Skip to content
2000
Volume 22, Issue 1
  • ISSN: 1573-4072
  • E-ISSN: 1875-6646

Abstract

Background

Globally large numbers of people are suffering from brain disorders for instance neurodegenerative disorders whereas treatments remain inadequate. The blood-brain barrier (BBB) serves as a major obstacle in transporting active therapeutic agents into the brain. Herbal medicines have the potency to treat these brain diseases however, it is having some limitations like poor brain permeability, and slow bioavailability.

Objective

The objective of this study was to assess the neuroprotective effects of andrographolide-loaded β-cyclodextrin-lactose nanoparticles on spatial learning and memory in experimental models, aiming to enhance cognitive function and brain health.

Methods

Swiss Albino mice (25-30 g, 2-3 weeks, both sexes) were sourced from Galgotias University, India (Reg. No. 94090). Andrographolide (AGL), a diterpenoid from was encapsulated in β-cyclodextrin (β-CD) combined with lactose using the solvent evaporation method, optimized Design-Expert software. To improve neuronal uptake, lactose was added to β-CD using the heating method, confirmed by 1H NMR spectra. The AGL-loaded β-CD-lactose nanoparticles were characterized for particle size, zeta potential, and entrapment efficiency, and their effects on brain tissue were assessed biochemically and histopathologically.

Results

Developed nanoparticles had particle size 247.9 ± 3.2 nm, PDI 0.5 ± 0.02, and zeta potential 26.8 ± 2.5 mV, while entrapment efficacy between 74 ± 0.45% to 82 ± 0.32% and drug release from the nanoparticles had 79.48 ± 0.74% in 24 hours. TEM analysis showed spherical morphology. Furthermore, behavioural assay and acute toxicity of nanoparticles were also analyzed which indicate that nanoparticles can reach into the brain and are safe for further use.

Conclusion

The lactose-modified β-cyclodextrin nanoparticles significantly improved the neuroprotective effect of AGL, offering an effective strategy for drug delivery to the brain and potential treatment for neurodegenerative disorders.

Loading

Article metrics loading...

/content/journals/cbc/10.2174/0115734072326670250119061702
2025-01-27
2026-02-16
Loading full text...

Full text loading...

