The identification and characterization of anti-oxidant compounds from plant sources has become an active research area because compounds such as polyphenols act as primary anti-oxidants and free radical scavengers. Polyphenols exhibit multiple pharmacological properties such as anti-microbial, anti-allergenic, anti-ulcerogenic, anti-neo-plastic, and anti-inflammatory activities [1Formica JV, Regelson W. Food Chem Toxicol 1995; 33: 1061.
[http://dx.doi.org/10.1016/0278-6915(95)00077-1] ]. The polyphenol family includes phenolic acids, stilbenes, chalcones, coumarins, cromones, lignans, flavonoids, isoflavonoids, neoflavonoids, and tannins. The polyphenol family has been shown to possess significant anti-oxidant capacities, while maintaining low toxicities [2So FV, Guthrie N, Chambers AF, Moussa M, Carroll KK. Nutr Cancer 1996; 26: 167.
[http://dx.doi.org/10.1080/01635589609514473] [PMID: 8875554] ].

The Piper aduncum L. species (Piperaceae), locally known as “Matico”, has long been used in alternative medicine as an antiseptic for wound healing, and as treatment for haemostasis, dysentery, chronic diarrhea, common cold and gastrointestinal pain in multiple countries [3Morandim AA, Bergamo DCB, Kato MJ, et al. Phytochem Anal 2005; 16: 282.
[PMID: 16042156] ]. Its essential oils have also shown biological activity including strong anti-bacterial, cytotoxic, fungistatic, insecticidal, and molluscidal activities [4Mikich SB, Bianconi GV, Maia BH, Teixeira SD. J Chem Ecol 2003; 29: 2379.
[http://dx.doi.org/10.1023/A:1026290022642] ]. From a chemical standpoint, a variety of phenolic secondary metabolites such as lignans, chalcones, dihydrochalcones, chromene, and benzoic acid derivatives have been isolated from P. aduncum L. [5Parmar VS, Jain SC, Bisht KS, et al. Phytochemistry 1997; 46: 597.]. Other well known plants in the Piper genus include black pepper (Piper nigrum) and kava-kava (Piper methysticum).

P. aduncum L. is one of the 429 species that belong to the Piperaceae family that have been identified in Peru alone [6Mostacero J, Mejia F, Gamarra O. Consejo Nacional de Ciencia, Tecnología e Innovacion Tecnologica; CONCYTEC. 2002; 1-2: p. 232.]. It is widespread in north and central eastern Peru and it remains as one of the most widely employed phytotherapy agents by local communities. The local herbal market also widely employs it in multiple compositions. Although used widely, no anti-oxidant assessment has been performed and little ethnobotanical or phytochemical research on this species has been conducted. In the present work, liquid chromatography coupled with mass spectrometry and electrospray ionization (LC-MS-ESI) was employed to identify individual polyphenols of ethanol extracts from leaves of P. aduncum L. collected from five sub-regions of the tropical Peruvian rainforest. The DPPH radical and hydrogen peroxide scavenging activity, reducing power, and total polyphenol content, were also assessed.

The validated LC-MS-ESI method had a limit of quantification (LOQ) of 50 ng/mL for all the compounds and a coefficient of variation (CV %) for the different compounds ranging from 3-7% inter-day and 5-8% intra-day. Standard curves ranged from 100 ng/mL to 50 μg/mL. As shown in Fig. (1), baseline separation and resolution was achieved for all the compounds and the total ion chromatograms were recorded in the negative ion mode. It was found that the predominant phenolic constituents in Piper aduncum L. were quercetin, phloridzin, and epicatechin followed by quercetin glycosides (quercitrin and rutin), catechin, benzoic acids (gallic acid and chlorogenic acid), and phloretin.

