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Pharmacological Investigation of Solanum Incanum Against P. Falciparum, L. infantum, T. cruzi and T. brucei : A Role of Antioxidant Effect and Clinical overview
Department of Medicinal Chemistry, Pharmacology and Toxicology unit, College of Clinical Pharmacy, Albaha University, P.O. Box 1988, Al-Aqiq, Al-Baha 65431, Saudi Arabia.
Corresponding Author E-mail: firstname.lastname@example.org
The in vitro antiprotozoal and cytotoxic activity of the Solanum incanum leaves and fruit extract of Albaha region was assessed against Plasmodium falciparum (chloroquine resistant K1 strain), Leishmania infantum, two Trypanosoma (T. cruzi and T. brucei) and MRC-5 cell-lines respectively. Additionally, ethnomedicinal studies by survey and interview, antioxidant potential by DPPH assay was studied for Solanum incanum. Results indicated that the S. incanum fruit was inactive (IC50: >64 µg/mL) against P.falcipuram but leaves had shown low activity (IC50: 47 µg/mL), against L.infantum both fruit (IC50: 27.3 µg/mL) and leaves (IC50: 27.3 µg/mL) had good activity, against T.brucei both fruit (IC50: 34.1 µg/mL) and leaves (IC50: 32.7 µg/mL) had moderate activity. S incanum fruits (IC50: 9.3 µg/mL) had pronounced activity against T. cruzi but leaves (IC50: 6.0 µg/mL) had pronounced activity against T .cruzi with selectivity index > 1. Solanum incanum fruits had stronger antioxidant activity (IC50: 98.7 µg/mL ) than leaves (IC50 : 293.2 µg/mL) but both fruit and leaves had lower antioxidant activity than standards (Ascorbic acid IC50: 19.1 µg/mL; Trolox IC50: 19.5 µg/mL). Our results demonstrate that S incanum leaves has promising activity against T .cruzi possibly active constituents like flavonoid, solasonine and solamargine are contributing for this effect. Furthermore previous reports demonstrate that T .cruzi infection is inhibited by antioxidant effects through NRF2 upregulation, possibly our extracts inhibited T .cruzi through antioxidant pathway.
Solanum Incanum; Antimalarial; Antileishmaniasis; Antitrypanosomal; Albaha; Saudi Arabia
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Anwar S. Pharmacological Investigation of Solanum Incanum Against P. Falciparum, L. infantum, T. cruzi and T. brucei : A Role of Antioxidant Effect and Clinical overview. Biomed Pharmacol J 2018;11(2).
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Anwar S. Pharmacological Investigation of Solanum Incanum Against P. Falciparum, L. infantum, T. cruzi and T. brucei : A Role of Antioxidant Effect and Clinical overview. Biomed Pharmacol J 2018;11(2). Available from: http://biomedpharmajournal.org/?p=20254
Artemisinin and quinine are two of the most effective antimalarial derived from plant sources that are used to treat fevers. Third Atovaquone, is a synthetic version of the natural product lapichol. 1 Researching antimalarial remedy from plant sources could yield a next-generation antimalarial drug. Henceforth screening of natural product becomes very important lead for discovery of new antimalarial drugs. Currently, Antimonials, Amphotericin-B, Miltefosine, Paromomycin drugs are used to treat leshmaniasis. Then, new oral agents like miltefosine and novel formulations of old antileishmanial drugs are being used. High cost, toxicity and resistance of these agents caused concerns in usage in poor countries. 2 Pentamidine, Suramin, Melarsoprol, Eflornithine and Nifurtimox-Eflornithine combination are currently used for typsonomas infection. In stage II T brucei gambience and stages of T brucei rhodesiense , Pentamidine , Suramin and Eflornithine proved to be ineffective. Melarsoprol have shown toxicity, around 5% of patients died due to post-treatment reactive encephalopathy (PTRE). Moreover 30 % patients have shown resistance to these drugs, particularly combination of Nifurtimox–Eflornithine.3
