OPEN. Lotus A Source of Food and Medicine: Current Status and Future Perspectives in Context of the Seed Proteomics

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Lotus A Source of Food and Medicine: Current Status and Future Perspectives in Context of the Seed Proteomics Carlo F. Moro, Masami Yonekura, Yoshiaki Kouzuma, Ganesh K.

1 Lotus A Source of Food and Medicine: Current Status and Future Perspectives in Context of the Seed Proteomics Carlo F. Moro, Masami Yonekura, Yoshiaki Kouzuma, Ganesh K. Agrawal, and Randeep Rakwal International Journal of Life Sciences ISSN No OPEN ACCESS DOIdx.doi.org/ /ijls.v7i This article is protected by copyright, and all rights are held exclusively by International Journal of Life Sciences. C IJLS

2 An Independent, Open Access, Peer Reviewed, Non-Profit Journal International Journal of Life Sciences Founded 2007 International Journal of Life Sciences Copyright c International Journal of Life Sciences Review Article Lotus A Source of Food and Medicine: Current Status and Future Perspectives in Context of the Seed Proteomics Carlo F. Moro, Masami Yonekura *, Yoshiaki Kouzuma, Ganesh K. Agrawal, 2, 3, 4 and Randeep Rakwal 1Laboratory of Molecular Food Functionality, College of Agriculture, Ibaraki University, Chuuo, Ami, Inashiki, Ibaraki , Japan; 2Research Laboratory for Biotechnology and Biochemistry (RLABB), GPO Box 13265, Kathmandu, Nepal; 3Graduate School of Life and Environmental Sciences, University of Tsukuba, Tennoudai, Tsukuba , Japan; 4Department of Anatomy I, School of Medicine, Showa University, Hatanodai, Shinagawa, Tokyo , Japan Article Information Submitted: January, 2012 Revised: May, 2012 Accepted: June, 2012 Key words: Nelumbo nucifera, lotus, proteome ABS TR ACT Nelumbo nucifera (Gaertn.), commonly known as the lotus, is an aquatic plant native to India and presently consumed as food, mainly in China and Japan. In addition to its use as food, the lotus plant is also widely used in Indian and Chinese traditional medicine. Extracts from different parts of the lotus plant have been reported to show several biological activities, such as antioxidant, free radical scavenging, anti-inflammatory, and immuno-modulatory activities. Despite this, little work has been done in isolating and identifying the proteins responsible for these activities. To date, there is no report on systematic protein analysis of the lotus plant. In this review, we discuss the medicinal value of the lotus plant and reported works on its biological activities. We also present a proteomics approach for systematic investigation of the lotus seed proteome. INTRODUCTION The lotus plant: historical, social and economical significance Nelumbo nucifera (Gaertn.) is an aquatic perennial belonging to the family of Nelumbonaceae, which has several common names (e.g. Indian lotus, Chinese water lily, and sacred lotus). Lotus is a large and rhizomatous aquatic herb with slender, elongated, branched, creeping stem consisting of nodal roots; leaves are membranous, peltate (60-90 cm and above), orbicular, and concave to cupshaped; petioles are long, rough with small distinct prickles; flowers are white to rosy (Fig. 1a), sweet-scented, solitary, hermaphrodite, cm diameter; ripe carpels are 12 mm long, ovoid and glabrous; fruits are ovoid having nut like achenes; seeds are black, hard and ovoid (Fig. 1b,c) (Sridhar and Rajeev, 2007). The lotus is native to India, but was widely spread through Persia, Egypt, and Asia in the ancient times. It was introduced in Europe as a type of water-lily in the 18th century, and nowadays it can be found in modern botanical gardens all over the world. Lotus plants are commonly cultivated in Australia, China, India, Iran, and Japan (Anonymous, 1966). The lotus flower is considered a symbol of beauty and purity in both Hindu and Buddhist religions and is widely depicted in religious texts and pictures, as well as in literature and oral traditions of several Asian cultures. In China during 1999, it served as an industrial crop grown over 40,000 ha. (Guo, 2009). Lotus was introduced from China to Japan and has been cultivated there for more than 1000 years (Komatsu et al., 1975). In India, it is wide spread and known even from Himalayan lakes at altitude up to 1400 m (Polunin and Stainton, 1984). Lotus seeds sold in the Indian markets ('kamal gatta') as vegetable or raw material for Ayurvedic drug preparation (Anonymous, 1992). Seeds and roots of lotus are regarded as popular health food and the alkaloid (liensinine) extracted from them is effective to treat arrhythmia (Ling et al., 2005). Nutritional value The lotus seeds and rhizome are extensively consumed as food in China and Japan. The seeds can be eaten raw, roasted, or ground and boiled into a syrup or paste and used as ingredient in a large number of traditional Chinese and Japanese pastries and desserts. The roasted seeds are also good coffee substitute and possess saponins, phenolics, * Correspondence to: Masami Yonekura,Laboratory of Molecular Food Functionality, College of Agriculture, Ibaraki University, Chuuo, Ami, Inashiki, Ibaraki , Japan. E mail: yonekura@mx.ibaraki.ac.jp; Phone No: (+81) , Copyright reserved c International Journal of Life Sciences doi:dx.doi.org/ /ijls.v7i1.6394

