Rhinacanthus Nasutus “Tea” Infusions and the Medicinal Beneﬁts of the Constituent Phytochemicals

nutrients

Review Rhinacanthus nasutus “Tea” Infusions and the Medicinal Beneﬁts of the Constituent Phytochemicals

James Michael Brimson 1,2 , Mani Iyer Prasanth 1,2, Dicson Sheeja Malar 1,2, Sirikalaya Brimson 3 and Tewin Tencomnao 1,2,*

1 Age-Related Inﬂammation and Degeneration Research Unit, Chulalongkorn University, Bangkok 10330, Thailand; [email protected] (J.M.B.); [email protected] (M.I.P.); [email protected] (D.S.M.) 2 Department of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok 10330, Thailand 3 Department of Clinical Microscopy, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok 10330, Thailand; [email protected] * Correspondence: [email protected]; Tel.: +662-218-1533

Received: 20 October 2020; Accepted: 3 December 2020; Published: 9 December 2020

Abstract: Rhinacanthus nasutus (L.) Kurz (Acanthaceae) (Rn) is an herbaceous shrub native to Thailand and much of South and Southeast Asia. It has several synonyms and local or common names. The root of Rn is used in Thai traditional medicine to treat snake bites, and the roots and/or leaves can be made into a balm and applied to the skin for the treatment of skin infections such as ringworm, or they may be brewed to form an infusion for the treatment of inflammatory disorders. Rn leaves are available to the public for purchase in the form of “tea bags” as a natural herbal remedy for a long list of disorders, including diabetes, skin diseases (antifungal, ringworm, eczema, scurf, herpes), gastritis, raised blood pressure, improved blood circulation, early-stage tuberculosis antitumor activity, and as an antipyretic. There have been many studies investigating the roles of Rn or compounds isolated from the herb regarding diseases such as Alzheimer’s and other neurodegenerative diseases, cancer, diabetes and infection with bacteria, fungi or viruses. There have, however, been no clinical trials to confirm the efficacy of Rn in the treatment of any of these disorders, and the safety of these teas over long periods of consumption has never been tested. This review assesses the recent research into the role of Rn and its constituent compounds in a range of diseases.

Keywords: cancer; neurodegenerative disease; diabetes; infectious disease; natural products; phytochemicals; snake jasmine

1. Introduction Rhinacanthus genus comprising of about 25 species belongs to the family Acanthaceae and distributed throughout tropical and subtropical regions [1]. Rhinacanthus nasutus (Rn), commonly referred to as snake jasmine because of the shape of its flower, and is a native of Southeast Asia. The plant is also referred to by the names (as well as others (Table1)) Rhinacanthus rottlerianus Nees and Justicia rottleriana Wall. It is a small, slender shrub growing to a height of about 0.6 to 1.2 m [2]. It is a woody based plant with sparsely branched club-shaped fruit and four seeds. The whole plant is widely used in traditional medicinal practices for the treatment of diverse disease conditions. Medicinal preparations of the plant in the form of decoctions and herbal tea have been given internally to the people for the treatment of hepatitis, diabetes, hypertension, while the external application in the form of paste has been used by the people who suffer from psoriasis, eczema, ringworm as well as inflammation. Traditionally seeds, roots and leaves of the plant have been used against scabies, eczema and various skin conditions. Leaves are used for prickly heat as well as scurf, and roots are being

Nutrients 2020, 12, 3776; doi:10.3390/nu12123776 www.mdpi.com/journal/nutrients Nutrients 2020, 12, 3776 2 of 18 boiled along with milk and used as an aphrodisiac. A decoction of roots is used as an antidote for snake bites. Stem and leaves of the plant are used to treat pulmonary tuberculosis and high blood pressure [3]. Several reports have revealed the folklore and traditional medicinal practices using Rn ranging from antioxidant, antimicrobial to anticancer and neuroprotectant. The rhinacanthin-rich leaf extract has been reported to have antimicrobial activity against S. mutans, S. epidermidis, P.acnes and S. aureus [4]. Traditional usage of Rn in the treatment of skin diseases caused by fungi has been reported, showing that the leaf extract has fungicidal activity by acting on the cell wall causing its degeneration and death [5]. The leaf extract of the plant also showed significant larvicidal activity against Culex quinquefasciatus, Anopheles stephensi, Aedes aegypti and prevented the emergence of larvae to adults [6,7]. Further, a formulation containing root extract of the plant has also been prepared for the control of mosquito vectors [8]. Methanolic extract of Rn was found to exhibit significant antioxidant activity in cell-free in vitro studies as well could ameliorate oxidative stress and improve the antioxidant status in experimental rats [9,10]. Additionally, methanol extract of the root was reported to exhibit a cytotoxic effect against oral squamous cell carcinoma cells and promyelocytic leukemia cells while showing lower-level cytotoxicity to normal cells [11]. Ethanolic extract of the plant was shown to exhibit hepatoprotective activity against aflatoxin B1 induced toxicity in Wistar rats by enhancing the glutathione level and modulating the activity of serum enzymes [12]. In addition, the root extract inhibited lipopolysaccharides (LPS)-induced inflammation by reducing the level of nitric oxide (NO) in RAW264.7 macrophage cells [11]. Further, Rn root extract has also been reported to have a neuroprotective effect against hypoxia as well as glutamate-induced cell death in HT22 cells by bringing back the redox homeostasis [13,14]. Bibliographical analysis of the publications (original research articles) related to Rn shows a significant increase in publications from the late 2000s onwards with 11 Rn related articles published in 2009, 70 articles in 2015 and 62 so far in 2020. These articles break down into the following subject areas; pharmacology and toxicology (19%), biochemistry, genetics and molecular biology (17%), agricultural and biological sciences (17%), chemistry (14%), medicine (13%), immunology (4%) and others (16%) (data obtained from SCOPUS). With the recent increase of publications related to Rn in mind, this review aims to cover the advances and research into Rn over the last decade in the fields of neurodegeneration, cancer, diabetes, as well as Rn’s proprieties as an antimicrobial, antiviral and antifungal agent, and assess whether there is enough evidence to warrant the use of Rn or compounds isolated from Rn in human trails. All plants described in this manuscript have had their names checked at The Plant List [15]. Nutrients 2020, 12, 3776 3 of 18 Nutrients 2020, 12, x FOR PEER REVIEW 3 of 16

Table 1. List of common/regional names of Rhinacanthus nasutus (L.) Kurz. Table 1. List of common/regional names of Rhinacanthus nasutus (L.) Kurz.

Synonyms

Rhinacanthus nasutus (L.) Kurz Rhinacanthus communis Nees Rhinacanthus osmospermus Bojer ex Nees Justicia dichotoma Rottler * Justicia nasuta L. Justicia macilenta E. Mey. * Justicia rottleriana Wall. * Justicia silvatica Nees * Justicia sylvatica Vahl * Pseuderanthemum connatum Lindau Dianthera paniculata Lour.

Common Name Common Names Origin

জূঈপান Juipana Bengali

Dainty Spurs, Snake jasmine English White Crane flower

पालकजूही Palakjuhi, Hindi जूहीपानी Juhipani

ಾಗಮಲ್ಗೆ Nagamallige, Kannada Doddapatike Dadmari Konkani Puzhukkolli Nagamulla Purukolli Orukaalmudanthi Malayalam Vellakkurunji Nagamulla (നാഗമുല്ല) Pushpakedal

गजकण Gajkarni Marathi

Yudhikaparni, Sanskrit Yoodhikaparni Uragamalli Tamil Nagamalli (நாகமல்லீ)

Nagamalle (నాగమల్లె) Telugu

Palakjuhi Urdu Thong Phan chang (ทองพนชั ง่ั ), Yaa man kai (หญาม นไกั ) (ตะ ซิ ช ี ซ)ี Ta si chi si Thai (กะเหรยงสะกอี่ )

* Name* Name is unresolved, is unresolved, but some but data some suggest data thatsuggest it is synonymous that it is synonymous with Rhinacanthus with nasutusRhinacanthus(L.) Kurz nasutus (Rn) (checked (L.) viaKurz the plant (Rn) list (checked [15]. via the plant list [15].

