Article pubs.acs.org/jnp
Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
Structural Characterization and Immunomodulatory Activity of a Polysaccharide from Eurycoma longifolia Ping He, Zhou Dong, Qian Wang, Qi-Ping Zhan, Meng-Meng Zhang,* and Hui Wu* College of Food Sciences and Engineering, South China University of Technology, Guangzhou 510640, People’s Republic of China
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ABSTRACT: A polysaccharide, Ali-1, was isolated from the roots of Eurycoma longifolia, a popular traditional medicinal herb in Malaysia. The structure of Ali-1 was characterized by monosaccharide, methylation, and NMR data analyses. The average molecular weight of Ali-1 is 14.3 ku, and it is composed of arabinose (14.31%), xylose (57.69%), galacturonic acid (13.03%), and glucuronic acid (14.86%). The main chain comprises (1→4)-linked xylose residues. It has branch points in the main chain; (1→2,4)-linked xylose residues, 1,2linked glucuronic acid residues, and 1,2-linked arabinose residues form the branches, and the branches are terminated with T-linked galacturonic acid residues and T-linked arabinose residues. Ali-1 significantly improves the pinocytic and phagocytic abilities of RAW264.7 cells and facilitates cytokine secretion according to an immunostimulation assay. These results demonstrate that Ali-1 has potential as a functional supplement for people with compromised immune systems. Eurycoma longifolia, a flowering plant belonging to the Simaroubaceae family, is native to Southeast Asia. Also known as tongkat ali, it is well known as an aphrodisiac and for possessing antimalarial effects and is therefore used as a traditional herbal medicine in Southeast Asia.1 Research on E. longifolia, particularly on the structure and function of its active compounds, has increased in recent years. The root extracts are mainly applied as herbal medicines to treat diseases such as erectile dysfunction,2 intermittent fever,3 and tumors.4 E. longifolia is known to possess an abundance of bioactive ingredients, such as quassinoids,5 alkaloids,6 tirucallane-type triterpenoids, and bioactive steroids.7 Among the important active ingredients in plant extracts, polysaccharides are known to have unique physiological activities. However, little is known about the polysaccharides of E. longifolia and their activities. It is widely believed that the main active substances responsible for the anticancer and aphrodisiac effects of E. longifolia are eurycomanones.8,9 Cancer, reproductive health, and human immunity are related, and plant polysaccharides are natural products with strong immunomodulatory activities.10,11 However, there is no research on the immunological activities of the polysaccharides from E. longifolia. Furthermore, various physiological activities of polysaccharides are associated with their unique structural features.12 The analyses of the structure of a polysaccharide is particularly important for understanding the mechanism of its physiological activity. However, the molecular weight, monosaccharide composition, glycosidic bond type, and other basic structural information on the polysaccharides of E. longifolia are still unknown. © XXXX American Chemical Society and American Society of Pharmacognosy
Macrophages are vital to immune response, as they can directly kill pathogens by phagocytosis and indirectly protect the immune system by releasing cytotoxic agents. In macrophage stimulation experiments, the release of NO, TNF-α, and IL-6 is generally used as immune response indexes for evaluating the immunomodulatory activity of a compound of interest, which in this case are the polysaccharides of E. longifolia.13,14 Herein, a new polysaccharide (Ali-1) was extracted and purified from E. longifolia. Ali-1 was structurally characterized, and its immunomodulatory activity was evaluated in RAW264.7 cells. These results may provide a foundation for future exploration and identification of the polysaccharides present in E. longifolia.
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RESULTS AND DISCUSSION
Separation and Purification of Polysaccharides. The crude polysaccharide fraction from the dried roots of E. longifolia was obtained by extraction with hot water and precipitation with EtOH in a 6.93% yield. A well-resolved peak was obtained after separation using a diethylaminoethyl (DEAE) column. The fraction was eluted with ultrapure water (Figure 1A). A Sephadex G-50 column was used for further separation and purification steps. Only one peak, named Ali-1, was obtained (Figure 1B). The polysaccharide
Received: March 21, 2018
A
DOI: 10.1021/acs.jnatprod.8b00238 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Article
Figure 1. (A) Chromatogram of the polysaccharides from E. longifolia on DEAE-Sepharose Fast Flow (3.6 × 20 cm). (B) Chromatogram of the polysaccharides from E. longifolia on Sephadex G-50 (1.6 × 85 cm).
showed no direct cytotoxicity toward human Burkitt’s lymphoma cells and showed more potent immunostimulating properties than high-molecular-weight carrageenan. In fact, botanical polysaccharides with low molecular weights have also been found in plants such as maca and Cordyceps militaris.17,18 Although Ali-1 has a low molecular weight, it may still have significant biological activities. IR Data. The IR spectrum of Ali-1 is shown in Figure 4. The bands in the 1200−1000, 1400−1200, 3000−2800, and 3600−
content in this fraction was found to be 95.4% after lyophilization. UV Spectra. As shown in Figure 2, the ultraviolet (UV) spectra of Ali-1 did not have absorption peaks at 260 and 280 nm, confirming that no nucleotide or protein impurities are present.15
Figure 2. UV spectra of Ali-1 in the 190−400 nm range.
Determination of Molecular Weight. The results of the molecular weight determination of Ali-1 are given in Figure 3.
Figure 4. IR spectrum of Ali-1.
Figure 3. Molecular weight of Ali-1 based on high-performance gel permeation chromatography.
3200 cm−1 regions are characteristic absorption peaks of polysaccharides. The absorption peaks were identified by comparison with reported data.19 The characteristic vibration at 3400 cm−1 corresponded to O−H functional groups, and that at 2921 cm−1 was indicative of C−H bonds. The absorption band at 1649 cm−1 could be attributed to CO stretching vibrations, and the band at 1414 cm−1 was indicative of CC stretching vibrations. The presence of a pyranose ring was confirmed by the intense and sharp peak at 1014 cm−1. Monosaccharides are structurally classified as α or β based on the configuration of the anomeric carbon. The stretching vibrations of the hemiacetal hydroxy group and the terminal −CH2OH differ if these groups are on the same face or on different faces, and the configuration could be assigned as α or β based on these vibrations. According to literature reports,20 the characteristic absorption peak at 851 ± 8 cm−1 is indicative of an α-pyranoside, and a band at 891 ± 7 cm−1 indicates a βpyranoside; for a polysaccharide, the absorption band indicative of the α-configuration appears at 574 ± 5 cm−1, and that of the β configuration at 591 ± 9 cm−1. Ali-1 has characteristic absorption peaks at 857 and 579 cm−1, indicating the presence of an α-pyranoside. Determination of the Monosaccharide Composition. HPLC with 1-phenyl-3-methyl-5-pyrazolone (PMP) was used for these analyses because of the possible presence of uronic
The presence of an intense sharp peak shows that Ali-1 is a pure polysaccharide. The molecular weight of Ali-1 was found to be 14.3 ku, making Ali-1 a low-molecular-weight polysaccharide. Typically, polysaccharides with high molecular weights tend to have more improved biological activities. However, higher masses are often associated with problems such as higher apparent density and viscosity, poor solubility, and low absorbability. Low-molecular-weight polysaccharides may also have stronger biological activities. Stephanie et al.16 reported that low-molecular-weight carrageenan (
