The chemistry and biological activities of Peperomia pellucida (Piperaceae): A critical review

Author’s Accepted Manuscript The chemistry and biological activities of Peperomia pellucida (Piperaceae): A critical review Nayara Sabrina F. Alves, William N. Setzer, Joyce Kelly R. da Silva www.elsevier.com/locate/jep

PII: DOI: Reference:

S0378-8741(18)32327-4 https://doi.org/10.1016/j.jep.2018.12.021 JEP11647

To appear in: Journal of Ethnopharmacology Received date: 4 July 2018 Revised date: 13 December 2018 Accepted date: 14 December 2018 Cite this article as: Nayara Sabrina F. Alves, William N. Setzer and Joyce Kelly R. da Silva, The chemistry and biological activities of Peperomia pellucida (Piperaceae): A critical review, Journal of Ethnopharmacology, https://doi.org/10.1016/j.jep.2018.12.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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The chemistry and biological activities of Peperomia pellucida (Piperaceae): A critical review Nayara Sabrina F. Alvesa, William N. Setzerb,c and Joyce Kelly R. da Silvaa a

Programa de Pós-Graduação em Biotecnologia, Universidade Federal do Pará, 66075-900

Belém, Brazil b

Department of Chemistry, University of Alabama in Huntsville, Huntsville, AL 35899, USA

c

Aromatic Plant Research Center, 230 N 1200 E, Suite 102, Lehi, UT 84043, USA

ABSTRACT Ethnopharmacological relevance: Peperomia pellucida (L.) Kunth is an annual weed with a preference to humid places with reduced solar radiation. This plant is mainly distributed in the Neotropics, Africa, Southeast Asia, and Australia. It is popularly employed in the treatment of a variety of health conditions such as abscesses, abdominal pain, skin sores, conjunctivitis, measles, and kidney troubles. Several studies have also described its antimicrobial, cytotoxic, antidiabetic and a variety of other bioactivities. The aim of the review: The aim of this work is to evaluate, using a critical review, the present ethnomedicinal applications, phytochemistry and pharmacological studies of P. pellucida essential oils (EOs) and extracts from different locations around the world. Materials and methods: This review was performed through an online survey of the ethnomedicinal practices, chemical compositions and pharmacological applications of P. pellucida EOs and extracts. The data were mainly obtained from online journals and books published in English, Portuguese and Spanish. The information was collected from websites such as Google, Google Scholar, PubMed, Science Direct, ResearchGate and other online databases that provided more information about this herb.

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Results: Peperomia pellucida bioactivities such as antimicrobial, cytotoxic, antioxidant, fracture healing, antidiabetic and anti-hypercholesterolemia have been described in several literature sources. Nonetheless, most reports only provide the phytochemical screening of extracts, which does not allow the identification of the active compounds. From these studies, some reported constituents are not included in the Dictionary of Natural Products (DNP), which raises questions toward their identification. In addition, some biological assays were even performed without standard controls for comparison which also makes these results questionable. Conclusion: This review evaluates data regarding the phytopharmaceutical potential of P. pellucida. In general, several important aspects were questionable or missing in these manuscripts, which points out the need of more investigation on the pharmacological properties and phytochemical compositions of this herb.

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Graphical abstract

Keywords: Peperomia pellucida; ethnomedicinal practices; phytochemistry; pharmacological applications.

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_______________ * Corresponding Author: Programa de Pós-Graduação em Biotecnologia, Universidade Federal do Pará, Rua Augusto Corrêa n°1, 66075-900, Belém, Brazil. Tel/Fax: +55-91-32017297. E-mail address: [email protected] (J. K. R. da Silva), [email protected] (N. S. F. Alves), [email protected] (W. N. Setzer).

Table of Contents 1.

Introduction ....................................................................................................................... 5

2.

Distribution and local names ............................................................................................ 7

3.

Local and traditional uses................................................................................................. 8

4.

Phytochemistry ................................................................................................................ 14 4.2 Volatile compounds......................................................................................................... 14 4.3 Non - volatile compounds ............................................................................................... 16

5.

Biological activities ........................................................................................................... 19 5.1 Antibacterial and Antifungal activity .............................................................................. 19 5.3 Antioxidant activity ......................................................................................................... 26 5.4 Fracture healing ............................................................................................................. 27 5.5 Anti-inflammatory and Analgesic activity ...................................................................... 28 5.6 Antidiabetic and Anti-hypercholesterolemia activities ................................................... 29 5.7 Toxicological studies ...................................................................................................... 31 5.8 Other activities ................................................................................................................ 31

6.

Conclusion ........................................................................................................................ 35

Acknowledgements ................................................................................................................. 36 Author contributions .............................................................................................................. 36 Conflict of interest statement................................................................................................. 36 REFERENCES ....................................................................................................................... 37

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1. Introduction The Piperaceae is a large family of angiosperms composed of around 3,700 species (Christenhusz and Byng, 2016). It is among the oldest of the pan-tropical plants and its species are mainly distributed in two genera: Piper with around 2,000 species (Quijano-Abril et al. 2006) and Peperomia with approximately 1,600 (Frenzke et al., 2015). The group Peperomia is one of the largest genera of basal angiosperms (Wanke et al., 2006) and is composed of herbs usually perennial (Shu, 1999). Several Peperomia species are cultivated as ornamentals because of the beauty of their foliage (Guimarães and Carvalho-Silva, 2012). Its species are dispersed pantropically, having the greatest biodiversity in the Neotropics followed by Southern Asia (about 100 species), Madagascar (about 40 species), Africa (about 20 species), Australia and New Zealand (less than 20 species) (Wanke et al., 2006). Peperomia pellucida (L.) Kunth (Figure 1) is known for its variety of pharmacological properties. This plant is an herbaceous herb with succulent, alternate oval leaves and inflorescences in terminal spikes, axillary and opposite to the leaves. The species grows well in humid and loose soils in places with reduced solar radiation with preference to rainy periods (Arrigoni-Blank et al., 2004). Stems are succulent, translucent green, erect or ascending, and internodes are usually 3-8 cm long, glabrous and hairless (Majumder, 2011). It is an annual, fasciculate short-rooted herb usually having a height of 15 to 45 cm (Majumder and Kumar, 2011). The plant is a weed with heart-shaped leaves and tiny seeds attaching to the cord-like spikes (Mosango, 2008; Ooi et al., 2008). Its scientific name is usually written in books and publications in South America as P. pellucida (L.) H.B.K. However, P. pellucida (L.) Kunth should be applied since Kunth

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was responsible for the present classification of this species (previously named by Linnaeus – 1753 – as Piper pellucidum L.) in the genus Peperomia (Mathieu and Posada, 2006). The abbreviation H.B.K. is possibly used because of its taxonomic information published in the book “Nova Genera et Species Plantarum” which was written by Humboldt, Bonpland and Kunth – H.B.K. in 1816. Traditionally, Peperomia pellucida has been utilized in the folk medicine of several pantropical countries to treat a wide spectrum of ailments and diseases such as skin sores (Arrigoni-Blank et al., 2002), gastrointestinal disorders (Mollik et al., 2010), dysentery, diarrhea, indigestion (Mollik et al., 2010), abscesses and injuries (Bojo et al., 1994). In the literature, numerous studies on chemical and pharmacological properties have been reported for its EO and extracts, which include cytotoxic (Xu et al., 2006), analgesic (Aziba et al., 2001), antibacterial (Khan and Omoloso, 2002), and anti-inflammatory potential (ArrigoniBlank et al., 2004). Peperomia pellucida essential oils (EOs) and extracts are typically composed of phenylpropanoids followed by sesquiterpenes (De Diaz et al. 1988). However, the chemical composition can vary significantly depending on its place of origin. The aim of this review is to evaluate the present ethnomedicinal applications, phytochemical and pharmacological studies of P. pellucida EOs and extracts from different locations around the world under a critical view. A similar review on P. pellucida was published by Raghavendra and Kekuda (2018). However, a more detailed survey with a critical analysis of the available data is still needed to understand the state of the research on this species. In the present work, there is a more extensive coverage of this plant’s ethnopharmacological and phytochemical characteristics. The biological activities and ethnopharmacological aspects reported also include new references. Furthermore, a critical selection of the material included was also done since several studies have described pharmacological properties with high concentrations of extracts and / or essential oils of P.

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pellucida. In general, the literature previously published failed to report several scientific details, which emphasizes the importance of conducting more complete studies focused on P. pellucida ethnomedicinal, pharmacological potential and phytochemical composition.