2. Phytochemicals Found in Rhinacanthus nasutus

Nutrients 2020, 12, 3776 4 of 18

2. Phytochemicals Found in Rhinacanthus nasutus Rn has been identified as an important medicinal plant due to its medicinal properties for various disease conditions. This has led to the characterizing of the active ingredients responsible for exhibiting these medicinal properties (Table2). Kodama et al. 1993 has reported the isolation of a naphthopyran derivative 3,4-dihydro-3,3-dimethyl-2H-naphtho[2,3-b]pyran-5,10-dione which also possess antifungal activity [16]. Several phytochemicals including β-amyrin, glutinol, lupeol, stimasterol, sitosterol, umbelliferone, 2-methylanthraquinone, wogonin, oroxylin A, (+)-praeruptorin, p-hydroxy benzaldehyde, methyl vanillate, syringaldehyde, Dehydro-α-lapachone and novel compounds rhinacanthin A-D, G-Q, (RnA-D and RnG-Q) rhinacanthone, 4-acetonyl-3,5-dimethoxy-p-quinol have been isolated by Wu et al. 1998 from the roots, stem and leaves of Rn [17–19]. Rhinacanthin S (RnS) and RnA isolated from the leaves of Rn showed acetylcholinesterase inhibitory activity and cytotoxicity, respectively [20]. RnC has also been found to attenuate renal oxidative stress and protect against streptozotocin-nicotinamide induced diabetic nephropathy in rats [21]. Based on the antiviral property of the plant, several research groups have succeeded in isolating and examining the activity of phytochemicals from the roots and aerial parts of R. nasutus. Sendl et al. 1996 has reported the isolation of two naphthoquinones, RnC and RnD, from the whole plant and their antiviral activity against human cytomegalovirus [22]. The lignans Rhinacanthin E and F (RnE and RnF) isolated from the aerial part of the plant have been reported to have significant activity against the influenza virus under in vitro conditions [23]. An antiviral study by Ngoc et al. 2019 with a novel naphthoquinone analog rhinacasutone and seven other known compounds like rhinacanthone, RnC, RnD, RnN, RnQ and RnE and heliobuphthalmin indicated that RnC, RnD, RnN and RnQ exhibit significant antiviral activity against influenza virus A/PR/8/34 (PR8), Anti-Human Rhinovirus 1B (HRV1B), and Coxsackievirus B3 (CVB3) [24]. Kwak et al. 2018 has isolated trans O-coumaric acid, 2,4-dihydroxycinnamic acid, 3,4-dimethoxyphenyl-O-β-D-glucopyranoside, p-hydroxy phenethyl trans-ferulate, 2,3-bis[(4-hydroxy-3,5-dimethoxyphenyl)methyl]-1,4-butanediol,8,80-bisdihydrosiringenin glucoside, dehydrodiconiferyl alcohol-4-β-D-glucoside and (-)-dehydrodiconiferyl alcohol from the aerial parts of the plant. Among which, 2,3-bis[(4-hydroxy-3,5-dimethoxyphenyl)methyl]-1,4-butanediol and 8,80-bisdihydrosiringenin glucoside were found to have significant neuraminidase inhibitory activity [25]. Nutrients 2020, 12, x FOR PEER REVIEW 4 of 16

plant and their antiviral activity against human cytomegalovirus [22]. The lignans Rhinacanthin E and F (RnE and RnF) isolated from the aerial part of the plant have been reported to have significant activity against the influenza virus under in vitro conditions [23]. An antiviral study by Ngoc et al. Nutrients 20202020,, 1122,, xx FORFOR PEERPEER REVIEWREVIEW 44 ofof 1616 Nutrients2019 with 2020 a, 1novel2, x FOR naphthoquinone PEER REVIEW analog rhinacasutone and seven other known compounds4 oflike 16 rhinacanthone, RnC, RnD, RnN, RnQ and RnE and heliobuphthalmin indicated that RnC, RnD, RnN plantplant andand theirtheir antiviralantiviral activityactivity againstagainst humanhuman cytomegaloviruscytomegalovirus [[2222]]... TheTheThe lignanslignanslignans RhinacanthinRhinacanthin EE and RnQ exhibit significant antiviral activity against influenza virus A/PR/8/34 (PR8), Anti-Human andand FF ((RnERnE andand RnFRnF)) isolatedisolatedisolated fromfromfrom thethethe aerialaerialaerial partpartpart ofofof thethethe plantplantplant havehavehave beenbeenbeen reportedreportedreported tototo havehavehave significantsignificantsignificant Rhinovirus 1B (HRV1B), and Coxsackievirus B3 (CVB3) [24]. Kwak et al. 2018 has isolated trans O- activityactivity againstagainst thethethe influenzainfluenzainfluenza virusvirusvirus underunderunder ininin vitrovitrovitro conditionconditionconditionss [[2323]]... AnAnAn antiviralantiviralantiviral studystudystudy bybyby NgocNgocNgoc etetet al.al.al. coumaric acid, 2,4-dihydroxycinnamic acid, 3,4-dimethoxyphenyl-O-β-D-glucopyranoside, p- 20192019 withwith aa novelnovel naphthoquinonenaphthoquinone analoganalog rhinacasutonerhinacasutone andand sevenseven otherother knownknown compoundscompounds likelike hydroxy phenethyl trans-ferulate, 2,3-bis[(4-hydroxy-3,5-dimethoxyphenyl)methyl]-1,4- rhinacanthone,rhinacanthone, RnC,RnC, RnD,RnD, RnN,RnN, RnQRnQ andand RnERnE andand heliobuphthalminheliobuphthalmin indicatedindicated thatthat RnC,RnC, RnD,RnD, RnNRnN butanediol,8,8′-bisdihydrosiringenin glucoside, dehydrodiconiferyl alcohol-4-β-D-glucoside and (-)- andand RnQRnQ exhibitexhibit significantsignificant antiviralantiviral activityactivity againstagainst influenzainfluenzainfluenza virusvirusvirus A/PR/8/34A/PR/8/34A/PR/8/34 (((PR8PR8)),,, AntiAnti--HumanHuman dehydrodiconiferyl alcohol from the aerial parts of the plant. Among which, 2,3-bis[(4-hydroxy-3,5- RhinovirusRhinovirus 1B1B ((HRV1BHRV1B)),,, andandand CoxsackievirusCoxsackievirus B3B3 ((CVB3CVB3)) [[2424]]... KwakKwakKwak etetet al.al.al. 201820182018 hashashas isolatedisolatedisolated transtranstrans OOO-- dimethoxyphenyl)methyl]-1,4-butanediol and 8,8′-bisdihydrosiringenin glucoside were found to coumariccoumaric acid,acid, 2,42,4--dihydroxycinnamicdihydroxycinnamic acid,acid, 3,43,4--dimethoxyphenyldimethoxyphenyl--OO--ββ--DD--glucopyranoside,glucopyranoside, pp-- have significant neuraminidase inhibitory activity [25]. hydroxyhydroxy phphenethylenethyl transtrans--ferulate,ferulate, 2,32,3--bis[(4bis[(4--hydroxyhydroxy--3,53,5--dimethoxyphenyl)methyl]dimethoxyphenyl)methyl]--1,41,4-- butanediol,8,8′butanediol,8,8′--bisdihydrosiringeninbisdihydrosiringenin glucoside,glucoside, dehydrodiconiferyldehydrodiconiferyl alcoholalcohol--44--ββ--DD--glucosideglucoside andand ((--))-- Table 2. Structures and activities of compounds found specifically in Rhinacanthus nasutus extracts. dehydrodiconiferyl alcohol from the aerial parts of the plant. Among which, 2,3-bis[(4-hydroxy-3,5- Nutrients 2020, 12, 3776 dehydrodiconiferyldehydrodiconiferyl alcoholalcohol fromfrom thethe aerialaerial partsparts ofof thethe plant.plant. AmongAmong which,which, 2,32,3--bibis[(4s[(4--hydroxyhydroxy--3,53,5-- 5 of 18 dimethoxyphenyl)methyl]dimethoxyphenyl)methyl]Name --1,41,4--butanediolbutanediolStructure and and 8,8′ 8,8′--bisdihydrosiringeninbisdihydrosiringeninReported glucosideglucoside Activities werewere foundfound toto havehave significantsignificant neuraminidaseneuraminidase inhibitoryinhibitory activityactivity [[2525]]... Table 2. Structures2-Hydroxy- and1,4- activities of compounds found speciﬁcallyAn interconversion in Rhinacanthus of pyridine nasutus nucleotidesextracts. to naphthoquinoneTableTable 2.2. StructuresStructures andand activitiesactivities ofof compoundscompounds foundfound specificallyspecificallycombat inin RhinacanthustheRhinacanthus effects of oxidativenasutunasutuss extractsextracts stress ...[ 26]

NameNameName StructureStructureStructure ReportedReported ActivitiesActivities Reported Activities 3,4-dihydro-3,3- 22-dimethyl--HydroxyHydroxy---2H-1,41,4---- AnAn interconversioninterconversionAn interconversion ofof pyridinepyridine of nucleotidesnucleotides pyridine nucleotides toto to combat the eﬀects of oxidative stress 2-Hydroxy-1,4-naphthoquinone Antitumor activity [27] naphtho[2,3naphthoquinonenaphthoquinone-b] pyran - combatcombat thethe effectseffects ofof oxidativeoxidative stressstress [[[2626]]] [26] 5,10-dione