References

  1. JellingerK.A. Basic mechanisms of neurodegeneration: A critical update.J. Cell. Mol. Med.201014345748710.1111/j.1582‑4934.2010.01010.x 20070435
    [Google Scholar]
  2. FeiginV.L. NicholsE. AlamT. BannickM.S. BeghiE. BlakeN. CulpepperW.J. DorseyE.R. ElbazA. EllenbogenR.G. FisherJ.L. FitzmauriceC. GiussaniG. GlennieL. JamesS.L. JohnsonC.O. KassebaumN.J. LogroscinoG. MarinB. Mountjoy-VenningW.C. NguyenM. Ofori-AsensoR. PatelA.P. PiccininniM. RothG.A. SteinerT.J. StovnerL.J. SzoekeC.E.I. TheadomA. VollsetS.E. WallinM.T. WrightC. ZuntJ.R. AbbasiN. Abd-AllahF. AbdelalimA. AbdollahpourI. AboyansV. AbrahaH.N. AcharyaD. AdamuA.A. AdebayoO.M. AdeoyeA.M. AdsuarJ.C. AfaridehM. AgrawalS. AhmadiA. AhmedM.B. AichourA.N. AichourI. AichourM.T.E. AkinyemiR.O. AkseerN. Al-EyadhyA. Al-Shahi SalmanR. AlahdabF. AleneK.A. AljunidS.M. AltirkawiK. Alvis-GuzmanN. AnberN.H. AntonioC.A.T. ArablooJ. AremuO. ÄrnlövJ. AsayeshH. AsgharR.J. AtalayH.T. AwasthiA. Ayala QuintanillaB.P. AyukT.B. BadawiA. BanachM. BanoubJ.A.M. BarbozaM.A. Barker-ColloS.L. BärnighausenT.W. BauneB.T. BediN. BehzadifarM. BehzadifarM. BéjotY. BekeleB.B. BelachewA.B. BennettD.A. BensenorI.M. BerhaneA. BeuranM. BhattacharyyaK. BhuttaZ.A. BiadgoB. BijaniA. BililignN. Bin SayeedM.S. BlazesC.K. BrayneC. ButtZ.A. Campos-NonatoI.R. Cantu-BritoC. CarM. CárdenasR. CarreroJ.J. CarvalhoF. Castañeda-OrjuelaC.A. CastroF. Catalá-LópezF. CerinE. ChaiahY. ChangJ-C. ChatziralliI. ChiangP.P-C. ChristensenH. ChristopherD.J. CooperC. CortesiP.A. CostaV.M. CriquiM.H. CroweC.S. DamascenoA.A.M. DaryaniA. De la Cruz-GóngoraV. De la HozF.P. De LeoD. DemozG.T. DeribeK. DharmaratneS.D. DiazD. DinberuM.T. DjalaliniaS. DokuD.T. DubeyM. DubljaninE. DukenE.E. EdvardssonD. El-KhatibZ. EndresM. EndriesA.Y. EskandariehS. EsteghamatiA. EsteghamatiS. FarhadiF. FaroA. FarzadfarF. FarzaeiM.H. FatimaB. FereshtehnejadS-M. FernandesE. FeyissaG.T. FilipI. FischerF. FukumotoT. GanjiM. GankpeF.G. Garcia-GordilloM.A. GebreA.K. GebremichaelT.G. GelawB.K. GeleijnseJ.M. GeremewD. GezaeK.E. Ghasemi-KasmanM. GideyM.Y. GillP.S. GillT.K. GirmaE.T. GnedovskayaE.V. GoulartA.C. GradaA. GrossoG. GuoY. GuptaR. GuptaR. HaagsmaJ.A. HagosT.B. Haj-MirzaianA. Haj-MirzaianA. HamadehR.R. HamidiS. HankeyG.J. HaoY. HaroJ.M. HassankhaniH. HassenH.Y. HavmoellerR. HayS.I. HegazyM.I. HeidariB. HenokA. HeydarpourF. HoangC.L. HoleM.K. Homaie RadE. HosseiniS.M. HuG. IgumborE.U. IlesanmiO.S. IrvaniS.S.N. IslamS.M.S. JakovljevicM. JavanbakhtM. JhaR.P. JobanputraY.B. JonasJ.B. JozwiakJ.J. JürissonM. KahsayA. KalaniR. KalkondeY. KamilT.A. KanchanT. KaramiM. KarchA. KarimiN. KasaeianA. KassaT.D. KassaZ.Y. KaulA. KefaleA.T. KeiyoroP.N. KhaderY.S. KhafaieM.A. KhalilI.A. KhanE.A. KhangY-H. KhazaieH. KiadaliriA.A. KiirithioD.N. KimA.S. KimD. KimY-E. KimY.J. KisaA. KokuboY. KoyanagiA. KrishnamurthiR.V. Kuate DefoB. Kucuk BicerB. KumarM. LaceyB. LafranconiA. LansinghV.C. LatifiA. LeshargieC.T. LiS. LiaoY. LinnS. LoW.D. LopezJ.C.F. LorkowskiS. LotufoP.A. LucasR.M. LuneviciusR. MackayM.T. MahotraN.B. MajdanM. MajdzadehR. MajeedA. MalekzadehR. MaltaD.C. ManafiN. MansourniaM.A. MantovaniL.G. MärzW. Mashamba-ThompsonT.P. MassenburgB.B. MateK.K.V. McAlindenC. McGrathJ.J. MehtaV. MeierT. MelesH.G. MeleseA. MemiahP.T.N. MemishZ.A. MendozaW. MengistuD.T. MengistuG. MeretojaA. MeretojaT.J. MestrovicT. MiazgowskiB. MiazgowskiT. MillerT.R. MiniG.K. MirrakhimovE.M. MoazenB. MohajerB. Mohammad