separations Article Capturing Dioclea Reflexa Seed Bioactives on Halloysite Nanotubes and pH Dependent Release of Cargo against Breast (MCF-7) Cancers In Vitro Srinivasan Balapangu 1,2, Emmanuel Nyankson 3, Bernard O. Asimeng 1, Richard Asiamah 1, Patrick K. Arthur 2,4 and Elvis K. Tiburu 1,2,* 1 Department of Biomedical Engineering, University of Ghana, Legon LG27, Ghana; ssbalapangu@ug.edu.gh (S.B.); boasimeng@ug.edu.gh (B.O.A.); rasiamah001@st.ug.edu.gh (R.A.) 2 West Africa Center for Cell Biology of Infectious Pathogens (WACCBIP), University of Ghana, Legon LG54, Ghana; parthur14@gmail.com 3 Department of Material Science & Engineering, University of Ghana, Legon LG27, Ghana; enyankson@ug.edu.gh 4 Department of Biochemistry, Cell & Molecular Biology, University of Ghana, Legon LG54, Ghana * Correspondence: etiburu@ug.edu.gh; Tel.: +233-559-585-194 Abstract: In this work, optimization parameters were developed to capture plant metabolites from Dioclea Reflexa (DR) seed ex-tracts onto halloysites nanotubes (HNTs). A one-step pool of the crude extracts at neutral pH from the HNT lumen failed to elicit a reduction in breast cancer, Michigan Cancer Foundation-7 (MCF-7) cell viability. However, the pH-dependent elution of metabolites   revealed that the acidic pH samples exhibited profound antiproliferative effects on the cancer cells compared to the basic pH metabolites using both trypan blue dye exclusion assay and 3-(4,5- Citation: Balapangu, S.; Nyankson, dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) viability test. pH~5.2 samples E.; Asimeng, B.O.; Asiamah, R.; Arthur, P.K.; Tiburu, E.K. Capturing demonstrated by half-maximal inhibitory concentration (IC50) of 0.8 mg and a cyclic voltammetry Dioclea Reflexa Seed Bioactives on oxidation peak potential and current of 234 mV and 0.45 µA, respectively. This indicates that the Halloysite Nanotubes and pH cancer cells death could be attributed to membrane polarization/depolarization effects of the sample. Dependent Release of Cargo against Fluorescence-activated cell sorting (FACS) studies confirmed that the plant metabolites affected Breast (MCF-7) Cancers In vitro. breast cancer apoptotic signaling pathways of cell death. The studies proved that plant metabolites Separations 2021, 8, 26. https:// could be captured using simplified screening procedures for rapid drug discovery purposes. Such doi.org/10.3390/separations8030026 procedures, however, would require the integration of affordable analytical tools to test and isolate individual metabolites. Our approach could be an important strategy to create a library and database Academic Editor: Marcello Locatelli of bioactive plant metabolites based on pH values. Received: 23 December 2020 Keywords: halloysite nanotubes; cyclic voltammetry; polarization/depolarization Dioclea Reflexa; Accepted: 14 February 2021 plant metabolites; anticancer metabolites Published: 27 February 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- 1. Introduction iations. Traditional herbal medicine practices continue to be inseparable in the lives of many people in the world, including the inhabitants of sub-Sahara Africa (sSA), due to the impact of plant metabolites for treating several diseases [1–3]. Over the years, the global accep- tance of herbal medicines in homes, as well as health clinics, has resulted in the growth Copyright: © 2021 by the authors. of the herbal products market in most countries in the subregion. However, there are Licensee MDPI, Basel, Switzerland. still challenges with understanding mechanisms of action due to the lack of standardized This article is an open access article procedures to rapidly prepare plant metabolites to meet pharmacological criteria [4–6]. distributed under the terms and This gap, especially in the subregion, has elevated the research community enthusiasm in conditions of the Creative Commons pursuing cheap and affordable functional materials to facilitate the development of simple Attribution (CC BY) license (https:// and effective separation technologies that have similar or even better performance efficien- creativecommons.org/licenses/by/ cies than the traditional and more expensive technologies. Such easy-to-use technologies 4.0/). Separations 2021, 8, 26. https://doi.org/10.3390/separations8030026 https://www.mdpi.com/journal/separations Separations 2021, 8, 26 2 of 14 will facilitate the capture, release, and compilation of plant metabolites in a library and database to support further scientific research in the field of plant medicine [7,8]. Recently, solid-phase microextraction (SPME) technology has been developed using TiO2 nanotube arrays in situ on Ti wires for selective removal of organic compounds [9]. However, this technology exhibits some drawbacks, including fragility, which limits longevity. Halloysites nanotubes (HNTs) are natural tubules of aluminosilicate minerals composed of different proportions of aluminum, silicon, hydrogen, and oxygen, often with the chemical formula Al2Si2O5(OH)4.nH2O [10,11]. They are empty cylinders with widths of about 100 nanometers and consist of two structures: the anhydrous structure with an interlayer dispersing of approximately 7Å and the hydrated structure with an augmented interlayer dividing of 10 Å, due to the presence of water in the lamellar spaces [12–14]. In each layer of the halloysite nanotubes (HNTs), the siloxane (SiOH) groups are found on the outer surface, while the aluminol (AlOH) groups are situated on the inner surfaces, making the outer and inner surfaces have different charges. The positive charge of the internal lumen is a consequence of protonation of the AlOH group at low pH, whereas the SiOH groups has overall negative charge due to the coordination of the atoms. These unique properties of HNTs have made it possible for it