Antimicrobial Properties of the Medicinal Plant Cardiospermum Halicacabum L: New Evidence and Future Perspectives

European Review for Medical and Pharmacological Sciences 2019; 23: 7135-7143 Antimicrobial properties of the medicinal plant Cardiospermum halicacabum L: new evidence and future perspectives

R. GAZIANO1, E. CAMPIONE2, F. IACOVELLI3, E.S. PISTOIA1, D. MARINO1, M. MILANI4, P. DI FRANCESCO1, F. PICA1, L. BIANCHI2, A. ORLANDI5, S. MARSICO6, M. FALCONI3, S. AQUARO6

1Department of Experimental Medicine, University of Rome Tor Vergata, Rome, Italy 2Department of Systems Medicine, University of Rome Tor Vergata, Rome, Italy 3Department of Biology, Structural Bioinformatics Group, University of Rome Tor Vergata, Rome, Italy 4Department Cantabria Labs Difa Cooper, Caronno Pertusella, Varese, Italy 5Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy 6Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Edificio Polifunzionale, Rende, Cosenza, Italy Roberta Gaziano and Elena Campione contributed equally to this work and should be considered co-first authors

Abstract. – The emergence and rapid spread Key Words: of multidrug-resistance in human pathogenic C. halicacabum, Antimicrobial, T. rubrum, Hsp90, microorganisms urgently require the develop- Molecular modeling, Antifungal therapeutic strate- ment of novel therapeutic strategies for the gies. treatment of infectious diseases. From this perspective, the antimicrobial properties of the natural plant-derived products may represent an Introduction important alternative therapeutic option to synthetic drugs. Among medicinal plants, the Car- diospermum halicacabum L. (C. halicacabum), The increasing resistance of the microorgan- belonging to Sapindaceae family, could be a isms towards conventional antimicrobial agents very promising candidate for its antimicrobial has led to serious health problems in recent years activity against a wide range of microorganisms, and even more it is be expected in the future. This including both Gram positive and Gram nega- global phenomenon encourages the development tive bacteria, as well as fungal pathogens. Al- of new agents which can effectively inhibit mi- though the antimicrobial properties of C. halicacabum have been intensively studied, the mech- crobial growth. An alternative and very promis- anism/s by which it exerts the inhibitory activi- ing approach to overcome this issue might be the ty towards the pathogenic microbes have not yet use of natural antimicrobial products. The Middle been completely understood. This review focus- East represents an important source of medicinal es on the main antimicrobial activities displayed plants, which have been used for treating diseases in vitro by the plant extract, with particular at- for thousands of years in traditional medicine. tention on our recent advances. We demonstrat- The bioactive aromatic products extracted from ed that C. halicacabum is able to exert in vitro a some of these plants have been shown to possess dose-dependent fungistatic effect against Try- 1 chophyton rubrum (T. rubrum) through molecu- potential antimicrobial properties . Nowadays lar interaction with the fungal heat shock protein people in developing countries prefer plant-de- (Hsp)-90 chaperone. These findings are sup- rived natural products limiting their exposure to ported by a growing body of research indicating the side effects and toxicity of synthetic drugs. the crucial role played by the Hsp90 in the viru- Among medicinal plants, Cardiospermum hali- lence of the pathogenic microorganisms, including fungal pathogens. The possible future use of cacabum L. (C. halicacabum) is an herbaceous C. halicacabum for treating a wide range of in- climber belonging to the Sapindaceae family, fectious diseases is also discussed. naturally distributed in tropical and subtropical

