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Extracted from The sacred lotus (Nelumbo nucifera)– phytochemical and therapeutic profile - Dr Pulok K. Mukherjee1,2,*, Debajyoti Mukherjee1, Amal K. Maji1, S. Rai1 and Michael Heinrich2  Article first published online: 8 JAN 2010  DOI: 10.1211/jpp.61.04.0001  2009 Royal Pharmaceutical Society of Great Britain

The fruit of this plant is an aggregate of indehiscent nutlets. Ripe nutlets are ovoid, roundish or oblongish, up to 1.0 m long and 1.5 cm broad, with a hard, smooth, brownish or greyish black pericarp which is faintly longitudinally striated, pedunculated and single seeded. Seeds fill in the ripe carpel. The seeds are sold as a vegetable in Indian markets, under the name of ‘kamal gatta’. The fruits show remarkable dormancy; indeed the longevity of its seeds exceeds that of any known species of flowering plants. Robert Brown, first keeper of Botany in the British Museum, experimented with fruits of Nelumbo at various times between 1843 and 1845 and showed that they retained the power of germination after 150 years of confinement in a glass-top box.

Traditional uses

The seeds and fruits are used as a health food in Asia and to treat many ailments, including poor digestion, enteritis, chronic diarrhoea, insomnia, palpitations, spermatorrhoea, leucorrhoea, dermatopathy, halitosis, menorrhagia, leprosy, tissue inflammation, cancer, fever and heart complaints, and as an antiemetic, poisoning antidote, diuretic and refrigerant. Lotus seedpods are sometimes used as a traditional medicine for haemostatic function.  The seed powder mixed with honey is useful in treating cough. Embryos of lotus seed are used in traditional Chinese medicine to overcome nervous disorders, insomnia, high fevers (with restlessness) and cardiovascular diseases (e.g. hypertension, arrhythmia).

Phytochemistry

The seeds of N. nucifera are rich in asparagin, fat, protein, starch and tannin. The lotus seed is composed of three parts – integuments, plumule and cotyledons, which comprise 3.74%, 3.03% and 93.23% of the mass, respectively. The average weight of 100 seeds is 87.35 g. A large amount of glutathione is contained in the plumule (l3 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 oxidised 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 l year. In general, the rate of germination of the stored seeds seems to be closely related to the content of reduced glutathione.

Normally, lotus seeds are rich in protein, amino acids, unsaturated fatty acids and minerals. Nelumbo seeds have also been found to contain a variety of minerals such as chromium (0.0042%), sodium (1.00%), potassium (28.5%), calcium (22.10%), magnesium (9.20%), copper (0.0463%), zinc (0.0840%), manganese (0.356%) and iron (0.1990%). Other relevant nutritional elements include total ash (4.50%), moisture (10.50%), crude carbohydrate (1.93%), crude fibre (10.60%), fat (72.17%), and protein (2.70%); its energy value is 348.45 cal per 100 g.

The major secondary metabolites present in the seeds  are alkaloids such as dauricine , lotusine, nuciferine , pronuciferine, liensinine , isoliensinine , roemerine, neferine  and armepavine. Procyanidin  was isolated form the seedpod of N. nucifera. Seeds also contain gallic acid (11), D(–)-3′-bromo-O-methyl-armepavine , D–1,2,3,4-tetrahydro-6-methoxy-1-(p-methoxy benzly)-2-methyl-7-isoquinolinol , saponins and carbohydrates. The seed polysaccharides have also been isolated and characterised. Acid hydrolysis and methylation showed that seed polysaccharides are mainly composed of four types of monosaccharide: D-galactose, L-arabinose, D-mannose and D-glucose.

Pharmacology and toxicology

Anti-ischaemic activity

The seed of N. nucifera shows potent anti-ischaemic effects in the isolated rat heart. The effective amount of seed extract against ischaemia induced in the isolated rat heart was assessed by measuring cardiac output; doses of 0.1–30 mg/ml were tested. Maximal recovery was seen at a dose of 10 mg/ml, although cardiac output was similar after treatment with 3 or 10 mg/ml doses (63.5 ± 3.2 and 65.8 ± 4.0 ml/min, respectively). Thus, the 3 mg/ml dose was determined to be the optimum dose for anti-ischaemic effects in the rat.