Solanum incanum commonly seen in Africa, Middle East and South Africa that belongs to subgenus Leptostemonum and Melongena section. It grows as a wild in Madagascar and Mauritius, fruits are used as edible. In Africa, treating for various diseases, entire plant of S. incanum is used as folk medicine. Roots consist of new spirostanol saponin, four known saponins, indioside D, dioscin, protodioscin, methyl-protodioscin and steroid glycoalkaloid solamargine. Aerial parts consist of two steroidal glycosidal alkaloids, solasonine and solamargine and non-steroidal components like three phenylalkanoic acids, benzyl-O-b-D-xylopyranosyl(1®2)-b-D-glucopyranoside, flavonoids , chlorogenic acid, adenosine and new compound kaempferol 3-O-(6²¢-O-2,5-dihydroxycinnamoyl)-b-D-glucopyranosyl (1®2) b-D-glucopyranoside. 4
S incanum have shown antibacterial effect against E coli, Strep pyogenes, Staphy aureus, and P aeuruginosa. Antipyretic, antinociceptive, hypoglycemic, anorexic effects, insect repellant properties and spasmolytic effects have been reported. Solamargine was found to induce apoptosis in HA549 lung adenocarcinoma cell and hepatoma cells. Solamargine was found to induce apoptosis in breast cancer by increasing the expression of external death receptors such as TNFR-1- related death domain (FADD), Fas, TNFR-1 and triggered death pathway mediated by mitochondria by augmenting the intrinsic proportion of Bax / Bcl-2. Solamargine was found to be effective against human K562 leukemia and squamous cell carcinoma by causing tumor cell bursting and injury by damaging the cell membrane. 5 Solanum incanum leaf extracts have shown antileishmanial activities against Leishmania amazonensis. 6 In present research, our aim was to investigate antioxidant, antimalarial activity against Plasmodium falciparum, antileishmanial activity against Leishmania infantum and antitrypanosomal activity against Trypanosoma cruzi and Trypanosoma brucei of the methanolic extracts of Solanum incanum fruits and leaves from Albaha region. To the best of our knowledge, these pharmacological activities have not been reported previously against above parasites.
The plant aerial parts were collected between the month of March – June from various sites in Albaha town and suburbs of Albaha region (Table 1). The plants were taxonomically studied, identified, numbered as voucher specimens and preserved at the pharmacology lab, College of Clinical Pharmacy, Albaha University, Saudi Arabia.
Preparation of Extracts
Plant aerial parts were air dried and finely powdered, at room temperature 10 g powder was subjected to extraction with 100 mL methanol 4 times with continuous shaking. The crude extract was filtered and evaporated under vacuum at 40°C until dryness. The percent yield was calculated for each dried extract. The collected dried crude extracts were retained at 4°C.
Antioxidant Activity: DPPH free Radical Scavenging Assay
The plant extract (500 μL) was added to 5 ml of DPPH solution (0.004% w/v in 80% methanol) in amber coloured bottle. The concentration of the plant extract tested was in the range between 1 µg/ml to 2.5 mg/ml. Ascorbic acid and trolox used as standard, 80% methanol as a blank and DPPH solution without plant extract was used as negative control. In the dark, the reaction complex was incubated at 37oC for 30 min and absorbance was read at 517 nm. The test was repeated 3 times and DPPH scavenging effect was calculated as per the following formula:
% DPPH scavenging effect = (Ao -A1) ×100/Ao
Where Ao is the measurement for the negative control, A1 is the measurement for the DPPH in presence of plant extract – the measurement of plant extract in 80% methanol. An IC50 value was calculated from the dose inhibition curve and results were calculated as average ± SD.