Corresponding Author: Mattia Falconi, Ph.D; e-mail: [email protected] 7135 R. Gaziano, E. Campione, F. Iacovelli, E.S. Pistoia, D. Marino, et al. regions of the world. It grows in the plains of action of its bioactive compounds with the fungal Africa, America, Bangladesh, India, Malacca, Hsp90, compromising the biological functions and Pakistan. Its roots, leaves, and seeds have of the chaperone24. These findings are support- been employed as herbal medication2. The phy- ed by recent reports demonstrating the pivotal tochemical characterization of the constituents of role played by the Hsp90 in the virulence of the the plant extracts revealed the presence of β-ara- pathogenic microorganisms like bacteria, fungi, chidic acid, apigenin, apigenin-7-O-glucuronide, and human protozoan. Based on our results, here- and chrysoeriol-7-O-glucuronide3,4. Various fatty in we firstly propose that the chaperone Hsp90 acids and volatile esters were also isolated from in pathogens could be a potential therapeutic seed oil5,6. Furthermore, other secondary me- target for C. halicacabum, defining a possible tabolites have been found in small quantities in mechanism of action underlying its antimicrobial the extracts including alkaloids, carbohydrates, properties. Finally, the future therapeutic utility proteins, saponins, lignin, steroids, flavonoids, of this herb in the treatment of infectious diseases terpenoids, and cardiac glycosides7,8. The plant- is also discussed. based herbal products are commercially available as a gel, cream, shampoo, spray, medical drops, and pills. They are effective especially Antimicrobial Properties of in the treatment of dry itchy skin and scalp and Cardiospermum Halicacabum for treating chronic skin inflammatory disorders, including psoriasis4,9. The plant has been used in Antibacterial Activity Ayurveda and folk medicine for a long time in It has been reported that Cardiospermum the treatment of rheumatism, lumbago, cough, halicacabum exerts an important antimicrobial and hyperthermia10. Furthermore, it has been em- activity against numerous bacteria responsible ployed to treat nervous diseases like epilepsy and for the most common human infectious diseases. anxiety disorders, as a demulcent in orchitis, and Depending on the different solvents or extraction in dropsy11-13. C. halicacabum also acts as a dia- techniques, a different degree of inhibition can phoretic, diuretic, laxative, and anti-diarrheal6,14. be detected. Both the aqueous and alcoholic Additionally, it exhibits antioxidant and anti-in- extracts exhibit a marked activity against Gram flammatory activities via suppression of TNF-a positive and Gram-negative bacterial strains, production, vasodepressant, and even anticancer although some reports7 suggest that the aqueous effect in vitro15-18. Remarkable antipyretic and extract displays more effective than the alcohol- antiulcer activities of C. halicacabum, as well ic one. In detail, among Gram positive species, as an antihyperglycemic effect against strepto- Bacillus subtilis and Bacillus cereus appeared zotocin-induced diabetes, have also been shown to be more susceptible than Staphylococcus au- in experimental animal models19-21. Its aqueous reus to the aqueous leaf extract, whereas among leaf extract increases fertility in male rats and Gram negative bacteria, Escherichia coli was exhibits a significant hepatoprotective effect22. more sensitive to aqueous than the alcoholic The herb has also been shown to have strong pro- extract7. On the other hand, the ethanolic frac- tective activity against diverse infectious diseases tion was found to be more effective than the for its antibacterial, antifungal, and antiparasitic aqueous extract against Gram positive bacterial activity3,23. Although the antimicrobial proper- strains, especially Staphylococcus epidermidis, ties of the plant have extensively been studied, Streptococcus faecalis, Staphylococcus aureus, the precise mechanisms underlying its inhibitory Bacillus subtilis and Bacillus cereus23,25-27. The effect against microbial pathogens have not yet ethanolic extract also displayed remarkable in- been completely clarified. This review summa- hibitory activity against various Gram-nega- rizes available scientific data reported by the tive bacteria, especially Escherichia coli23 and most recent studies describing the antimicrobial Citrobacter freundii26. By contrast, it exerted activity of C. halicacabum. Furthermore, the re- only moderate inhibition of the multidrug resis- view highlights our recent results showing that C. tant Shigella spp., Salmonella typhi, Klebsiella halicacabum exerted an inhibitory effect against pneumoniae, and Enterobacter25 while exerted the dermatophytic fungus Trychophyton rubrum. minimal or no activity against Pseudomonas In addition, by molecular docking simulations, spp.23,25. The last one was found to be more we showed that the fungistatic effect of C. hal- susceptible to benzene followed by acetone, eth- icacabum may be related to the molecular inter- anol, and aqueous extracts23,25-27. A considerable