Fig. 1. Peperomia pellucida L. inflorescences (A) and leaves (B) (Photographed by NAYARA ALVES).

2. Distribution and local names Peperomia pellucida is mainly distributed in countries of Central and South America, Africa, Southeast Asia, and Australia (Loc et al., 2010). The plant is commonly known as ‘erva-de-jaboti’ and ‘língua de sapo’ in Brazil (Arrigoni-Blank et al., 2004), ‘lochi pata’ and ‘mashitandu chedi’ in India (Flowers of India, 2005), but it also receives a variety of different popular names in countries such as Puerto Rico, Suriname, Malaysia, Thailand and many others as shown on Table 1. Table 1 Peperomia pellucida traditional names in different countries. Local name Country/Region

Continent

Reference

Nigeria

Africa

୎ⲡ (Ding Cao) Diya Thippili, Wathura Gas

Singapore Sri Lanka

Asia Asia

Sirih cina, ketumpangan air, tumpang angin or căo hú jīao

Malaysia

Asia

Soladoye et al. (2010), Sonibare and Adesanya (2012) Siew et al. (2014) Barberyn Ayurveda Resorts and the University of Ruhuna (2017), Ediriweera (2007) Mosango (2008), De Padua et al. (1999)

Rinrin, Renren

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Ketumpangan air, sasaladaan, suruh-suruhan Pak-krasang, chaa kruut, phak krasang, phak haak kluai Càng cua Usuba sunakosho Lochi pata, mashitandu chedi, pononoa, toyakandha, varshabhoo, latapate, panpatta, charkouma, ponownowa

Luchi pata, paneri Olasiman-bato, Pansit-pansitan or ulasimang bato, Sinaw-sinaw, ulasimang bato, olasiman-ihalas, tangon-tangon Kaca-kaca, surukan and sasaladaan Corazón de hombre Prenetaria Zèb kourès Sumu mairen, Hierba de sapo, sumu yal Alumbre or erva-de-vidro Pépéromie or herbe à couleuvre Pepper elder, silverbush, rat-ear, man-to-man, clearweed Coraçãozinho, erva-de-jaboti, língua de sapo, erva-de-vidro, favaquinha Konsaka wiwiri, eagoe ragoe, kosaka wi, sapoe rakei

Indonesia Thailand

Asia Asia

Vietnam Japan India

Asia Asia Asia

Bangladesh

Asia

Philippines

Asia

Indonesia

De Padua et al. (1999) Mosango (2008), De Padua et al. (1999) Mosango (2008) Mosango (2008) Ghani (1998), Flowers of India (2005), Panghal et al. (2010), Shil et al. (2014), Buragohain, (2011) Mosango (2008), Uddin (2014) Manalo et al. (1983), Mosango (2008), Morilla et al. (2014), Batugal et al. (2004) Mosango (2008), Susilawati et al. (2017) Cano and Volpato (2004)

Asia Oceania Cuba Caribbean, Latin America Puerto Rico Caribbean, Bosque (2014) Latin America Martinique Caribbean, Longuefosse and Nossin (1996) Latin America Central Coe and Anderson (1995), Nicaragua America Coe and Anderson (2005), Coe and Anderson (1999) Spain Europe Mosango (2008) France Europe Mosango (2008) North America North America Mosango (2008) Brazil

South America

Suriname

South America

Arrigoni-Blank et al. (2004), Albuquerque et al. (2007) Ruysschaert et al. (2009), Flora of the Guianas (2018)

3. Local and traditional uses The traditional use of plants has been associated with medical application for millennia (Lowe et al., 2001). In folk medicine, P. pellucida is widely reported as a medicinal species in many cultures (Telban, 1988). The plant is distributed in several countries in South America, including Argentina, Bolivia, Brazil, Colombis, Ecuador, French Guiana, Guyana, Paraguay, Peru, Suriname, Uruguay, and Venezuela (Tropicos, 2018). In the Northeast region of Brazil, for example, this species is usually used to treat abscesses, furuncles, skin sores and conjunctivitis (Bojo et al., 1994; Arrigoni-Blank et al., 2002). In the Caatinga, a Brazilian semi-arid biome, P. pellucida leaves are used to treat inflammation in general and

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hypertension (Albuquerque et al., 2007). In addition, it is applied as an emollient, taken orally as a diuretic agent as well as for the treatment of cough and cardiac arrhythmia (Van Den Berg, 1993; Pimentel, 1994). In Suriname, the plant is applied topically to treat inflammations and skin bruises and taken orally as a diuretic agent (Flora of the Guianas, 2018). The extract, mixed with oil of Cocos nucifera, is rubbed on the body of babies to treat pain (Ruysschaert et al., 2009). In this country, an infusion is employed as both an eye bath to improve the sight and as eye drops to aid with sore eyes (Heyde, undated; Morton, 1981). In Bolivia, this herb is applied to stop hemorrhages and to treat fever and wounds (Muñoz et al., 2000). Besides this, in both South and Central America, it is widely used in food preparation by the local community because it stimulates appetite and digestion (De Padua et al., 1999). Aerial parts are also applied to treat inflammation, dermatological and various kidney diseases in Central America and Mexico (Heinrich et al., 1998). A decoction of the whole plant is orally administered in Garífuna and Miskitu of eastern Nicaragua against animal bites and stings (snakes, scorpions and insects), infections, venereal diseases and menstrual disorders associated with hemorrhage (Coe and Anderson, 1996; Coe and Anderson, 1997). Furthermore, a decoction of the whole plant is taken orally to treat the side effects caused by snakebites in eastern Nicaragua (Coe and Anderson, 2005). In addition, Rama midwives in eastern Nicaragua indicated that a decoction of the whole plant is orally taken to treat vaginal infections and alleviate menstrual pain (Coe, 2008). The same uses and mode of application were reported in Sumu (Ulwa) of Southeastern Nicaragua (Coe and Anderson, 1999). In the Caribbean region, which is part of the Neotropics, P. pellucida is applied against several health problems. For instance, the plant is employed as diuretic, hypotensive, coolant, depurative, anti-poisonous as well as antibiotic and fungicide in Puerto Rico (Bosque, 2014). In Cuba, a plant decoction is administrated orally against calculus and as a

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diuretic agent (Cano and Volpato, 2004). An infusion is also taken orally to relieve constipation and rectal inflammation (Roig y Mesa, 1945; Morton, 1981). Besides this, aerial parts of P. pellucida are used in the village Bwa Mawego of Dominica to treat inflammation and skin eruptions (Quinlan and Quinlan, 2007). In the Grenadines, a tea is used as a treatment for undernourished children (Howard, 1952; Asprey and Thornton, 1953a) In addition, a decoction of the plant is used as a cold remedy in Jamaica, Caribbean (Asprey and Thornton, 1953a; Asprey and Thornton, 1953b; Asprey and Thornton, 1955a; Asprey and Thornton, 1955b; Morton, 1981). In this island it is also seen as a valuable children’s remedy. An informant who had lived in Cuba mentioned it as an excellent blood cooler and sleeping herb (Dalziel, 1937; Beckwith, 1927). In Martinique, a decoction or juice made from the herb is instilled to treat ocular infection (Longuefosse and Nossin, 1996). The plant is used as a condiment to help with appetite and digestion in Puerto Rico. A decoction is also employed as a diuretic and to expel kidney stones (Nuñez-Melendez, 1964; Morton, 1981). In Barbados it is applied to treat children with marasmus, a serious malnutrition. Besides this, the herb is taken to treat kidney complaints in adults and is used externally on sores and skin eruptions in children in Barbados (Gooding, 1940; Morton, 1981). Furthermore, in Trinidad and Tobago, Caribbean, P. pellucida is applied as a blood cleanser and is used to treat common cold symptoms and general health conditions (Clement et al., 2007). An infusion or a decoction of the whole plant is also employed to treat coughs, colds, influenza, fever, diarrhea, and for cooling, stoppage of water, and as a cleanser (Lans, 2006; Clement et al., 2015). The fresh plant is also ingested for sore throat (Williams and Williams, 1951; Wong, 1976; Morton, 1981). The herb also plays an important role in the popular medicine of several Asian countries. In Sri Lanka, its use is usually associated with the Ayurvedic medicine for the treatment of diarrhea, dysentery, naso-pharyngeal infections, paralysis, epilepsy, convulsions,

