molecules Article
Cytotoxic, Antitumor and Immunomodulatory Effects of the Water-Soluble Polysaccharides from Lotus (Nelumbo nucifera Gaertn.) Seeds Yafeng Zheng 1 , Qi Wang 1,2 , Weijing Zhuang 1 , Xu Lu 1 , Anca Miron 3 , Tsun-Thai Chai 4 , Baodong Zheng 1, * and Jianbo Xiao 5, * 1 2 3 4 5
*
College of Food Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China;
[email protected] (Y.Z.);
[email protected] (Q.W.);
[email protected] (W.Z.);
[email protected] (X.L.) Institute of Agricultural Engineering, Fujian Academy of Agriculture Sciences, Fuzhou 350003, China Faculty of Pharmacy, Grigore T. Popa University of Medicine and Pharmacy Iasi, Universitatii Street, No. 16, Iasi 700115, Romania;
[email protected] or
[email protected] Department of Chemical Science, Faculty of Science, Universiti Tunku Abdul Rahman, Jalan Universiti Bandar Barat, Kampar 31900, Perak, Malasia;
[email protected] Institute of Chinese Medical Sciences, State Key Laboratory of Quality Research in Chinese Medicine, University of Macau, Taipa, Macau, China Correspondence:
[email protected] (B.Z.);
[email protected] (J.X.); Tel.: +853-6571-8163 (J.X.)
Academic Editor: Maurizio Battino Received: 10 October 2016; Accepted: 28 October 2016; Published: 2 November 2016
Abstract: Lotus is an edible and medicinal plant, and the extracts from its different parts exhibit various bioactivities. In the present study, the hot water–soluble polysaccharides from lotus seeds (LSPS) were evaluated for their cancer cell cytotoxicity, immunomodulatory and antitumor activities. LSPS showed significant inhibitory effects on the mouse gastric cancer MFC cells, human liver cancer HuH-7 cells and mouse hepatocarcinoma H22 cells. The animal studies showed that LSPS inhibited tumor growth in H22 tumor-bearing mice with the highest inhibition rate of 45.36%, which is comparable to that induced by cyclophosphamide (30 mg/kg) treatment (50.79%). The concentrations of white blood cells were significantly reduced in cyclophosphamide-treated groups (p < 0.01), while LSPS showed much fewer side effects according to the hematology analysis. LSPS improved the immune response in H22 tumor-bearing mice by enhancing the spleen and thymus indexes, and increasing the levels of serum cytokines including tumor necrosis factor-α and interleukin-2. Moreover, LSPS also showed in vivo antioxidant activity by increasing superoxide dismutase activity, thus reducing the malondialdehyde level in the liver tissue. These results suggested that LSPS can be used as an antitumor and immunomodulatory agent. Keywords: lotus seeds; polysaccharides; antitumor; immunomodulatory
1. Introduction Cancer is a malignant disease characterized by an abnormal cell growth and proliferation with the potential to invade surrounding tissues and metastasize to other tissues in the body [1]. As cancer incidence increases every year, more effective therapies are urgently needed. However, currently many available chemotherapy drugs cause serious side effects [2]. Previous studies showed that different non-toxic polysaccharides extracted from natural sources exhibit promising antitumor and immunomodulatory activities [3]. Lotus (Nelumbo nucifera Gaertn.), an aquatic perennial plant of economic importance, has been cultivated in China for about 3000 years. Being both an edible and medicinal plant, all the parts of the lotus are used, especially the rhizomes and seeds [4]. Lotus seeds (Figure 1), called ‘Lian Zi’ in Chinese,
Molecules 2016, 21, 1465; doi:10.3390/molecules21111465
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the lotus are used, especially the rhizomes and seeds [4]. Lotus seeds (Figure 1), called ‘Lian Zi’ in Chinese, aresources valuable sources of proteins, starch, and fatty unsaturated fattyhave acids, which have aamino wellare valuable of proteins, starch, and unsaturated acids, which a well-balanced balanced amino acid as wellofassugar, low levels of sugar, sodium and[5,6]. cholesterol Besides acid composition, ascomposition, well as low levels sodium and cholesterol Besides[5,6]. being used being used as food, lotus seeds also have a very long history as a traditional medicine in China and as food, lotus seeds also have a very long history as a traditional medicine in China and are used arediuretic, used as diuretic, antiemetic, anti-inflammatory and anticancer agents [7,8]. Variousactive activecomponents components as antiemetic, anti-inflammatory and anticancer agents [7,8]. Various extracted from different part of lotus seeds (seedpod, plumule and cotyledons) were characterized to be mainly flavonoids, polyphenols polyphenols and and alkaloids alkaloids [9], [9], which which have have many many pharmacological pharmacologicaland and physiological physiological activities, including antioxidant activity [10–13], DNA- and cell-protective effects [14,15], anti-obesity [17], and and anti-cancer anti-cancer activity activity [18,19]. [18,19]. activity [16], immunomodulatory effects [17],