7136 Antimicrobial properties of C. halicacabum L activity against Streptococcus faecalis, Bacillus Antiparasitic Activity cereus, Bacillus steadher, and some Gram-nega- Among medicinal plants, C. halicacabum holds tive species, including Salmonella typhi, Shigel- promising antiparasitic properties. The leaves la boydii, Klebsiella pneumoniae, and Entero- ethyl acetate extract of the plant was found to bacter, was also obtained with the oil fraction25. have moderate antiplasmodial activity against a Furthermore, the butanol extraction showed a Chloroquine (CQ)-resistant strain of Plasmodium strong inhibitory effect against Micrococcus lu- falciparum, whereas the dichloromethane metha- teus and a modest activity against Staphylococ- nol (1:1) whole balloon plant extract showed good cus epidermidis and Streptococcus faecalis25. in vitro antiplasmodial activity28,29. Furthermore, The ethyl acetate fraction was found to exert a its shoot water extract displayed a weak antiplas- remarkable antibacterial activity against Strep- modial activity in vitro, while there was no evi- tococcus faecalis, Staphylococcus epidermidis, dence of its protective effect in a murine model Micrococcus luteus, and Klebsiella pneumoniae. of malaria30. The in vitro antifilarial activity of The same fraction also displayed a significant ethanol and aqueous extracts of C. halicacabum inhibition against Shigella boydii, Salmonella against Brugia pahangi was also investigated31. typhi and paratyphi B, and Enterobacter25. Fi- The aqueous extracts showed a mild reduction nally, the chloroform was seen to be more effec- of the motility of adult worms in a concentration tive against Staphylococcus aureus than alcohol and time dependent manner, and significantly re- and ether extractions but showed no inhibition duced the pattern of microfilarial release from fe- against Escherichia coli26. Altogether these da- male worms. Furthermore, the ethanol extract, at ta suggest that the natural extract of this plant the highest concentrations used, rapidly inhibited is able to exert antibacterial activity against a both the motility of adult worms and the release broad spectrum of bacterial pathogens. The type of microfilariae from females. of the extracting solvent can significantly influence its antimicrobial properties, probably by Hsp90 Chaperone as a Potential affecting the content of the bioactive compounds Molecular Target of Cardiospermum present in the plant. Halicacabum in Pathogens While the antimicrobial activity of C. hali- Antifungal Activity cacabum has been well established, there is no C. halicacabum displays in vitro strong an- evidence regarding the cellular/molecular mech- tifungal properties against the most common anisms underlying its inhibitory activity against pathogenic fungi responsible for opportunistic pathogenic microorganisms. By using the mo- infections in immunocompromised individuals. lecular docking analysis we recently proposed, As reported by Shareef et al25 the ethanol ex- for the first time, that Hsp90 chaperone could be tract showed a consistent inhibitory effect against involved in the fungistatic activity of C. halicaca- the opportunistic fungal species Saccharomyces bum against the fungus Trychophyton rubrum24. cerevisiae and Aspergillus niger. However, the Targeting the Hsp90 chaperone, C. halicacab- ethanol, aqueous, and oil extracts displayed only um could interfere with the biological functions a moderate inhibition against the opportunistic of the protein, similarly to the Hsp90 inhibitor yeast Candida albicans25. On the other hand, geldanamycin, a benzoquinone ansamycin antibi- the ethanol extract exhibited an important anti- otic, which targets the ADP/ATP binding pocket fungal activity against Candida albicans ATCC in the Hsp90 chaperone32,33. 22723. Additionally, we previously demonstrated Hsp90, a highly conserved and ubiquitous that the methanol extract exerts a clear-cut and ATP-dependent molecular chaperone, has been dose-dependent inhibitory effect against the an- identified from bacteria to mammals. In eukary- thropophilic dermatophyte species Trichophyton otes, Hsp90 is essential for cell viability under all rubrum24. Furthermore, seed oil has been shown tested growth conditions34. Its activity requires to be active against zoophilic pathogenic derma- collaboration with several Hsp90 co-chaperones. tophytes Micosporillum gypsiccus and Trycho- Together with Hsp70 chaperone, it plays a crucial phyton mentagrophytes25. By contrast, ethanol, role in the activation and stabilization of a vari- aqueous, and oil extracts did not influence the ety of different client proteins, including many in vitro growth of dermatophytes Trichophyton kinases involved in cell-signalling pathways. As longifusis, Trichophyton tonsurans, and Micros- for other classes of molecular chaperones, Hsp90 porum canis25. has a critical ATPase activity. Consistently, the