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skin and mucosae tumors and, cancers (Barberyn Ayurveda Resorts and the University of Ruhuna, 2017). In addition, In Western and Sabaragamuwa provinces in Sri Lanka, the plant is applied against bites of different types of snakes such as cobras, vipers, kraits, hump-nosed vipers and scant venomous snakes. The two main treatment types employed are paththu (for bandaging) and peyawa (for drinking) (Dharmadasa et al., 2016). In Ayurveda, P. pellucida is also used in the treatment of several other health problems such as constipation, kidney diseases, urinary infections, emaciation, edema and general weakness. Furthermore, the Karens of Middle Andaman Islands use the complete herb to treat cuts and wounds (Sharief et al, 2005). Additionally, the whole plant is crushed and rubbed on burned areas to minimize burning sensation and prevent blisters (Ediriweera, 2007). In Bangladesh, Asia, the plant is commonly consumed to treat gastrointestinal disorders such as dysentery, diarrhea, indigestion, colic, acidity, constipation, bloating, lack of appetite and stomachache (Mollik et al., 2010). Furthermore, a paste is prepared from P. pellucida leaves and is popularly applied against boils in the country (Faruque and Uddin, 2011). In the Kanda tribe, one of the lesser known small tribes of Bangladesh, the whole plant is cleaned, macerated and turned into a paste to be applied adjacent to the site of poisonous snakes, insects or reptiles bites. This alternative treatment seems unique to the Kanda healers. The interesting fact is that the paste cannot be applied on the top of bitten areas (Rahmatullah et al., 2013). In the Katakhali Pouroshova of Rajshahi district, the whole plant is used as a treatment for eczema, abdominal pains, headache and fever (Rahman and Sarker, 2015). The whole plant in Vietnam is employed as a warm poultice against malaria, headache, injuries and burns (Vo, 2012). Besides this, the juice from its leaves is used to treat colic (Vo, 2012) and as an antihypertensive agent (Pham, 2002). In addition, Asian peoples also employ P. pellucida against various respiratory tract disorders and skin diseases (Mollik et al., 2010). Leaf extracts are also applied by local

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people to treat mental disorders (Khan et al., 2008). A decoction of the plant is used to treat bone aches and pains in the Malay community (Ong and Nordiana, 1999). The plant is used in Philippines to treat several diseases and disorders. Peperomia pellucida is boiled and then used to reduce uric acid levels and in the treatment of renal disease while a topical solution is applied against acne and boils (Egwuche et al., 2011). In the Philippines, the whole herb is applied as a warm poultice to treat abscesses, boils and pimples. Furthermore, a decoction is employed against gout, kidney troubles and rheumatic pain (De Padua et al., 1999). An infusion of the whole plant is also used to treat kidney stones in Subanens in the municipality of Dumingag, province of Zamboanga del Sur (Morilla et al., 2014). Furthermore, in Peninsular Malaysia, Asia, P. pellucida is boiled and the decoction is drunk to treat rheumatism and fatigue. In addition, the leaf juice is applied against colic and abdominal pains while crushed leaves are used to cure headache in Java (De Padua et al., 1999). It is also popularly applied against several dermatological diseases in Tigbauan, Iloilo (Tantiado, 2012). Besides this, a decoction of the whole plant is employed to treat joint pain in Singapore (Siew et al., 2014). In Asia, specifically among the Nicobarese of Car Nicobar Island, India, the plant juice is administered orally against diarrhea, dysentery and to stop frequent urination (Verma et al., 2010). The Karens of Middle Adaman, India, use the plant as a treatment for cuts, wounds, headache, fever and weakness. In this process, a paste of the whole plant is applied as poultice and bruised leaves are added to the head at the temporal region (Sharief et al., 2005). In the North-Kamrup district of Assam, India, a plant paste is employed externally to reduce little pimples and white spots of the body (Das et al., 2006). In the Saperas community of Khetawas, Jhajjar District, Haryana, India, the herb is used to treat fistula (Panghal et al., 2010). In the Reang tribe of Tripura State of India, a paste of P. pellucida leaves is applied externally to heal wounds (Shil et al., 2014). The whole plant paste from

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Jalpaiguri district, West Bengal, India, is applied against boils (Bose et al., 2015). A paste of the complete plant is even applied on burns for quick relief in Tinsukia District of Assam, India (Buragohain, 2011). Traditional healers in African countries have also reported the uses of P. pellucida as a medicinal herb. In Western Africa, it is locally used by local medicine to treat convulsions (De Padua et al., 1999). The plant is employed in the treatment of measles, small pox, male impotence, mental disorders, and breast cancer in Nigeria (Aziba et al., 2001). Infusion associated with milk is further applied to boost the immune system of sick people (Idris et al., 2016). In Cameroon, the aqueous extract is commonly applied for fracture healing (Florence et al., 2017). The plant is locally applied in Gabon for the treatment of fever and hypertension (Mengome et al., 2010). The whole plant is used in the treatment of hemorrhoids in southwestern, Nigeria (Soladoye et al., 2010). A recipe combination of leaves of P. pellucida, Ocimum gratissimum and Vernonia amygdalina are squeezed with water, filtered and taken with glass cup thrice daily to treat diabetes in Lagos State, Nigeria (Gbolade, 2009). In Okaakoko, Nigeria, the herb is used as a treatment against eczema and skin diseases. The plant is ground to a paste and applied to the affected parts to soften boils and treat skin bumps (Obata and Aigbokhan, 2012). Besides this, it is employed in Papua New Guinea, Oceania, against many health problems, which include wounds, boils, pimples, abscesses, abdominal pain, colic, gout, kidney troubles, rheumatic pain, fatigue, headache, measles, small-pox, male impotence and mental disorders (Khan and Almoloso, 2002). P. pellucida leaves are also applied as a postpartum remedy after heating (not in water) or pounding by healers in Sukajadi, Indonesia (Roosita et al., 2008). Additionally, leaves and stems can be used as food in many cultures (Philippine Medicinal Plants, 2018; Ade-Ademilua et al., 2017; De Padua et al., 1999). In

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salads, the plant has the crispness of carrot sticks and celery (Philippine Medicinal Plants, 2018).

4. Phytochemistry 4.2 Volatile compounds

The essential oils of P. pellucida are typically composed of phenylpropanoids as major compounds followed by sesquiterpenes (De Diaz et al., 1988). The most predominant chemotype is characterized by the presence of dillapiole with amounts ranging of 20.7 to 55.3% (Verma et al., 2014; De Lira et al., 2009). The chemical composition of specimens collected in the Brazilian Amazon is marked by dillapiole (39.7 – 55.3%), b-caryophyllene (10.7 – 14.3%) and carotol (0 – 8.1%) (Da Silva et al., 1999; De Lira et al., 2009). Likewise, a specimen collected near to Rio de Janeiro (Brazil) had as major constituents dillapiole (36.9%) and carotol (13.4%). In addition, the authors reported the presence of 5-hydroxy-3,4methylenedioxyallylbenzene (10.6%), but this compound is not found in the Dictionary of Natural Products (DNP) (Moreira et al., 1999). Dillapiole (37.8%) was also detected in the EO of P. pellucida from Bamboutos Mountain (Cameroon), followed by myristicin (11.3%) and germacrene D (10.3%) (François et al., 2013). However, the major compounds of the EO of the whole plant from Uttarakhand (India) were carotol (26.6 - 32.0%), dillapiole (25.1 - 30.2%), pygmaein (5.5 - 10.5%) and bcaryophyllene (5.6 - 8.3%). The root and aerial parts oils contained dillapiole and apiole in the proportions of 63.9% and 9.2%, and 20.7% and 1.1%, respectively (Verma et al., 2014). Atypically, a specimen collected in Nigeria displayed trans-3-pinanone (32.6%), 1methylethylidenepropane dinitrile (18.6%), which is also not found in the DNP, and 3-octyl acetate (13.1%) as the main constituents (Oloyede, 2010).

