5.3. Antipyretic Activity
Sinha et al., 2000, reported the antipyretic potential of ethanol extract of lotus stalks on normal body temperature as well as yeast-induced pyrexia using rat as in vivo model. Lotus extract, at a dose of 200 mg/kg, markedly decreases the body temperature for a period of 3 hr after administration, while at 400 mg/kg dose it decreases for up to 6 hr. In this yeast model, elevation of body temperature was dose dependently lowered up to 4 hr at both doses by lotus extract and these results were comparable to standard medicine for fever: paracetamol at 150 mg/kg [29]. Similar result was reported by Mukherjee et al., 1996, in yeast-induced pyrexia using rat model [30].
5.4. Antiviral Activity
Kashiwada et al., 2005, isolated quercetin 3-O-β-D-glucuronide, (+)-1(R)-coclaurine, and (−)-1(S)-norcoclaurine from Nelumbo nucifera leaves. The latter two compounds possessed therapeutic activity against HIV with EC50 values of 0.8 and <0.8 μg/mL and therapeutic index (TI) values of >125 and >25, respectively, while the first compound was less potent (EC50, 2 μg/mL). Other active principles like aporphine, benzylisoquinoline, and bisbenzylisoquinoline alkaloids (liensinine, isoliensinine, and neferine) isolated from leaves and embryo of lotus exhibited potent anti-HIV activities (EC50, <0.81 μg/mL; TI of >9.9, >8.6, and >6.5, resp.). An aporphine alkaloid, nuciferine also showed EC50, 0.8 μg/mL, and TI of 36 μg/mL [31]. Kuo et al., 2005, reported the antiviral activity of lotus seed ethyl alcohol extract on herpes simplex type 1 (HSV-1). At a dose of 100 μg/mL, ethyl alcohol extract of lotus markedly inhibited HSV-1 replication with IC50 of 50.0 μg/mL for replication. Various subfractions of Nelumbo nucifera seeds butanol (NN-B) extract were investigated for the HSV-1 inhibitory effects indicating that NN-B-5 out of major nine fractions, NN-B-1 to NN-B-9, had the highest suppresser activity. However, ethyl alcohol extracts obtained from fresh lotus seeds showed anti-HSV-1 activity with IC50 62.0 ± 8.9 μg/mL. Furthermore, to get deeper insight of NN-B-5 potency to suppress the acyclovir-resistant HSV-1 replication, the TK HSV-1 strain was targeted and plaque reduction assay was carried out. Results showed 85.9% inhibition of TK HSV-1 replication in HeLa cells by 50 μg/mL of NN-B-5. These results provide evidence that NN-B-5 inhibit the acyclovir-resistant HSV-1 replication [32].
5.5. Immunity
Liu et al., 2004, study the effect of ethyl alcohol extracts of lotus in primary human peripheral blood mononuclear cells (PBMC) stimulated by phytohemagglutinin (PHA: a specific mitogen for T lymphocytes) to inhibit the cell proliferation and cytokines production [33]. Liu et al., 2006, also studied the effects of (S)-armepavine from Nelumbo nucifera to suppress T cells proliferation. The therapeutic benefit of (S)-armepavine on immune disease, systemic lupus erythematosus (SLE), was investigated on MRL/MpJ-lpr/lpr mice as an in vivo model having disease feature similar to human SLE. For this study, (S)-armepavine was treated orally for 6 weeks to MRL/MpJ-lpr/lpr mice and their SLE features were evaluated. The results showed that (S)-armepavine was successful to prevent lymphadenopathy as well as extend the life span of mice. Also, (S)-armepavine treatment resulted in significant decrease of T lymphocyte-mediated cytokines production in dose-dependent manner. Likewise, (S)-armepavine impaired interleukin-2 (IL-2) and interferon (IFN-γ) transcripts in human PBMC. This study concluded that (S)-armepavine could be potential therapeutic option as an immunomodulator for the management of SLE [34]. In another study, Liu et al., 2007, revealed the suppressive action on PHA-induced PBMC proliferation and genes expression of IL-2 and IFN-γ by (S)-armepavine without direct cytotoxicity [35].