3,43,4---dihydrodihydro---3,33,3--- Inhibited the growth of S. aureus with an Rhinacanthindimethyldimethyl---2H2H -A-- 3,4-dihydro-3,3-dimethyl-2H-naphtho[2,3-b] pyran-5,10-dione inhibitionAntitumor Antitumorzone of 16 activitymmactivity (25 [[mm[2727]]] disk)Antitumor [28] activity [27] naphtho[2,3naphtho[2,3---b]b] pyranpyran--- 5,10-dione 5,105,10---dionedione

Inhibition of cytochrome enzymes CYP6AA3 Rhinacanthin B InhibitedInhibitedInhibited thethethe growthgrowthgrowth ofofof S.S. aureusaureus withwith anan Rhinacanthin A RhinacanthinRhinacanthin AA Inhibitedand CYP6P7 the growth in vitro of [29S.] aureus with an inhibition zone of 16 mm (25 mm disk) [28] inhibitioninhibitioninhibition zonezonezone ofofof 161616 mmmm (25(25 mmmm disk)disk) [[[2828]]]

Prevented Aβ-induced toxicity in rat hippocampal neurons and attenuated InhibitionInhibitionInhibition ofofof cytochromecytochromecytochrome enzymesenzymesenzymes CYP6AA3CYP6AA3 Rhinacanthin B RhinacanthinRhinacanthin BB lipopolysaccharidesInhibition (LPS of)- cytochromeactivated nitric enzymes CYP6AA3 and CYP6P7 in vitro [29] andand CYP6P7CYP6P7 inin vitrovitro [[[2929]]] oxide (NO) production, inducible NO

synthase (iNOS) expression, and nuclear factor kappaPreventedPrevented-light-chainPrevented AβAβ--enhancer--inducedinducedinduced Aβ -induced toxicitytoxicityoftoxicity activated ininin toxicity ratratrat B cells in rat hippocampal neurons and attenuated hippocampalhippocampal(NF-lipopolysaccharidesκB) signaling neuronsneurons in andand rat attenuatedgliaattenuated (LPS)-activated [30]; nitric oxide (NO) production, inducible NO Exhibitedlipopolysaccharideslipopolysaccharideslipopolysaccharides neuroprotectivesynthase (iNOS) (((LPSLPS )))effect---activated expression,activated by reducing nitricnitric and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling in rat glia [30] cleavedoxideoxide (NO)(NO)caspase production,production,-3- and caspase inducibleinducible-9a-related NONO ;Exhibited neuroprotective effect by reducing cleaved caspase-3- and synthasesynthaseapoptosis (iNOS)(iNOS) and expression,expression,anti-inflammatory andand nuclearnuclear effect factorfactor by Rhinacanthin C Rhinacanthin C caspase-9a-related apoptosis and anti-inflammatory effect by decreasing High kappakappa--lightlight--chainchain--enhancerenhancer ofof activatedactivated BB cellscells kappadecreasing-light-chain Highmobility-enhancer mobility group of activatedgroup box 1box (HMGB-1) B 1 cells mRNA and protein expression [31]; (NF-κB) signaling in rat glia [30]; (HMGB(((NFNF-1)-- -κBmRNAκBInhibits))) signalingsignaling and cholangiocarcinoma protein inin ratrat gliaexpressionglia [[[3030]]];;; cell [31 migration]; and invasion by decreasing matrix ExhibitedExhibitedInhibits metalloproteinase-2cholangiocarcinoma neuroprotectiveneuroprotective effecteffect (MMP-2),cell byby migration reducingreducing Urokinase-Type Plasminogen Activator (uPA), focal Nutrients 2020, 12, x FOR PEER REVIEW 5 of 16 cleavedcleavedand caspaseinvasioncaspaseadhesion---33 b--- kinase yandand decreasing caspasecaspase (FAK)--- 9amatrix and9a---relatedrelated mitogen-activated protein kinase (MAPK) pathways, metalloproteinaseapoptosisapoptosis andandalong aantinti-2 --(-inflammatoryinflammatoryMMPinflammatory with- inhibition2), Urokinase effecteffecteffect of cell - bybyTypeby migration and antiproliferative effects [32] RhinacanthinRhinacanthin CC Inhibitory activity against cytomegalovirus Plasminogendecreasingdecreasing Activator HighHigh mobilitymobility (uPA ) group,group focal boxadhesionbox 11 [22]; (((HMGBHMGBkinase-- -1(1FAK))) mRNAmRNA) and andand mitogen proteinprotein-activated expressionexpression protein [[[3131]]] ;;; antiviral activities againstInhibitory influenza activityvirus against cytomegalovirus [22]; Rhinacanthin D InhibitsInhibitsInhibitskinase cholangiocarcinomacholangiocarcinomacholangiocarcinoma (MAPK) pathways, cellcellcell along migrationmigrationmigration with Rhinacanthin D A/PR/8/34antiviral (PR8), Anti activities-Human against Rhinovirus influenza 1B virus A/PR/8/34 (PR8), Anti-Human Rhinovirus andandinhibition invasioninvasion1B of bb y (HRV1B),celly decreasingdecreasing migration and matrixmatrix Coxsackievirusand B3 (CVB3)-infected Vero cells [24] (HRV1B), and Coxsackievirus B3 (CVB3)- metalloproteinasemetalloproteinaseantiproliferative---22 (((MMPMMP effects---22))),,, UrokinaseUrokinase [32] ---TypeType infected Vero cells [24] PlasminogenPlasminogen ActivatorActivator ((uPAuPA))),,, focalfocalfocal adhesionadhesionadhesion kinasekinase (((FAKFAK))) andand mitogenmitogen---activatedactivated proteinprotein kinasekinase (((MAPKMAPK))) pathways,pathways, alongalong withwith inhibitioninhibitioninhibition ofofof cellcellcell migrationmigrationmigration andandand antiproliferative effects [[32]] Antiviral antiproliferativeantiproliferativeactivity against influenza effectseffects [[3232 virus]] type Rhinacanthin E A [23]

Antiviral activity against influenza virus type Rhinacanthin F A [30]

Inhibition of cytochrome enzyme CYP6P7 in Rhinacanthin G vitro [29]

Inhibition of cancer cell lines KB, HeLa, and Rhinacanthin M HepG2 with IC50 values of 1.5, 3.0 and 4.6 µM, respectively [33]

Rhinacanthin-N caused G2/M arrest of HeLaS3 cells after 24 h incubation and increased the proportion of sub-G1 hypodiploid cells, apoptotic cells [34]; Rhinacanthin N Antimetastatic activity as it inhibited the metastatic pulmonary colonization of the melanoma cells in C57BL/6 male mice [35]; Antiproliferative activity against HeLaS3 cells and suppressed tumor growth in vivo [36]

Rhinacanthin O Unknown activities

Induction of apoptosis in tumor cells that may be associated with the activation of the Rhinacanthin Q caspase-3 pathway [34]; Antiviral activities against PR8, HRV1B, and CVB3-infected Vero cells [24]

Nutrients 2020, 12, x FOR PEER REVIEW 5 of 16 NutrientsNutrients 20 2020202020,, , 1 11222,, , x xx FOR FORFOR PEER PEERPEER REVIEW REVIEWREVIEW 555 of ofof 16 1616 Nutrients 2020, 12, 3776 Inhibitory activity against cytomegalovirus 6 of 18 IIInhibitorynhibitorynhibitory activity activityactivity against againstagainst[22]; cytomegalovirus cytomegaloviruscytomegalovirus antiviral activities[ [against[222222]]];; ; influenza virus Rhinacanthin D A/PR/8/34antiviralantiviralantiviral activitiesactivities(activitiesPR8), Anti against againstagainst-Human influenza influenzainfluenza Rhinovirus virus virusvirus 1B RhinacanthinRhinacanthin D D Table 2. Cont. A/PR/8/34A/PR/8/34(HRV1B )( ((,PR8PR8 PR8and))), , ,Coxsackievirus Anti Anti---HumanHuman Rhinovirus Rhinovirus B3 (CVB3 )1B -1B (((HRV1BHRV1B))),, , infectedand andand Coxsackievirus Coxsackievirus Vero cells [24 B3 B3] ( (CVB3CVB3)))--- Name Structureinfectedinfectedinfected V Veroeroero cells cellscells [ [[242424]]] Reported Activities

Antiviral activity against influenza virus type Rhinacanthin E AAntiviralntiviralntiviral activity activityactivity against againstagainstA [23 influenza ]influenzainfluenza virus virusvirus type typetype Rhinacanthin E RhinacanthinRhinacanthin E E Antiviral activity against inﬂuenza virus type A [23] AA [ [[232323]]]

Antiviral activity against influenza virus type Rhinacanthin F AAntiviralntiviralntiviral activity activityactivity against againstagainstA [30 influenza ]influenzainfluenza virus virusvirus type typetype Rhinacanthin F RhinacanthinRhinacanthin F F Antiviral activity against inﬂuenza virus type A [30] AA [ [[303030]]]

