
Apply to the affected area 2-3 times a day or when in pain until the disappearance of symptoms.

Flowers or roots
Wound healing, muscle pain and rheumatism.
Apply to the affected area 2 times a day until disappearance of symptoms.

Administration mode of extraction Part of plant indication Posology-mode of use
Oral use
Drink 1-2 teaspoons of extract diluted in coffee or water, 1-2 times a day until disappearance of symptoms.

Maceration
Expectorant and in the treatment of sinusitis.
Drink 1-3 cups of infusion at night until disappearance of symptoms.

Decoction
Anti-inflammatory, wound healing, swelling, rheumatism and after surgical removal of the ovaries.
Drink half to 1 cup, 1-3 times a day for 7 days.

Flowers and roots
Colic and bladder pain.
Drink 10-15 drops diluted in coffee, 2 times a day until disappearance of symptoms.Infusion Colic and headache.Drink 1 cup, 2 times a day until disappearance of symptoms.
Drink 2 tablespoons diluted in tea/ coffee or water, 2 times a day until disappearance of symptoms.
Drink 1 liter per day in small doses, every day.

Roots and leaves
Flu, infection and cold.
Drink 1 teaspoon of extract diluted in coffee or water, once a day.

Decoction
Urinary infection.Drink 1 cup, 3 times a day, for 7 days.

Infusion Leaves Wound healing, infection and rheumatism
Drink 1 teaspoon, 3 times a day, for 15 days.
In relation to the recommendation of use of C. uniflora by health professionals, only 6.2% of users reported using the plant on the advice of these sources.Concerning the perceptions of effectiveness with C. uniflora, 97% of informants who used the plant said they experienced improvement with its use.When questioned about the safety of the plant, 98.5% of respondents said that it has no toxicity and approximately 85% that it has no contraindications or drug interactions.

diSCuSSion
The demographic characteristics of the participants in this study have many parallels with published work.Age profiles of respondents in this study match closely with those in the literature; in the work of Gbolade (2012), 43% of respondents were between 30 and 50 years, and 49% were over 50 years (compared to 37% and 47% respectively).Interestingly however, it was found in this study that approximately 30% of who use C. uniflora were less than 40 years, showing the use of this species by newer generations also.Ethnobotanical studies describing self-selection of medicinal plants often show that the female sex predominates with such use, as observed herein where 74% participants were female.This may be related to the fact that men are working outside the home and women more involved with domestic responsibilities (Al-Adhroey et al. 2010, Jesus et al. 2009, Liu et al. 2014, Sivasankari et al. 2014) claim that women have a greater interest in the use of medicinal plants.Moreover, it is common for them to have accumulated more information on plant use than men, reflecting their traditional roles in the care of home and children, in order to preserve family and community health (Abe and Ohtani 2013).In the present study, 34.1% of participants identified as female homemakers and 22.3% were retired, including some women.In contrast, in ethnobotanical studies of plant use by traditional healers, in some cultures it may be more common for the repository of knowledge to lie predominantly with men (Ngarivhume et al. 2015).