Figure nucifera Gaertn.). Gaertn.). Figure 1. 1. Seeds Seeds and and seedpods seedpods of of lotus lotus (Nelumbo (Nelumbo nucifera
Isolation and characterization of polysaccharides with antitumor activity, an immunomodulatory Isolation and characterization of polysaccharides with antitumor activity, an immunomodulatory effect or other pharmacology have emerged as one of the important research fields in chemistry and effect or other pharmacology have emerged as one of the important research fields in chemistry and biology [20]. However, there is little data on the lotus polysaccharides. Jiang and coworkers reported biology [20]. However, there is little data on the lotus polysaccharides. Jiang and coworkers reported that aa polysaccharide-protein that polysaccharide-protein complex complex from from the the lotus lotus rhizome rhizome exhibited exhibited aa certain certain immunoregulatory immunoregulatory effect and anti-HIV-1 activity [21]. Moreover, the lotus plumule polysaccharide effect and anti-HIV-1 activity [21]. Moreover, the lotus plumule polysaccharide was was purified, purified, characterized and proved to exert anti-inflammatory activity and anti-diabetic activity [22–26]. characterized and proved to exert anti-inflammatory activity and anti-diabetic activity [22–26]. In In our our previous study, a novel water-soluble polysaccharide from lotus seed was extracted, characterized previous study, a novel water-soluble polysaccharide from lotus seed was extracted, characterized and found and found to to exhibit exhibit antioxidant antioxidant potential potential [27]. [27]. Further Further investigation investigation is is required required to to reveal reveal the the potency potency of this novel lotus seed polysaccharide. Therefore, the present study was carried out to evaluate the of this novel lotus seed polysaccharide. Therefore, the present study was carried out to evaluate the cytotoxic, antitumor and immunomodulatory effects of the hot water–soluble polysaccharides from cytotoxic, antitumor and immunomodulatory effects of the hot water–soluble polysaccharides from lotus seeds. seeds. lotus 2. Results Results 2.1. Extraction of Lotus Seed Polysaccharides LSPS were extracted extracted from from the thedried driedlotus lotusseeds seedsby byhot hotwater waterextraction. extraction.The The extraction yield extraction yield of of LSPS was 0.95%, which is lower than the yield (1.51%) of the lotus seeds’ polysaccharides using LSPS was 0.95%, which is lower than the yield (1.51%) of the lotus ultrasound-assisted extraction as as reported reported in our previous study [27]. However, However, lotus seeds are a ultrasound-assisted extraction traditional polysaccharides obtained by by hothot water extraction are traditional food food ingredient ingredientininvarious varioussoups. soups.The The polysaccharides obtained water extraction more similar to the dietary ones present in lotus seed soups. A comparative analysis of the structural are more similar to the dietary ones present in lotus seed soups. A comparative analysis of the properties the lotus of seed extracted by various methods will be methods performedwill in our structural of properties thepolysaccharides lotus seed polysaccharides extracted by various be future studies. performed in our future studies. 2.2. Cytotoxic Effects on MFC, HuH-7 and H22 Cells 2.2. Cytotoxic Effects on MFC, HuH-7 and H22 Cells To evaluate the cytotoxicity of LSPS on tumor cells in vitro, MFC, HuH-7 and H22 cells were To evaluate the cytotoxicity of LSPS on tumor cells in vitro, MFC, HuH-7 and H22 cells were incubated with various concentrations of LSPS (0, 50, 100 and 200 µg/mL) for 48 h. According to incubated with various concentrations of LSPS (0, 50, 100 and 200 μg/mL) for 48 h. According to Figure 2, compared with the NC group, incubation of various concentrations of LSPS inhibited the Figure 2, compared with the NC group, incubation of various concentrations of LSPS inhibited the growth of tumor cells. However, all the doses of LSPS in tumor cell culture media are not sufficient for growth of tumor cells. However, all the doses of LSPS in tumor cell culture media are not sufficient half-maximal inhibition of tumor cell proliferation, suggesting that LSPS could only weakly inhibit for half-maximal inhibition of tumor cell proliferation, suggesting that LSPS could only weakly tumor cell proliferation in vitro [28]. It is noticeable that LSPS exhibited more potent inhibitory effects inhibit tumor cell proliferation in vitro [28]. It is noticeable that LSPS exhibited more potent inhibitory
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effects on viability in and HuH-7 cells in comparison to MFC cells, which asuggests a certain on viability in HuH-7 H22and cellsH22 in comparison to MFC cells, which suggests certain antitumor effects onespecially viability in HuH-7 and H22 cells cells. in comparison to MFC cells, which suggests a certain antitumor potential, especially on cells. hepatoma potential, on hepatoma antitumor potential, especially on hepatoma cells.