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The EO from southwest Nigeria had linalool (17.1%), limonene (14.3%) and βcaryophyllene (12.5%) in the leaves, while the stem EO was mainly composed of linalool (12.6%) and β-caryophyllene (11.5%) and limonene (10.7%) (Okoh et al., 2017). Some of the GC-MS results are not correct. The compound phenylethyl alcohol is listed in the manuscript with RI of 856 and coumarin with 879. However, according to the Adams library, they should have RI values of 1107 and 1434, respectively. The RI value attributed to 3phenylpropanoic acid was 897, which is considered very low. According to molecular weight it should be between 1329 and 1356 (Peng et al., 1988; Yuceer et al., 2001; NIST 2011). EO compositions of P. pellucida from different locations in the world are summarized in Table 2. Table 2 Essential oil compositions of Peperomia pellucida collected from different sites around the world. Plant part Compounds Country Continent Ref. extracted Leaf trans-3-Pinanone (32.6%), 1-methylethylidene propane Nigeria Africa Oloyede (2010) dinitrilea (18.6%) and 3-octyl acetate (13.1%) Leaf Germacrene D (10.3%), Western Africa François et al. (2013) myristicin (11.3 %) Cameroon and dillapiole (37.8 %) Leaf Linalool (17.1%), Southwest Africa Okoh et al. (2017) limonene (14.2%) Nigeria and β-caryophyllene (12.5%) Stem Linalool (12.6%), Southwest β-caryophyllene (11.5%) Nigeria Africa Okoh et al. (2017) and limonene (10.7%). Whole Carotol (26.6–32.0%), plant dillapiole (25.1–30.2%), Northern Asia Verma et al. (2014) pygmaein (5.5–10.5%), India and β-caryophyllene (5.6–8.3%) Carotol (32.1%), Northern Leaf Asia Verma et al. (2014) dillapiole (20.7%), India and β-caryophyllene (7.6%) Dillapiole (63.9%), apiole (9.2%), Northern Asia Verma et al. (2014) Roots and β-caryophyllene (4.4%) India Dillapiole (39.7%), β-caryophyllene (10.7%), and bicyclogermacrene (4.9%) Dillapiole (36.9%), Leaf carotol (13.4%) and 5-hydroxy-3,4-methylenedioxy allylbenzenea (10.6%) Whole Dillapiole (55.3%), β-caryophyllene (14.3%) plant and carotol (8.1%) a The identification of this compound is in doubt. Leaf

Northern Brazil

South America

Da Silva et al. (1999)

Southeast Brazil

South America

Moreira et al. (1999)

Northern Brazil

South America

De Lira et al. (2009)

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4.3 Non - volatile compounds

The chemical composition of extracts of P. pellucida is dominated by phenylpropanoid pathway derivatives with a large diversity of lignans with different skeletons (C6C3 dimers) (see Fig. 2) (Parmar et al. 1997). Methylenedioxy lignans such as sesamin (1) were isolated from leaves of P. pellucida and some members of a specific compound class called peperomins (2-9) and the norlignan 7-oxopachypostaudin A (10) (Xu et al. 2006). In addition, cyclobutane dimers such as pellucidin A (11) (Bayma et al. 2000) and tetrahydrofuran lignans (12-15) have been reported (Fig. 2).

Fig. 2. Lignans identified in P. pellucida

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The structural diversity of flavonoids produced by Peperomia species is limited to flavones and C-glycosylated flavones (exclusively on the A-ring) (16, 17) (Aqil et al., 1993; Xu et al., 2006). A methylated flavan-3-ol called pellucidatin (18) has been reported only in P. pellucida (Aqil et al. 1993). A flavonoid inhibitor of angiotensin-converting-enzyme (ACE) was isolated from aerial parts using chromatography. Its structure was determined as caryatin-7-O-β-rhamnoside (19) (Kurniawan et al., 2016). In addition, the isolation of a xanthone glycoside from P. pellucida called patuloside A (20) has been reported in the literature (Figure 3) (Khan et al., 2010).

Fig. 3. Flavonoids present in P. pellucida

The occurrence of steroids as another compound class has been reported for P. pellucida collected in Asia. The ether-soluble neutral fraction of the whole plant from the Philippines had apiole, 2,4,5-trimethoxystyrene, and three phytosterols: campesterol, stigmasterol and β-sitosterol (Manalo et al., 1983). The isolation compounds from the hexane and ethyl acetate fractions of P. pellucida, collected in Indonesia and obtained by maceration with methanol, resulted in the identification of stigmasterol, an analogue of pheophytin and βsitosterol-D-glucopyranoside (Hartati et al., 2015). The extract of P. pellucida from Malaysia was obtained by extraction with 70%

18

methanol, and the authors reported GC-MS analysis of the extract to show phytol (37.88%), decahydro-2-naphthalenol, (26.20%), which is not in the Dictionary of Natural Products and therefore raises questions regarding its identification, methyl palmitate (18.31%), and methyl linoleate (17.61%) as the only compounds in the extract (Wei et al., 2011). Unfortunately, GC-MS analysis of a polar extract will likely miss many non-polar volatiles as well as the higher molecular weight non-volatile components. The other problem with this work is that retention indices (RI) and retention times (RTs) values were not reported, so there is nothing to compare with. The fact that four compounds made up 100% of the extract is also improbable. Ethanolic leaf extract of P. pellucida from Nigeria was rich in apiole (15.0%), methyl 10-octadecenoate (15.3%) and phytol (16.6%) (Igwe and Mgbemena, 2014). As above, GC-MS was used to analyze the ethanol extract, so the non-polar EO components as well as the non-volatiles were likely missed. In addition, several reported components are doubtful. The identification of 10,12-octadecadiynoic acid (eluting between β-caryophyllene and bicyclogermacrene) is dubious. The butylated hydroxytoluene (BHT) is most likely a contaminant and not a natural product, 1,2-dimethoxy-4-(2-methoxyethenyl)benzene might be 2,4,5-trimethoxystyrene, which had been previously reported in P. pellucida (Da Silva et al., 1999), and the MS shown in the manuscript for methyl 10-octadecenoate is more consistent with methyl oleate (methyl 9-octadecenoate). Phytochemical screening of crude extracts from different tissues of P. pellucida has been described in the literature. The extracts and fractions were mainly composed of: alkaloids, flavonoids (Ibibia, 2012), sterols, tannins, reducing sugars (Mengome et al., 2010), saponins, triterpenoids (Bialangi et al., 2016), carbohydrates, phenols (Oloyede et al., 2011), azulenes, carotenoids, depsides (Mendes et al., 2011), and quinones (Mazroatul et al., 2016).

19

5. Biological activities

5.1 Antibacterial and Antifungal activity

Bioprospecting effective compounds against antibiotic- and antifungal-resistant microorganisms has become a major priority in the last few years (Arunachalam and Gayathri, 2010; Chandra et al. 2017). The potential of P. pellucida EOs and extracts against a large spectrum of Gram-positive, Gram-negative and acid-fast microorganisms has been reported in several works (Table 3). Antimicrobial assays have also been performed against human pathogenic fungi (Candida albicans, Penicillium notatum) and phytopathogenic organisms, which include Rhizopus stolonifera (also known as Rhizopus stolon), Fusarium moniliforme, Aspergillus niger and Aspergillus flavus (Table 3).

Plant Part Leaf

Leaf

Stem and Leaf

Leaf

--Leaf

Leaf

Whole plant

Leaf

Material EO

EO

EO

Extract

Extract Extract

Extract

Extract

Extract

alkaloids, tannins and flavonoids (type of extract not indicated)

HE: alkaloids, tannins, flavonoids, antraquinones, glycosides. ME: alkaloids, saponins, tannins, flavonoids, glycosides.EAE: tannins, flavonoids, antraquinones. CE: steroids. AE and ME: alkaloids and phenolics.

HF: steroid, alkaloids, tannins, flavonoids; CF and EF: carbohydrates, alkaloids, tannins, flavonoids

---

Germacrene D (10.3%), myristicin (11.3 %) and dillapiole (37.8 %) Stem: linalool (12.6%), β caryophyllene (11.5%) and limonene (10.7%). Leaves: linalool (17.1%), limonene (14.2%) and β-caryophyllene (12.5%) EE: Alkaloids, tannins, saponins, terpenoids, flavonoids and cardaic glycosides

Compounds trans-3-pinanone (32.6%), 1-methylethylidene propane dinitrilea (18.6%) and 3-octyl acetate (13.1%)

CE: B. Subtilis and E. coli. AE and ME: B. subtilis, E. coli and P. aeruginosa ME: S. aureus, E. coli, P. aeruginosa, K. pneumoniae, B. subtilis and C. albicans

--1,000 to 5,000 µg/mL

DE, EAE and ME: P. aeruginosa EAF (S. aureus, B. subtilis, E. coli, P. mirabilis, P. fluorescens, P. aeruginosa, S. typhi); EF (E. coli, P. mirabilis, P. fluorescens, P. aeruginosa, S. typhi) and HF (S. aureus, B. subtilis, B. cereus, E. coli, P. mirabilis, P. fluroescens, P. aeruginosa, S. typhi); aqueous (no activity); CF (E. coli, P. fluorescens, P. aeruginosa And S. typhi) HE, ME and EAE: S. aureus, S. typhi, E. coli, K. pneumoniae, P. aeruginosa

Kalaiarasi et al. (2016)

Abere et al. (2012)

25 to 50 μg/mL

Idris et al. (2016)

Mohamad et al. (2015) Zubair et al. (2015)

Ojo et al. (2012)

Okoh et al. (2017)

François et al. (2013)

Ref. Oloyede (2010)

---

25,000 to 200,000 µg/mL

12.5 x 106 and 200 x 106 µg/mL

1,250 to 5,000 µg/mL 150 to 200 µg/mL

MIC 100,000 to 200,000 µg/mL

EE: Klebsiella spp., Enterobacter, P. aeruginosa, E. coli

E. coli, E. cloacae, M. smegmatis, L. ivanovii, S. aureus, S. uberis, and V. paraheamolyticus

Microorganisms Bacteria: E. coli, S. aereus, B. subtilis, P. aeruginosa, K. pneumoniae and S. typhi. Fungi: C. albicans, R. stolon, A. niger and P. notatum R. stolonifera and F. moniliforme

Peperomia pellucida essential oil, extract composition and antimicrobial activity.