Administration mode of extraction
One of the factors commonly cited to explain the ongoing widespread use of medicinal plants in the world is the affordable cost of such therapy, which is usually associated with developing countries and low income (Yabesh et al. 2014, Maroyi 2011).Individuals with lower educational levels may also have more contact with the use of medicinal plants when compared to people with a higher education level (Kayani et al. 2014).In the present study, almost 80% of participants did not proceed to secondary education.These percentages were very similar to those observed by Gakuya et al. (2013) and Kaval et al. (2014) in analogous studies with other plant species.The economic situation of the majority of respondents (Table I) was similar to that found by Santos et al. (2009); however the economic status does not directly predict use of C. uniflora as clearly, as 21.5% reported their monthly income as between 3 and 5 wages.
As with other studies (Juárez-Vázquez et al. 2013, Jesus et al. 2009), a majority of participants collected C. uniflora directly from its natural habitat.Mondin (2015) describes that C. uniflora is appearing on the southern coast of Santa Catarina in sandy well-drained soils.Flowering usually occurs from September to March and May to July, predominantly in October and November, matching the most common time of harvesting reported by users.Such information is very valuable, as the production of chemical substances can vary depending on stages of growth and collection period, in addition to other environmental factors, exposure to pesticides and heavy metal contamination (Padma 2005).Furthermore, the availability of nutrients, carbon dioxide, ozone, light and appropriate temperature, also can change the production of secondary metabolites (McKiernan et al. 2014).
A lack of standardization in the preparation and use of plant extracts seems to be one of the main drawbacks of traditional medicine, as there is often no well-defined ratio between the quantity of plant material and solvent to be used (Al-Adhroey et al. 2010).In the present study this was also observed, since many different forms of preparation of C. uniflora extracts were documented (Tables III and  IV).Regarding maceration, many said that the plant should stay in contact with the liquid (alcohol or rum) for a few days or until the liquid is yellowish.After this period, the extract can then be used, and may be subsequently stored until it runs out.In contrast, if employing a decoction or infusion, users leave the plant in contact with the solvent (water) only for a few minutes, with such extracts being completely consumed after preparation.Clearly, use of different solvents and preparation methods may likely afford extracts with varying phytochemical fingerprints.
Studies performed with both aerial and underground parts of C. uniflora have revealed interesting biological activities (Nascimento et al. 2002(Nascimento et al. , 2004(Nascimento et al. , 2007)).These investigations, coupled with the observations of this study regarding methods of use of C. uniflora, may suggest a higher concentration of active compounds in inflorescences and roots.However, more studies are needed to afford a complete chemical profile of the species.
Constituents responsible for the therapeutic action of plants are typically secondary compounds such as alkaloids, phenolic compounds, essential oils and others (Halberstein 2005, Gurib-Fakim 2006, Wink 2013).These metabolites, in most cases, exhibit some biological activity.Polyphenols, for example, are generally pharmacologically active, with diverse actions including antioxidant, antiinflammatory, antibacterial, antifungal and antiviral activities (Wink 2013).
In the present study, none of the biological activities described by the literature were specifically described by C. uniflora users, although the reference to use in vaginal infection may correlate with observed anti-fungal effects.But this affirmation is merely speculative.Interestingly however, in our laboratory, a study showed that alcoholic extracts of the inflorescences of C. uniflora have antinociceptive activity in mice (unpublished data), which may pharmacologically validate the use of this plant as an anti-inflammatory and analgesic.In light of these observations, further studies are needed to clarify whether the therapeutic actions mentioned by the user population of C. uniflora in souther Brazil are due to reported or as yet uncharacterized constituents.
Other published ethnobotanic studies show that other plants of the family Asteraceae are used for the same therapeutic purposes as C. uniflora in southern Brazil (Rokaya et al. 2010, Abe and Ohtani 2013, Gakuya et al. 2013, Juárez-Vázquez et al. 2013, Bulut and Tuzlaci 2013, Leto et al. 2013, Kayani et al. 2014).It remains to be ascertained whether the active constituents of these species overlap with those of C. uniflora or are distinct.Besides, southern region of Brazil is colonized by European immigrants who were decisive in the cultural habits of the region.So it is important to note that Arnica-da-praia may have been confused by Spanish immigrants with Arnica montana L., since they are very similar morphologically in size and inflorescence, because both are small herbs and have chapters with yellow flowers.This idea is supported by the therapeutic indications of A. montana, which is known worldwide and used for the same medicinal purposes described in this survey for C. uniflora as analgesic, for contusion, bowels ache, common cold, cough, hematoma, headache, phlebitis, rheumatism and others applications (for review, see Obón et al. 2012).
In this study, only a minority of participants reported the use of C. uniflora pursuant to recommendation from a healthcare professional.A lack of access to modern health services is often cited as a factor that leads people to choose medicinal plants as a primary resource for the treatment of their health problems, which may partly account for this finding (Kayani et al. 2014, Simbo 2010).Another factor that may explain the low rate of use of the plant as directed by healthcare professionals is the fact that many have acquired knowledge of the species through their family.However, as emphasized by Rossato and Chaves (2012) of this species.It is estimated that 70-80% of people worldwide rely mainly on traditional medicines, usually herbal medicines, to meet their basic health needs (Hamilton 2004).Many people prefer the use of medicinal plants to allopathic drugs as they consider that they have fewer side effects than synthetic drugs (Gakuya et al. 2013, Kayani et al. 2014).However, the secondary metabolites of plants do not always provide benefits, and may cause toxicity depending on the concentration present or through inappropriate use (Turolla and Nascimento 2006).Furthermore, they can trigger adverse reactions by interacting with other drugs or food, or due to patient characteristics such as age, gender, physiological conditions, genetic traits, etc (Balbino and Dias 2010).Thus, care in the therapeutic use of plants is essential due to a lack of knowledge about the interactions between plant extracts and synthetic drugs (Rossato andChaves, 2012, Juárez-Vázquez et al. 2013); this is particularly of concern for little-studied species such as C. uniflora.

