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SOLA
Apoptosis


INGREDIENTS
solanigrine & solanine from Solanum nigrum (Eggplant)






INTRODUCTION
Solanine is found mainly in the tuber of the potato (Solanum tuberosum L.) [1-5] and in the whole plant of the nightshade (Solanum nigrum Linn) of the family Solanaceae. The content of this substance is rather high in the green peel and the sprouts of potato and is the main toxic substance[6]. The whole plant of the nightshade contains many steroid alkaloids, including solamargine, solasonine, and solanine, as well as saponin and other substances. It can be used for anti-tumor purposes, with a strong inhibitory effect on tumors in animals and a clearly toxic effect on tumor cells[7]. Its ethanol extract is capable of inhibiting the growth of breast cancer and induce apoptosis in tumor cells[8]. The extract from the nightshade also has a strong anti-inflammatory effect because it can facilitate the formation of antibodies[9]. The anti-tumor effect of solamargine has been reported[10], but there is as yet no report about any anti-tumor effect of solanine. From our past experience in both in vivo and in vitro experiments, we have found that solanine is cytotoxic to cells, especially for the hepatocarcinomatic cell HepG2.

Through morphological observation, as well as DNA ladder and flow cytometry, we discovered that solanine exerted its anti-tumor effect by inducing apoptosis in HepG2.

The concept "apoptosis" was officially proposed by Kerr in 1972[11]. In the three decades since it was proposed, it has always been the focus of biological researches.

Especially since 1990s, the study of apoptosis has suddenly been propelled to the frontier of life science, leading to an upsurge of research activities involving almost all the fields of biomedical studies. As a result, a great deal of literature accumulated in a few years about the.

Solanine opens up the PT channels in the membrane by lowering the membrane potential, leading to Ca being transported down its concentration gradient, which in turn leads to the rise of the concentration of Ca in the cell, turning on the mechanism for apoptosis.

RESEARCH

Effect of solanine on the membrane 2+ potential of mitochondria in HepG2 cells and [Ca ]i in the cells

AIM: To observe the effect of solanine on the membrane 2+ potential of mitochondria in HepG2 cells and [Ca ]i in the cells, and to uncover the mechanism by which solanine induces apoptosis.

METHODS: HepG 2 cells were double stained with AO/EB, and morphological changes of the cells were observed using laser confocal scanning microscopy
(LCSM). HepG 2 cells were stained with TMRE, and change in the membrane potential of mitochondria in the cells were observed using LCSM. HepG2 cells were 2+ double stained with Fluo-3/AM, and change of [Ca ]i in the cells were observed using LCSM. HepG2 cells were double stained with TMRE and Fluo-3/AM, and both the change in membrane potential of mitochondria and that 2+ of [Ca ]i in the cells were observed using LCSM.

RESULTS: Cells in treated groups showed typical signs of apoptosis. Staining with TMRE showed that solanine could lower membrane potential; staining with
Fluo-3/AM showed that solanine could increase the concentration of Ca in tumor cells; and those of double staining with TMRE and Fluo-3/AM showed that solanine could increase the concentration of Ca in the cells at the same time as it lowered the membrane potential of mitochondria.

CONCLUSION: Solanine opens up the PT channels in the membrane by lowering the membrane potential, leading to Ca being transported down its concentration gradient, which in turn leads to the rise of the concentration of Ca in the cell, turning on the

Gao SY, Wang QJ, Ji YB. World J Gastroenterol 2006; 12(21): 3359-3367

Induction of apoptosis in HepG2 cells by solanine and Bcl-2 protein

The nightshade (Solanum nigrum Linn.) has been widely used in Chinese traditional medicine as a remedy for the treatment of digestive system cancer. The anti-tumour activity of solanine, a steroid alkaloid isolated from the nightshade has been demonstrated. To observe the effect of anti-tumour and mechanism of solanine. The MTT assay was used to evaluate the IC50 on the three digestive system tumour cell lines. The effect on the morphology was observed with a laser confocal microscopy; the rate of apoptosis and the cell cycle were measured using flow cytometry (FCM); the expression of Bcl-2 protein was measured by Western blot. The results show that the IC50 for HepG2, SGC-7901, and LS-174 were 14.47, >50, and >50 μg/ml, respectively; the morphology of cells in the negative control was normal; for the treated groups, typical signs for apoptosis were found. The rate of apoptosis in HepG2 cells induced by solanine was found to be 6.0, 14.4, 17.3, 18.9, and 32.2%, respectively. Observation of the cell cycle showed that cells in the G2/M phases disappeared while the number of cells in the S phase increased significantly for treated groups. Western blot showed that solanine decreased the expression of Bcl-2 protein. Therefore, the target of solanine in inducing apoptosis in HepG2 cells seems to be mediated by the inhibition in the expression of Bcl-2 protein.