5.6. Anti-Inflammatory Activity
Tissue inflammation is harmful response that produces tissue injury and may cause serious diseases such as asthma, atopic dermatitis, and rheumatoid arthritis [36]. There is now convincing evidence that cytokines secreted by T cells such as IL-4, IL-10, and INF-γ in response to antigen stimulation play a role in atopic dermatitis, lung inflammation, and asthma [37]. Phytochemical (NN-B-4) identified by a bioassay-based screening procedure from ethanol extract of Nelumbo nucifera extracts significantly attenuated proliferation of PMBC induced by phytohemagglutinin, expression of IL-4, IL-10, and INF-γ, and cdk-4 gene. NN-B-4 arrest progression of cell cycle of activated PBMC by blocking PBMC from the G1 transition to S phase [32]. Triterpenoid betulinic acid isolated from methanol extract of Nelumbo nucifera rhizome was evaluated for the marked anti-inflammatory activity against edema in rat paw caused by carrageenan and serotonin. Methanol extract showed anti-inflammatory activity at doses of 200 and 400 mg/kg p.o. Similarly betulinic acid demonstrated significant anti-inflammatory effect in inflammatory experimental models at doses of 50 mg/kg and 100 mg/kg p.o. Extract and betulinic acid produce similar effect as compared to two potent anti-inflammatory drugs, phenylbutazone and dexamethasone [38].
5.7. Diabetes and Complications
The effect of ethanolic extract of N. nucifera rhizome was studied in glucose-fed hyperglycemic and streptozotocin-induced diabetic rats. The extract showed improved tolerance action of glucose. Similarly, the potentiating action of extract on the exogenous insulin was seen. The antidiabetic potential of the extract was compared and found to be similar with that of tolbutamide in normal and diseased rat [39]. Such effect could be due to, in part, attenuated absorption of glucose from intestine, as noted, for example, when plant fiber is given orally with glucose [40]. Key enzyme aldose reductase of the polyol pathway has been suggested to play central role in the diabetes and its complications. A methanol extract from Nelumbo nucifera stamens revealed inhibitory activity against rat lens aldose reductase. Out of 13 isolated flavonoids from the extract, compounds having 3-O-alpha-1-rhamnopyranosyl-1(1→6)-beta-d-glucopyranoside groups in their carbon ring, including kaempferol 3-O-alpha-1-rhamnopyranosyl-1(1→6)-beta-d-glucopyranoside and isorhamnetin 3-O-alpha-1-rhamnopyranosyl-1(1→6)-beta-d-glucopyranoside, demonstrate highest inhibitory effect of rat lens aldose reductase in vitro [41].
5.8. Treatment for Erectile Dysfunction
Chen et al. in 2008 investigated the effects of extract neferine on basal concentration of the cyclic adenosine monophosphate and cyclic guanosine monophosphate. Neferine potentiated the cAMP concentration dose dependently; however, this effect was not suppressed by inhibitor of adenylyl cyclase in rabbit corpus cavernosum in vitro. Neferine dose dependently increased cAMP accumulation catalyzed by a stimulator of cAMP production, namely, prostaglandin E1 (PGE1). The level of cGMP was not affected by guanylyl cyclase inhibitor and sodium nitroprusside. Neferine enhances the concentration of cAMP in tissue of rabbit corpus cavernosum notably by suppressing activity of phosphodiesterase [42]. In another study, Chen et al. further highlighted treatment of erectile dysfunction of the in vitro relaxation mechanisms of neferine on the rabbit corpus cavernosum tissue [43] and in vitro effects of neferine on cytosolic free calcium concentration in corpus cavernosum smooth muscle cells of rabbits [44].
5.9. Restenosis and Atherosclerosis
Proliferation and expression of matrix metalloproteinase 2 (MMP-2) by smooth muscle cells after percutaneous transluminal coronary angioplasty (PTCA) are responsible for degradation of extracellular matrix (ECM) and migration of smooth muscle cells from tunica media to tunica intima, thereby narrowing the luminal area. Administration of leaf and root extract of N. nucifera for 4 weeks significantly suppressed the narrowing of luminal area and stenosis rate in rat model. Similarly, proliferation of vascular smooth muscle cell (VSMC) and expression of MMP-2 in VSMC was dose dependently inhibited by different parts of N. nucifera extracts [8]. Similarly, in rabbit model of atherosclerosis induced by high cholesterol diet, leaf extract of N. nucifera showed potent antiatherosclerotic activity via inhibition of VSMC proliferation and migration [45] and improved plasma cholesterol level [46]. The active principle of N. nucifera, neferine, inhibits angiotensin II-stimulated proliferation in VSMC through heme oxygenase-1 [47] as well as downregulating fractalkine gene expression [48].