ConCLuSionS
In this study, it was found that C. uniflora is widely known and used in Brazil, considering that of the 94.1% of respondents who recognized it, 74.3% utilize it as a medicinal plant.
In relation to popular indications, it was noted that the plant is most commonly used for wound healing, muscle pain, bumps and hematomas, flu and cold, insect bites and toothache.Furthermore, it was found that people have great confidence in the use of this plant, 98.5% considering it to be nontoxic and 85% asserting that it has no contraindications and drug interactions.
In conclusion, in light of its widespread recognition and utilization as a medicinal plant for a range of diverse indications, future phytochemical, pharmacological and toxicological analyzes informed by the results of this study are warranted, in order to ensure safe use of the species and validate its popular use.

Figure 1 -
Figure 1 -Location of the city of Balneário Rincão.Font: Adapted Google Maps.

Figure 2 -
Figure 2 -Main therapeutic indications cited by C. uniflora users.
, only those medicinal plants that have been validated with the National Agency of Sanitary Vigilance (ANVISA) should be indicated by health professionals in Brazil.No information was found on C. uniflora in RDC No. 26 of May 13, 2014, or in Instruction No. 2 of May 13, 2014, or in the monographs on herbal traditional use of the European Community (Community herbal monographs with traditional use).Notably however, the vast majority of C. uniflora users expressed confidence in its efficacy without fear of significant side effects.Such beliefs undoubtedly motivate ongoing confidence in the medicinal use LUAN S. RAMOS et al.
Rossato et al. (2012)ndRossato et al. (2012)between December 2013 and March 2014.We conducted a pilot survey to test the interviews.This form was composed of questions related to socio-demographic characteristics of participants (gender, age, education, etc.), and also the plant C. uniflora (part used, time and place of collection, method of preparation, administration, etc.).An exemplar of C. uniflora was collected at Vereador Custódio Borges, Bairro Pedreira, city of Balneário Rincão, identified botanically by Dr. Vanilde Citadini-Zanette and Dr. Mara Rejane Ritter, and a herbarium sample deposited in the Herbarium of Pe.Raulino Reitz at the Universidade do Extremo Sul Catarinense (UNESC) as CRI 10.304.

tABLe i economic status and educational level of C. uniflora users.
POPULAR MEDICINAL USES OF Calea uniflora LESS 2323

tABLe iii (continuation) tABLe iV indications for popular use orally, otologically and via inhalation of C. uniflora.
LUAN S. RAMOS et al.Macerations are made with rum or alcohol.Infusions are prepared through aqueous decoction.Poultices are prepared with lard.Note: Information about the plant parts used, methods of preparation, therapeutic indications and methods of use present in this Table are described as reported by the study participants.