3 and carbohydrates in appreciable quantities (Anonymous, 1992; Ling et al., 2005). Likewise, the rhizomes can be eaten raw, pickled, stewed, and fried. Stems, leaves, and petals can also be eaten, and the stamens are traditionally used to make herbal tea. The lotus rhizomes consist of 1.7% protein, 0.1% fat, 9.7% carbohydrate, and 1.1% ash (Reidm,1977). The stems contain 6, 2.4, and 0.2 mg/100 g of calcium, iron, and zinc, respectively (Ogle et al., 2001). The mature seeds of N. nucifera consist of around 8-10% moisture, 25% protein, 3.7% crude fat, 65% carbohydrate, 3-4% crude fibre, 4% ash, and contain 388 cal/100 g of energy. Mineral composition of 100 g of lotus seeds consists of sodium (7.86 mg), potassium (48.5 mg), calcium (313 mg), phosphorus (6.25 mg), magnesium (43.9 mg), copper (2.51 mg), zinc (7.72 mg), manganese (16.6 mg), iron (16.4 mg), and selenium (1.04 mg). Lotus seed flour has a lipid composition of mg/g of polyunsaturated fatty acids and 5.05 mg/g of saturated fatty acids oleic, where linoleic acid is the most abundant unsaturated fatty acid, and palmitic acid is the most abundant saturated acid. Total phenolic content of the lotus seed cotyledon is 4%, and tannin content is 3.9% (Bhat and Sridhar, 2008). Table 1. Lotus nutritional content (seeds) (MEXT, 2000). MEDICINAL USE OF THE LOTUS PLANT PARTS Leaves Seeds Lotus leaves are used in traditional medicine to treat hypertension, diarrhea, fever, weakness, infection, skin inflammation, and body heat imbalance (Sridhar and Rajeev, 2007). They are also an effective treatment against abnormal bleeding such as hematemesis, epistaxis, hemoptysis, hematuria, and metrorrhagia (Ou, 1989). Extracts from the lotus leaves have shown a strong antioxidant and radical scavenging ability, as well as nhibitory activity of diabetic complication factors (Wu et al., 2003; Saengkhae et al., 2007, Jung et al. 2008, Huang et al., 2011). Extracts of the lotus leaf have been used to treat obesity, and have had reported anti-obesity and anti-hyperlipidemia effects on rodents (Lacour et al., 1995, Onishi et al., 1984, Ono et al., 2006). Additionally, leaf extracts were found to modulate lipolysis-activity and decreased adipogenesis in human pre-adipocytes (Siegner et al., 2010) as well as to lower elevated cholesterol levels in mice and reducing levels of phospho-lipids and triglycerides (Onishi et al., 1984). Two anti-hiv principles have been isolated from the ethanolic extract of the lotus leaves (Kashiwada et al., 2005). Roots/Rhizomes Hydro-alcoholic extract from N. nucifera seeds showed potent free-radical scavenging ability when tested by the 2,2-diphenyl-1-picrylhydrazyl free radical (DPPH) and nitric oxide methods, showing a half maximal inhibitory concentration (IC50) value of 16.12±0.41 g/ml for the DPPH test and 84.86± 3.56 g/ml for the nitric oxide method, values which were similar or above that of rutin (IC50 values of 18.95±0.49 g/ml and ±5.01 g/ml, respectively). Rats treated with the extract for 4 days prior to injection with CCl4 showed a dose-dependent increase in superoxide dismutase and catalase, and a decrease in thiobarbituric acid reactive substances when compared with control rats also subjected to oxidative damage by CCl4 (Rai et al., 2006). It was observed that ethanolic lotus seed extract caused significant increase in total and differential leucocyte and lymphocyte population in treated mice, indicating an immunological effect. Treatment with the extract was also shown to inhibit foot paw edema. Unlike with similar treatment with rhizome extract, neutrophil adhesion wasn't significantly altered (Mukherjee et al., 2010). (S)-armepavine, an extract from lotus seeds, has been shown to possess immunomodulatory effects, inhibiting the symptoms in MRL/MpJ-lpr/lpr mice (similar to those of human systemic lupus erythematosus) and significantly increasing survival rates, with an effect comparable to that of cyclosporin A (Liu et al., 2006). Aqueous extracts from the lotus rhizome presented different phenolic, tannin, and flavonoid content than the lotus leaves, but have also shown antioxidant activity, albeit not as pronounced as the leaf extracts (Huang et al., 2011). Ethanolic extracts from the rhizome knots have shown more antioxidant activity than those from the whole rhizome, also correlating to their phenolic content (Hu and Skibsted, 2002). ACTIVE COMPOUNDS All parts of the lotus plant have yielded significant amounts of phenolic and flavonoid compounds (Bhat et al., 2008; Hu et al., 2002; Huang et al., 2011; Jung et al.,2008; Mukherjee et al., 2009; 2