5.10. Antiaging
Sacred lotus (Nelumbo nucifera) seed extract contains antiaging agent that has beneficial effect to reduce symptoms like loss of elasticity, acne, pores, wrinkles, fine lines, blemishes, and so forth. A suitable vehicle possesses the formulations which has potent antiaging agent. It promotes younger looking skin [49]. Mahmood and Akhtar, 2013, study the efficacy of green tea and lotus extract cosmetic formulations for the treatment of facial wrinkles in healthy Asian using a noninvasive device, the Visioscan VC, along with software for surface evaluation of living skin [50] and a noninvasive photometric device (Sebumeter) for the measurement of casual sebum secretions on both sides of the face [51]. Result showed that green tea and lotus combined in multiple emulsions demonstrated a synergistic antiaging effect. The active constituents having antioxidant activity in both herbal plants may possess a beneficial effect on skin surface, thus recommending these plants as the future of new antiaging products.
5.11. Antiarrythmia
Phytochemicals, dauricine and neferine, obtained from N. nucifera seed have cardiovascular pharmacological effect. Phytochemicals of N. nucifera blocked the Na+K+ and Ca+2 cardiac transmembrane current. Notable compound neferine has antiarrhythmic effect and also significantly inhibits aggregation of platelet in rabbits [52]. The antiarrhythmic potency of principle alkaloid from N. nucifera, neferine, has been demonstrated in several in vivo experimental studies [53]. In a guinea pig papillary muscles and atria, the activity of neferine was investigated on the electrical and mechanical activity. Neferine, at a dose of 0.1 mmol/L, decreased the force of contraction, lowered the amplitude and also V max of action potential (AP), and prolonged the action potential duration at 50% (APD50), action potential duration at 90% (APD90), and effective refractory period (ERP). Furthermore, neferine (30 μmol/L) partly antagonized the effect of acetylcholine 10 μmol/L [54]. In guinea pig atria, automaticity inducing effect of adrenaline was attenuated by neferine 30 μmol/L. Similarly, neferine at dose of 30 μmol/L inhibited and shifted dose-effect curve of isoprenaline, an action varied from propranolol action. Also, neferine effect on the Ca2+ dose response curve in guinea pigs left atria was studied. Neferine, similar to verapamil, revealed dualistic action in antagonism of Ca2+ [55]. Neferine (8 mg/kg, i.v.) electrophysiological effects on ischemic ventricular tachyarrhythmias were studied in both normal and ischemic myocardium by using programmed electrical stimulation (PES) in open chest during 5–8 days after acute myocardial infarction. Neferine was effective in preventing ventricular tachycardias and sudden cardiac death after damage due to myocardial ischemia [56].