Table 3

20

Leaf Leaves, stems, flowers Whole plant Leaf Leaf

Leaf

---

---

Leaf

Leaf Leaf

Leaf

Extract Extract

Extract

Extract

Extract

Extract

Extract Extract

Extract

ME: alkaloids, tannins, resins, steroids, phenols and carbohydrates

EE: proteins, aminoacids, phenols and tannins, flavonoids, steroids and triterpenoids, azulenes, carotenoids, depsides and depsidones -----

Patuloside A (3-β-D-glucopyranosyloxy-1,5,6trihydroxy-9H-xanthene-9-one) –isolated from EE

BF: flavonoids, saponins and tannins

EE: Apiole (15%), methyl 10-octadecenoatea (15.3%) and phytol (16.6%)

Pachypophyllin (isolated from CE) ME: Phytol (37.9%), decahydro-2-naphthalenola (26.2%) and methyl palmitate (18.3%)

The positive identification of this compound is in doubt.

Extract Extract

ME: flavonoids and alkaloids. EAE: flavonoids alkaloids, saponins, tannins, flavonoids and cardiac glycosides (type of extract not indicated) --alkaloids, saponin, tannins, flavanoids, steroids, and glycosides (type of extract not indicated) --200 µg/mL

EAE: S. aureus, B. subtilis, P. aeruginosa, and E. coli E. coli ME: E. tarda, E. coli, Flavobacterium sp., P. aeruginosa, V. cholerae, Klebsiella sp., A. hydrophila, V. alginolyticus, Salmonella sp. EE: S. aureus, E. faecalis, B. cereus, S. typhi, P. mirabilis and E. coli

EE: C. albicans EE and AE: E. coli, P. mirabilis, P. aeruginosa and E. coli MF, HF, EAF, BE, AE: E. coli, S. aereus, B. subtilis, P. aeruginosa, K. pneumonae, S. typhi, C. albicans, R. stolon, A. niger and P. notatum

BF: B. coagulans, M. roseus, S. faecalis, E. coli and P. aeruginosa B. subtilis, B. megaterium, S. aureus, S. haemolyticus, E. coli, S. dysenteriae, S. sonnei, S. flexneri, P. aeruginosa, S. typhi EE: S. aureus and P. aeruginosa

--6,200 to 12,500 µg/mL

EE: S. dysenteriae ME: A. hydrophila, S. agalactiae

50,000-200,000 µg/mL

---

---

62,500 µg/mL

8 to 64 µg/mL

-----

--31.2 to 125 μg/mL

1,250 to 2,500 µg/mL ---

ME and EAE: P. aeruginosa, S. typhii, S. dysenteriae Alcohol: S. aureus, S. mutans, E. coli, K. pneumoniae

Hastuti et al. (2017) Akinnibosun et al. (2008) Oloyede et al., 2011

Mendes et al. (2011)

Khan and Omoloso (2002) Khan et al. (2010)

Igwe and Mgbemena (2014)

Ragasa et al. (1998) Wei et al. (2011)

Bojo et al. (1994)

Uddin (2014) Manaf and Daud (2016)

Mensah et al. (2013)

Ibibia (2012)

Legend: Hexane extract (HE); Chloroform extract (CE); Dichloromethane extract (DE); Ethyl acetate extract (EAE); Ethanolic extract (EE); Methanol extract (ME); Aqueous extract (AE); Hexane fraction (HF); Butanol fraction (BF); Chloroform fraction (CF); Ethyl acetate fraction (EAF).

a

Leaf

Extract

Extract

Leaf

Extract

21

22

The antimicrobial activity of P. pellucida EO from Nigeria was evaluated against Gram-negative

bacteria,

which

include

Escherichia

coli,

Klebsiella

pneumoniae,

Pseudomonas aeruginosa and Salmonella typhi, Gram-positive microorganisms such as Staphylococcus aureus and Bacillus subtilis, and against the fungi Candida albicans, Rhizopus stolon, Aspergillus niger and Penicillium notatum. A complete list with the names and morphological characteristics of the bacteria inhibited by P. pellucida can be found in Table 4. Minimum inhibitory concentrations (MIC) ranged from 25,000 to 200,000 µg/mL in comparison to standards gentamicin and tioconazole for bacteria and fungi, respectively (Oloyede, 2010). Usually, the antimicrobial activity of extracts is classified according to MIC values as good (MIC < 100 µg/mL), moderate (MIC from 100 to 500 µg/mL) and weak (MIC from 500 to 1000 µg/mL) (Holetz et al., 2002). Extracts with MIC values over 1000 µg/mL are considered inactive. Table 4 General characterization of the bacteria strains inhibited by Peperomia pellucida essential oils and extracts Bacteria Staining Shape Bacteria Bacillus cereus Gram + Rod Enterobacter aerogenes Bacillus coagulans Gram + Rod Enterobacter cloacae Bacillus megaterium Gram + Rod Escherichia coli Bacillus subtilis Gram + Rod Flavobacterium sp. Enterococcus faecalis Gram + Spherical Klebsiella sp. Listeria ivanovii Gram + Rod Proteus mirabilis Micrococcus roseus Gram + Spherical Pseudomonas aeruginosa Mycobacterium smegmatis AF Rod Salmonella sp. Staphylococcus aereus Gram + Spherical Salmonella typhi Streptococcus agalactiae Gram + Spherical Shigella flexneri Streptococcus haemolyticus Gram + Spherical Shigella sonnei Streptococcus uberis Gram + Spherical Vibrio alginolyticus Aeromonas hydrophila Gram Rod Vibrio cholera Edwardsiella tarda Gram Rod Vibrio paraheamolyticus Legend: Gram-positive (Gram +); Gram negative (Gram -); Acid-fast (AF).

Staining Gram Gram Gram Gram Gram Gram Gram Gram Gram Gram Gram Gram Gram Gram -

Shape Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Curved-rod Curved-rod Curved-rod

EO from southwest Nigeria has been reported as effective to combat pathogenic bacteria by microdilution method. Escherichia coli, Enterobacter cloacae, Mycobacterium smegmatis, Listeria ivanovii, Staphylococcus aureus, Streptococcus uberis, and Vibrio paraheamolyticus had MIC values ranging from 150 to 200 µg/mL considering ciprofloxacin (MIC 25-500 µg/mL) as the standard (Okoh et al., 2017).