Inhibition of cytochrome enzyme CYP6P7 in Rhinacanthin G InhibitionInhibitionInhibition of ofof cytochrome cytochromecytochromevitro [29 enzyme enzymeenzyme] CYP6P7 CYP6P7 in in Rhinacanthin G RhinacanthinRhinacanthin G G Inhibition of cytochrome enzyme CYP6P7 in vitro [29] vitrovitrovitro [ [[292929]]]

Inhibition of cancer cell lines KB, HeLa, and Rhinacanthin M HepGInhibitionInhibitionInhibition2 with of ofof IC cancer cancercancer50 values cell cellcell of lines lineslines 1.5, KB, KB,KB,3.0 HeLand HeLHeL a4.6aa,, , and and andµM , Inhibition of cancer cell lines KB, HeLa, and HepG2 with IC50 values of 1.5, 3.0 and Rhinacanthin M RhinacanthinRhinacanthin M M HepGHepG222 with with IC IC5050respectively50 values valuesvalues of ofof 1.5, 1.5,1.5, [33 3.0 3.0]3.0 and andand 4.6 4.64.6 µM µMµM,, , 4.6 µM, respectively [33] respectivelyrespectivelyrespectively [ [[333333]]] Rhinacanthin-N caused G2/M arrest of HeLaS3 RhinacanthinRhinacanthincells after 24---N Nh causedincubationcaused G2/M G2/M and arrest arrest increased of of HeLaS3 HeLaS3 the cellscellscellsproportion after afterafter 24 2424Rhinacanthin-N h h hof incubation incubationincubation sub-G1 hypodiploid and andand caused increased increasedincreased G2 cells,/M the thethe arrest of HeLaS3 cells after 24 h incubation and increased the proportion of sub-G1 hypodiploid cells, apoptotic cells [34]; proportionproportionproportionapoptotic of ofof sub subsub---G1G1 cells hypodiploid hypodiploid [34]; cells, cells, Antimetastatic activity as it inhibited the metastatic pulmonary colonization of the Rhinacanthin N Rhinacanthin N Antimetastaticapoptoticapoptoticapoptotic activity cells cellscells as [ [[34it3434 inhibited]]];; ; the apoptotic cells [34melanoma]; cells in C57BL/6 male mice [35]; RhinacanthinRhinacanthin N N AntimetastaticmetastaticAntimetastatic Antiproliferativepulmonary activity activity colonizationas as it it inhibited activityinhibited of against the the HeLaS3 cells and suppressed tumor growth melanomametastaticmetastatic pulmonary cellspulmonary in C57BL/6 colonization colonizationcolonization male mice of ofof the [thethe35 ] ; in vivo [36] Antiproliferativemelanomamelanoma cells cells inactivityin C57BL/6 C57BL/6 against male male HeLaS3 mice mice [ [[353535 cells]]];; ; AntiproliferativeAntiproliferativeand suppressed activity activity tumor against growthagainst HeLaS3 inHeLaS3 vivo [cells 36cells] andandand suppressed suppressedsuppressed tumor tumortumor growth growthgrowth in inin vivo vivovivo [ [[363636]]]

Rhinacanthin O Unknown activities RhinacanthinRhinacanthin O O UnknownUnknown activities activities

Induction of apoptosis in tumor cells that may InductionInductionInductionbe associated of ofof apoptosis apoptosisapoptosis with in theinin tumor tumortumor activation cells cellscells thatof thatthat the m mm ayayay Rhinacanthin Q bebebe associated associatedassociatedcaspase with withwith-3 pathway the thethe activation activationactivation [34]; of ofof the thethe RhinacanthinRhinacanthin Q Q Antiviral activitiescaspasecaspasecaspase---33 3against pathway pathwaypathway PR8, [ [[343434 HRV1B,]]];; ; and AAntiviralntiviralntiviralCVB3 activities activitiesactivities-infected against againstagainst Vero PR8, PR8,PR8, cells HRV1B, HRV1B,HRV1B, [24] and andand CVB3CVB3---infectedinfectedinfected Vero VeroVero cells cellscells [ [[242424]]]

NutrientsNutrients 20202020,, 1122,, xx FORFOR PEERPEER REVIEWREVIEW 55 ofof 1616

IInhibitorynhibitory activityactivity againstagainst cytomegaloviruscytomegalovirus [[2222]];; antiviralantiviral activitiesactivities againstagainst influenzainfluenza virusvirus RhinacanthinRhinacanthin DD A/PR/8/34A/PR/8/34 ((PR8PR8)),, Anti Anti--HumanHuman RhinovirusRhinovirus 1B1B (HRV1B), and Coxsackievirus B3 (CVB3)- (HRV1B), and Coxsackievirus B3 (CVB3)- infectedinfected VVeroero cellscells [[2424]]

AAntiviralntiviral activityactivity againstagainst influenzainfluenza virusvirus typetype RhinacanthinRhinacanthin EE AA [[2323]]

AAntiviralntiviral activityactivity againstagainst influenzainfluenza virusvirus typetype RhinacanthinRhinacanthin FF AA [[3030]]

InhibitionInhibition ofof cytochromecytochrome enzymeenzyme CYP6P7CYP6P7 inin RhinacanthinRhinacanthin GG vitrovitro [[2929]]

InhibitionInhibition ofof cancercancer cellcell lineslines KB,KB, HeLHeLaa,, andand RhinacanthinRhinacanthin MM HepGHepG22 withwith ICIC5050 valuesvalues ofof 1.5,1.5, 3.03.0 andand 4.64.6 µMµM,, respectivelyrespectively [[3333]]

RhinacanthinRhinacanthin--NN causedcaused G2/MG2/M arrestarrest ofof HeLaS3HeLaS3 cellscells afterafter 2424 hh incubationincubation andand increasedincreased thethe proportion of sub-G1 hypodiploid cells, Nutrients 2020, 12, 3776 proportion of sub-G1 hypodiploid cells, 7 of 18 apoptoticapoptotic cellscells [[3434]];; RhinacanthinRhinacanthin NN AntimetastaticAntimetastatic activityactivity asas itit inhibitedinhibited thethe metastaticmetastatic pulmonarypulmonary colonizationcolonization ofof thethe Table 2. Cont . melanomamelanoma cellscells inin C57BL/6C57BL/6 malemale micemice [[3535]];; AntiproliferativeAntiproliferative activityactivity againstagainst HeLaS3HeLaS3 cellscells Name Structureandand suppressedsuppressed tumortumor growthgrowth inin vivovivo [[3636 Reported]] Activities

Rhinacanthin O RhinacanthinRhinacanthin OO UnknownUnknown activitiesactivities Unknown activities

Nutrients 2020, 12, x FOR PEER REVIEW InductionInduction ofof apoptosisapoptosis inin tumortumor cellscells thatthat6 of mm ay16ay bebe associatedassociatedInduction withwith of thethe apoptosis activationactivation in tumorofof thethe cells that may be associated with the activation of Rhinacanthin Q RhinacanthinRhinacanthin QQ caspasecaspase--33 pathwaypathway [[3434]];; the caspase-3 pathway [34]; Antiviral activities against PR8, HRV1B, and CVB3-infected Vero cells [24] NutrientsNutrients 20 202020, ,1 122, ,x x FOR FOR PEER PEER REVIEW REVIEW AAntiviralntiviral activitiesactivities againstagainst PR8,PR8, HRV1B,HRV1B,66 andof andof 16 16 CVB3-infected Vero cells [24] CVB3-infected Vero cells [24] Rhinacasutone Unknown activities

Rhinacasutone RhinacasutoneRhinacasutone UnknownUnknown activities activities Unknown activities

Strong inducer of apoptosis in HuH-7 cells [37]; Heliobuphthalmin High antineoplasic activities against the classicalSStrongtrong inducer inducer(multi- drugof of apoptosis apoptosis-resistant) in in MDRHuH HuH -subline7-7 cells cells derived from gastric[37[37];] ; carcinomaStrong inducer [38] of apoptosis in HuH-7 cells [37];