4 Ono et al., 2006; Rai et al., 2006). After methanolic extraction, the phenolic content of each part of the plant, determined in terms of gallic acid equivalents per g of dried extract (GAE, mg/g extract), was found to follow the order of: leaves (177.7) > de-embryonated seeds (92.7) > stamens (83.4) > embryos (41.0) > rhizomes (21.6). For the flavonoid contents, determined as mg of (+)-catechin equivalent per g of dried extract (CE, mg/g extract), the order was: leaves (125.6) > de-embryonated seeds (82.9) > stamens (50.3) > embryos (18.9) > rhizomes (8.5) (Jung et al. 2008). Table 2. Biological effects and extracts obtained from the Lotus plant. Upon fractionation of methanolic leaf extract of lotus, different amounts of phenolic and flavonoid compounds were found depending on the solvent used, the highest content being found in the etyl acetate and n-butanol extracts, which were also shown to possess the greatest radical scavenging effect (Jung et al., 2008). A large amount of glutathione is contained in the plumule (13 g per plumule) and cotyledons (164 g per cotyledon) of N. nucifera; the amount of total plumule increases gradually in the maturing seed. The reduced form of glutathione is dominant in the early stages, while the amount of oxidized form exceeds that of the reduced form at the end of maturation. The amount of the reduced form of glutathione in the unripe fruit decreases markedly upon storage for one year (Mukherjee et al., 2009). The lotus seeds contain a large number of alkaloids, notably dauricine, lotusine, nuciferine, pronuciferine, liensinine, isoliensinine, roemerine, neferine, and armepavine. Procyanidin was also isolated from the seed pod. Seeds also contain gallic acid, isoquininolinol, saponins, and carbohydrates (Mukherjee et al., 2009 ). Seed polysaccharides are mainly composed of D-galactose, L-arabinose, D-mannose, and D-glucose (Das et al., 1992). 13C-NMR and in-source pyrolysis-mass spectroscopy analysis showed that the fruit wall and seed coat of the N. nucifera seed are composed of a complex of polysaccharide, based primarily on galactose and mannose units and insoluble tannins. PROTEOMICS EVIDENCES Considering its status as food and medicine, remarkably little research has been done concerning the proteins expressed in the lotus plant. There are works regarding the identification and subsequent cloning and expression of copper-zinc superoxide dismutase genes (Dong et al., 2010), small heat shock proteins (Zhou et al., 2012) and a phytochelatin synthase (Liu et al., 2012), those are, however, primarily at the genome and transcriptome, rather than proteome level. A notable exception is a proteomic and functional analyses of lotus annexins (Chu et al., 2012). However, the lack of a comprehensive proteome research is regrettable, considering the large role of proteins in nutrition and in the physiological activity of foods. Therefore, we are currently engaged in a work to develop a proteome analysis of the lotus seed, since the seed is arguably the most concentrated source of protein in the plant. Furthermore, the seed is the most resilient part of the lotus plant, and can be stored indefinitely before opened. Research Objectives The objectives of our work are to develop suitable methods of protein extraction for proteomic and biological assay purposes; develop the seed proteome using parallel complementary approaches, such as one- and two-dimensional gel electrophoresis (2-DGE)-based proteomics approaches including N-terminal sequencing, and shotgun proteomics. Fig. 1 illustrates the experimental workflow for sample preparation and 1-DGE approach as an example. 3 Current Work Sample Preparation Lotus seeds were cracked. The husk was removed, endosperm and embryo fragments were separated, and endosperm sheet was scrapped off. Endosperm and embryo portions were separately ground into fine powder in liquid nitrogen with mortar and pestle and stored at -80o C until used. Extraction Five different extraction protocols were tested, the main variation being the extraction buffer. Those were: Tris/thiourea-containing lysis buffer (LB-TT) extraction, Tris-HCl buffer saline (TBS) extraction, TBS crude extraction (without a protein precipitation and cleaning step), Trichloroacetic acid(tca)-acetone buffer extraction, and Tris-buffered phenol extraction. All extraction protocols, except TBS-crude and phenol, included a protein precipitation and clean-up step using the ProteoExtract kit (Calbiochem). All extracts except the TBS-crude were resuspended in LB-TT buffer after precipitation (Agrawal and Rakwal, 2006, 2011). Protein Concentration Protein concentration in the extracts was measured by the Bradford method using bovine serum albumin standards. Extracts were diluted to 10 times in ultrapure water and the same amount of LB-TT buffer as the extracts was applied to the dilution of the standards to compensate for any potential interference of LB-TT in the absorbance. Determination of protein concentration by A280 absorbance assays were also attempted, but found to be unreliable for extracts suspended in LB-TT.