5.12. Hepatoprotective Activity
Sohn et al., 2003, reported Nelumbo nucifera hepatoprotective effect. The protective effects of the ethanol extract of seeds of Nelumbo nucifera (ENN) against cytotoxicity induced by CCl4 in primary cultured rat hepatocytes were evaluated by the cellular leakage of aspartate transaminase (AST) and cell survival rate. The cellular leakage of AST and the cell death caused by CCl4 were significantly inhibited in dose-dependent manners by ENN concentrations between 10 and 500 μg/mL. Similarly, the hepatoprotective effect of ENN against hepatotoxin Aflatoxin B1 (AFB1) was also tested. ENN showed significant hepatoprotective activity at concentrations of both 250 and 500 μg/mL by 74.5% and 94.6%, respectively, as compared with AFB1 controls (48.9%) [57]. Je and Lee investigated the in vitro hepatoprotective effect of Nelumbo nucifera leaves butanol extract (NNBE) on hepatic damage in cultured hepatocytes induced by H2O2. The hepatoprotective effect of NNBE was related to the upregulation of various enzymes: superoxide dismutase-1 (SOD-1) by 0.62-fold, catalase (CAT) by 0.42-fold, and heme oxygenase-1 (HO-1) by 2.4-fold. Likewise, pretreatment of NNBE increased the accumulation of nuclear factor erythroid 2-related factor 2 (Nrf2) in nucleus by 8.1-fold signifying that increased SOD-1, CAT, and HO-1 expressions are mediated by Nrf2 [58]. In another study by Yuan et al., ethyl acetate (NUEA) and n-butanol (NUBU) extracts of N. nucifera leaves were evaluated for hepatoprotective effect on CCl4 induced acute liver injury in mice. Except for NUEA group, the levels of aspartate aminotransferase (AST/GOT) and alanine aminotransferase (ALT/GPT) in each treatment group significantly decreased. Moreover, the contents of malondialdehyde (MDA) and the level of SOD in liver of each group were significantly decreased [59]. Likewise, Tang et al. evaluated the mechanisms of hepatoprotective effect of Nelumbo nucifera leaves extract (NLE) in in vivo model of experimental alcoholic steatohepatitis. The result showed the inhibitory action of NLE on lipid accumulation by altering the levels of triglycerides (TG) and cholesterol (TC) in both plasma and hepatic content analysis. Furthermore, NLE increased the expression of the 5′ adenosine monophosphate-activated protein kinase (p-AMPK/AMPK) ratio and peroxisome proliferator-activated receptor- (PPAR-) α responsible for fatty acid oxidation and transport via carnitine palmitoyltransferase-1 (CPT1) and microsomal triglyceride transfer protein (MTP). These results clarify that the NLE can prevent alcoholic steatohepatitis by multiple pathways [60].
5.13. Pulmonary Fibrosis
Xiao et al. evaluated the effect of bisbenzylisoquinoline alkaloid isoliensinine isolated from the seed embryo of Nelumbo nucifera, on pulmonary fibrosis induced by bleomycin in mice. This study demonstrated that isoliensinine significantly inhibited the hydroxyproline content. Also, isoliensinine subsided histological injury in lungs due to enhancing superoxide dismutase (SOD) activity and malondialdehyde (MDA) level by bleomycin. Moreover, it also inhibits, tumor necrosis factor- (TNF-) α and TGF-β1 overexpression [61]. Similarly evidence was provided by Zhao et al., 2010, on antifibrosis property of neferine by reversing the decrease in SOD activity, increased in MDA levels and myeloperoxidase activity. Neferine also alleviated bleomycin-induced increase of TNF-α, interleukin- (IL-) 6, and endothelin-1 in plasma or in tissue [62]. Niu et al., 2013, showed inhibitory effect of neferine on amiodarone-induced pulmonary fibrosis, due to its potency of anti-inflammation, inhibition of surfactant protein-D (SP-D), and balancing of the increased CD4+CD25+ regulatory T cells (Tregs) which may modulate Th1/Th2 imbalance by suppressing Th2 response [63].