Gholi MezerjiN. MohammadiM. Mohammadi-KhanaposhtaniM. MohammadibakhshR. Mohammadnia-AfrouziM. MohammedS. MohebiF. MokdadA.H. MonastaL. MondelloS. MoodleyY. MoosazadehM. MoradiG. Moradi-LakehM. MoradinazarM. MoragaP. Moreno VelásquezI. MorrisonS.D. MousaviS.M. MuhammedO.S. MuruetW. MusaK.I. MustafaG. NaderiM. NagelG. NaheedA. NaikG. NajafiF. NangiaV. NegoiI. NegoiR.I. NewtonC.R.J. NgunjiriJ.W. NguyenC.T. NguyenL.H. NingrumD.N.A. NirayoY.L. NixonM.R. NorrvingB. NoubiapJ.J. Nourollahpour ShiadehM. NyasuluP.S. OgahO.S. OhI-H. OlagunjuA.T. OlagunjuT.O. OlivaresP.R. OnwujekweO.E. OrenE. OwolabiM.O. PaM. PakpourA.H. PanW-H. Panda-JonasS. PandianJ.D. PatelS.K. PereiraD.M. PetzoldM. PillayJ.D. PiradovM.A. PolanczykG.V. PolinderS. PostmaM.J. PoultonR. PoustchiH. PrakashS. PrakashV. QorbaniM. RadfarA. RafayA. RafieiA. RahimF. Rahimi-MovagharV. RahmanM. RahmanM.H.U. RahmanM.A. RajatiF. RamU. RantaA. RawafD.L. RawafS. ReinigN. ReisC. RenzahoA.M.N. ResnikoffS. RezaeianS. RezaiM.S. Rios GonzálezC.M. RobertsN.L.S. RoeverL. RonfaniL. RoroE.M. RoshandelG. RostamiA. SabbaghP. SaccoR.L. SachdevP.S. SaddikB. SafariH. Safari-FaramaniR. SafiS. SafiriS. SagarR. SahathevanR. SahebkarA. SahraianM.A. SalamatiP. Salehi ZahabiS. SalimiY. SamyA.M. SanabriaJ. SantosI.S. Santric MilicevicM.M. SarrafzadeganN. SartoriusB. SarviS. SathianB. SatpathyM. SawantA.R. SawhneyM. SchneiderI.J.C. SchöttkerB. SchwebelD.C. SeedatS. SepanlouS.G. ShabaninejadH. ShafieesabetA. ShaikhM.A. ShakirR.A. Shams-BeyranvandM. ShamsizadehM. SharifM. Sharif-AlhoseiniM. SheJ. SheikhA. ShethK.N. ShigematsuM. ShiriR. ShirkoohiR. ShiueI. SiabaniS. SiddiqiT.J. SigfusdottirI.D. SigurvinsdottirR. SilberbergD.H. SilvaJ.P. SilveiraD.G.A. SinghJ.A. SinhaD.N. SkiadaresiE. SmithM. SobaihB.H. SobhaniS. SoofiM. SoyiriI.N. SposatoL.A. SteinD.J. SteinM.B. StokesM.A. SufiyanM.B. SykesB.L. SylajaP.N. Tabarés-SeisdedosR. Te AoB.J. Tehrani-BanihashemiA. TemsahM-H. TemsahO. ThakurJ.S. ThriftA.G. Topor-MadryR. Tortajada-GirbésM. Tovani-PaloneM.R. TranB.X. TranK.B. TruelsenT.C. TsadikA.G. Tudor CarL. UkwajaK.N. UllahI. UsmanM.S. UthmanO.A. ValdezP.R. VasankariT.J. VasanthanR. VeisaniY. VenketasubramanianN. ViolanteF.S. VlassovV. VosoughiK. VuG.T. VujcicI.S. WagnewF.S. WaheedY. WangY-P. WeiderpassE. WeissJ. WhitefordH.A. WijeratneT. WinklerA.S. WiysongeC.S. WolfeC.D.A. XuG. YadollahpourA. YamadaT. YanoY. YaseriM. YatsuyaH. YimerE.M. YipP. YismaE. YonemotoN. YousefifardM. YuC. ZaidiZ. ZamanS.B. ZamaniM. ZandianH. ZareZ. ZhangY. ZodpeyS. NaghaviM. MurrayC.J.L. VosT. Global, regional, and national burden of neurological disorders, 1990–2016: A systematic analysis for the Global Burden of Disease Study 2016.Lancet Neurol.201918545948010.1016/S1474‑4422(18)30499‑X 30879893
    [Google Scholar]
  3. ZhangS. GanL. CaoF. WangH. GongP. MaC. RenL. LinY. LinX. The barrier and interface mechanisms of the brain barrier, and brain drug delivery.Brain Res. Bull.2022190698310.1016/j.brainresbull.2022.09.017 36162603
    [Google Scholar]
  4. GondeD.P. BholeB.K. KakadK.S. Andrographolide, diterpenoid constituent of Andrographis paniculata: Review on botany, phytochemistry, molecular docking analysis, and pharmacology.Ann. Pharm. Fr.2024821154310.1016/j.pharma.2023.10.001 37813330
    [Google Scholar]