7137 R. Gaziano, E. Campione, F. Iacovelli, E.S. Pistoia, D. Marino, et al. structural changes induced by the ATP binding ticancer agents has raised considerable interest in Hsp90 play a key role in co-chaperone and in this protein also as a target for new antifungal chaperone client protein interactions. Hsp90 is a therapies. Recently, many investigations suggest conformationally dynamic dimeric protein (Fig- the crucial role played by Hsp90 in the microbial ure 1). The N-terminal domain (NTD) contains virulence and pathogenicity41-43. Studies on the a unique ATP-binding pocket and co-chaperone interrelationship among antifungal drug resis- interacting motifs35,36. The ATP binding to the tance, survival under stressful conditions, and the N-terminal domain and its subsequent hydrolysis signalling pathways involved in these processes by Hsp90 triggers a conformational cycle that in fungi, highlighted Hsp90 as a novel potential is crucial for the chaperone activity37. The heat molecular target for antifungal therapy. Its in- shock proteins have been extensively studied volvement in the pathogenicity and resistance to in cancer because they are important regula- azole and echinocandin antifungal drugs of the tors of cellular proliferation and differentiation. most clinically important fungi, i.e., Aspergil- Moreover, they are strongly implicated in the lus fumigatus, Candida albicans, Cryptoccoccus molecular orchestration, which leads to the ini- neoformans, and Trychophyton rubrum, is well tiation and/or progression of cancer38. This fact established41,42,44,45. In particular, the genetic or conditioned an increasing development of several pharmacological inhibition of Hsp90 by geldana- Hsp90 and other Hsps inhibitors that have shown mycin blocks the emergence of azole resistance in promising results, both in pre-clinical and clinical both fungal yeasts Saccharomyces cerevisiae and studies, in the treatment of tumor diseases39,40. Candida albicans44. Furthemore, Hsp90 governs The application of some Hsp90 inhibitors as an- the phenotypic switching of Candida albicans

Figure 1. ATP-coupled functioning cycle of Hsp90. A, In the resting state Hsp90, through the dimerization of its C-terminal domain (cyan), forms a homodimer with an open V-shaped conformation. B, Upon ATP (red) binding to the N-terminal ATPase domain, Hsp90 undergoes significant conformational changes and through an intermediate-state the N-terminal lids close and subsequently ATP is incorporated in the nucleotide-binding pocket. C, This results in a dimerization of the N-terminal domains (green) of each homodimer followed by closure of Hsp90 (D) and recruitment of the M domain (orange) for ATP hydrolysis. Finally, ATP is hydrolyzed and Hsp90 reverts to the open conformation where the N-terminal domains dissociate, allowing repetition of the cycle. Hsp90 inhibitors like geldanamycin (grey), radicicol, and purine derivatives bind to the N-terminal domain competing with ATP for binding. This picture has been produced using the program Chimera35 using X-ray structures (PDB IDs: 2CG9, 2IOQ) deposited in the Protein Data Bank36.