Heliobuphthalmin HeliobuphthalminHeliobuphthalmin HHighigh antineoplasic antineoplasicHigh antineoplasic activities activities against against activities the the against the classical (multi-drug-resistant) MDR Rhinacanthone-induced apoptosis in HeLa classicalclassical (multi (multi-drug-drug-resistant)-resistant)subline MDR MDR subline subline derived from gastric carcinoma [38] cells is mediated primarily through the derivedderived from from gastric gastric carcinoma carcinoma [ 38[38] ] mitochondrial-dependent signaling pathway asRhinacanthone Rhinacanthoneit inhibitedRhinacanthone-induced proliferation-induced-induced apoptosis apoptosisof HeLa cellsapoptosisin in HeLa HeLa along in HeLa cells is mediated primarily through the cellscells withis is mediated mediatedmitochondrial-dependent chromatin primarily primarily condensation, through through signaling the the pathway as it inhibited proliferation of HeLa cells along with chromatin condensation, internucleosomal DNA fragmentation, mitochondriamitochondriainternucleosomall-ldependent-dependent DNA signaling fragmentation,signaling pathway pathway increase in the proportion of sub G(1) apoptotic cells, and externalization of Rhinacanthone Rhinacanthone as itincrease inhibited in proliferation the proportion of HeLaof sub cells G(1) along as it inhibited proliferationannexin-V. Increase of HeLa in cells the levelalong of Bax and a decrease in the level of Bcl-2 and apoptoticwith cells, chromatin and externa condensation,lization of annexin - with chromatin condensation,activation of caspase 3 and 9 [39]; internucleosomal DNA fragmentation, V. Increaseinternucleosomal inInhibited the level DNA tumorof Bax fragmentation, celland growtha decrease in Dalton’s in ascitic lymphoma (DAL) in Swiss albino RhinacanthoneRhinacanthone theincrease levelincrease of Bclin in the -the2 and proportion proportion activation of of ofsub sub caspase G(1) G(1) 3 mice [40] apoptoticapoptotic cells, cells, and andand externa externa 9 [39lization]lization; of of annexin annexin-- V.InhibitedV. Increase Increase tumor in in the the celllevel level growth of of Bax Bax inand and Dalton’s a a decrease decrease ascitic in in thelymphomathe level level of of Bcl (DAL)Bcl-2-2 and and in activationSwiss activation albino of of caspasemice caspase [40 3 ]3 andand 9 9 [ 39[39];] ; 3. Rhinacanthus nasutus and Neurodegenerative DiseasesInhibitedInhibited tumor tumor cell cell growth growth in in Dalton’s Dalton’s ascitic ascitic lymphomalymphoma (DAL) (DAL) in in Swiss Swiss albino albino mice mice [ 40[40] ] Neurodegenerative diseases pose a significant health issue since the average ages of the world’s populations are increasing, and there are more older generations alive than at any time in previous 3.3. Rhinacanthus Rhinacanthus nasutus nasutus and and Neurodegenerative Neurodegenerative Diseases Diseases decades. Age brings numerous health problems, including neurodegenerative diseases such as Alzheimer’sNeurodegenerativeNeurodegenerative disease, Parkinson diseases diseases’s disease,pose pose a a significant significant multiple sclerosis,health health issue issue and since sinceamyotrophic the the average average lateral ages ages sclerosis. of of the the world’s world’s There populationsarepopulations currently are are44 increasing,increasing,million people and and there livingthere are arewith more more Alzheimer’s, older older generations generations 7 million alive alive living than than with at at any anyParkinson’s time time in in previous previous disease decades.worldwidedecades. AgeAge [41 ,brings42brings]. With numerousnumerous the rise in hehe populationalthalth problems,problems, and life includingincluding expectancy neurodegenerativeneurodegenerative around the world, diseasesdiseases these numbers suchsuch asas Alzheimer’sareAlzheimer’s predicted disease, disease,to rise toParkinson Parkinson 76 million’s’s disease, bydisease, 2030 multipleand multiple 135 million sclerosis, sclerosis, by and 2050and amyotrophic amyotrophic for Alzheimer’s lateral lateral disease, sclerosis. sclerosis. and There upThere to are20are million currentlycurrently by 2050 4444 millionmillion for Parkinson’s peoplepeople livingliving diseases withwith [ 42 Alzheimer’s,Alzheimer’s,,43]. 77 millionmillion livingliving withwith Parkinson’sParkinson’s didiseasesease worldwideworldwideTreatments [ [4141,42,42 for]]. .With With neurodegenerative the the rise rise in in population population diseases and and falllife life expectancy intoexpectancy three generalaround around the categories;the world, world, these thesepreventative numbers numbers, arerestorative,are predicted predicted and to to riseregenerative. rise to to 76 76 million million The by byrestorative 2030 2030 and and category135 135 million million covers by by 2050 2050most for for drugs Alzheimer’s Alzheimer’s available disease, disease,for the andtreatment and up up to to 20of20 neurodegenerativemillion million by by 2050 2050 for for Parkinson’sdiseases, Parkinson’s such diseases diseases as levodopa [ [4242,43,43 ]for]. . Parkinson’s and acetylcholinesterase inhibitors for Alzheimer’sTreatmentsTreatments disease. forfor neurodegenerativeneurodegenerative These drugs are oftendiseasesdiseases prescribed fallfall intointo after threethree symptoms generalgeneral have categories;categories; begun topreventativepreventative show, long, , restorative,afterrestorative, the initial and and neurodegeneration regenerative. regenerative. The The restorative restorativehas taken place,category category with covers covers the aim most most of drugs replacingdrugs available available neurotransmitters for for the the treatment treatment that ofareof neurodegenerative neurodegenerativelost from the neurodegeneration. diseases, diseases, such such as Theseas levodopa levodopa treatments for for Parki Parki oftennson’snson’s only andimproveand acetylcholinesterase acetylcholinesterase the symptoms but inhibitors inhibitors do not forslowfor Alzheimer’s Alzheimer’s or reverse thedisease. disease. progression These These drugs drugsof the are aredisease often often in prescribed prescribed any way. after Regenerativeafter symptoms symptoms treatments have have begun begun are to theto show, show, holy graillong long afterofafter neurodegenerative the the initial initial neurodegeneration neurodegeneration diseases, drugs has has that taken taken stimulate place, place, with thewith growth the the aim aim of of of new replacing replacing neurons neurotransmitters neurotransmitters to replace those that that arehaveare lost lost been from from lost, the the thusneurodegeneration. neurodegeneration. restoring function These These or treatments treatmentsthe implantation often often only only of improve stemimprove cells the the that symptoms symptoms would but growbut do do andnot not slowdifferentiateslow or or reverse reverse into the the the progression progression desired neuronal of of the the disease cellsdisease to replacein in any any way. thoseway. Regenerative Regenerativethat were lost. treatments treatments For now, aredespite are the the holy advancesholy grail grail ofinof neurodegenerativethe neurodegenerative field, these kind sdiseases, diseases, of drugs drugs drugsand therapies that that stimulate stimulate are still the the a longgrowth growth way of of from new new being neurons neurons brought to to replace replace to clinical those those trials that that have[have44]. Preventative beenbeen lost,lost, thusthus treatments restoringrestoring tend functionfunction to cover oror dietary thethe implantationimplantation and behavioral ofof stemstem changes cellscells thatthat reducewouldwould thegrowgrow risk andand of differentiateneurodegenerativedifferentiate into into the the diseases. desired desired neuronal Examplesneuronal cells cellsof preventative to to replace replace those those measures that that were were that lost. lost.can ForreduceFor now now the, ,despite despite relative advances advances risk of indevelopingin the the field, field, these Alzheimer’sthese kind kindss o o fincludef drugs drugs and andnot therapies smoking,therapies are maintainingare still still a a long long an way way active from from lifestyle, being being brought brought maintaining to to clinical clinical a healthy trials trials [weight,[4444]]. . PreventativePreventative and treating treatmentstreatments diseases such tendtend as toto depression covercover dietarydietary and and anddiabetes. behavioralbehavioral changeschanges thatthat reducereduce thethe riskrisk ofof neurodegenerativeneurodegenerativeHerbal remedies diseases. diseases. and traditional Examples Examples medicines of of preventative preventative for the treatmentmeasures measures ofthat that cog can cannitive reduce reduce decline the the haverelative relative been risk risk used of of developingfordeveloping centuries, Alzheimer’s Alzheimer’s and many haveinclude include been not not shown smoking, smoking, to have maintaining maintaining positive effectsan an active active on neuronslifestyle, lifestyle, inmaintaining maintaining a laboratory a a healthy settinghealthy, weight,suchweight, as Curcumaand and treating treating longa diseases diseases [45], Mucuna such such as as pruriens depression depression [46], andand diabetes. Bacopadiabetes. monnieri [47]. Rn is lesser known for its HerbalHerbal remedies remedies and and traditional traditional medicines medicines for for the the treatment treatment of of cog cognitivenitive decline decline have have been been used used for for centuries, centuries, and and many many have have been been shown shown to to have have positive positive effects effects on on neurons neurons in in a a laboratory laboratory setting setting, , suchsuch as as Curcuma Curcuma longa longa [ [4545]], ,Mucuna Mucuna pruriens pruriens [ [4646]], ,and and Bacopa Bacopa monnieri monnieri [ [4747]]. .Rn Rn is is lesser lesser known known for for its its