  5. ZhongR. MiaoL. ZhangH. TanL. ZhaoY. TuY. Angel PrietoM. Simal-GandaraJ. ChenL. HeC. CaoH. Anti-inflammatory activity of flavonols via inhibiting MAPK and NF-κB signaling pathways in RAW264.7 macrophages.Curr. Res. Food Sci.202251176118410.1016/j.crfs.2022.07.007 35941847
    [Google Scholar]
  6. LiJ. WuY. DongS. YuY. WuY. XiangB. LiQ. Research progress on neuroprotective effects of isoquinoline alkaloids.Molecules20232812479710.3390/molecules28124797 37375352
    [Google Scholar]
  7. ParamanickD. SinghV.D. SinghV.K. Neuroprotective effect of phytoconstituents via nanotechnology for treatment of Alzheimer diseases.J. Control. Release202235163865510.1016/j.jconrel.2022.09.058 36191675
    [Google Scholar]
  8. CiureaA.V. MohanA.G. Covache-BusuiocR.A. CostinH.P. GlavanL.A. CorlatescuA.D. SaceleanuV.M. Unraveling molecular and genetic insights into neurodegenerative diseases: Advances in understanding Alzheimer’s, Parkinson’s, and Huntington’s diseases and amyotrophic lateral sclerosis.Int. J. Mol. Sci.202324131080910.3390/ijms241310809 37445986
    [Google Scholar]
  9. SongvutP. BoonyarattanasoonthornT. NuengchamnongN. JunsaiT. KongratanapasertT. SupannapanK. KhemawootP. Enhancing oral bioavailability of andrographolide using solubilizing agents and bioenhancer: Comparative pharmacokinetics of] Andrographis paniculata formulations in beagle dogs.Pharm. Biol.202462118319410.1080/13880209.2024.2311201 38351624
    [Google Scholar]
  10. LeeM.K. Liposomes for enhanced bioavailability of water-insoluble drugs: In vivo evidence and recent approaches.Pharmaceutics20201226410.3390/pharmaceutics12030264
    [Google Scholar]
  11. NachonF. CarlettiE. RoncoC. TrovasletM. NicoletY. JeanL. RenardP.Y. Crystal structures of human cholinesterases in complex with huprine W and tacrine: Elements of specificity for anti-Alzheimer’s drugs targeting acetyl- and butyryl-cholinesterase.Biochem. J.2013453339339910.1042/BJ20130013 23679855
    [Google Scholar]
  12. PettersenE.F. GoddardT.D. HuangC.C. CouchG.S. GreenblattD.M. MengE.C. FerrinT.E. UCSF Chimera—A visualization system for exploratory research and analysis.J. Comput. Chem.200425131605161210.1002/jcc.20084 15264254
    [Google Scholar]
  13. O’BoyleN.M. BanckM. JamesC.A. MorleyC. VandermeerschT. HutchisonG.R. Open babel: An open chemical toolbox.J. Cheminform.2011313310.1186/1758‑2946‑3‑33 21982300
    [Google Scholar]
  14. MorrisG.M. HueyR. LindstromW. SannerM.F. BelewR.K. GoodsellD.S. OlsonA.J. AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility.J. Comput. Chem.200930162785279110.1002/jcc.21256 19399780
    [Google Scholar]
  15. LiH. LeungK-S. WongM-H. idock: A multithreaded virtual screening tool for flexible ligand docking.IEEE Symposium on Computational Intelligence in Bioinformatics and Computational Biology (CIBCB)San Diego, CA, USA778410.1109/CIBCB.2012.6217214
    [Google Scholar]
  16. FarheenM. AkhterM.H. ChitmeH. AkhterM.S. TabassumF. JaremkoM. EmwasA.H. Harnessing folate-functionalized nasal delivery of dox–erlo-loaded biopolymeric nanoparticles in cancer treatment: Development, optimization, characterization, and biodistribution analysis.Pharmaceuticals202316220710.3390/ph16020207 37259356
    [Google Scholar]