7138 Antimicrobial properties of C. halicacabum L from yeast to hyphal growth. The morphoge- Table I. Cardiospermum halicacabum compounds are netic plasticity of the fungus is implicated in its potential molecular interactors of Trychophyton rubrum Hsp90. In a previous work the docking energies of all listed virulence, since mutants defective in the yeast C. halicacabum compounds have been obtained through to filament transition are attenuated in terms of a molecular docking simulation, using a structural model virulence46,47. of Trichophyton rubrum Hsp90 as a receptor. The results These reports prompted us, in a previous work, indicated that all 13 bioactive compounds tested were to speculate about the potential interaction of C. potential Hsp90 interactors, although the 2 flavonoids, rutin, and luteolin-7-O-glucoside, showed the highest affinity for halicacabum with the Hsp90 of Trychophyton the ATP site-binding of fungal Hsp9024. rubrum, the main fungus involved in superficial mycoses. Using molecular docking simulations, Compound name we analyzed the interaction of 13 compounds of Rutin C. halicacabum (Table I) with the ATP-binding Luteolin-7-O-glucoside 24 site of the fungal Hsp90 . Intriguingly, among Kaempferol the 13 plant compounds tested, the flavonoids Apigenin luteolin-7-O-glucoside and rutin were identified Cholecalciferol as the most important potential Hsp90 inhibitors, All-trans-retinoic acid (ATRA) Quercetin although all tested compounds showed an affinity Chrysoeriol for the fungal Hsp90 ATP-binding pocket. These Calycosin-7-O-beta-D-glucopyranoside results were supported by in vitro agar diffu- 1-Hentriacontanol sion tests showing a direct remarkable antifungal Pentadecanoic acid activity of the single flavonoid components on 3,4-Dihydroxybenzoic acid Trychophyton rubrum. A considerable antifungal 3,4-Dihydroxybenzaldehyde activity of luteolin-7-O-glucoside against other fungi like Aspergillus fumigatus, Aspergillus niger, and Alternaria alternata, has already been chophyton rubrum (Figure 2). These results are in described by Chiruvella et al48. Furthermore, line with our previous analyses showing that AT- herein we demonstrate that among the numer- RA inhibited in vitro the germination of Candida ous components of C. halicacabum, also the albicans and Aspergillus fumigatus in a dose-de- all-trans-retinoic acid (ATRA), an isomer of the pendent manner49. Of note, when the compounds retinoic acid representing the oxidized form of luteolin-7-O-glucoside, rutin, or ATRA were in- vitamin A, is capable of exerting a clear dose-de- dividually tested against the Trychophyton ru- pendent fungistatic activity in vitro against Try- brum growth, their antifungal action resulted in

Figure 2. Disc diffusion assay of antifungal activity of ATRA against Trichophyton rubrum. Fungal cultures were incubated at 30°C with all trans retinoic acid ATRA at 1, 0.5, 0.25, and 0.125 mM/disc. After 24 hours of incubation, a clear-cut zone of inhibition was observed in the area treated with 1 mM of ATRA. One of the 3 representative experiments is shown.