Following standard drugs were used for different assays as positive control: tamoxifen – cytotoxicity test against MRC-5, chloroquine – antiplasmodial test against P. falciparum, miltefosine – antileishmanial test against L. infantum, benznidazole – antitrypanosomal test against T. cruzi and suramin for T. brucei. All standard drugs were either purchased from Sigma-Aldrich (tamoxifen, suramin) or from WHO-TDR (chloroquine, miltefosine, benznidazole).
Standard screening methodologies and the integrated board for microbial screening was applied as described previously. 7 All experimental tests were repeated thrice at the Microbiology, Parasitology and Hygiene laboratory, University of Antwerp, Belgium. To estimate IC50 (50% inhibitory concentration), the plant extract was studied at five different concentrations (64, 16, 4, 1 and 0.25 μg/mL). The DMSO concentration in final in-test was 0.5%. Simultaneously, fibroblast (MRC-5) cell line cytotoxicity evaluation was performed for estimating antiprotozoal selectivity. For positive activity the criteria was with an IC50<10 μg/mL and selectivity index (SI) of >1.
P. falciparum (K 1-strain ; chloroquine resistant) was grown in human erythrocytes O+ in RPMI-1640 medium with human serum supplement (10%) at 37°C with atmosphere with low oxygen (3% – O2, 4% – CO2, and 93% – N2). In multiwall plate, infected human RBCs (200 μL, 1% – parasitaemia, 2% -hematocrit) were added to each well and followed by 72h incubation. Test plates were frozen at -20°C after incubation. The Malstat assay was used to measure parasite multiplication, a colorimetric test based on the 3-acetylpyridine adenine dinucleotide (APAD) reduction by parasite specific lactate dehydrogenase (pLDH). 8
Primary peritoneal macrophages of mouse was infected with L. infantum (MHOM/MA(BE)/67) amastigotes procured from infected donor hamster spleen. In 96 multiwell plates, macrophages (3 × 104 ) were seeded in each well to determine in vitro antileishmanial effect. After 48 hours of growth, amastigotes (5 × 105 per well) were reseeded and kept for incubation for 2h at 37°C. Simultaneously pre-diluted plant extracts were added to each well and further kept for incubation for 5 days at 37°C with CO2 (5%). Giemsa staining was done on 500 cells; parasite burden (mean number of amastigotes/macrophage) was determined microscopically and expressed as percentage of the blank control without plant extract.
Trypanosoma brucei (Squib-427 strain; suramin sensitive) was grown in hirumi 9 medium with fetal calf serum supplement (10 %) at 37°C with CO2 (5% ) .9 In 96-multiwell plate, around 1 .5 × 104 trypomastigotes were seeded in each well and after 72h at 37°C, parasite multiplication was determined by adding resazurin. 10 To study effect of S incanum on Chagas disease, T. cruzi (Tulahuen CL2 ; benznidazole sensitive) was grown on MRC-5 cells in minimal essential medium with glutamine (20 mML) supplement, sodium hydrogen carbonate (16.5 mM) and fetal calf serum (5%). In 96-multiwell plate, MRC-5 cells (4 × 103) and parasites (4 × 104) were seeded in each well, incubated for 7 days at 37°C and parasite multiplication was determined by addition of chlorophenol red β-D-galactopyranoside a β-galactosidase substrate .11 After 4 hrs, the reaction mixture color was read at 540 nm and the readings were depicted as the percentage blank control.
Cytotoxicity Testing Against MRC-5 Cells
MRC-5 SV2 cells were grown in minimum essential medium with L-glutamine (20 mM) supplement, sodium hydrogen carbonate (16.5 mM) and fetal calf serum (5%). For the study, MRC-5 cells (104 per well) were seeded onto multiwell plates containing pre-diluted sample and incubated at 37°C with CO2 (5%) for 72 h. After 4 h resazurin was added and cell viability was determined by flourometry. Fluorescence was determined at excitation 550 nm, emission 590 nm and the results were expressed as percent reduction in cell viability compared to control.