Antioxidant activity

The ethanol extract of the seed has been evaluated for its antioxidant activity using the 2,2-diphenyl-1picrylhydrazyl (DPPH) free radical assay. Potent free radical scavenging effects were seen, with a median inhibition concentration (IC50) of 6.49 μg/ml.  Furthermore, the antioxidant activity of the hydroalcoholic extract of seed has been reported by Rai et al. using the DPPH and nitric oxide methods. The hydroalcoholic extract exhibited strong free radical scavenging activity, with IC50 values of 6.12 ± 0.41 μg/ml in the DPPH assay and 84.86 ± 3.56 μg/ml in the nitric oxide assay. These values were lower than those of rutin, a standard free radical scavenger. Administration of the hydroalcoholic extract of seed to Wistar rats at 100 and 200 mg/kg for 4 days before carbon tetrachloride treatment caused significant dose-dependent increases in the levels of superoxide dismutase (SOD) and catalase, and a significant decrease in the level of thiobarbituric acid reactive substances. These changes observed at 100 mg/kg were comparable to those observed with vitamin E at 50 mg/kg.

Procyanidin and condensed tannin isolated from the seed pod of N. nucifera have several pharmacological activities, including lipid auto-oxidation, lipoxygenase inhibition and free radical scavenging comparable to butylated hydroxytoluene (0.1%). At a concentration of 62.5 μg/ml, procyanidin inhibited lipoxygenase activity by more than 90%, with an IC50 value of 21.6 μg/ml.47

Hepatoprotective activity

An ethanol extract of the seed was studied for hepatoprotective effects in carbon tetrachloride and aflatoxin B1-induced hepatotoxicity models. Cell death caused by carbon tetrachloride was significantly inhibited in a dose-dependent manner by the ethanolic extract at concentrations between 10 and 500 μg/ml. The same extract reduced the genotoxicity of aflatoxin B1, showing complete inhibition at a concentration of 250 μg per plate.

Antiproliferative activity

The ethanolic extract of N. nucifera seed suppressed cell cycle progression, cytokine gene expression and cell proliferation in human peripheral blood mononuclear cells (PBMC). To study the effects on PBMC proliferation, resting cells or cells activated with phytohaemaglutinin (PHA) were treated with 100 μg/ml of an ethanolic extract of N. nucifera seed.  Cell proliferation was determined on the basis of uptake of tritiated thymidine. PBMC proliferation was not affected by DMSO treatment. Ciclosporin blocked PHA-activated PBMC proliferation. Ethanolic extract of N. nucifera seed (100 μg/ml) significantly suppressed PBMC proliferation stimulated with PHA. The ethanol extract of N. nucifera suppressed proliferation in PHA-activated PBMC. The stimulated cell cycle progression in PHA-activated PBMC was significantly arrested at G0/G1 stage, and gene expression and production of interleukin(IL)-2, IL-4, IL-10, interferon gamma (IFN-γ) and cyclin-dependent kinase 4 in activated PBMC were also decreased by N. nucifera extract.  Liu and co-workers have isolated (S)-armepavine (C19H23O3N; molecular weight 313) from N. nucifera seed extract. (S)-Armepavine inhibited the proliferation of human PBMCs activated with PHA and gene expression of IL-2 and IFN-γ without direct cytotoxicity, which leads to the improvement of autoimmune diseases in MRL/MpJ-lpr/lpr mice. The mechanism involved in these inhibitions is blockade of membrane-proximal effectors such as IL-2-inducible T cell kinase and phospholipase C γ in a phosphatidylinositol 3-kinase-dependent manner.

An isoliensinine alkaloid isolated from the seed embryo had inhibitory effects on the proliferation of porcine coronary arterial smooth muscle cells induced by angiotensin II. Its mechanisms were investigated by counting cultured cell number, the MTT assay, immunohistochemistry and Western blotting. Angiotensin II (0.1 μM) significantly evoked cell proliferation by 42%, which could be dose-dependently inhibited by 0.01–3 μM isoliensinine; the percentage of inhibition of isoliensinine was 25% at 0.01 μM. These results suggest that isoliensinine possesses antiproliferative effect, which is related to a decrease in the over-expression of platelet-derived growth factor, basic fibroblast growth factor and proto-oncogenes c-fos, c-myc and hsp70.

The effect of neferine on platelet aggregation, thromboxane A2/prostaglandin (PG) I2 and cAMP/cGMP balance were studied using turbidimetry and radioimmunoassay. It significantly inhibits rabbit platelet aggregation induced by ADP, collagen, arachidonic acid and platelet-activating factor with IC50 values of 16, 22, 193 and 103 μM, respectively. Neferine was found to increase vascular 6-keto-PGF1α and platelet cAMP levels in a dose-dependent manner, but inhibited arachidonic acid-stimulated thromboxane A2 release from platelets.