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The methanol leaf extract from Malaysia, which contained phytol, decahydro-2naphthalenol and methyl palmitate, was screened for antimicrobial activity. Edwardsiella tarda, Escherichia coli, Flavobacterium sp., Pseudomonas aeruginosa and Vibrio cholerae were inhibited with a MIC of 31.2 μg/mL; Klebsiella sp., Aeromonas hydrophila and Vibrio alginolyticus at 62.5 μg/mL; and Salmonella sp. as well as V. parahaemolyticus were inhibited at 125 μg/mL (Wei et al., 2011). Phytol was previously described as active against eight bacterial (MIC 3 – 38 μg/mL) and eight fungal strains (MIC 8 – 16 μg/mL) (Pejin et al., 2014). However, methyl palmitate is not described as antimicrobial agent. The identification of MeOH extract was carried out by GC-MS and these substances do not necessarily represent its composition. Furthermore, the compound decahydro-2-naphthalenol is not found in the DNP. The ethanolic leaf extract of P. pellucida from Avodim Ubakala (Nigeria) was evaluated against S. aureus, Enterococcus faecalis, B. cereus, S. typhi, Proteus mirabilis and E. coli by disc diffusion technique. The MIC values were very high, ³ 25%, indicating the extract to be inactive. Additionally, no positive control was reported. The major components reported for this extract were apiole (15.0%), methyl 10-octadecenoate (15.3%) and phytol (16.6%) (Igwe and Mgbemena, 2014). Note, however, that these compounds are the major volatile compounds in the EtOH extract and not necessarily the main components of the extract. The antifungal potential of apiole is well-known (Meepagala et al., 2005; RazzaghiAbyaneh et al., 2007), but there are no reports on its antibacterial activity. Fatty acids (as their conjugate bases) tend to be antimicrobial because they are surfactants, but methyl esters tend not to be active (Thormar, 2011). Methanolic extract of different tissues of P. pellucida collected in Papua New Guinea was fractionated into petroleum ether (P), dichloromethane (DCM), ethyl acetate (EtOAc) (E) and butanol (BuOH). The fractions were mainly composed of alkaloids,

24

flavonoids, saponins, sterols, tannins, triterpenoids. Butanol fractions (4 mg/disc) were the most effective against Bacillus coagulans, Micrococcus roseus, E. faecalis, E. coli and P. aeruginosa. Chloramphenicol was used as the positive control (Khan and Omoloso, 2002). The xanthone glycoside patuloside A (3-β-D-glucopyranosyloxy-1,5,6-trihydroxy-9Hxanthene-9-one) isolated from P. pellucida displayed a high effect against four Gram-positive bacteria (B. subtilis, Bacillus megaterium, S. aureus, Staphylococcus haemolyticus) and six Gram-negative bacteria (E. coli, Shigella dysenteriae, Shigella sonnei, Shigella flexneri, P. aeruginosa, S. typhi) with MIC values ranging from 8 to 64 μg/mL where kanamycin (2-16 μg/mL) was used as the standard antibiotic (Khan et al., 2010). Phytochemical screening of ethanolic leaf extract of plants from the Brazilian Amazon revealed the presence of phenols and tannins, flavonoids, steroids and triterpenoids, azulenes, carotenoids, depsides and depsidones. The extract exhibited antimicrobial potential against S. aureus and P. aeruginosa with MIC of 625,000 µg/mL, but no reference standard was described (Mendes et al., 2011). This assay was performed by disc diffusion technique and the MIC value was greater than 1000 µg/mL, which can be considered inactive. Phytochemical screening of aerial parts of individuals from Ogbomoso, Nigeria revealed the presence of alkaloids and flavonoids in the methanol extract. Antimicrobial assays were performed by the disc diffusion test. The extract showed activity against S. typhi, P. aeruginosa, and S. dysenteriae with MIC values varying from 1200 to 2500 µg/mL in comparison to the standard ciprofloxacin (Ibibia, 2012). Furthermore, ethanol leaf extract diluted in water at 70% was active against C. albicans using agar diffusion method and Nystatin as the positive control (Hastuti et al., 2017). Antimicrobial assays were also performed by applying the agar well diffusion method. Proteus mirabilis, P. aeruginosa and E. coli showed the highest susceptibility to the aqueous extract of leaf of P. pellucida (17.4mm–21.2mm) while P. aeruginosa (13.4mm–19.6mm), displayed the highest

25

vulnerability to the ethanolic extract (Akinnibosun et al., 2008). Nonetheless, this method does not allow comparison since MIC values are not provided in the manuscript. Additional information regarding the antimicrobial potential of P. pellucida EOs and extracts can be found in Table 4. 5.2 Cytotoxic activity The use of anticancer drugs requires a balance between medical safety and efficacy in patients (Hait, 2010). For this reason, the scientific community’s major goal has been to search for new efficient and safe natural compounds against the variety of cancers present nowadays (Poonam and Chandana, 2015; Seca and Pinto, 2018). Wei and co-workers (2011) have screened the methanol extract of P. pellucida against

the human breast

adenocarcinoma (MCF-7) cell line and reported an IC50 of 10.4 μg/mL. However, the data shown in their paper reveals an IC50 > 30 μg/mL. Furthermore, the analysis of the extract by GC-MS showed phytol, methyl palmitate, and decahydro-2-naphthalenol (which is not found in the Dictionary of Natural Products). Phytol had previously shown in-vitro cytotoxic activity against MCF-7 cells (Satyal et al., 2012; Pejin et al., 2014). A MeOH extract of P. pellucida exhibited a significant cytotoxic activity against human cervical cancer cell line (HeLa) and the human hepatic carcinoma cell line (Hep G2) and showed no toxicity against normal human kidney cells (HEK 293) (Pappachen and Chacko, 2013). The α-methylene lactones, 2-methylene-3-[(3ʹ,4ʹ,5ʹ-trimethoxyphenyl)(5″methoxy-3″,4″-methylenedioxyphenyl)methyl] butyrolactone (ent-7,8-didehydropaperomin B), and peperomin E, isolated from the EtOAc extract of the whole plant, inhibited growth of human promyelocytic leukemia (HL 60, IC50 1.4 and 1.8 µM), breast cancer (MCF-7, IC50 of 3.8 and 3.9 µM) and cervical cancer (HeLa, IC50 9.1 and 11.1 µM) cells having etoposide (IC50 0.21, 0.19 and 3.6 µM) as the reference standard (Xu et al. 2006).

26

5.3 Antioxidant activity

Antioxidants are molecules capable of neutralizing free radicals, which are molecules characterized by the presence of an unpaired electron. These radicals act by attacking important macromolecules causing damage to the cell. Due to their instability and reactive characteristic, they are usually associated with diseases such as cancer, atherosclerosis, neurodegenerative syndromes and aging (Lobo et al., 2010; Pisoschi and Negulescu, 2011). Several studies have reported the potential of P. pellucida against free radicals. The EO from Nigeria at concetrations of 0.1 mg/mL and 0.2 mg/mL showed 97.9% and 98.6% of inhibition of free radical 2,2-diphenyl-1-picryhydrazyl radical (DPPH) being more active than the standards ascorbic acid (90.9% and 68.7%), butylated hydroxyl anisoleBHA (95.4% and 94.3%) and a-tocopherol (15.4% and 12.4%) (Oloyede, 2010); the antioxidant activity the essential oil better than ascorbic acid, BHA and a-tocopherol is very surprising. The authors did not determine IC50 values for the DPPH assay; the concentrations used were relatively high (100 and 200 μg/mL), so that the percent inhibitions were above 90%. In general, BHA, ascorbic acid, and α-tocopherol are much better antioxidants and freeradical scavengers than essential oils or essential oil components (Ruberto and Baratta, 2000; Agnaniet et al., 2005; Wang et al., 2010; Tsai et al., 2011; Amorati et al., 2013; Sharopov et al., 2015a; Sharopov et al., 2015b). Note that reported IC50 values for DPPH activity are BHA, 3.09 μg/mL (Mimica-Dukić et al., 2010); ascorbic acid, 7 μg/mL (Sharopov et al., 2015a); and α-tocopherol, 0.041 μg/mL (Tsai et al, 2011). Peperomia pellucida raw samples were exposed to g-irradiation at doses of 0, 2.5, 5, 7.5 and 10 kilogray (kGy). The results indicated that ethanol crude extract of 7.5 kGy significantly increased activity, promoting a decrease of IC50 value from IC50 244.9 μg/mL to 166.2 μg/mL using the DPPH method. Quercetin was applied as the reference drug, but no chemical composition was indicated (Mun’im et al., 2017).

27

The effect of extraction methods (maceration and reflux) with different solvents (methanol, butanol and ethylacetate) on DPPH scavening was investigated. The extract obtained by refluxing EtOAc showed the best IC50 value (74.0 µg/mL) in comparison to the methanol extract (150.0 µg/mL). However, the chemical composition was not idenfied, and the standard drug was not reported (Phongtongpasuk, 2014). The plant methanol extract from Malaysia had the strongest free radical scavenging (IC50 83.0 µg/mL) in comparison to the reference stantard BHT (IC50 27.0 µg/mL). However, the chemical composition was not described (Mutee et al., 2010). Wei and co-workers had reported that the methanol leaf extract of P. pellucida from Malaysia showed significant DPPH radical inhibitory activity of 30% inhibition at a concentration of 625 μg/mL (Wei et al., 2011). However, a 30% inhibition of DPPH radical at 625 μg/mL cannot be considered “significant” for a methanol extract. Freeradical scavenging activities of IC50 < 100 μg/mL should be considered good activity in a DPPH assay (Kukić et al., 2006; Jeong et al., 2008; Atmani et al., 2009). Furthermore, it is not clear what the inhibitory components in the extract may be; these workers used only GCMS to analyze the methanol extract.