In table 1, the plants name, family, investigated parts, voucher specimen numbers, local names, percent yields, and their traditional uses are listed. The DPPH assay is directly measures the ability of bioactive molecules to scavenge free radicals. The DPPH scavenging activity ( IC50 ) of Solanum incanum fruits and leaves, ascorbic acid and trolox as standard are presented in Table 2. Solanum incanum fruits had stronger antioxidant activity (IC50 : 98.7 µg/mL ) than leaves (IC50 : 293.2 µg/mL) but both fruit and leaves had lower antioxidant activity than standards (Ascorbic acid IC50 : 19.1 µg/mL; Trolox IC50 : 19.5 µg/mL).
Table 1: Ethnomedicinal uses and characteristics
|Species||Plant Family (Voucher specimen no)||Part testeda
(Yield in %)
|Local name||Traditional uses|
|Solanum incanumL.||Solanacea (CP-131)||F (7.6%)||Al-hadak||Antiseptic, Leishmaniasisa|
|Solanum incanumL.||Solanaceae (CP-131)||L (3.9%)||Al-hadak||Leaves as dressing for healing wounds, paste of fruits for treating leishmaniasisa|
F: fruits; L: leaves. 1most information obtained from a interviewing with local people.
Table 2: Antioxidant activity of extracts
|Plant||DPPH scavenging activity|
|1||Solanum incanum (fruits)||98.7|
|2||Solanum incanum (L)||293.2|
In table 3, antiprotozoal and cytotoxic activity of standards and S. incanum (fruits and leaves) extracts of Albaha region against tested parasites are presented. The listed IC50 values are the means of three determinations. The following criteria were applied to express their level of activity, when IC50 ≤ 5 μg/ml considered as pronounced activity, 5 < IC50 ≤ 20 considered as good activity, 20 < IC50 ≤ 30 considered as moderate activity, 30 < IC50 ≤ 60 considered as low activity, IC50 > 64 μg/ml considered as inactive. For cytotoxicity, test sample is considered as cytotoxic when CC50 < 32 μg/ml. For the samples tested, the cytotoxicity of MRC-5 cell lines and activity have been compared using the selectivity index (SI) ration (SI = CC50 MRC-5 cells/IC50 protozoa). Selectivity against protozoa is considered selective when the value is >1 whereas if the value is <1 is considered selective against MRC-5 cell lines.13,14 As per above criteria S. incanum fruit was inactive (IC50: >64 µg/mL) against P.falcipuram but leaves had shown low activity (IC50: 47 µg/mL), against L.infantum both fruit (IC50: 27.3 µg/mL) and leaves (IC50: 27.3 µg/mL) had good activity, against T.brucei both fruit (IC50: 34.1 µg/mL) and leaves (IC50: 32.7 µg/mL) had moderate activity. S incanum fruits ((IC50: 9.3 µg/mL) had pronounced activity against T. cruzi but leaves (IC50: 6.0 µg/mL) had pronounced activity against T .cruzi with selectivity index > 1. Our results demonstrate that S incanum leaves have shown promising activity against T .cruzi.
Table 3: Antiprotozoal effect of the Solanum incanum and its cytotoxicity against MRC-5 cell lines
|P. falciparum||L. infantum||T. cruzi||T. brucei||MRC-5|
|IC50 b||SId||IC50||SId||IC50 b||SId||IC50b||SId||CC50 c|
|Chloroquine||0.3 ± 0.1||–||–||–||–|
|Miltefosine||–||3.3 ± 0.7||–||–||–|
|Benznidazole||–||–||2.2 ± 0.5||–||–|
|Suramin||–||–||–||0.03 ± 0.02||–|
|Tamoxifen||–||–||11.0 ± 2.3|
aAP, aerial parts; F: fruits; L: leaves.
bIC50: Concentration of extract showing 50% growth inhibition.