Ji, Y.B.,Gao, Sy.Y., Ji, C.F. & Zou, X. Journal of Ethnopharmacology. Volume 115, Issue 2, 17 January 2008, Pages 194-202. doi:10.1016/j.jep.2007.09.023


Evaluation of activities of Solanum nigrum fruit extract

It has hepatoprotective activity against CCl4-induced hepatic damage in rats. The activity was evaluated using biochemical parameters such as serum aspartate amino transferase (AST), alanine amino transferase (ALT), alkaline phosphatase (ALP) and total bilirubin (decreases), while increases the activity of aminopyrine  N-demethylase, uridine diphosphate, glucuronyl transferase, and glutathione S-transferase1.

Solanum nigrum fruit extract could be used as an anti-oxidant and cancer chemo-preventive material2.

Solanum nigrum is an herbal plant that has been used as hepatoprotective and anti-inflammation agent. The results of the study suggest that Solanum nigrum protects liver against the CCl4 induced oxidative damage in rats, and this hepatoprotective effect might be contributed to its modulation on detoxification enzymes and its antioxidant and free radical scavenger effects8.

Oral administration of SNE significantly reduces Thioacetamide -induced hepatic fibrosis in mice, probably through the reduction of transforming growth factor-β1 secretion9. It also protects against hepatitis B virus infection B10.   

Kaushik, D., Jogpal, V., Kaushik, P., Lal, S. et al. Archives of Applied Science Research; 2009, 1 (1): 43-50                                                      

Solanum nigrum Linn.- A review.

Phytochemical investigation of whole plant reported that which contain alkaloids, flavonoids, tannins, saponins, glycosides, proteins, carbohydrates, coumarins & phytosterols. It has been found that Solanum nigrum contains the substances, such as total alkaloid [6], steroid alkaloid [7], steroidal saponins [8] and glycoprotein [9], exhibiting anti-tumor activity [10]. Researchers studied the chemical characterization of osmotin - like protein from this plant [11]. New glycoprotein (150 KDa) has been isolated from this plant which consist carbohydrate content (69.74%) and protein content (30.26%) which contain more than 50% hydrophobic aminoacids such as glycine and proline [12]. Small unripe fruits of Solanum nigrum had a high concentration of solasodine, but both the concentration and the absolute amount per fruit decreases with fruit maturation [13]. The berries of Solanum nigrum from New Zealand have recently been studied and found to contain 4 steroidal alkaloid glycosides, Solamargine, Solasonine, α and b- solanigrine. The berries of Solanum nigrum have been found to contain a saturated steroidal genin, which has been identified as tigogenin by mixed melting point and IR spectroscopy [14]. One spirosestanol glycoside and two furostanol glycosides have been isolated from a methanol extract of the stems and roots of Solanum nigrum [15]. Some researchers found the presence of ascorbic acid in the fruits of Solanum nigrum and the concentration of ascorbic acid is more in fruit than root [16]. Six new steroidal saponins, solanigrosides C-H, and one known saponin, degalactotigonin, were isolated from the whole plant of Solanum nigrum [17]. Some researchers isolated two new steroidal saponins, named nigrumnins I and II, together with two known saponins were obtained from the whole plant of Solanum nigrum [18]. Recently phytochemical analysis of Solanum nigrum has resulted in the isolation of two novel disaccharides. Their structures were determined as ethyl b - D -thevetopyranosyl-(1-4)- b- D -oleandropyranoside and ethyl b- D -thevetopyranosyl-(1-4)- a - D-oleandropyranoside, respectively, by chemical and spectroscopic methods [19]. Solanum nigrum seeds have high lipid content. Their protein content and minerals elements (Mg being prominent) are considerable and Solanum nigrum oil is an important source of linoleic acid [20]. Chemical structures of some phytoconstituents from Solanum nigrum present in [Figure 2].
Many investigations showed that extracts of Solanum nigrum suppressed the oxidant mediated DNA-sugar damage, and the plant exerted cytoprotection against gentamicin- induced toxicity on Vero cells and anti-neoplastic activity against Sarcoma 180 in mice. More recent studies revealed an inhibitory effect of extracts of Solanum nigrum on 12-O - tetradecanoylphorbol 13-acetate (TPA)- induced tumor promotion in HCT-116 cells, and a remarkable hepatoprotective effect of the  ethanol extract of dried fruits of Solanum nigrum against CCl 4 - induced liver damage. Recently, Lin et al., demonstrated that the water extract of Solanum nigrum contains several antioxidants, such as gallic acid, PCA, catechin, caffeic acid, epicatechin, rutin and narigenin, and possesses strong antioxidative activity in vitro [21],[22].

The ethanol extract of the fruit of Solanum nigrum was studied for its neuropharmacological properties on experimental animals. On intraperitoneal injection, the extract significantly prolonged pentobarbital induced sleeping time, produced alteration in the general behavior pattern, reduced exploratory behavior pattern, suppressed the aggressive behavior, affected locomotor activity and reduced spontaneous motility. The observations suggest that the fruit of Solanum nigrum possesses potential CNS-depressant action [23].

The protective effects of water extract of Solanum nigrum against liver damage were evaluated in carbon tetrachloride (CCl 4 ) - induced chronic hepatotoxicity in rats. The results of this study suggest that Solanum nigrum could protect liver against the CCl 4 induced oxidative damage in rats, and this hepatoprotective effect might be contributed to its modulation on detoxification enzymes and its antioxidant and free radical scavenger effects [22]. Other research reported that Oral administration of Solanum nigrum significantly reduces thioacetamide-induced hepatic fibrosis in mice, probably through the reduction of TGF-1 secretion[10].

