Optimisation of a Naviglio-assisted extraction followed by determination of piperine content in Piper longum extracts

Abstract Studies were made to increase the yield of piperine extraction using Naviglio Extractor® solid–liquid dynamic extractor (SLDE) from fruits of Piper longum. The effects of ratio w/v were investigated and optimised for the best method. The maximum yield of piperine (317.7 mg/g) from P. longum fruits was obtained in SLDE 1:50 ethanol extract. Extraction yields of piperine obtained from Soxhlet extraction, decotion (International Organization for Standardization) and conventional maceration extraction methods were found to be 233.7, 231.8 and 143.6 mg/g, respectively. The results of the present study indicated that Naviglio Extractor® is an effective technique for the extraction of piperine from long pepper. Graphical Abstract


Introduction
The genus Piper (Piperaceae) contains approximately 1000 species of herbs, shrubs and small trees. Several Piper spp. are of economic importance since they are used as spices and in traditional medicines (Simpson & Ogorzaly 2000). Among these, Piper nigrum is one of the most studied and its biological activities have been largely reviewed (Srinivasan 2007;Butt et al. 2012;Meghwal & Goswami 2013).
Long pepper (Piper longum L.), sometimes called Javanese, Indian or Indonesian long pepper, is cultivated for its fruit, which is usually dried and used as a spice. In Ayurvedic medicine, it is considered as rejuvenator. P. longum stimulates the appetite and dispels gas from the intestines. The extract of the fruits (Piper longis Fructus) is used as anti-inflammatory and insecticidal agent (Kapoor et al. 2009;Kumar et al. 2011). The biological activities of long pepper were recently reviewed (Kumar et al. 2011). The most characteristic secondary metabolites of Piper spp. are N-alkylamides that possess several biological properties (Lee et al. 2006(Lee et al. , 2008Boonen et al. 2012).
Piperine [1-(5-(1,3-benzodioxol-5-yl)-1-oxo-2,4, pentadienyl) piperidine] is the pungent compound of pepper and the most abundant alkylamide (alkaloid) in P. longum, together with other alkaloids that are responsible of the pharmacological properties of long pepper (Dyer et al. 2004;Kumar et al. 2011;Meghwal & Goswami 2013). Piperine is heat-and light-sensitive and care has to be taken in pepper storage to avoid its loss. Due to its biological importance, several papers dealing with its extraction have been reported.
It is well known that the extraction methods used to process plant material can highly affect the quantity of biologically active compounds in the extract. The efficiency of the extraction methods is of a particular importance as bioactive compounds are often present in low concentrations. Conventional techniques to prepare Piper extracts include maceration (Namjoyan et al. 2012), heat-reflux, Soxhlet extraction (SE) (Subramanian et al. 2011). Used solvents are alcohols (Borges & Pino 1993), water (Nabi et al. 2013), or methylene chloride (Shingate et al. 2013). However, these approaches suffer several disadvantages, such as the use of large volumes of solvents, low yields, lengthy procedures and, for heat-reflux extractions, the potentially degradation of metabolites.
In recent years, newer techniques, such as ultrasound (Rathod & Rathod 2014) and microwave-assisted extraction (Raman & Gaikar 2002) or extraction with super-critic (Dutta & Bhattacharjee 2015) or ionic fluids (Cao et al. 2009) have been applied to Piper extraction, but, to the best of our knowledge, the use of Naviglio Extractor® has not been reported.
Naviglio Extractor® is an innovative technology that works by alternating a static phase with a dynamic one and in these last years it has been used to prepare plant extracts (Biagi et al. 2014;Ferrara et al. 2014;Caprioli et al. 2016). The principles at the basis of Naviglio extraction have been described (Naviglio 2003).
The aim of this work was to compare P. longum extracts produced by four solid−liquid extraction methods, namely decotion according International Organization for Standardization (ISO) rules (ISO 1982), conventional maceration (CM), SE and Naviglio Solid-Liquid Dynamic Extrction (SLDE), using aqueous ethanol (96%) as a solvent and to study their chemical composition in terms of piperine concentration and secondary metabolites profile. Furthermore, the influence of the Piper fruits/solvent ratio was studied.

Results and discussion
Four extraction systems (ISO, CM, SE and SLDE) were tested with the same drug/solvent ratio (1:100 w/v) using the same time of extraction (3 h) and 96% ethanol as the extraction solvent. Extractions yields are reported in Table S1. Extracts were qualitatively characterised by HPLC analysis. As it is possible to note ( Figure S1), the chromatograms are qualitatively very similar, and SLDE 1:100 showed a higher amount of piperine. To confirm these data, a spectrophotometric analysis of piperine concentration in the prepared extract was undertaken.
The analytical UV method was validated in terms of linearity, accuracy, intraday and interday precision. Data are reported in Tables S2-S4. To evaluate the piperine content in each extract, 0.02 mg/mL solutions in ethanol of each extract were prepared and analysed by UV. For each solution absorbance at 343 nm was determined, and the corresponding concentration of piperine against respective absorbance value was evaluated using the piperine calibration curve.
The two classical high-temperature methods (ISO and SE) showed more or less the same amount of piperine (231 vs. 233 mg/g, respectively) and the same qualitative composition ( Figure S1), while the two room temperature methods (CM and SLDE 1:100) resulted very different (143 vs. 277 mg/g, respectively). From these data, SLDE 1:100 resulted the best method. At this point, we studied the influence of the drug/solvent ratio and we observed an increment of the piperine extraction in SLDE 1:50 and a decrease in SLDE 1:10, and this last had still higher amount of piperine compared to CM.

Conclusion
In this work, a comparison among different extraction procedures was studied. Naviglio Extractor® resulted the best method for piperine extraction particularly in the w/v 1:50 compared to conventional techniques such as maceration, decotion and SE. Quantitative analysis of the piperine concentration in each extract was performed using the simple, reliable and not expensive UV-VIS technique. This is not the first report about the superiority of Naviglio as extraction system. Biagi and coll. studied ten common medicinal plants containing polyphenols and for each analysed specific markers such as flavonoids, anthocyanosides and caffeic derivatives in addition to total polyphenols content comparing CM and SLDE (Biagi et al. 2014) and they found that Naviglio Extractor® guaranteed a significant improvement in the chemical quality of extracts.