cCC50: Concentration of extract showing 50% of mortality of MRC- 5 cells
dSI: Selectivity index. —: Not done
Solanum Incanum aerial parts consist of steroidal components such as two biologically active glycosidal alkaloids, solasonine and solamargine whereas non-steroidal constituents such as benzyl-O-b-D-xylopyranosyl(1®2)-b-D-glucopyranoside, chlorogenic acid, flavonoid, phenylalkanoic acids and adenosine . 4 Auta et al reported Solanum incanum contained 26.7 % of flavonoid additionally they identified antioxidant elements such as riboflavin, ascorbic acid and tocopherol. 36 Flavonoid compounds have been widely attributed for antioxidant properties. Previously reported antioxidant effect of Solanum incanum fruits varied from our findings may be due to geographical origin since we found that Mytrus communis L essential oil varied its pharmacological properties as per different origin. 15,16 Steroidal alkaloids tomatidine and solasodine from Solanum aculeastrum has been reported for antioxidant properties. 18 Flavonoid has been reported for wider role in counteracting free radicals, it is attributed to inhibit ros, reactive species scavenging, block free radical production and activate antioxidant protection signaling pathways. 19 Furthermore, we previously demonstrated that phenolics compounds like flavonoid as cyanidin-3-O-glucoside are able to upregulate Nrf2 transcriptional factor. 20,21,22 Until now there is no report for direct upregulation of Nrf2 by any active constituents of solanum incanum but Meybodi et al reported that steroidal alkaloids upregulate Nrf2 and have shown anticancer effect. 23
The position of oxidative insult in parasitic infection is ambiguous and widely debated, it is said that oxidative generation helps in combating infection, while it is also said that it is contributing in pathophysiology of infection. Furthermore, oxidative insult markers are found to be elevated in infected rats and humans rather in uninfected groups. 24 Appreciation to upregulation of a redox-sensitive gene regulatory system mediated by the transcription factor (Nrf2) which is involved in response to oxidative insult and xenobiotics, communicated through Antioxidant Responsive Element (ARE). 25 Nrf2 stimulates HO-1 (heme oxygenase -1) enzyme expression that counteracts pro-oxidant heme by balancing cellular redox status. Surprisingly, the stimulation of Nrf2 and upregulation of HO-1 expression significantly decreased parasite population in isolated macrophages and in infected animals. This process did not involve in eradicating of apoptotic infected cells and did not depend on immunity mediated by T-lymphocytes. Nrf2 regulated pathway induced infection inhibition did not depend on other effectors as IFN-1, TNF, NO. Particularly, it was proved in iNOS-deficient mice that Nrf2 is able to reduce high levels of T.cruzi burden independent of other pathways. 12
There are about sixteen species in Saudi Arabia profoundly seen in West and Southwest part of the country. 26,27 Extracts of S. incanum were investigated phytochemically and biologically, but there is no reports regarding antiprotozoal activity so far. 28,29,30 Another possibility for antitrypanosomal compounds in S. incanum extract is steroidal alkaloids or steroid derivatives such as cilistol-A because it was reported that steroidal alkaloids such as solasonine, solamargine and a-chaconine from Solanum species were found to have antitrypanosomal activity against Trypanosoma cruzi. 31,32 . Furthermore, Pavia et al reported that macrophage specific mechanism, since upregulation of antioxidant pathway decreased T. cruzi burden in only macrophages not in other type of cells. Particularly, this trend is important because macrophage is involved in in vivo iron storage. Iron is mobilized and controlled by regulated expression of a particular iron exporter protein-ferroportin. Antioxidant transcription factor Nrf2 transcribes ferroportin and ferritin proteins that are responsible for storage of iron in an inert redox form. 34, 35 So most of the iron is stored, and free iron is made unavailable to intracellular pathogens, predicting this may be a possible mechanism by which antioxidant pathway upregulation is inhibiting T cruzi.