Anti-inflammatory activity

At  a dose of 10 mg/kg, the seed extract of N. nucifera inhibited the production of pro-inflammatory cytokine tumour necrosis factor-α (TNF-α) and increased anti-inflammatory cytokine IL-10 in BALB/c mice with systemic inflammation induced by an intraperitoneal injection of lipopolysaccharide (LPS). Studies in LPS-challenged mice showed that a high dose (20 mg/day) of seed extract significantly decreased TNF-α levels in the serum and significantly increased the levels of IL-10 produced by peritoneal macrophages. This result demonstrated that administration of the seed extract before systemic inflammation attenuates acute inflammation in vivo.

Anti-fertility activity

The petroleum ether extract of the seed has been reported to possess anti-fertility activity in female albino mice – at a dose of 3 mg/kg. It blocked the oestrus cycle at the metoestrus stage compared with ethyl oleate (0.1 ml/20 g). The extract significantly reduced uterine weight and affected the oestrus cycle by blocking biogenesis of ovarian steroids at an intermediate stage.

Anti-arrhythmic activity

Neferine, an alkaloid isolated from the seed embryo of N. nucifera, has been reported to have anti-arrhythmic effects on rabbit sinoatrial nodes and clusters of cultured cardiac myocytes from neonatal rats.  Neferine inhibits the slow transmembrane Na+ and/or Ca2+ current of the myocardium, which leads to its anti-arrhythmic action.  Neferine causes non-specific inhibition of the Na+, Ca2+ and K+ cardiac transmembrane currents in guinea-pig papillary muscles and atria, which relates to its anti-arrhythmic activity.  Experiments in anaesthetised cats showed that intravenous neferine at concentrations of 1–10 mg/kg dose-dependently decreased the amplitude and prolonged the duration of the monophasic action potential, decreased left ventricular pressure and prolonged the sinus cycle length. These effects demonstrated that neferine has similar electromechanical properties in the heart as quinidine.

Liensinine is another alkaloid isolated from the seed of the lotus which has been reported to have anti-arrhythmic effect; its mechanism may be related to blockade of Ca2+ and Na+ influx. Intravenous liensinine (3 mg/kg) temporarily inhibited all parameters of haemodynamics in anaesthetised and pithed rats. Its effects were slightly stronger than those of quinidine (3 mg/kg); the inhibitory effects of liensinine (12 mg/kg) on all haemodynamic parameters were comparable to those of verapamil (1 mg/kg). The haemodynamic effects of liensinine may be similar to verapamil but different from quinidine.63 Liensinine at 10–100 μM was shown to concentration-dependently decrease the amplitude of the action potential. The effects of liensinine on slow action potentials and slow inward currents have also have been studied and suggest that liensinine possesses calcium antagonistic effects.

Anti-fibrosis activity

The inhibitory effect of isoliensinine isolated from the seeds was studied on bleomycin-induced pulmonary fibrosis in mice.  Administration of isoliensinine remarkably suppressed the increase in hydroxyproline content and abated the lung tissue injury induced by bleomycin. It enhanced SOD activity and decreased the malondialdehyde level in a concentration-dependent manner. Moreover, isoliensinine significantly inhibited the over-expression of TNF-α and transforming growth factor-β (TGF-β) induced by bleomycin. These results indicated that isoliensinine possesses significant inhibitory activity against bleomycin-induced pulmonary fibrosis, probably due to its antioxidant and/or anti-inflammatory activities and inhibitory effect on TNF-α and TGF-β induced by bleomycin.

Antiviral activity

Ethanol extract of the seed (100 μg/ml) significantly suppressed replication of herpes simplex virus-1 (HSV-1), with an IC50 of 50 μg/ml. Furthermore, a sub-fraction of N. nucifera (NNFR) has an inhibitory effect on HSV-1. NNFR at a concentration of 50 μg/ml inhibited HSV-1 replication in HeLa cells by up to 85.9%, attenuating aciclovir-resistant HSV-1 propagation.  In a bioassay-guided fractionation, NNFR significantly blocked HSV-1 multiplication in HeLa cells without apparent cytotoxicity. The production and mRNA transcription of infected cell protein was found to be decreased in NNFR-treated HeLa cells. The antiviral actions of NNFR is therefore likely to be mediated through inhibition of immediate early transcripts, such as infected cell protein (ICP) 0 and ICP4 mRNA and then blocking the downstream accumulation of all viral products.