# Guineensine: Isolation, Synthesis, and Biological Activity
## 2 Isolation Methods of Guinea from _Piper_ Species  
Guineensine is collected from _Piper_ spp. using different extraction procedures and several system solvents, as presented in Table 1. Fruits of the plants are used to obtain the extracts, from which guineensine is isolated via TLC and preparative HPLC methods [22; 43; 48; 49; 50]. Extraction methods are accompanied by strengths and weaknesses. The first authors who isolated guineensine from _P. guineens_ using dried, powdered fruits were [PERSON] et al. [22]. Soxhlet extraction with petroleum, chloroform, and methanol was used. Guineainse was isolated from petroleum and chloroform fractions and characterized by its IR and NMR spectrum [22]. In the study of [PERSON] and [PERSON] [51], an acetone extract was prepared by stirring from black pepper fruits. The extract was further fractionated on a column of silica gel. Thin layer chromatography followed where the yield of guineensine was 12.8%. Fruits of _P. nigrum_ were extracted using methanol for 48 h [43]. The organic solvent was evaporated under reduced pressure and the obtained extract was partitioned. Of the fractions prepared, the chloroform fraction was selected for further study. Guineainse, alongside pellitorine, pipercle, and retrofractamide A, were isolated using preparative chromatography. Guineainse yielded the highest amount, namely 2.1 mg. In the study of [PERSON] et al. [48], the authors prepared extracts from _P. nigrum_ fruits using different solvents. The chloroform extract was chosen to isolate guineensine using preparative HPLC. This fraction was purified, to finally yield 341.5 mg extract of unknown purity. The authors obtained analytically pure guineensine (11.7 mg) by the further purification of crude guineainse (12.2 mg) using a Sephadex column. \begin{table}
\begin{tabular}{c c c c c c c} \hline \hline
**No.** & **Extraction Method** & **Extraction Time** & **Yield/Extraction Solvent (s)** & **Pros** & **Cons** & **Reference** \\ \hline \multirow{4}{*}{1} & Soxhlet extraction & \multirow{4}{*}{-} & 30 g/750 g of fresh fruits & \multirow{4}{*}{Mechanically efficient technique, inexpensive} & \multirow{4}{*}{-} & [22] \\  & & & (petroleum and chloroform extracts) & & & \\  & & & extracts) 20 g/750 g of fresh fruits (methanotextract) & & & \\  & Conventional & & & & & \\  & extraction & 30 min & 12.8\% (acetone extract) & Good reproducibility, less time consuming & - & [51] \\  & with stirring & & & & & \\  & & 48 h & 11\% (methanol extract) & Low cost & Time consuming & [43] \\  & & & & High solvent & \\  & & & 11.7 mg/1 kg of fruits & & & \\  & & & (n-hexane, chloroform, and methanol extracts) & - & consumption, time consuming & [48] \\  & & & & methanol extracts & & \\  & & & & & \\  & Ultrasound extraction & 30 min & 2.47 g (12.4\%) & Low cost, less time consuming, low solvent consumption & - & [49] \\  & Accelerated solvent & & & 209.7 mg/100 g of dry black pepper (ethyl acetate extract) & Good reproducibility, high yield & - & [50] \\  & extraction (ASE) & & & & & \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Methods for isolation of guineensineine. In another study by [PERSON] et al. [49], fruits of different _Piper_ spp. were extracted with \(n\)-hexane in an ultrasound water bath. The organic solvent was evaporated under reduced pressure and extracts were then analyzed using liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS). Several piperamides were detected among which guineensine was found in most of the extracts. Its quantity varied from 11.58 \(\pm\) 0.11 mg (piperine equivalents/g extract) in _P. guineense_ sample to 55.52 \(\pm\) 2.15 mg in _P. nigrum sample_. Fruits from _P. nigrum_ cultivated in Costa Rica were extracted with ethyl acetate by [50], using an accelerated solvent extraction (ASE) technique. Identification of the compounds presented in the extracts was performed on a UPLC QTOF-ESI-MS system and isolation of the selected alkamides was achieved using a preparative and semipreparative HPLC method.