Developing and testing new drugs in humans that target parasitic protozoa is challenging. In humans, parasitic infections are in different clinical forms, drug resistance and genetic manipulation by parasites are observed and pharmacokinetic requirements of a new drug are complex, 37 Around 1200 medicinal plants are used against malaria and fever throughout the world but most of them did not undergo clinical trials.38 Many medicinal plants and its isolated secondary metabolite like saponins, phenolics, alkaloids, cardiac glycosides, polyacetylenes and terpeniods screened for anti-trypanosomal activity. Among these metabolites, some have shown promising activity in sub-micromolar concentration but very few established in vivo studies, lack clinical data and not translated in clinical practices. 39 The minimum requirement for herbal drug safety depends on duration of disease state. Pre-clinical and control clinical trial are required for acute diseases whereas for preclinical may be required and clinical trial may be or may not be required for chronic diseases. 40 Challenges for clinical trial for herbal products are many, to name few of them, herbal drugs have improper standardization and quality control, dosage forms are not uniform, inadequate randomization studies, low patient numbers and lack of significant data and very long duration of treatment. USFDA since 1994, do not evaluate herbal medicines under “Food and Drug administration” regulation but evaluated under “Dietary Supplement Health and Education Act of 1994”, by this regulation herbal product as mere dietary supplement to improve and prevent diseases. 41 All these parameters should be considered while testing S incanum extracts in humans.
Solanum incanum antiprotozoal effect has been reported for the first time against P. falciparum, L. infantum, T. brucei and T. cruzi. S. incanum fruits had stronger antioxidant profile. Among tested protozoa, S.incanum have shown promising activity against T.cruzi, may be antioxidant properties is contributing molecular mechanism to inhibit T.cruzi. Further gene expression studies are needed to confirm and establish molecular mechanism of T.cruzi inhibition. Appropriate in vivo animal screening studies and clinical trial data should be established for human use.
The author extend his appreciation to the Paul Cos and Louis Maes of Laboratory of Microbiology, Parasitology and Hygiene (LMPH), Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, Antwerp University, Belgium ; Prof Nasser A. Awadh Ali , College of Clinical Pharmacy, Albaha University for technical support.
- Wells, T.N. Natural products as starting points for future anti-malarial therapies: going back to our roots. Malar. J., 2011; 10:S3. [PubMed: 21411014].
- Sundar,S., Chakravarty,J ., Rai,V.K et al. AmphotericinB treatment for Indian visceral leishmaniasis: response to15 daily versus alternate-day infusions. Clinical Infectious Diseases., 2007; 45 (5) :556–561.
- Babokhov, P., Sanyaolu ,A.O., Oyibo,W.A., Adetayo, F.et al .A current analysis of chemotherapy strategies for the treatment of human African trypanosomiasis. Pathogens and Global Health.,2013;107 (5): 242-252.
- Manase, M.J., Mitaine-Offer, A.C., Pertuit, et al. Solanum incanum and S. heteracanthum as sources of biologically active steroid glycosides: confirmation of their synonymy. Fitoterapia. , 2012 ; 83(6):1115-9.
- Mwonjoria, J.K., Ngeranwa, J.J., Kariuki, H.N., et al. Ethno medicinal, phytochemical and pharmacological aspects of solanum incanum (lin.). International Journal of Pharmacology and Toxicology, 2014; 2(2):17-20.
- Al-Sokari, S.S. Ali,N.A. Monzote, L. Al-Fatimi, M.A. Evaluation of Antileishmanial Activity of Albaha Medicinal Plants against Leishmania amazonensis. Biomed. Res. Int., 2015;938747.
- Cos, P., Vlietinck, A.J., Berghe, D.V., Maes, L. Anti-infective potential of natural products: How to develop a stronger in vitro proof-of-concept. J. Ethnopharmacol., 2006; 106: 290–302.
- Makler, M.T., Ries, J.M., Williams, J.A., Bancroft, J.E., Piper, R.C., Hinrichs, D.J. Parasite lactate dehydrogenase as an assay for Plasmodium falciparum drug sensitivity. Am. J. Trop. Med. Hyg., 1993; 48: 739–741.