Nutrients 2020, 12, 3776 8 of 18

3. Rhinacanthus nasutus and Neurodegenerative Diseases Neurodegenerative diseases pose a significant health issue since the average ages of the world’s populations are increasing, and there are more older generations alive than at any time in previous decades. Age brings numerous health problems, including neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, and amyotrophic lateral sclerosis. There are currently 44 million people living with Alzheimer’s, 7 million living with Parkinson’s disease worldwide [41,42]. With the rise in population and life expectancy around the world, these numbers are predicted to rise to 76 million by 2030 and 135 million by 2050 for Alzheimer’s disease, and up to 20 million by 2050 for Parkinson’s diseases [42,43]. Treatments for neurodegenerative diseases fall into three general categories; preventative, restorative, and regenerative. The restorative category covers most drugs available for the treatment of neurodegenerative diseases, such as levodopa for Parkinson’s and acetylcholinesterase inhibitors for Alzheimer’s disease. These drugs are often prescribed after symptoms have begun to show, long after the initial neurodegeneration has taken place, with the aim of replacing neurotransmitters that are lost from the neurodegeneration. These treatments often only improve the symptoms but do not slow or reverse the progression of the disease in any way. Regenerative treatments are the holy grail of neurodegenerative diseases, drugs that stimulate the growth of new neurons to replace those that have been lost, thus restoring function or the implantation of stem cells that would grow and differentiate into the desired neuronal cells to replace those that were lost. For now, despite advances in the field, these kinds of drugs and therapies are still a long way from being brought to clinical trials [44]. Preventative treatments tend to cover dietary and behavioral changes that reduce the risk of neurodegenerative diseases. Examples of preventative measures that can reduce the relative risk of developing Alzheimer’s include not smoking, maintaining an active lifestyle, maintaining a healthy weight, and treating diseases such as depression and diabetes. Herbal remedies and traditional medicines for the treatment of cognitive decline have been used for centuries, and many have been shown to have positive effects on neurons in a laboratory setting, such as Curcuma longa [45], Mucuna pruriens [46], and Bacopa monnieri [47]. Rn is lesser known for its neuroprotective effects; however, there has been much research suggesting that it could be beneficial in the prevention and treatment of neurodegenerative disease [48]. Previous work from our laboratory has shown that Rn has potent antioxidant effects in cultured neuronal cell lines subjected to hypoxia [13] and that Rn can counteract the cytotoxic effects of glutamate and amyloid-β (Aβ)[14]. We, therefore, hypothesized a reactive oxygen species inhibiting model (Figure1) that could explain the protective effects of Rn [49]. Many other studies have shown that Rn possesses potent antioxidant effects, with the crude leaf extract showing similar free radical scavenging properties to the standard Trolox and prevent reactive oxygen species (ROS) induced hemolysis of red blood cells [50,51]. Furthermore, active compounds such as RnC, RnD and RnN being potent superoxide scavengers [52]. There is increasing evidence to suggest that neurodegenerative disease is not restricted to a neurological component; rather, there is a strong interaction with immunological mechanisms in the central nervous system (CNS). In particular, diseases such as Alzheimer’s [53], Parkinson’s [54], multiple sclerosis [55], and ALS [56] have evidence for immunological components. Recent work investigating the neuroprotective effects of RnC (one of the major active compounds found in Rn) has shown that RnC is able to attenuate neuroinflammation brought on by LPS, Aβ, and interferon-γ in BV-2 and primary rat glial cells [30]. In particular, this study showed that RnC was able to reduce the release of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) from BV-2 cells in response to LPS, Aβ, and interferon-γ, as well as reducing NO production and inducible NO synthase (iNOS), IL-1β, C-C Motif Chemokine Ligand 2 (CCL-2), and CCL-5 mRNA levels in primary rat microglial cells. Furthermore, the Chuang et al. 2018 study showed that RnC could prevent damage to primary rat neurons caused by Aβ suggesting that RnC may be a potential in Alzheimer’s disease treatment. The findings from this study are in line with other studies investigating RnC on neuroinflammation [31]. The Chang et al. 2016 study showed that RnC could prevent increases in IL-1β, IL-6, and TNF-α Nutrients 2020, 12, x FOR PEER REVIEW 7 of 16 neuroprotective effects; however, there has been much research suggesting that it could be beneficial inNutrients the prevention2020, 12, 3776 and treatment of neurodegenerative disease [48]. Previous work from 9our of 18 laboratory has shown that Rn has potent antioxidant effects in cultured neuronal cell lines subjected to hypoxia [13] and that Rn can counteract the cytotoxic effects of glutamate and amyloid-β (Aβ) [14]. mRNA expression in animals subjected to subarachnoid hemorrhage. This study suggests that RnC We, therefore, hypothesized a reactive oxygen species inhibiting model (Figure 1) that could explain may be of benefit to patients who have suffered from delayed ischemic neurologic deficit following an the protective effects of Rn [49]. Many other studies have shown that Rn possesses potent antioxidant aneurysm by preventing neuroinflammation, and could explain the neuroprotective effects of Rn seen effects, with the crude leaf extract showing similar free radical scavenging properties to the standard in cultured HT-22 cells that were exposed to hypoxic, low glucose conditions and then reperfused with Trolox and prevent reactive oxygen species (ROS) induced hemolysis of red blood cells [50,51]. oxygen and glucose [13]. The neuroprotective effects of Rn could further be explained by the finding Furthermore, active compounds such as RnC, RnD and RnN being potent superoxide scavengers that RnS has acetylcholinesterase activity [20]. [52].

FigureFigure 1.1. RhinacanthusRhinacanthus nasutus nasutus(Rn ) and(Rn) its and protective its protective effects in pathways effects involvedin pathways in neurodegenerative involved in neurodegenerativediseases. Rn inhibits diseases. tumor Rn necrosis inhibits factor tumor alpha necrosis (TNF αfactor) release alpha thus (TNF preventing) release its thus interaction preventing with itsthe interaction TNFα receptor with (TNFthe TNFαR). Rnreceptoralso prevents (TNF R). nuclear Rn also factor prevents kappa-light-chain-enhancer nuclear factor kappa of-light activated-chain- B ⍺ enhancercells (NF- ofκ B)activated activation, B cells which (NF in-κB) turn activation, leads a reduction which in inturn oxidative leads a stress.reduction in oxidative stress. ⍺ ⍺ ThereAltogether, is increasin these studiesg evidence have to shown suggest that thatRn has neurodegenerative neuroprotective properties, disease is withnot potentialrestricted for to thea neurologicaltreatment of component Alzheimer’s; rather, and aneurysm there is patients.a strong interaction The crude extractwith immunological or its active components mechanisms have in boththe centralshown nervous potential system for reducing (CNS) neuroinflammation. In particular, diseases and providing such as Alzheimer’s protection against [53], P reperfusionarkinson’s injury.[54], multiple sclerosis [55], and ALS [56] have evidence for immunological components. Recent work 4. Rhinacanthus nasutus and Cancer investigating the neuroprotective effects of RnC (one of the major active compounds found in Rn) has shownCancer that is RnC the termis able given to attenuate to a group neuroinflammation of diseases that brought involve on abnormal by LPS, cellAβ, growth,and interferon with the-γ inpotential BV-2 and to primary spread and rat invadeglial cells other [30] parts. In particular of the body., this The study 2014 showed World Healththat Rn OrganizationC was able to statisticsreduce theshow release that of there tumor were necrosis approximately factor alpha 18 million(TNF-α new) and cases interleukin and 9 million-6 (IL-6) deaths from BV worldwide.-2 cells in response to LPS,In A normalβ, and healthyinterferon cells,-γ, as there well are as signalsreducing that NO prevent production programmed and inducible cell death NO (apoptosis).synthase (iNOS There), ILare-1β, also C- signalsC Motif that Chemokine lead to apoptosis, Ligand 2 for (CCL example,-2), and when CCL a- cell5 mRNA is damaged levels or in has primary reached rat the microglia end of itsl cells.lifespan. Furthermore, There are the two Chuang classes of et genes, al. 2018 which study under showed the rightthat Rn circumstanceC could prevent can cause damage cancers. to primary The first ratgroup neurons is the caused oncogenes, by Aβ with suggesting roles in that the Rn promotionC may be of a cellpotential division in Alzheimer’s and cell survival. disease Well-studied treatment. Theexamples findings include from this the study GTPase are Ras in line [57 ]with tyrosine other kinase studies receptors investigating such R asnC epidermal on neuroinflammation growth factor [31receptor]. The Chang (EGFR) et [ 58al.] 2016 and transcriptionstudy showed factors that RnC such could as the preventC-Myc increasesgene [59]. in The IL- second1β, IL-6, class and ofTNF genes-α mRNAthat can expression lead to cancers in animals are known subjected as tumor to subarachnoid suppressor genes. hemorrhage. A typical This example study would suggests be p53that, whichRnC mayis mutated be of benefit in 50% to of pa humantients who tumors have [60 suffered]. from delayed ischemic neurologic deficit following an aneurysmThe principal by preventing treatment neuroinflammation methods are a combination, and could of explain surgery, the chemotherapy, neuroprotective and effects radiotherapy. of Rn seenThe latterin cultured two do HT not-22 specifically cells that target were cancer exposed cells. to Instead,hypoxic, any low fast glucose proliferating conditions cell, resultingand then in several undesirable side effects. Ideal cancer treatment would target a specific property of a cancer cell, preventing its growth and inducing apoptosis without harming healthy cells.