5.4 Fracture healing

Fractures are the most common large-organ injury in humans. Their healing represents a postnatal regenerative process that resembles embryonic bone development (Einhorn and Gerstenfeld, 2015). The effect of the whole P. pellucida aqueous extract on fracture healing was evaluated in female Wistar rats at doses of 100, 200, and 400 mg/kg. The negative control received distilled water, and a second control group of rats with no fractures was administered with 400 mg/kg of extract. The dry extract revealed the presence of some minerals such as potassium (K), phosphorus (P), magnesium (Mg), calcium (Ca) and sodium (Na). The effects on body weight, the relative weights of organs (femurs, uteri and ovaries)

28

and on hematology were evaluated. Peperomia pellucida extract increased body weight at 400 mg/kg, the amounts of white blood cells (WBCs) by 60% at 200 mg/kg, and the amounts of calcium, phosphorus and bone alkaline phosphatase (Florence et al., 2017). Ethanol extract was administered at doses of 100 and 200 mg/kg in adult females Sprague-Dawley rats having a drill hole injury (0.80 mm) in the femur diaphysis. Control groups received gum-acacia in distilled water. The extract stimulated bone regeneration at the injury site and increased mineral deposition to 9.4% at 100 mg/kg dose and to 84.8% at 200 mg/kg. Furthermore, micro-computed tomography (mCT) was performed to assess the internal microstructure of the mineralized tissue. The results indicated that both doses increased bone volume, trabecular thickness, trabecular number and decreased trabecular separation and structure model index. The extract also induced the expression of osteogenic genes and increased mineralized nodule formation of bone marrow stromal cells (BMCs) over control, but the chemical composition was not reported (Ngueguim et al., 2013).

5.5 Anti-inflammatory and Analgesic activity

Pathogenic microrganisms, temperature changes and chemicals can cause a series of damages to body tissues. Thus, the first step is to activate the defense mechanism. In the initial stage, immune cells are released into the affected area, causing inflammation which creates a hostile environment against invading microorganisms. However, if inflammation is not controlled, it can become a chronic problem, causing a series of damages to health (DeWit et al., 2017). Petroleum ether, chloroform and methanol extracts of P. pellucida were evaluated in regards to their effect on paw edema inhibition. The petroleum ether extract significantly reduced inflammation at a dose of 1000 mg/kg in comparison to the positive control indomethacin (10 mg/kg) (Mutee et al., 2010). Similarly, 200 and 400 mg/kg of aqueous leaf extract showed anti-inflammatory activity of 49.1% and 51.1% on edema

29

inhibition in rats (Arrigoni-Blank et al., 2004). The anti-inflammatory potential was assessed by using the carrageenan-induced rat hind paw edema method. Additionally, variations on the anti-inflammatory potential of P. pellucida were evaluated during the four seasons of the year in east-central Brazil, considering the vegetative (phenophase 1), beginning of bloom (phenophase 2), complete bloom (phenophase 3), and seed set (phenophase 4) phases. In the winter, the extract exhibited anti-inflammatory activity of 41.60% (phenophase 1), 36.70% (phenophase 2), and 36.90% (phenophase 4). In the summer, plants displayed potential mainly on phenophases 1 and 2 with inhibition of 43% and 42%, applying indomethacin (10 mg/kg) as the reference standard (Arrigoni-Blank et al., 2002). The analgesic potential of P. pellucida has also been assayed. This activity refers to the property of relieving pain (Shipton, 1997). Aqueous extract of aerial parts was evaluated by the abdominal writhing test (induced by acetic acid) and the hot-plate method in mice. The results showed that in the first test 400 mg/kg inhibited pain by 50.1% while in the hot-plate test 100 mg/kg was enough to inhibit pain by 53.4% (Arrigoni-Blank et al., 2004). Likewise, the methanol extract of aerial parts of plants from Nigeria was also reported as an analgesic agent. MeOH extract exhibited a significant effect on acetic acid-induced writhing in mice at doses of 70, 140 and 210 mg/kg with inhibition values of 37.7%, 66.4% and 78.3%, correspondingly (Aziba et al., 2001).

5.6 Antidiabetic and Anti-hypercholesterolemia activities

The plant antidiabetic activity was evaluated in alloxan-induced diabetic rats. The blood glucose level had a 62%, 64% and 68% reduction in rats fed with glibenclamide (600 µg/kg body weight), P. pellucida at 10% and 20% diet supplementation, in comparison to the normal control group, which were fed with water. Treatment with glibenclamide and the

30

plant at 10% and 20% also led to increased activities of superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH). The levels of aspartate transaminase (AST), alanine transaminase (ALT) and alkaline phosphate (ALP) were reduced in rats fed with the plant compared to the diabetic control group (Hamzah et al., 2012). The Oral Glucose Tolerance Test (OGTT) of the whole plant extract in alloxaninduced diabetic mice indicated that animals fed with 500 mg/kg of extract significantly decreased blood glucose concentration at 90 and 120 minutes compared to diabetic control groups. Phytochemical screening of leaf and stem extracts showed the presence of flavonoids, glycosides, saponins and carbohydrates but was negative for tannins (Sheikh et al., 2013). A new antidiabetic compound, 8,9-dimethoxy ellagic acid, was isolated from the ethyl acetate fraction of P. pellucida leaves from Indonesia by chromatographic separations on silica gel 60 (Merck). The compound exhibited 33.7% of blood glucose reduction in a normoglycemic model at 100 mg/kg body weight in alloxan-induced hyperglycemic Swiss Webster mice in comparison to the diabetic control group (Susilawati et al., 2017). The anti-hypercholesterolemia potential of an ethanol extract was also tested with respect to total cholesterol, HDL, LDL, and triglycerides present in the serum of Wistar rats. The ethanol extract at a dose of 300 mg/kg of body weight reduced total cholesterol and LDL, increased HDL levels, but had no significant effect on triglycerides. The phytochemical screening showed flavonoids, tannins, alkaloids, steroids and quinones as major constituents of the extract (Mazroatul et al., 2016). The anti-cholesterol activity was further evaluated in alloxan-induced diabetic rats fed with P. pellucida 10% and 20% (w/w) diet supplementation as well as mouse chow. The levels of total serum cholesterol, triglycerides and LDLcholesterol were reduced compared to untreated diabetic rats. In addition, HDL-cholesterol was increased in these groups having the diabetic control group as a reference (Hamzah et al., 2012).

31

5.7 Toxicological studies

Peperomia pellucida (L.) Kunth toxicity has been evaluated in several studies. Its aqueous extract at a dose of 5,000 mg/kg in Swiss mice for 14 days indicated low toxicity because there were no changes in their behavior or weight (Arrigoni-Blank et al., 2004). The plant ethanol extract had mild toxicity with LD50 of 15.13 g/kg for male and 11.87 g/kg for female mice (Dewijanti et al., 2014). The methanol (LC50 260.89 μg/mL), hexane (LC50 333.91 μg/mL), and ethyl acetate (LC50 45.85 μg/mL) fractions of the plant leaves were toxic, but the most polar fractions (butanol and aqueous fractions) with lethal doses greater than 1,000 μg/mL were non-toxic using the brine shrimp lethality assay (Oloyede et al., 2011). The plant methanol extract had LD50 values higher than 4,000 mg/kg of body weight with no sign of toxicity on the skin and hair, respiration system, defecation, feed intake and behavior using the acute toxicity tests in Deutschland, Denken, and Yoken mice – DDY mice (Waty et al., 2017). The acute toxicity of petroleum ether at 1,200 mg/kg and ethyl acetate fractions at 1,000 mg/kg were found to be safe and no mortality was observed for albino mice (Khan et al., 2008). Peperomia pellucida methanol extract showed LC50 of 2.4 ± 0.5 μg/mL while the oil indicated LC50 of 8.3±0.2 μg/mL employing the brine shrimp bioassay (De Lira et al., 2009). When applying this method, crude extracts and isolated substances are classified as toxic with values of LC50 < 1000 μg/mL and non-toxic with LC50 > 1000 μg/ mL (Meyer et al., 1982). The compound Patuloside A, isolated from the plant leaves, had its toxicity determined by the same method, and the results showed a LC50 value of 18.24 µg/mL which can be considered toxic (Khan et al., 2010).

