Extraction, Isolation and Characterization of Bioactive Compounds from Ethanol and Chloroform Extracts of Piper sarmentosum Roxb. Leaves

MATERIALS AND METHODS

Study area: This research was conducted from March to August, 2020 at Plant Physiology Laboratory, Department of Biology, Faculty of Science and Technology, Universitas Airlangga, Surabaya, Indonesia.

Materials, tools and instrumentation: Piper sarmentosum leaves were purchased from Kayon flower market, Surabaya, Indonesia, ethanol (Fulltime, China), chloroform (Fulltime, China), Whatman No. 14 filter paper (Merck, United States), glass jars, vials, glass spatula, micropipette (Thermo Fisher, United States), Gass Chromatography-Mass Spectrometry (GC-MS) (Agilent Technologies 7890A, United States) and analytical balance (Ohaus, United States).

Extraction: After cleaning and removal any materials under tap water, the P. sarmentosum leaves were stored in an oven at 60°C and dried in the sunlight and then stored at room temperature until further use. The 100 g of the plant sample powdered were soaked in 1000 mL of ethanol and chloroform separately for 24 hrs. Whatman No. 14 filter paper was used to separate the extract of the plant. The filtrates were used for further phytochemical analysis using GC-MS.

Determination of the ethanol and chloroform compounds using GC-MS: The GC-MS was used to identify the bioactive compound from ethanol and chloroform extracts of P. sarmentosum. The analysis of bioactive compounds by a Gas Chromatograph (GC) model 7890A equipped with a capillary column model number Agilent 19091S-433 (30 m×250 μm×0.25 μm). The injection of the sample in splitless mode is about one μL. The injection temperature was 300°C. A carrier gas with a constant flow of 20 mL min^–1 is Helium. The GC oven program started at 100°C and held for 2 min, then increased to 300°C and hold for 24 min. The component was determined by the retention time and mass spectrum with the Automated Mass Spectral Deconvolution and Identification System "AMDIS" software. Compounds were identified using the mass spectra database and library Wiley and NIST MS Search software version 2.0.

RESULTS

Identification of bioactive compounds on ethanol and chloroform extracts of Piper sarmentosum Roxb. leaves: In this study, the identification of bioactive compounds in the ethanol and chloroform extracts of P. sarmentosum leaves was carried out using GC-MS. Table 1 shows that ethanol extract from P. sarmentosum leaves has 13 bioactive compounds, which were three higher compounds, 1,3-benzodioxole, 4-methoxy-6-(2-propenyl) (50.65%), 1,3-benzodioxole,5-(2-propenyl)-(25.19%) and alpha-cubebene (3.15%). While Table 2 shows that chloroform extract from P. sarmentosum leaves has 34 bioactive compounds, which were also three higher compounds, such as trans-isocroweacin (62.94%), 1,3-benzodioxole, 5-(2-propenyl)-(13.92%) and 4-Methoxy-N-methyl phenylethylamine (4.25%).

DISCUSSION

Dry leaves of P. sarmentosum in the form of powder after 24 hrs were extracted using ethanol and chloroform using the maceration method resulting in various compounds by GC-MS. The results show that the ethanol and chloroform extracts of P. sarmentosum contained 13 and 34 compounds,

respectively. Table 1 shows that the ethanol extract of P. sarmentosum contains 3 main compounds, such as 1,3-benzodioxole, 4-methoxy-6-(2-propenyl), 1,3-benzodioxole, 5-(2-propenyl) and alpha-cubebene, as much as 50.65, 25.19 and 5.15%, respectively. Meanwhile, Table 2 shows that the chloroform extract of P. sarmentosum also contains 3 main compounds, such as trans-isocroweacin or the common name is myristicin, 1,3-benzodioxole, 5-(2-propenyl) and 4-Methoxy-N-methylphenylethylamine, as much as 62.94, 13.92 and 4.25%, respectively. A previous study reported that P. sarmentosum in methanol extract contains 12 compounds by UV-Vis spectrometry and15 compounds by LC-MS^10,19.

This shows that the different types of solvents used for extraction can affect the content identified using GC-MS, LC-MS or UV-Vis spectrometry. Usually, organic solvents tend to extract more polar bioactive compounds as well. On the other hand, the number of compounds identified in the chloroform extracts from the leaves of P. sarmentosum was higher than the ethanol extract. The majority of compounds in these plants were indicated as non-polar. Whereas in the ethanol extract were 13 compounds were identified. Ethanol is a type of solvent which is polar.

The 1,3-Benzodioxole, 5-(2-propenyl) and alpha-cubebene were including compounds that can be found in both types of solvents used. The types of compounds identified in each ethanol and chloroform extract are also different. This is because the types of solvents are not the same, so the compounds that come out during the maceration process are different.

Myristicin or 1,3-benzodioxole, 4-methoxy-6- (2-propenyl) were the majority compound in ethanol and chloroform extract of P. sarmentosum. This compound was usually used for insecticidal activity²⁰. It is also contained in many various leaves such as P. mullesua, Carum roxburghianum Benth. and Cinnamomum champhora root wood^20-22. Myristicin contains in chloroform extract of P. sarmentosum about 62.94%. It is highly potential to use myristicin as an inducer in the anti-inflammatory, antiproliferative and antioxidant pathway mechanisms²³. Another compound identified in this research was alpha-cubebene, a waxy herbal odour type with medium odour strength²⁴. In a previous study, this compound is also known as part of terpenes (sesquiterpenes) for the biochemical mechanisms underlying drought stress tolerance in thyme²⁵. It is also identified in many plant extracts, such as seeds of Schisandra chinensis and leaves of Solanum verbascifolium^26,27.

CONCLUSION

The ethanol and chloroform extracts of P. sarmentosum leaves contain various types of bioactive compounds that have high potential and can be prospectively developed to be utilized for biological activities. The ethanol extract from P. sarmentosum leaves has 13 bioactive compounds with the highest percentage area of the bioactive compound being 1,3-benzodioxole, 4-methoxy-6-(2-propenyl) (50.65%). The chloroform extract from P. sarmentosum leaves have 34 bioactive compounds with the highest percentage area of the bioactive compound is trans-isocroweacin or myristicin (62.94%).

SIGNIFICANCE STATEMENT

The various bioactive compounds of P. sarmentosum Roxb. leaves that can be beneficial for providing information in the form of bioactive compounds that have the potential to be used as an alternative to developing drug-based sources in the pharmaceutical field. This study will help the researchers to uncover the critical areas of biological activities of P. sarmentosum from Indonesia that many researchers were not able to explore.

ACKNOWLEDGMENT

The authors thanked the Faculty of Science and Technology Universitas Airlangga for providing the necessary support for this research. This research was supported by a Faculty Superior Research Scheme 2020 (Ref. No. 346/UN3/2020) from Universitas Airlangga Surabaya, Indonesia.