Fig. (1) Chromatograms of ethanol extract of P. aduncum L. A) Total ion chromatogram. B) HPLC/UV chromatogram (280 nm). Peak assignment: 1, gallic acid; 2, catechin; 3, chlorogenic acid; 4, epicatechin; 5, quercetin-3-rutinoside (rutin); 6, quercetin-3-rhamnoside (quercitrin); 7, phloridzin; 8, daidzein (internal standard); 9, quercetin; and 10, phloretin.

It was observed that the composition in individual phenolics varied depending on the geographical area. However, the content of catechin, epicatechin, and quercetin-3-rhamnoside (quercitrin) remained constant independent of the geographical area (Table 1). The total sum of individual phenolics from highest to lowest was San Isidro > Honolulo > Tingo María > Tulumayo > Marona Baja, which correlated with the DDPH radical scavenging, H₂O₂ scavenging activity, reducing power, and total polyphenol content (Table 2). The sub-regions from where P. aduncum L. was sampled are within a 10 mile-ratio, but based on the statistically significant differences in the measured parameters, geographical location plays an important role. This observation correlates with previous reports that demonstrated that the absence or presence of pharmacologically active substances can vary within the same specimen depending on the collection place, weather conditions, soil characteristics, time of the year when the collection is performed, and farming conditions [14Machado LR, Sharapin N. Segundo Congreso Internacional y Segundo Congreso Peruano de Plantas Medicinales y Fitoterapia. Lima, Peru 2003; pp. 13-20.].

Table 2

DPPH Radical Scavenging Activities, Hydrogen Peroxide (H₂O₂) Scavenging Activities, Reducing Powers, and Total Polyphenol Content of P. aduncum L. Ethanol Extracts (mean ± SEM, n = 4) from Different Geographical Sub-Regions. All, Except the DDPH Radical Scavenging Assay (Concentration Range 0-300 ug/mL), were Performed at Extract Concentration of 100 µg/mL Including the Controls. AAE = Ascorbic Acid Equivalent, GAE = Gallic Acid Equivalent, NA = Not Applicable

The P. aduncum L. ethanol extracts exhibited DPPH IC₅₀ values of 85-220 μg/mL, which correlate to the values for green tea (100-300 μg/mL) and are slightly lower than the values for black tea (200-800 μg/mL) [15du Toit R, Volsteedt Y, Apostolides Z. Toxicology 2001; 166: 63.
[http://dx.doi.org/10.1016/S0300-483X(01)00446-2] ]. Ability to act as a scavenger may be responsible to some extent for the reported biological activities of P. aduncum L. The different polyphenols detected (most of them for the first time) in P. aduncum L. may be responsible to some extent for its anti-oxidant capacity since gallic acid, chlorogenic acid, catechin, and quercetin have reported IC₅₀ values of 1-8 μg/mL [16Cos P, Ying L, Calomme M, et al. J Nat Prod 1998; 61: 71.
[http://dx.doi.org/10.1021/np970237h] [PMID: 9461655] ]. However, since P. aduncum L. exhibited greater IC₅₀ values than any polyphenolic compound alone, there might be some combinational effects or other factors (e.g. extraction solvent or procedure) yet to be determined. Furthermore, it was observed that P. aduncum L. from all the tested sub-regions contains higher hydrogen peroxide scavenging capacity than cat’s claw (Uncaria tomentosa) [17Goncalves C, Dinis T, Batista MT. Phytochemistry 2005; 66: 89.
[http://dx.doi.org/10.1016/j.phytochem.2004.10.025] [PMID: 15649515] ] and greater total polyphenol content than garlic (Allium sativum L.) [18Tapsell LC, Hemphill I, Cobiac L, et al. Med J Aust 2006; 185: S4.], which are two well characterized medicinal plants.

This is the first report that has quantified various well-characterized polyphenols in P. aduncum L. and the first study in assessing its anti-oxidant capacity, reducing power, and capacity to scavenge hydrogen peroxide, and reducing power, which may be a rational explanation for the therapeutic application of the herb in Latin American medicine.