- Hirumi,H., Hirumi, K. Continuous cultivation of Trypanosoma bruce in blood stream forms in a medium containing a low concentration of serum protein without feeder cell layers. J. Parasitol., 1989; 75: 985–989.
- Raz, B., Iten, M., Grether-Buhler, Y., Kaminsky, R., Brun, R. The Alamar Blue asssay to determine drug sensitivity of African trypanosomes (T. b. rhodesiense, T. b. gambiense) in vitro. Acta Trop., 1997; 68: 139–147.
- Buckner, F.S., Verlinde, C.L., la Flamme, A.C., van Voorhis, W.C. Efficient technique for screening drugs for activity against Trypanosoma cruzi using parasites expressing beta-galactosidase. Antimicrob. Agents Chemother., 1996; 40: 2592–2597.
- Andrews, N.W. Oxidative stress and intracellular infections: more iron to the fire. J.Clin. Invest., 2012; 122: 2352–2354.
- Harborne,J.B. Phytochemical Methods. A Guide to Modern Techniques of Plants Analysis. 1998 ;Chapman & Hall, London.
- Tona, L. G., Mesia, K. G., Nanga, T. H., Cimanga, K. R., Apers, S., Cos, P., et al.In Vitro Antiprotozoal and Cytotoxic Activities of Plant Ex-tracts from Democratic Republic of Congo. Recent Re-search Development in Plant Science., 2007; 4 (1):41-60.
- Indhumathi, T and Suja, S. Study on In-vitro Antioxidant Potential of Solanum Incanum Fruit Extract. Int. J. Curnt. Tren. Pharm, Res., 2015; 3(3): 873-877.
- Anwar, S., Crouch, R.A., Awadh Ali, N.A., Al-Fatimi, M.A., Setzer, W.N., Wessjohann, L. Hierarchical cluster analysis and chemical characterisation of Myrtus communis L. essential oil from Yemen region and its antimicrobial, antioxidant and anti-colorectal adenocarcinoma properties. Nat .Prod. Res. 2017 ;1-6.
- Paiva,C.N. et al. Oxidative stress fuels Trypanosoma cruzi infection in mice. J. Clin. Invest.,2012;122(7):2531–2542.
- Srinivas, K., Jimoh, F.O., Grierson,D.S., Afolayan, A.J. Antioxidant activity of two steroid alkaloids extracted from Solanum aculeastrum. J. Pharmacol. Toxicol., 2007;2:160-167.
- Soobrattee, M.A., Neergheen, V.S., Luximon-Ramma, A., Aruoma, O.I., Bahorun, O.T. Phenolics as potential antioxidant therapeutic agents: mechanism and actions. Mutat. Res. Fundam. Mol. 2005;579:200–213.
- Speciale, A., Anwar, S., Canali, R., Chirafisi, J., Saija, A., Virgili, F., Cimino, F. Cyanidin-3-O-glucoside counters the response to TNF-alpha of endothelial cells by activating Nrf2 pathway. Mol.Nutr.Food.Res., 2013 ;57(11):1979-87.
- Anwar, S., Speciale, A., Fratantonio, D., Cristani, M., Saija, A., Virgili, F., Cimino, F. Cyanidin-3-O-glucoside modulates intracellular redox status and prevents HIF-1 stabilization in endothelial cells in vitro exposed to chronic hypoxia. Toxicol. Lett., 2014;226(2):206–213. 22. Anwar, S., Fratantonio, D., Ferrari, D., Saija, A. Cimino,F. Speciale, A. Berry anthocyanins reduce proliferation of human colorectal carcinoma cells by inducing caspase-3 activation and p21 upregulation. Mol. Med .Rep., 2016;14(2):1397-403.
- Meybodi, N.M., Mortazavian,A.M., Monfared, A.B., Sohrabvandi, S., Meybodi, F.A. Phytochemicals in cancer prevention: A review of the evidence. Iran. J. Cancer. Prev., 2017; 10: e7219.