Some research reported that a glycoprotein isolated from Solanum nigrum has a strong scavenging effect against reactive oxygen radicals, and growth inhibition effects against JA221 and XL1-Blue. Moreover, it has been reported that glycoprotein has a cytotoxic effect against MCF-7 and HT-29 cells, even at low concentrations [9], [24]. Glycoprotein has a strong scavenging activity against lipid peroxyl radicals and hypolipidemic activity by increasing the detoxicant enzymes activity through the inhibition of hepatic HMG-CoA reductase in mice [25]. It has been reported that glycoprotein has a cytotoxic effect on MCF-7 cells[26] and that the ethanolic extract has anti-proliferative, apoptotic and cytotoxic effects on MCF-7 cells [27]. Glycoprotein induces apoptosis through the NF-κb activation and inducible nitric oxide (iNO) production in HCT-116 cells [12]. Glycoprotein has the capacity to modulate the TPA-induced DNA-binding activities of transcription factors and NO production, which play a critical role with respect to cytotoxicity in MCF-7 cells. Therefore, glycoprotein from Solanum nigrum might be one of the agents that blocks TPA-mediated signal responses in tumor cells [9].

The 50% ethanol extract of the whole plant of Solanum nigrum was tested in vitro for its cytoprotection against gentamicin induced toxicity on Vero cells. Cytotoxicity was significantly inhibited as assessed by the Trypan blue exclusion assay and mitochondrial dehydrogenase activity (MTT) assay. The test extract also exhibited significant hydroxyl radical scavenging potential, thus suggesting its probable mechanism of cytoprotection [28].

Previous reports indicated that Solanum nigrum fruits possess beneficial activity as antiulcer, antioxidant and antitumor promoting agent in rats [29]. It has been reported earlier that aerial parts of Solanum nigrum is believed to offer its antiulcer action through acid and peptic suppression in aspirin induced ulcerogenesis in rats [30]. Oral administration of Solanum nigrum displayed a significant antiulcer activity without any apparent toxicological effects, which supports the use of Solanum nigrum in herbal medicine of India for ulcer therapy. The antisecretory activity of Solanum nigrum appears to be mainly related to the inhibition of H+K+ATPase and suppression of gastrin release, while its ulcer protective and ulcer healing activities may be primarily related to an antisecretory effect of Solanum nigrum [31].

The antioxidant potential of Solanum nigrum leaves extract was evaluated on the modulation of restraint induced oxidative stress. The post treatment of crude extract was found more effective in restoring restraint stress induced oxidative changes in rat plasma than pretreatment. In order to reduce oxidative stress, observed in many pathological conditions, the Solanum nigrum leaves extract can be given both as a prophylactic and therapeutic supplement for scavenging free radicals [32]. Antioxidant potential of isolated glycoprotein has been evaluated by several methods like DPPH, superoxide radical & hydroxyl radical assay, from these results it has been suggested that glycoprotein has potent antioxidative potential [33]. It has been reported that the extracts of berries of Solanum nigrum having significant larvicidal, anti-inflammatory and anti convulsant activity [34],[35],[36]. These studies suggest that Solanum nigrum possesses a beneficial activity as an antioxidant, and antitumor promoting, and hepatoprotective agent, although the mechanism for the activity remains to be elucidated

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24. K. Lim, H. Heo and S. Lee. Cytotoxic effect of glycoprotein isolated from Solanum nigrum L. through the inhibition of hydroxyl radical induced DNA-binding activities of NF-kappa B in HT-29 cells. Environmental Toxicology and Pharmacology. 17: 45-54 (2004).   
25. S. Lee, J. Koa, K. Lim and K.T. Lim. 150 kDa glycoprotein isolated from Solanum nigrum Linne enhances activities of detoxicant enzymes and lowers plasmic cholesterol in mouse. Pharmacological Research. 51: 399-408 (2005).   
26. S.J. Lee, and K.T. Lim. Anti-oxidative effects of glycoprotein isolated from Solanum nigrum Linne on oxygen radicals and its cytotoxic effects on the MCF-7 cells. Journal of Food Science. 68: 466-470 (2003).   
27. Y.O. Son, J. Kim, J.C. Lim, Y. Chung, G.H. Chung and J.C. Lee. Ripe fruits of Solanum nigrum L. inhibit cell growth and induce apoptosis in MCF-7 cells. Food and Chemical Toxicology. 41: 1421-1428 (2003).   
28. V.P. Kumar, S. Shashidhara, M.M. Kumar and B.Y. Sridhara. Cytoprotective role of Solanum nigrum against gentamicin-induced kidney cell (Vero cells) damage in vitro. Fitoterapia. 72: 481-486 (2001).   
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Saleem, T.S.M., Chetty, C.M. Ramkanth, S., Alagusundaram, M., Gnanaprakash, K., Rajan, V.S.T. & Angalaparameswari, S. Pharmacognosy Review. (2009) Vol 3, Issue : 6. Pp. 342-45.
 
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