Figure cells. The Figure 2. 2. Inhibitory Inhibitory effect effect of of LSPS LSPS on on MFC MFC (A); (A); HuH-7 HuH-7 (B) (B) and and H22 H22 (C) (C) cells. The OD OD values values are are Figure 2. Inhibitory effect of LSPS on MFC (A); HuH-7 (B) and H22 (C) cells. The OD values are represented as means ± SD. Compared with negative control (NC), * p < 0.05, ** p < 0.01. L-LSPS, represented as means ± SD. Compared with negative control (NC), * p < 0.05; ** p < 0.01.incubated L-LSPS, represented as means ± SD. Compared with negative control (NC), * p < 0.05, ** p < 0.01. L-LSPS, incubated with lotus seed solution (50 μg/mL); incubated withincubated lotus seedwith polysaccharide incubated withpolysaccharide lotus seed polysaccharide solutionM-LSPS, (50 µg/mL); M-LSPS, lotus seed with lotus seed polysaccharide solution (50 μg/mL); M-LSPS, incubated with lotus polysaccharide solution (100 μg/mL); H-LSPS, lotusincubated seed polysaccharide (200seed μg/mL). polysaccharide solution (100incubated µg/mL); with H-LSPS, with lotussolution seed polysaccharide solution solution (100 μg/mL); H-LSPS, incubated with lotus seed polysaccharide solution (200 μg/mL). (200 µg/mL).
2.3. Effect of LSPS on Tumor Growth and Body Weight 2.3. Effect of LSPS on Tumor Growth and Body Weight 2.3. Effect of LSPS Tumor Growth andofBody Weight To assess theon antitumor activity LSPS in vivo, LSPS was orally administered to H22 tumorTo assess the antitumor activity of LSPS in vivo, LSPS was orally administered to tumorbearing mice for 14 days. A significant reduction in vivo, the tumor was detected in H22 the to treated To assess the antitumor activity of LSPS in LSPSweight was orally administered H22 bearingatmice for 14 days. significant reduction in results the tumor weight was detected in the treated groups the end of the period, andreduction the in Figure 3. Compared tumor-bearing mice forexperimental 14Adays. A significant inare thesummarized tumor weight was detected in the groups at the end experimental period, and the results are summarized in Figure 3.in Compared treated groups at of thethe end of the period, and the results aretumor summarized Figure to the negative control group, theexperimental CTX-treated group showed significant inhibition (50.79%,3. to the negative control group, the CTX-treated group showed significant tumor inhibition (50.79%, Compared to the negative control group, the CTX-treated group showed significant tumor inhibition p < 0.01). Meanwhile, the different doses of LSPS (50, 100 and 200 mg/kg) resulted in tumor inhibition p < 0.01). Meanwhile, the different doses of LSPS (50, 100 and 200 mg/kg) resulted in tumor inhibition (50.79%, p < 0.01). Meanwhile, the different doses of LSPS (50, 100 and 200 mg/kg) resulted in tumor in H22 tumor-bearing mice with rates of 17.90%, 39.60% and 45.36%, respectively. in H22 tumor-bearing mice with mice rates with of 17.90%, 39.60% and 45.36%, respectively. inhibition in H22 tumor-bearing rates of 17.90%, 39.60% and 45.36%, respectively.
Figure 3. Cont.
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Figure 3. In vivo antitumor activities of LSPS in H22 tumor-bearing mice. Experiment design of H22 mouse hepatocellular carcinoma model (A); Effect oftumor-bearing LSPS on tumor weight (B) and average Figure LSPS in in H22 mice. Experiment design oftumor H22 Figure 3. 3. In Invivo vivoantitumor antitumoractivities activitiesofof LSPS H22 tumor-bearing mice. Experiment design of inhibition rate (C); Effect of LSPS on thymus index (D) and spleen index (E). The results are expressed mouse hepatocellular carcinoma model (A); Effect of LSPS on tumor weight (B) and average tumor H22 mouse hepatocellular carcinoma model (A); Effect of LSPS on tumor weight (B); and average as means ±rate SD (C); of 10Effect separateLSPS experiments. Compared with negative control (NC), * p < 0.05, ** p < 0.01. inhibition onLSPS thymus (D)index and spleen index (E). index The results areresults expressed tumor inhibition rate (C);ofEffect of on index thymus (D); and spleen (E). The are CTX, cyclophosphamide (30 mg/kg) treated group; L-LSPS, lotus seed polysaccharide (50 as means ±as SD of 10 separate experiments. Compared Compared with negative (NC), * p < (NC), 0.05, *** ppmg/kg)