7139 R. Gaziano, E. Campione, F. Iacovelli, E.S. Pistoia, D. Marino, et al. being lower than that obtained with the total plant an inhibitory action on Staphylococcus aureus, extract. Therefore, the antimicrobial effect of C. Staphylococcus epidermidis, and Enterococcus halicacabum could be the result of a synergistic faecalis, which are the major agents implicated interaction between the individual bioactive com- in nosocomial infections. Over the past two pounds, which altogether can work in a concerted decades mainly multi-drug resistant bacterial way, enhancing the antimicrobial properties of strains such as methicillin (MRSA) or vanco- the whole plant extract. mycin (VRSA) resistant staphylococci or van- In addition to fungi, the role of the heat shock comycin resistant enterococci (VRE), represent proteins as virulence factors in human protozoan a global concern61-63. So, there has been an in- parasites and bacteria has been speculated. Par- creasing need for the discovery and development ticularly, Hsp90 is involved in critical cellular of new antimicrobial agents. Natural products, processes such as stage development in important from this perspective, could represent an alter- human pathogens like Leishmania donovani50, native therapeutic strategy, overcoming the drug Trypanosoma cruzi51, Toxoplasma gondii52,53, and resistance in pathogens. Furthermore, C. halica- Plasmodium falciparum54. In bacteria, similar- cabum is also effective against the Gram neg- ly with their eukaryotic counterparts, Hsp90 is ative Escherichia coli, Klebsiella pneumoniae, involved in protein folding and it has also been Citrobacter, Enterobacter, and Pseudomonas implicated in the virulence of these microorgan- spp. These bacteria are considered the emerging isms55. It has been reported that the antibacterial nosocomial pathogens responsible for different activity of the selected tropical plant extracts types of infections, ranging from uncomplicated may be attributed to their high content of phe- urinary tract infections to life-threatening sep- nols and flavonoids56,57. In particular, flavonoids sis in health care settings. The latter is to date have multiple bacterial cell targets. One of their still uncontrollable, especially those caused by mechanisms of action consists in their ability to extended spectrum beta-lactamase (ESBL)-pro- interact with bacterial proteins either by non- ducing bacteria. In fact, these strains are resis- specific forces or by covalent bond formation, tant to the most beta-lactam antibiotics and often inactivating microbial adhesins, enzymes, and also exhibit co-resistance to many other classes cell envelope transport proteins23,58,59. Lipophilic of antimicrobial drugs, such as aminoglycosides flavonoids may also disrupt microbial membranes and quinolones, resulting in a marked limitation reducing the fluidity of outer and inner layers60. of the therapeutic options64. In addition to its Based on our results, it may be assumed that the antibacterial action, an important fungistatic antibacterial activity of the C. halicacabum total activity of C. halicacabum has been shown extract could be due, not only to the ability of against Candida albicans and Aspergillus fu- flavonoids to form complexes with extracellular migatus, the two most common opportunistic soluble proteins and cell wall, but also to its fungi which can be harmful to health. Indeed, ability to interfere with the Hsp90 function by they are responsible for systemic and fatal in- competing with ATP. Therefore, the Hsp90 might fections in immunocompromised individuals, be a novel and very promising therapeutic target especially those undergoing solid organ or bone of C. halicacabum against clinically important marrow transplantation, with diabetes mellitus, pathogens. haematological malignancy, human immunode- ficiency disorder, or long-term corticosteroid therapy recipient. The plant also exhibits an- Conclusions tifungal activity against Trychophyton spp., a group of pathogenic fungi that are involved in Our results suggest that the crude plant ex- superficial mycoses but can also cause deep tract of C. halicacabum may be an important and invasive infections, especially in severely natural source of biologically active compounds immunocompromised patients65. Despite the in- able to exert an efficient antimicrobial activity crease in the spectrum of available antifungal against a wide range of microorganisms. Par- drugs, the choice of suitable therapies for treat- ticularly, the antibacterial property of the plant ing systemic or superficial mycoses remains involves a broad spectrum of Gram positive relatively limited. This is due to multiple factors, and Gram negative microbes responsible for such as the emergence of drug-resistant fungal hospital-acquired/associated infections. Among strains and the heavy side effects and toxicity Gram positive bacteria, C. halicacabum exerts associated with current antifungal agents. Over-