5.14. Antiobesity Activity
Ohkoshi et al., 2007, reported the antiobesity efficacy of active constituents isolated from the leaves of Nelumbo nucifera via stimulated lipolysis in mice adipose tissue. Constituent obtained showed antiobesity activity via beta-adrenergic receptor pathway. Nelumbo nucifera leaves reduced body weight in A/J mice given high fat diet significantly. Several flavonoids were identified and isolated from NN by fractionation and chromatography. Flavonoids like quercetin, isoquercitrin, catechin, hyperoside, and astragalin showed lipolytic activity in visceral adipose tissue [64]. Ono et al., 2006, outlined the potential pharmacological mechanism of extract isolated from leaves of N. nucifera (NNE) in diabetic mice and rats. NNE demonstrated concentration dependent suppression of α-amylase, lipase activity, lipid metabolism, and expression of UCP3 mRNA in C2C12 myotubes in mice administrated extract for five weeks. The NNE also exhibited inhibitory activity of α-amylase and lipase in vitro. Lipase inhibitory activity by NNE (IC50, 0.46 mg/mL) was shown to be stronger when compared with α-amylase (IC50, 0.82 mg/mL). The extract revealed significant suppression in weight gain of body and parametrial adipose tissue. Also extract suppresses concentration of liver triacylglycerol in high fat diet induced obese mice and inhibits UCP3 mRNA expression in skeletal muscle [65]. Similarly, Velusami et al., 2013, study the antiobesity efficacy of methyl alcohol and successive aqueous extracts of N. nucifera petals. Parameter studied was effect of those extracts on adipogenesis, adipolysis, lipase, serotonin (5-HT2C), cannabinoid (CNR2), melanocyte concentrating hormone (MCHR1), and melanocortin (MC4R) receptors. Both methyl alcohol and successive aqueous extracts of N. nucifera petals inhibited lipid storage in adipocytes and increased lipolysis. Also, N. nucifera petal's methyl alcohol extract showed dose-dependent inhibition of lipase activity (IC50 value: 47 μg/mL). Furthermore, N. nucifera petal extracts possess antagonist and agonist activity on CNR2 and 5-HT2C receptors, respectively. However, it does not show any effect on MCHR1 and MC4R receptors. Overall result summarizes that methyl alcohol extract of N. nucifera petals showed better promising activity than successive aqueous extract to control obesity [66].
5.15. Anticancer Activity
Various extracts and isolated compound from different parts of N. nucifera possess anticancer activity both in vitro and in vivo. Among the three major alkaloids, isoliensinine possesses the most potent cytotoxic effect, primarily by inducing apoptosis on triple-negative breast cancer cells through ROS generation and p38 MAPK/JNK activation [67]. N. nucifera markedly suppressed the proliferation of non-small-cell lungs cancer (NSCLC) cells in the presence of nicotine, inhibited Wnt/β-catenin signaling activity, promoted the stabilization of Axin, and induced apoptosis. N. nucifera decreased the levels of β-catenin and its downstream targets including c-myc, cyclin D, and vascular endothelial growth factor- (VEGF-) A. N. nucifera also decreased the ratio of B-cell lymphoma 2/Bcl-2-associated X protein (Bcl-2/Bax), which may explain the proapoptosis effect of nuciferine [68]. Out of 15 compounds isolated from leaves from Nelumbo nucifera, 7-hydroxydehydronuciferine significantly inhibited the proliferation of melanoma, prostate, and gastric cancer cells [69]. Reversal effect of neferine on multidrug resistance human gastric carcinoma cell line (SGC7901) was studied by Cao et al. SGC7901 and its vincristine- (VCR-) resistant variant (SGC7901/VCR) were treated in the presence or absence of neferine and/or VCR. Neferine reverses multidrug resistance of human gastric carcinoma SGC7901/VCR cells by inhibiting permeability glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) expression in SGC701/VCR cells [70]. In another study, apoptosis inducing effect of neferine was proposed in lung cancer cells (A549 cells). Neferine treatment leads apoptosis by downregulation of nuclear factor kappaB and B-cell lymphoma 2 (Bcl2), upregulation of Bcl-2-associated X protein (Bax) and Bcl-2-associated death promoter (BAD), release of cytochrome C, activation of apoptosis regulator caspase cascade, and DNA fragmentation. Furthermore, neferine caused G1 cell cycle arrest by inducing p53 and its effector protein p21 as well as downregulation of cell cycle regulatory protein cyclin D1 [71]. In addition, neferine possessed growth-inhibitory effect due to cell cycle arrest at G1 on human osteosarcoma cells. It was observed that G1 arrest induction was dependent on p21(WAF1/CIP1); however, it was independent of p53 or RB (retinoblastoma-associated protein). Neferine caused upregulation of p21 due to rise in p21 protein half-life. Researchers examined four kinases that are supposed to affect the stabilization of p21 and found that neferine activated p38 MAPK and JNK. They demonstrated that inhibitor of p38 (SB203580), but not the inhibitor of JNK (SP600125), could decrease p21 upregulation of p21 in response to neferine. Neferine increased phosphorylation of p21 at Ser130 dependent on p38. These results highlighted a direct antitumor action of neferine revealing that neferine possesses cancer-preventive and cancer-therapeutic potential [72].