5.8 Other activities

Angiotensin-converting enzyme (ACE) is a component of the renin-angiotensin system (RAS). This system controls the blood pressure and sodium homeostasis (Sparks et al.,

32

2014). The enzyme increases blood pressure by causing the constriction of blood vessels. For this reason, ACE inhibitors have an important role in the treatment of hypertension, heart failure as well as other hypertension-related end-organ damage (Miller et al., 2006). ACE inhibition activitiy was evaluated after P. pellucida samples were treated with gamma irradiation at doses of 0, 2.5, 5, 7.5 and 10 kGy. The data indicated that doses of 10 kGy increased ACE inhibitory effect from 50.1% to 57.6% (Mun’im et al., 2017). Moreover, a flavonoid inhibitor of ACE was isolated from the aerial parts using chromatography. Its structure was determined as 3¢,4¢-dihydroxy-3-5-dimethoxyflavone-7-O-β-rhamnose. Later, its inhibitory activity was evaluated by using the in vitro hippuryl-L-histidyl-L-leucine (HHL) substrate degradation method and showed IC50 of 7.7 μg/mL (Kurniawan et al., 2016). The thrombolytic activity of P. pellucida has also been reported. Thrombosis is an arterial disease associated with myocardial infarction and stroke. It occurs when pathologic processes disregulate hemostasis and excessive quantities of thrombin form in the site (Furie and Furie, 2008). Thrombolytic activity was evaluated by the Daginawala method and streptokinase (SK) was used as the standard drug. The crude ethanolic extract of leaves was partioned into solvents of different polarities. Phytochemical screening of the hexane-soluble fraction (HXSF) showed steroid, alkaloids, tannins and flavonoids as the main components while the chloroform extract (CSF) and ethanol-soluble fraction (ESF) had carbohydrates, alkaloids, tannins and flavonoids. ESF exhibited the highest activity (50.6%) against thrombosis when compared to SK 62.7% (Zubair et al., 2015). This plant species has also been popularly used to treat gastric ulcers. Scientifically, its gastroprotective activity has been identified by applying the ethanol-induced gastric ulcer experimental in a rat model. The highest activity was reported to be the dichloromethane extract (82.3%) at 100 mg/kg, with dillapiole being the most active compound present in the extract. Rats treated with dillapiole at 3, 10, 30 and 100 mg/kg

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indicated gastroprotective activity of 23.1, 56.1, 73.2 and 85.5% in comparison to the negative control Tween 80 0.5% (Rojas-Martínez et al., 2013). Besides this, P. pellucida extract has also been described as an antipyretic agent. This means that this herb has the potential to reduce fever, which is a physiologic response triggered by infectious or aseptic stimuli (Aronoff and Neilson, 2001). Petroleum ether and ethyl acetate-soluble fractions of the ethanol leaf extract significantly reduced body temperature in rabbits by 90% and 70% at 80 mg/kg body weight, respectively. Pyrexia or fever had been previously induced in albino rabbits by intraperitoneal administration of boiled milk at 0.5 mL/kg body weight. These results were compared to the potential of aspirin (positive control) which reduced fever by 92.5% (Khan et al., 2008). The antisickling activity of aqueous methanol extract of P. pellucida leaves from Benin City, Nigeria was evaluated in the HbSS (homozygous form) red blood cells collected from sickle cell patients who were not in crises (Abere and Okpalaonyagu, 2015). Sickle-cell disease (SCD) is an inherited condition that causes abnormalities in erythrocytes, by not allowing an appropriate oxygen circulation (Rees et al., 2010). The maximum inhibition was 57.5% at 90 min. of incubation with 500 mg/mL of the extract. The presence of alkaloids, tannins, flavonoids, saponins and cardiac glycosides was reported in the extract (Abere and Okpalaonyagu, 2015). The problem with most of these reports is that phytochemical screening was performed, but not phytochemical analysis. For this reason, it is not possible to know what the active compounds are. Furthermore, antidiarrheal activity of leaves extract was assayed by using the castor oil-induced method. Diarrhea was caused by oral administration of castor oil in mice (1.0 mL/mice). The positive control group received Loperamide (50 mg/kg), the negative control group received distilled water (2 mL/mice) and the test groups were treated with 250 mg and 500 mg/kg body weight of the ethanol leaf extract. Phytochemical screening indicated

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that the ethanolic soluble fraction (ESF) had carbohydrates, alkaloids, tannins and flavonoids as major components. The ESF reduced the number of excretions by 41.8% at 250 mg/kg and 60.2% at 500 mg/kg body weight (Zubair et al., 2015). Hair-growth-promoting activity of ethanolic extract of plants from Indonesia has also been investigated. Rabbits were shaved and treated with extracts at 25% (S1), 50% (S2), 75% (S3) and 100% (S4) (v/v). Carboxymethyl cellulose (CMC) gel without the plant and 2% minoxidil were used as negative and positive control, respectively. The results indicated that topical CMC gel containing the ethanolic extracts increased hair growth by approximately 8.68 mm (S1), 9.42 mm (S2), 10.50 mm (S3) and 11.02 mm (S4). Although these data were not statistically significant when compared to the positive control (11.3 mm), this herb can still be a potent alternative for hair growth promotion (Kanedi et al., 2017). The effectiveness of the plant has been investigated against malaria, a mosquitoborne infectious illness caused by the protozoan Plasmodium falciparum. Malaria has been a major health problem because it has caused illness and death in many children and adults around the world (WHO, 2015). Antimalarial activity of P. pellucida extracts from Gorontalo, Indonesia, indicated that fractions of n-hexane, ethyl acetate and water had IC50 values of 12.8, 2.9 and 10.7 mg/mL against P. falciparum, respectively. Phytochemical screening of the methanol extract indicated the presence of alkaloids, flavonoids, steroids, saponins and triterpenoids (Bialangi et al., 2016). The plant has also been popularly applied as an antihypertensive remedy. Intravenous administration of aqueous extract (30 mg/kg) from Jamaica caused a dosedependent reduction in systolic blood pressure (SBP) by 63.3% (40 ± 10 mmHg), diastolic blood pressure (DBP) by 81.7% (15 ± 7 mmHg), heart rate (HR) by 91.6% (20 ± 7 beats/min) and mean arterial pressure (MAP) by 67.7% (23.3 ± 5 mmHg) (Nwokocha et al., 2012). The phytochemical screening of the methanol extract from Nigeria was positive for cardiac

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glycosides and alkaloids and showed hypotensive activity on arterial blood pressure. Intravenous administration of the extract reduced Mean Arterial Blood Pressure—MABP (-75 ± 4.1%) and HR (-200 ± 20.4%) compared to control (108 ± 2.1mmHg and 420 ± 10 beats /min) (Fasola and Adeboye, 2015).

6. Conclusion Peperomia pellucida has been shown to be an important species in traditional herbal medicine, which can be confirmed by its biological potential presented in the literature surveyed. Nevertheless, some studies reporting biological assays were performed using sample at very high concentrations or lacking in comparison with standard samples and even without determining MIC values, potentially leading to false positive results. Several reports on the essential oil composition have appeared in the literature as well as studies with only phytochemical screening which does not allow the identification of the components. However, some of these reports identified compounds that are not present in the Dictionary of Natural Products, which raises questions regarding their identification since there may be new substances without their chemical structure elucidated. The next steps on studying P. pellucida should be to carry out a comprehensive phytochemical analysis of the plant and to more accurately define its biological activities so that bioactivities can be correlated to phytochemical components. Furthermore, antimicrobial and anticancer essays, for example, need to be further explored in order to comprehend the species potential as a whole. Additionally, new biological tests are still needed to scientifically validate some of its ethnopharmacological applications so that the plant can be used as a future resource for disease treatment.

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Acknowledgements The authors are grateful to the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES - Coordination for the Improvement of Higher Education Personnel) for providing a scholarship to N.S.F. Alves. They also thank Prof. Huy Hùng Nguyễn for providing information on traditional medicinal uses of Peperomia pellucida in Southeast Asia.

Author contributions N.S.F. Alves and J.K.R. da Silva wrote the manuscript. N.S.F. Alves designed the main structure of the manuscript. W.N. Setzer and J.K.R da Silva reviewed the chemical composition and biological activities reported by the literature surveyed. W.N. Setzer proposed the critical analysis and contributed in the manuscript editing. All authors reviewed and approved the final version of this study.

Conflict of interest statement The authors have no conflict of interest.

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