- Percario, S., Moreira, D.R., Gomes, B.A.Q., Ferreira, M.E.S., Gonçalves, A.C.M., Laurindo, P.S.O.C. et al.Oxidative stress in malaria. International Journal of Molecular Sciences, 2012; 13, 16346–16372.
- Speciale, A., Anwar, S., Ricciardi, E., Chirafisi, J. et al. Cellular adaptive response to glutathione depletion modulates endothelial dysfunction triggered by TNF-α. Toxicol. Lett. 2011;207:291–297.
- Chaudhary, S.A., Flora of the Kingdom of Saudi Arabia. Ministry of Agriculture and Water, Kingdom of Saudi Arabia, 2001; Volume 2 (Part 2).
- Collenette, S., Wildflowers of Saudi Arabia. National Commission for wildlife Conservation and Development, Kingdom of Saudi Arabia, 1999; pp. 703-707.
- Lin , Y.L., Wang, W.Y., Kuo, Y.H., Chen, C.F. Nonsteroidal Constituents from Solanum incanum L. Journal of the Chinese Chemical Society, 2000; 47: 247-251
- Indhumathi, T. and Mohandass, S. Efficacy of ethanolic extract of Solanum incanum fruit extract for its antimicrobial activity. Int.J.Curr.Microbiol.App.Sci., 2014; 3(6): 939-949.
- Mwonjoria, J. K., Kariuki, H. N., and Waweru F. N .The antinociceptive antipyretic effects of Solanum incanum (linneaus) in animal models. International Journal of Phytopharmacology., 2011;2(1): 22-26.
- Chataing, B., Concepción, J.L., Lobatón, R., Usubillaga, A. Inhibition of Trypanosoma cruzi growth in vitro by Solanum alkaloids: a comparison with ketoconazole. Planta Med., 1998; 64 (1): 31-6
- Abreu Miranda, M., Tiossi, R. F., da Silva, M. R., Rodrigues, K. C., Kuehn, C. C., Rodrigues Oliveira, L. G., et al. In Vitro leishmanicidal and cytotoxic activities of the glycoalkaloids from Solanum lycocarpum (Solanaceae) fruits. Chemistry & Biodiversity.,2013; 10 (4): 642–648.
- Kaplan J. Mechanisms of cellular iron acquisition: another iron in the fire. Cell., 2002; 111(5): 603–606.
- Marro, S., et al. Heme controls ferroportin1 (FPN1) transcription involving Bach1, Nrf2 and a MARE/ARE sequence motif at position -7007 of the FPN1 promoter. Haematologica., 2010; 95(8):1261–1268.
- Hintze, K.J., Theil, E.C. DNA and mRNA elements with complementary responses to hemin, antioxidant inducers, and iron control ferritin-L expression. Proc. Natl. Acad. Sci.,2005; 102(42):15048–15052.
- Auta, R and Ali, I .Nutritional and chemical value of Solanum incanum (bitter garden egg), Intern. J. Trop. Med. Pub. Health 2011;1(1): 96-107.
- Andrews, K. T., Fisher, G., Skinner-Adams, T. S. Drug repurposing and human parasitic protozoan diseases. Int. J. Parasitol. Drugs Drug Resist., 2014; 4: 95–111.
- Willcox, M. Improved traditional phytomedicines in current use for the clinical treatment of malaria. Planta Med.,2011; 77: 662–671.
- Wink, M. Medicinal plants: a source of anti-parasitic secondary metabolites. Molecules., 2012; 17: 12771–12791.
- Parveen, A., Parveen, B., Parveen, R and Ahmad, S. Challenges and guidelines for clinical trial of herbal drugs. J Pharm Bioallied Sci., 2015 ;7(4):329–33.
- Calixto, J.B. Efficacy, safety, quality control, marketing and regulatory guidelines for herbal medicines (phytotherapeutic agents). Braz J Med Biol Res., 2000; 33: 179-189.