  17. YokoyamaR. TaharabaruT. NishidaT. OhnoY. MaedaY. SatoM. IshikuraK. YanagiharaK. TakagiH. NakamuraT. ItoS. OhtsukiS. ArimaH. OnoderaR. HigashiT. MotoyamaK. Lactose-appended β-cyclodextrin as an effective nanocarrier for brain delivery.J. Control. Release202032872273510.1016/j.jconrel.2020.09.043 33002523
    [Google Scholar]
  18. HussainK. AliI. UllahS. ImranM. ParveenS. KanwalT. ShahS.A. SaifullahS. ShahM.R. Enhanced antibacterial potential of naringin loaded β cyclodextrin nanoparticles.J. Cluster Sci.202233133934810.1007/s10876‑020‑01972‑8
    [Google Scholar]
  19. BrandbuE. Nursing practice-a conflict-filled reality. How can qualifications be met?.Vard Nord. Utveckl Forsk1994141242810.1177/010740839401400106 7941215
    [Google Scholar]
  20. OseniB.A. AzubuikeC.P. OkubanjoO.O. IgwiloC.I. PanyamJ. Encapsulation of andrographolide in poly(lactide-co-glycolide) nanoparticles: Formulation optimization and in vitro efficacy studies.Front. Bioeng. Biotechnol.2021963940910.3389/fbioe.2021.639409 33681172
    [Google Scholar]
  21. KulsiriratT. SathirakulK. KameiN. Takeda-MorishitaM. The in vitro and in vivo study of novel formulation of andrographolide PLGA nanoparticle embedded into gelatin-based hydrogel to prolong delivery and extend residence time in joint.Int. J. Pharm.202160212061810.1016/j.ijpharm.2021.120618 33887393
    [Google Scholar]
  22. ChaigneauT. PallottaA. BenaddiF.Z. SanceyL. ChakirS. BoudierA. ClarotI. Monitoring of gold biodistribution from nanoparticles using a HPLC-visible method.Separations202181121510.3390/separations8110215
    [Google Scholar]
  23. VogelH.G. VogelW.H. SchölkensB.A. SandowJ. MüllerG. VogelW.F. Psychotropic and neurotropic activity1. Drug Discovery and Evaluation.Berlin, HeidelbergSpringer Berlin Heidelberg200238559310.1007/3‑540‑29837‑1_6
    [Google Scholar]
  24. HazzaaS.M. EldaimM.A.A. FoudaA.A. MohamedA.S.E.D. SolimanM.M. ElgizawyE.I. Intermittent fasting ameliorated high-fat diet-induced memory impairment in rats via reducing oxidative stress and glial fibrillary acidic protein expression in brain.Nutrients20201311010.3390/nu13010010 33375195
    [Google Scholar]
  25. KaurA. AlamM.A. MahmoodT. AhsanF. Probing the memory-enhancing potential of kiwi fruit against scopolamine-induced memory impairment in experimental rats.Biomed. Pharmacol. J.20241721165117610.13005/bpj/2931
    [Google Scholar]
  26. ManiV. ParleM. RamasamyK. Abdul MajeedA.B. Reversal of memory deficits by Coriandrum sativum leaves in mice.J. Sci. Food Agric.201191118619210.1002/jsfa.4171 20848667
    [Google Scholar]
  27. ShangariN. O’BrienP.J. Catalase activity assays.Curr. Protoc. Toxicol.20062717.11510.1002/0471140856.tx0707s2720954160
    [Google Scholar]
  28. AkhtarM. MaikiyoA. NajmiA. KhanamR. MujeebM. AqilM. Neuroprotective effects of chloroform and petroleum ether extracts of Nigella sativa seeds in stroke model of rat.J. Pharm. Bioallied Sci.20135211912510.4103/0975‑7406.111825 23833517
    [Google Scholar]
  29. FabianO. BajerL. Histopathological assessment of the microscopic activity in inflammatory bowel diseases: What are we looking for?World J. Gastroenterol.202228365300531210.3748/wjg.v28.i36.5300 36185628
    [Google Scholar]
  30. Organisation for economic co-operation and development. OECD Guidelines for Multinational Enterprises.2011 EditionParisOECD Publishing201110.1787/9789264115415‑en
    [Google Scholar]