Nutrients 2020, 12, 3776 10 of 18

There have been several studies investigating the eﬀects of Rn and its constituent compounds on tumor cell growth. The early studies showed that the crude extracts from either the leaves or the roots and some of the extracted compounds could prevent the growth of tumor cell lines (Table3) such as KB, HEP-2, MCF-7, HepG2, HeLa, SiHa, C-32, LLC, Colon-26, P-388, A-549, HT-29, HL60 and Vero [17,38,61]. Rhinacanthone isolated from the roots inhibited the proliferation of HeLa cells and induced apoptosis via the mitochondrial dependent signaling mechanism [39]. RnC displayed potent cytotoxic and antimetastatic activity against cholangiocarcinoma cells (KKU-M156) by inhibiting invasion regulating focal adhesion kinase (FAK) and interfering with mitogen-activated protein kinase (MAPK) pathway [32]. RnA, isolated from the leaves of Rn, was found to be cytotoxic to MCF7 breast cancer cells (50 % inhibition concertation (IC50) value of 8.79 µM), and RnN was cytotoxic in NCI-H187 cell line (IC50 value of 2.24 µM) and Vero cells (IC50 value of 3.00 µM) [20].

Table 3. IC50 values for various extracts and isolated compounds from Rn in a range of cancer cell lines. Data extracted from Wu et al. 1998 [17], Siripong et al. 2006 [61] and Siripong et al. 2009 [39].

Chloroform Methanol Cell RnC RnD RnG RnO RnM RnN RnQ Rhinacanthone Extract Extract Line µM µM µM µM µM µM µM µM µg/mL µg/mL KB 0.55 3.9 0.46 0.47 4.7 5.5 2.6 0.33 1.4 3.8 Hep-2 0.3 4.0 0.8 7.6 3.3 3.7 6.1 1.2 3.6 4.4 MCF-7 0.8 20.0 0.88 14.7 8.7 8.1 8.9 2.6 10.6 4.9 HepG2 0.95 8.5 0.41 1.9 1.2 6.5 3.8 0.37 3.0 4.9 HeLa 0.39 4.4 0.29 0.49 4.7 6.1 4.3 0.87 3.8 4.2 SiHa 1.5 21.0 0.49 6.6 18.8 7.4 39.1 3.9 6.1 2.9 C-32 5.0 30.0 9.8 14.7 16.4 30.7 37.0 39.1 8.4 2.1 LLC 1.8 40 0.98 6.1 6.1 8.2 54.4 5.4 8.3 2.9 Colon-26 0.3 5.0 0.44 2.3 1.5 6.6 5.4 1.1 1.1 3.4 P-388 0.3 7 1.5 9.3 3.3 8.9 8.1 3.7 10.3 4.4 Vero 0.2 2.5 11.0 34.3 16.4 12.3 36.1 12.7 41.1 4.2

5. Rhinacanthus nasutus and Diabetes Worldwide, diabetes is regarded as one of the leading causes of death. According to the international diabetes federation, 463 million adults are living with the disease, and the figure is expected to increase by 700 million in 2045 [62]. Diabetes is a cluster of metabolic diseases resulting in a hyperglycemic condition. The increase in blood sugar level may result from impaired insulin secretion and action along with disruption in the normal metabolism of carbohydrates, proteins and lipids, leading to detrimental health effects. Diabetes is also characterized by a rise in fatty acids and triglycerides along with a decline of high-density lipoprotein cholesterol (HDL-C), leading to the deposition of fat and obesity [63]. Hyperglycemia can also increase the level of oxidative stress inside the system, which also contributes to the prevalence of diabetes [10]. Diabetic patients are also prone to suffer from other mild to severe complications like diabetic retinopathy, diabetic nephropathy, diabetic neuropathy, myocardial infarction and multiple organ failure. Among the several antidiabetic mechanisms, inhibition of α-glucosidase has been reported as the main event in exhibiting the effect, and α-glucosidase inhibitors have been widely prescribed for treating type II diabetes. α-glucosidase is involved in the conversion of starch and disaccharides to simple sugars, thereby increasing the glucose level in circulation [64]. In vitro studies have demonstrated that a rhinacanthin rich extract and its active constituent RnC exhibit noncompetitive inhibition against the α-glucosidase enzyme. In silico studies also showed the positive interaction and binding between RnC and α-glucosidase by hydrogen and hydrophobic bonding [65]. In addition, the rhinacanthin rich extract and RnC also exhibit anti-adipogenetic activity, suggesting that it can also be used effectively against obesity associated-type II diabetes [66]. Nutrients 2020, 12, 3776 11 of 18

One of the complex events leading to diabetes is the formation of advanced glycation end products (AGE). AGE’s are the products of the Maillard reaction wherein the proteins are glycated when exposed to sugar molecules, as well as from the degradation products of glucose like methylglyoxal, glycolaldehyde, 3-deoxyglucosone and acts as an important biomarker in diabetes. Elevated levels of AGEs are considered as one of the risk factors for the damage occurring to pancreatic beta cells that secrete insulin [67,68]. Aqueous extract of Rn under in vitro conditions has been reported to signiﬁcantly trap methylglyoxal, thereby inhibiting the formation of advanced glycation end products in a dose-dependent manner [69]. In silico studies also supported the anti-glycation property of RnC by masking lysine and arginine residues in human serum albumin, thereby preventing glycation [52]. Oxidative stress plays a major role in aggravating the progression of diabetic complications (Figure2), as the metabolic abnormalities occurring during the process induces the generation of superoxide radicals. This toxic phenomenon further exaggerates the generation of AGE and induces the expression of its receptor [70]. Rhinacanthin-rich extract and RnC were found to scavenge superoxide free radical in cyclic voltammogram analysis [52]. Additionally, they could also reduce the level of lipid peroxidation and improve the levels of antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), indicating the eﬀect of the plant in reducing oxidative stress [10,21]. Nutrients 2020, 12, x FOR PEER REVIEW 10 of 16

FigureFigure 2. Rhinacanthus2. Rhinacanthus nasutus nasutus(Rn (Rn)) and and its its beneﬁcial beneficial properties properties regarding regarding diabetes. diabetes.Rn Rninhibits inhibits the the productionproduction of of Advanced Advanced glycation glycation end-product end-product (AGEs), (AGEs), prevents prevents the the increase increase in in very very low-density low-density lipoproteinlipoprotein (VLDL) (VLDL) and low-densityand low-density lipoprotein, lipoprotein, and prevents and prevents the decrease the in decrease high density in lipoproteinhigh density (HDL).lipoproteinRn prevents (HDL). reactive Rn prevents oxygen speciesreactive (ROS) oxygen damage species by (R activatingOS) damage superoxide by activating dismutase superoxide (SOD) catalasedismutase and glutathione (SOD) catalase peroxidase and (GPx).glutathioneRn also regulatesperoxidase peroxisome (GPx). Rn proliferator-activated also regulates p receptoreroxisome (PPARproliferatorα) and glucose-activated transporter receptor (PPAR type 4 (GLUT-4).) and glucose transporter type 4 (GLUT-4). ⍺ OralOral administration administration of methanolof methanol extract extract of Rn of atRn 200 at mg200/kg mg/kg body body weight weight in adult in maleadult Wistarmale W ratsistar forrats 30 daysfor 30 reduced days reduced the levels the of levels total cholesterol, of total cholesterol, triglycerides triglycerides and phospholipids and phospholipids in the liver in of the diabetic liver of ratsdiabetic to a level rats like to thata level of non-diabeticlike that of non control.-diabetic Further, control. the extractFurther, also the reduced extract thealso level reduced of low-density the level of lipoprotein-cholesterollow-density lipoprotein (LDL-C)-cholesterol and very-low-density (LDL-C) and very lipoprotein-cholesterol-low-density lipoprotein (VLDL-C)-cholesterol in the diabetic(VLDL-C) ratsin apartthe diabetic from improving rats apart the from levels improving of HDL-C, the thereby levels exhibitingof HDL-C hypolipidemic, thereby exhibiting activity hypolipidemic in diabetic rats.activity More in importantly, diabetic rats. the More plant didimportant not exhibitly, the any plant hypoglycemic did not exhibit activity any in hypoglycemic normal rats indicating activity in thatnormal it was rats non-toxic indicating to normal that ratsit was [63]. non Impaired-toxic to lipoprotein normal rats and lipid[63]. metabolismImpaired lipoprotein are a characteristic and lipid featuremetabolism of type are II diabetes.a characteristic Pathological feature changes of type includeII diabetes. increased Pathological hepatic changes secretion include and impaired increased clearancehepatic ofsecretion VLDL [ 71and]. Oneimpaired of the clearance key players of inVLDL lipid [7 metabolism1]. One of isthe insulin, key players which in helps lipid in metabolism the storage is insulin, which helps in the storage of triacylglycerols (TAG) and reduces the generation of VLDL by declining the level of non-esterified fatty acids in circulation. Similar to Rn extract, oral administration of rhinacanthin‑rich extract and RnC significantly normalized the water and food intake in diabetic rats, along with the bodyweight, without causing any significant changes in the normal rats. It also decreased the hyperglycemic levels and destruction of beta cells in the pancreas and increased the serum insulin gradually in diabetic rats. The elevated levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) along with blood urea nitrogen (BUN) and creatinine in diabetic rats were significantly reduced by the extract treatment indicating the normalization of the liver as well as kidneys functioning in diabetic rats [72]. Similar results were also reported by Rao et al. 2013, where the key markers of liver functioning were normalized in diabetic rats upon treatment with the plant extract [73]. Further, RnC also modulated the levels of fasting blood glucose (FBG) and glycated hemoglobin (HbA1c) in diabetic rats. HbA1c is a form of hemoglobin that is in bonding with sugar moiety and is formed when there is an excessive circulation of glucose in the bloodstream. Moreover, RnC could also reduce the insulin resistance index, modulate antioxidant enzymes, and prevent the shrinking of the islet of Langerhans in diabetic rats [74]. The reported health benefits of the plant extract against diabetic condition could probably be mediated by the activity of cytosolic and mitochondrial enzymes. The level of cytosolic enzymes like glucose-6-phosphate dehydrogenase (G6PDH), glutamate dehydrogenase (GDH) and mitochondrial enzyme succinate dehydrogenase (SDH) is reduced in individuals with diabetes, where G6PDH plays a vital role in antioxidant defense against oxidative stress by the production of NADPH [75] while GDH and SDH are involved in