1-DGE I n t e r n a t i o n a l J o u r n a l o f L i f e S c i e n c e s 7 ( 1 ) : 2 0 1 3 ; 1-5 1-Dimentional Gel Electrophoresis (basically sodium dodecyl sulfate polyacrilamide gel

5 1-DGE I n t e r n a t i o n a l J o u r n a l o f L i f e S c i e n c e s 7 ( 1 ) : ; Dimentional Gel Electrophoresis (basically sodium dodecyl sulfate polyacrilamide gel electrophoresis, SDS-PAGE) assays were performed to compare band separation profile between different extracts and to identify bands suitable for N-terminal sequencing. Tricine-SDS-PAGE protocol was also used to attempt better separation of lower molecular weight protein bands. Usual length of the separation gel employed was approximately 10 cm. The usual method of staining was a modified Coomasie Brilliant Blue staining (Wang et al., 2007). An assay with fluorescent Flamingo stain (BioRad) was also performed. In average, approximately 35 distinct bands were observed, with more likely present but not immediately distinguishable. FUTURE WORK Immediate and future research works will include 2-DGE analysis of the prepared lotus seed protein, with aims to develop reference map, followed by trypsin digestion and mass spectroscopy analysis of distinct spots. Identification of proteins will also be concurrently performed by shotgun proteomics techniques. Furthermore, predicting that an abundance of seed storage proteins (SSPs) contained in the extracts might mask the presence of lower-abundance functional proteins, SSP depletion will be tested using a protamine sulfate precipitation protocol by (Sun Tae Kim, personal communication). Figure 1. Outline of work to extract and identify lotus seed proteins. 4

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Pink flower. Water lily. Cosmos. Prunus Mume Flower

Pink flower. Water lily. Cosmos. Prunus Mume Flower PINK FLOWER Complex Pink flower Prunus Mume Flower Water lily Cosmos Rose Camellia Lotus Flower Japanese apricot s flower Common name Latin name INCI name Efficacy Mume Flos Prunus mume Siebold & Zucc.