  31. Ballesteros-RamírezR. LassoP. UrueñaC. SaturnoJ. FiorentinoS. Assessment of acute and chronic toxicity in Wistar rats (Rattus norvegicus) and New Zealand rabbits (Oryctolagus cuniculus) of an enriched polyphenol extract obtained from Caesalpinia spinosa.J. Toxicol.2024202411010.1155/2024/3769933 38633362
    [Google Scholar]
  32. ZhangQ. CuiQ. Biodistribution of andrographolide to assess the interior‐exterior relationship between the lung and intestine using MICROPET.Thorac. Cancer202011113365337410.1111/1759‑7714.13682 33017514
    [Google Scholar]
  33. SanatiM. KhodagholiF. AminyavariS. GhasemiF. GholamiM. KebriaeezadehA. SabzevariO. HajipourM.J. ImaniM. MahmoudiM. SharifzadehM. Impact of gold nanoparticles on amyloid β-induced Alzheimer’s disease in a rat animal model: Involvement of STIM proteins.ACS Chem. Neurosci.20191052299230910.1021/acschemneuro.8b00622 30933476
    [Google Scholar]
  34. AskarK.A. KudiA.C. MoodyA.J. Comparative analysis of cholinesterase activities in food animals using modified Ellman and Michel assays.Can. J. Vet. Res.2011754261270 22468023
    [Google Scholar]
  35. PudlarzA.M. Ranoszek-SoliwodaK. CzechowskaE. TomaszewskaE. CelichowskiG. GrobelnyJ. SzemrajJ. A study of the activity of recombinant Mn-superoxide dismutase in the presence of gold and silver nanoparticles.Appl. Biochem. Biotechnol.201918741551156810.1007/s12010‑018‑2896‑y 30284207
    [Google Scholar]
  36. ZhangH.M. CaoJ. TangB.P. WangY.Q. Effect of TiO2 nanoparticles on the structure and activity of catalase.Chem. Biol. Interact.201421916817410.1016/j.cbi.2014.06.005 24931876
    [Google Scholar]
  37. NiyomchanA. ChatgatW. ChatawateeB. KeereekochT. IssuriyaA. JaisamutP. ChusriS. KunworarathN. Safety evaluation of the polyherbal formulation nawatab: Acute and subacute oral toxicity studies in rats.Evid. Based Complement. Alternat. Med.202320231941345810.1155/2023/9413458 37528898
    [Google Scholar]
  38. MotoyamaK. NishiyamaR. MaedaY. HigashiT. IshitsukaY. KondoY. IrieT. EraT. ArimaH. Synthesis of multi-lactose-appended β-cyclodextrin and its cholesterol-lowering effects in Niemann–Pick type C disease-like HepG2 cells.Beilstein J. Org. Chem.201713101810.3762/bjoc.13.2 28179943
    [Google Scholar]
  39. DashputreN.L. LaddhaU.D. DarekarP.P. KadamJ.D. PatilS.B. SableR.R. UdavantP.B. TajanpureA.B. KakadS.P. KshirsagarS.J. Potential therapeutic effects of naringin loaded PLGA nanoparticles for the management of Alzheimer’s disease: In vitro, ex vivo and in vivo investigation.Heliyon202399e1937410.1016/j.heliyon.2023.e19374 37662728
    [Google Scholar]
  40. LinC.H. ChenC.H. LinZ.C. FangJ.Y. Recent advances in oral delivery of drugs and bioactive natural products using solid lipid nanoparticles as the carriers.J. Food Drug Anal.201725221923410.1016/j.jfda.2017.02.001 28911663
    [Google Scholar]
  41. PoudelP. ParkS. Recent advances in the treatment of Alzheimer’s disease using nanoparticle-based drug delivery systems.Pharmaceutics202214483510.3390/pharmaceutics14040835 35456671
    [Google Scholar]
/content/journals/cbc/10.2174/0115734072326670250119061702
Loading
/content/journals/cbc/10.2174/0115734072326670250119061702
Loading

Data & Media loading...

Supplements

Supplementary material is available on the publisher’s website along with the published article.

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error
Please enter a valid_number test