Nutrients 2020, 12, 3776 12 of 18 of triacylglycerols (TAG) and reduces the generation of VLDL by declining the level of non-esterified fatty acids in circulation. Similar to Rn extract, oral administration of rhinacanthin-rich extract and RnC significantly normalized the water and food intake in diabetic rats, along with the bodyweight, without causing any significant changes in the normal rats. It also decreased the hyperglycemic levels and destruction of beta cells in the pancreas and increased the serum insulin gradually in diabetic rats. The elevated levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) along with blood urea nitrogen (BUN) and creatinine in diabetic rats were significantly reduced by the extract treatment indicating the normalization of the liver as well as kidneys functioning in diabetic rats [72]. Similar results were also reported by Rao et al. 2013, where the key markers of liver functioning were normalized in diabetic rats upon treatment with the plant extract [73]. Further, RnC also modulated the levels of fasting blood glucose (FBG) and glycated hemoglobin (HbA1c) in diabetic rats. HbA1c is a form of hemoglobin that is in bonding with sugar moiety and is formed when there is an excessive circulation of glucose in the bloodstream. Moreover, RnC could also reduce the insulin resistance index, modulate antioxidant enzymes, and prevent the shrinking of the islet of Langerhans in diabetic rats [74]. The reported health benefits of the plant extract against diabetic condition could probably be mediated by the activity of cytosolic and mitochondrial enzymes. The level of cytosolic enzymes like glucose-6-phosphate dehydrogenase (G6PDH), glutamate dehydrogenase (GDH) and mitochondrial enzyme succinate dehydrogenase (SDH) is reduced in individuals with diabetes, where G6PDH plays a vital role in antioxidant defense against oxidative stress by the production of NADPH [75] while GDH and SDH are involved in insulin secretion [76,77]. Administration of Rn extract significantly restored the levels of these enzymes, thereby ameliorating the diabetic conditions in diabetic rats [73]. Pioneering studies suggest the relationship between chronic inflammation and the pathophysiology of diabetes, and immunomodulatory drugs have been advised in the treatment of individuals with type II diabetes [78,79]. Free fatty acids and cholesterol induce inflammation in diabetic individuals and subsequently induces the release of proinflammatory cytokines and chemokines mediated by NFκB[80,81]. Rhinacanthins-rich extract and RnC administration reduced the levels of free fatty acids and cholesterol, thereby attenuated proinflammatory cytokines TNF-α, IL-1β, and IL6 in diabetic rats. In addition, the renal concentrations of caspase-3 and cytochrome C were also reduced in the kidneys of diabetic rats. Diabetes induced histological changes in the kidney such as thickening of the basement membrane of the renal tubules and necrosis of renal tubules were also reduced by the treatment of extract, indicating that the plant extracts can also be used as immunomodulatory agents in alleviating the complications of diabetes [21]. Other than reducing the glucose, triglyceride and cholesterol level, administration of Rn can also stimulate the expression of peroxisome proliferator-activated receptor (PPARα), fat adiponectin, glucose transporter type 4 (GLUT4) protein expression [82]. Expression of PPARα and GLUT4 are highly downregulated in individuals affected with diabetes, and both play a major role in the regulation of glucose, lipid metabolism and transports glucose from the circulation into insulin-sensitive cells, respectively [83,84]. PPAR agonists were also found to improve against insulin resistance and to reduce the level of TG and glucose [85]. The stimulation of these vital proteins upon Rn extract administration could be the reason behind its antidiabetic effect. Further, the pharmacokinetic and toxicity profile of RnC by in silico approach suggests that the phytochemical is devoid of carcinogenicity, mutagenicity and non-toxic, suggesting it is safe to use as an antidiabetic drug [72].

6. Antibacterial, Anti-Fungal, and Anti-Viral Activities of Rhinacanthus nasutus Since the discovery of penicillin and its derivatives nearly a century ago, the world’s population has grown, and deaths caused by bacterial infections has fallen dramatically. However, in recent years inappropriate and overuse has resulted in the emergence of antibiotic-resistant strains of bacteria, resulting in the need to find novel antibiotics. Extracts of Rn have been tested against a range of bacterial strains, and it has been shown to have effective antibiotic activities against Gram-positive strains, including Bacillus cereus, Bacillus globigii, Bacillus subtilis, and Staphylococcus aureus. However, Nutrients 2020, 12, 3776 13 of 18 it was not effective against Gram-negative strains such as Salmonella typhi, Pseudomonas aeruginosa or Escherichia coli [86], presumably due to the difference in the cell wall made up of Gram-positive and negative bacteria. The rhinacanthin-rich ethanol extract of Rn leaves has been shown to have activity against the ulcer and cancer-causing Gram-negative bacteria, Helicobacter pylori, with a minimum inhibitory concentration (MIC50) of 0.5 mg/mL [4]. Further studies have been carried out on RnC extracted from Rn, showing that the MIC50 for RnC against S. mutans, P.acnes, S. aureus and S. epidermidis are two, eight, two and eight µg/mL, respectively [4]. Studies, including RnN and RnQ, showed that the MIC50 ranged from 4.9 to 19.53 µg/mL for Gram-positive bacteria but had no anti-biotic effects on Gram-negative bacteria. Interestingly RnN was shown to be more potent than gentamycin in the inhibition of the Gram-positive bacteria [87]. Traditionally the extracts from Rn have been used for the treatment of skin infections with fungi, such as ringworm and tinea cruris [88]. Rn has been shown to be effective against a range of clinically relevant fungi, including C. albicans (of the most common hospital-acquired infections), T. mentagrophytes (a common cause of ringworm), C. tropicalis (a common cause of oral thrush, and particular risk to HIV patients) and C. parapsilosis (Cause of endocarditis in patients with prosthetic valves) [86]. Post-2019 and the Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic that has spread around the world at an alarming rate, it has become clear that there is a need for new and effective antiviral drugs. Rn and its constitutive compounds have been shown to be effective against various viruses, including Herpes [22,89,90] and Influenza [23]. Recent research has shown that compounds isolated from the aerial parts of Rn have neuraminidase inhibitory activity. Neuraminidase is a target on the surface of the influenza virus that plays an important role in the entry of the virus into the cell. It has been postulated that neuraminidase inhibitors may be of benefit in the treatment of SARS-CoV-2 [91]. However, any Rn compound would still need to be tested clinically, and there are already several neuraminidase inhibitors that have been tested for safety and would be ahead of any Rn compound with regards to clinical testing against SARS-CoV-2.

7. Conclusions Rn is a medicinal herb that contains several compounds that are potentially useful medically. RnC is of interest, particularly in the treatment of diabetes and neurodegeneration, where preclinical studies have shown it to be effective in controlling diabetes by inhibiting α-glucosidase and preventing neurodegeneration caused by Aβ. There is strong evidence for the effectiveness of RnC for it to be entered in clinical trials for diabetes and dementia. Other compounds extracted from Rn appear to be effective against cancer cell lines. Although these studies are less advanced and the mechanisms yet to be fully understood, there is still potential for Rn in cancer treatment.

Funding: The APC was funded by Chulalongkorn University. Acknowledgments: J.M.B. and M.I.P.were funded by the Rachadapisek Sompote Fund for Postdoctoral Fellowship, Chulalongkorn University. D.S.M. was funded by The Second Century Fund (C2F) for Postdoctoral Fellowship, Chulalongkorn University. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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