7140 Antimicrobial properties of C. halicacabum L all, these investigations make C. halicacabum References a very promising candidate for novel antimicrobial therapeutic strategies, overcoming the 1) Tajkarimi MM, Ibrahim SA, Cliver DO. Antimicrobial major problems related to the drug resistance in herb and spice compounds in food. Food Control microbes and to the side effects of conventional 2010; 21: 1199-1218. antimicrobial therapies. Furthermore, the Hsp90 2) Urdampilleta JD, Coulleri JP, Ferrucci MS, Forni-Mar- tins ER. Karyotype evolution and phylogenetic chaperone of microbial pathogens could be a analyses in the genus Cardiospermum L. (Paul- possible therapeutic target for C. halicacabum. linieae, Sapindaceae). Plant Biol (Stuttg) 2013; The potential use of chaperones as molecular 15: 868-881. drug targets could be considered unattractive 3) Raza SA, Hussain S, Riaz H, Mahmood S. Review of because of the similarity of the molecular struc- beneficial and remedial aspects of Cardiosper- ture between human and microbial chaperones. mum halicacabum L. Afr J Pharmacy Pharmacol However, they exhibit different dependencies 2013; 7: 3026-3033. on chaperone-dependent pathways and could 4) Subramanyam R, Newmaster SG, Paliyath G, New- master CB therefore display differences in the sensitivity of . Exploring ethnobiological classifi- 43 cations for novel alternative medicine: a case their inhibition . In conclusion, Hsp90 could not study of Cardiospermum halicacabum L. (Mo- be the only possible target for C. halicacabum, dakathon, Balloon Vine) as a traditional herb since multiple plant bioactive compounds may for treating rheumatoid arthritis. Ethnobotany interact with different molecular targets both 2007; 19: 1-16. in a single or multiple intracellular pathways. 5) Mikolajczak KL, Smith CR, Tjarks LW. Cyanolipids of Additional studies are required to fully define Cardiospermum halicacabum L. and other sapin- the precise mechanisms underlying the antimi- daceous seed oils. Lipids 1970; 5: 812-817. crobial effect of the plant. Furthermore, despite 6) Rao NV, Prakash KC, Kumar SM. Pharmacologi- cal investigation of Cardiospermum halicacabum the great body of evidence regarding the anti- Linn in different animal models of diarrhoea. Indi- microbial activity of C. halicacabum, most of an J Pharmacol 2006; 38: 346-349. the studies reported in the literature have been 7) Deepan T, Alekhya V, Saravanakumar P, Dhanaraju conducted in vitro. The traditional practitioners MD. Phytochemical and anti-microbial studies on in India currently prescribe the leaves of the the leaves extracts of Cardiospermum halicacab- plant to the patients, in consideration of their um Linn. Adv Biol Res 2012; 6: 14-18. many beneficial uses without any toxic effect16. 8) Menichini F, Losi L, Bonesi M, Pugliese A, Loizzo MR, Therefore, further investigations are required, Tundis R. Chemical profiling and in vitro biological in in vivo experimental models, to assess the effects of Cardiospermum halicacabum L. (Sap- indaceae) aerial parts and seeds for applications efficacy and safety of this plant, for its potential in neurodegenerative disorders. J Enzyme Inhib future research translation into clinical practice. Med Chem 2014; 29: 677-685. 9) Jong MC, Ermuth U, Augustin M. Plant-based oint- ments versus usual care in the management of Conflict of Interest chronic skin diseases: a comparative analysis The Authors declare that they have no conflict of interests. on outcome and safety. Complement Ther Med 2013; 21: 453-459. 10) The Wealth of India. A dictionary of Indian raw ma- Sources of Funding terials and industrial products, raw materials. Vol. 3. New Delhi: Council of Scientific and Industrial This work was supported by PRIN (Progetti di Rilevante Research 1992; pp. 269-271. Interesse Nazionale) Grant 2015W729WH_007 from the MIUR, Italy. 11) Kumar R, Murugananthan G, Nandakumar K, Tal- war S. Isolation of anxiolytic principle from ethanolic root extract of Cardiospermum halicacabum. Acknowledgements Phytomedicine 2011; 18: 219-223. We want to thank Graziano Bonelli for his excellent tech- 12) Jayasilan D. Anticonvulsant activity in herbal ex- nical help. tract. Inter J Sci Res 2017; 6: 123-124. 13) Neuwinger HD. African traditional medicine: a dictionary of plant use and applications. Medpharm Authors’ Contribution GmbH Scientific Publishers, 2000. Stuttgart, Ger- All authors contributed toward drafting and critically re- many. vising the manuscript, gave final approval of the version to 14) Dhar LM, Dhar MM, Dharwan NB, Mehrotra NB, be published, and agreed to be accountable for all aspects Ray C. Screening of Indian plants for biological ac- of the work. tivity. Indian J Exp Biol 1968; 6: 232-247.

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