Extraction of hydrolysable tannins from Phyllanthus niruri Linn.: Effects of solvents and extraction methods
Introduction
Phyllanthus niruri (Euphorbiacea) is an herbal plant indigenous to Malaysia and is locally known as ‘dukung anak’. It is commonly found in tropical regions and in other countries, it is known as ‘chanca piedra’ (Spanish), ‘paraparai mi’ (Paraguay), ‘quebra pedra’ (Brazil) or ‘punarnava’ (India). P. niruri is a popular folk medicine for treating kidney and gallbladder stones, liver related diseases such as jaundice and liver cancer, viral infections such as hepatitis and tuberculosis, malaria, diabetes and fever [1].
In scientific studies, P. niruri was found to exhibit antispasmodic, hypotensive, analgesic, antihepatotoxic, antihepatitis, antimutagenic, antiviral and antibacterial properties. The aqueous and/or alcohol extracts were found to inhibit activity of hepatitis B virus in vitro and in vivo [2], [3], [4], HIV-1 reverse transcriptase virus [5], [6], [7], enzymes processes peculiar to cancer cell's replication and growth [8] and the formation of kidney stones [9], lower the blood glucose levels [10], have the liver-protecting (antihepatotoxic) properties in vivo and in vitro [11] and produce analgesic effects in mice [12]. Other documented properties are antimalarial [13] and lipid lowering activity [14].
The medicinal effects are attributed to the active components present in P. niruri such as lignans, glycosides, flavonoids, alkaloids, ellagitannins, terpenes, and phenylpropanoids [1]. Common lipids, sterols, and flavonols also occur in the plant. Two tannin groups identified in P. niruri are hydrolysable tannins (ellagitannins) and condensed tannins (flavonoids). The final hydrolysis of ellagitannins yields ellagic acid and gallic acid [15]. Chemical structures of active hydrolysable tannins, namely geraniin and corilagin, gallic acid and ellagic acid, and condensed tannins such as flavon-3-ol and flavonol in P. niruri are as shown in Fig. 1, Fig. 2, respectively [5], [16], [17].
Most research on P. niruri was on the chemical screening, identification and isolation, and the biological assay and pharmacological studies [10], [18], [19]. However, not much study on the effects of solvents have on the extraction of active components from P. niruri has been reported. Notka et al. reported the effects of three solvents (water, methanol, 50% ethanol) for the pharmacological study on anti-HIV [5]. The researchers found that the 50% ethanol extract was the most active in inhibiting the replication of the reverse transcriptase virus. The 50% ethanol extract was assayed to consist of 1.10% geraniin and 2.28% (w/w) corilagin. De Souza et al. had reported on the quantity of active gallic acid on the water extract using high performance liquid chromatography (HPLC) technique, but no study on other solvent or analysis of other components besides gallic acid were carried out [20].
Therefore, the study of the solvent effects is very important for the screening and solvent selection of the extraction, fractionation and purification steps in the herbal processing. By understanding the solvent properties, component (solute) properties and solvent–solute interaction, rapid fractionation and isolation of desired components can be achieved. This paper presents the results on the effects of various organic and aqueous solvents with different polarities on the extract yield and the content of three hydrolysable tannins, namely gallic acid, ellagic acid and corilagin. Qualitative and quantitative effects of solvents using different extraction methods were investigated. The extraction methods utilized are the solvent extraction and the high-pressure extraction (supercritical fluid extraction and pressurized water extraction).
Section snippets
Chemicals and standards
The reference standards (gallic acid and ellagic acid) were both purchased from Sigma Chemicals (USA) at purity of 98%. Isolated geraniin (unknown purity) was supplied by Prof. H. Wagner (University of Munich, Germany). Analysis of corilagin was carried out by Nova Laboratories Sdn. Bhd. (Malaysia) using their isolated and patented standard (purity of 98%). The commercial P. niruri product, HEPAR-P™ (standardized to 4% corilagin and 18% total flavonoid content) was obtained from the same
Solvent effects on extract yield
The effects of organic and aqueous solvents on the extraction yield and content of bioactive gallic acid, ellagic acid and corilagin in P. niruri were studied using standard Soxhlet extraction method. The amount of extract after removal of solvent is listed in Table 1.
The results indicate that the P. niruri is most soluble in polar solvents namely water (26.2%) and aqueous ethanol (20.8–27.1%). This shows that most components in P. niruri are hydrophilic or water-soluble. The color of the
Conclusions
The screening by various extraction solvents showed that most components in P. niruri are hydrophilic or water-soluble. The active hydrolysable tannins (gallic acid, ellagic acid and corilagin) were preferably extracted using water or ethanol–water mixtures. Due to the nature of water at sub-critical conditions, it was found that pressurized water extraction (PWE) had the highest overall extract and corilagin yield in the shortest extraction time compared to Soxhlet or SFE method. However, SFE
Acknowledgements
The research was funded by Malaysian Ministry of Science, Technology and Innovation (MOSTI) under Intensified Research in Priority Area (IRPA) grant no. 09-02-02-0091-EA234 and by University Malaya under Vote F (0168/2003A). The main author also thank Mr. N.L. Phang (Nova Laboratories Sdn. Bhd., Malaysia) and Prof. H. Wagner (University of Munich, Germany) for providing free HEPAR-P™ capsules and geraniin standard, respectively.
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