to be used in various biomedical applications, such as the development of biohybrid materials for health applications [15]. The charge disparity has drawn interest from the research community, whereby overall negatively charged proteins taken above their isoelectric points were mostly loaded into the positively charged nanotube lumen [16]. Therefore, in a pool of organic compounds, HNTs can facilitate the formation of a transient bond between selected bioactive compounds and the AlOH or SiOH as a function of pH conditions and can be very effective as a nano drug carrier for different applications [17–22]. The loading efficiency is influenced by the charge characteristics of the active agents, as well as vacuum pressure [23]. The species Dioclea Reflexa (DR) hook belong to leguminoase plants, which include legume, pea, and the bean families. There are certain classes of compounds in Dioclea reflexa (DR) that have clinical usefulness in both temperate and tropical regions [24–26]. Extract of DR seed has been shown to boost hematological parameters and antioxidant activities which protect the kidney and blood from oxidative and related injuries under acute and chronic toxicological challenges [1,2,24,25,27–31]. In addition, the aqueous extract of the seeds produces 100% mortality in third stage mosquito larvae of Aedes aegypti. The seed is a potential food source which contains around 14% protein, 8% fats, and 58% carbohydrates [26]. Though these metabolites in the pool continue to show promise in disease treatment, there is very limited data in the literature of the properties of single isolates and their medicinal relevance, albeit due to the difficulties in pursuing systematic separation of the complex mixtures in a single separation method. Thus, the current work describes the use of a simplified method to systematically isolate bioactive compounds from extracted complex mixtures from DR and test their inhibitory effects on breast Michigan Cancer Foundation-7 (MCF-7) cells. The rationale is that the larger surface area coupled with the differential polarity of the lumen and the surface of the HNTs will be sufficient to bind selectively with the plant metabolites in the crude extracts of DR. The authors hypothesized that: (1) the pH dependent elution of the plant metabolites can identify therapeutic bioactive compounds against cancer cells and that (2) specific HNT could isolate structurally and functionally related metabolites from complex mixtures in a single step. The evidence of the entrapped species onto the HNTs was monitored using X-ray diffractometry (XRD) and Fourier transform infrared spectroscopy (FTIR) to determine the degree of aluminol (AlOH) and the siloxane (SiOH) groups modification since these two functional groups will be key sites for bioactive compounds interaction. pH-dependent eluted samples have been tested on breast (MCF-7) cancer cell lines to investigate both their inhibitory and the mechanism using cyclic voltammetry and flow cytometry analyses [32–35]. The results are reported here and show evidence of differential inhibitory effects of the bioactive compounds from the various pH conditions. Separations 2021, 8, 26 3 of 14 2. Materials and Methods 2.1. Materials N,N-dimethyl sulfonamide, sodium hydroxide (NaOH, >99%), acetic acid (CH3COOH, >99%), sulfuric acid (H2SO4, >99%), hydrochloric acid (HCl), and propidium iodide were purchased from Fisher Scientific, Altrincham, UK. Samples of natural halloysite (cat. no. 685 445) were purchased from Sigma Aldrich, St. Louis, USA. All chemicals were analytical grade and were, therefore, used without further purification. Breast (MCF-7) cancer cells (HTB-22) were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA) and maintained in Dulbecco’s modified eagle medium (DMEM-F12) complete media supplemented with 10% fetal bovine serum (FBS), minimum essential medium (MEM) nonessential amino acids, gentamicin, and 10 µg/mL insulin in a 5% CO2 incubator at 37 ◦C. All culturing media were obtained from ATCC (Manassas, VA). RNase A from Sigma Aldrich, St. Louis, MO, USA. 2.2. Methods 2.2.1. Loading and pH-Dependent Release of DR Metabolites A 5 g quantity of Dioclea Reflexa (DR) seed powder was suspended in 30 mL of 70% ethanol (pH 7.4) for 24 h to extract the plant metabolites [6]. A volume of 30 mL of the supernatant was used for the immobilization using 120 to 1320 milligram quantities in intervals of 120 mg/mL of halloysite nanotubes(HNTs). A UV–Vis spectrophotometer (Shimadzu UV/Vis 1601 spectrophotometer, Shimadzu Corporation, Tokyo, Japan) was used to determine the concentration of the crude extracts before and after loading onto the halloysites nanotubes. A standard curve was then constructed to determine the amount of entrapped bioactive compounds from the DR seed extracts. The percentage loading capacity, LC was obtained from Equation (1): LC = Mm/Mh × 100 (1) where Mm and Mh are the masses of the entrapped metabolites and the amount of hal- loysites used for the entrapment, respectively. The loaded HNTs were weighed and stored at 20 ◦C, and the entrapped metabolites were released using a buffer of pH (4.1–9.6) for 24 h. 2.2.2. Characterization of HNTs and DR Loaded HNTs FTIR spectra of empty halloysites nanotubes (HNTs) and Dioclea Reflexa (DR) loaded HNTs were recorded with a Nicolet MAGNA-IR 750 Spectrometer (Nicolet Instrument Co., Madison, WI, USA). The spectra were recorded from 500 to 4000 cm−1 wavenumber with 16 scans and spectral resolution of 4 cm−1. XRD (Empyrean, Malvern Panalytical B.V, Almelo, The Netherlands) of the HNTs and loaded HNTs were performed using a Pan Analytical diffractometer with CuKα radiation. A 2θ scan was performed from 5 to 35◦ in steps of 0.05◦, with a tube voltage of 45 kV and a current of 40 mA. The Thermogravimetric Analyzer (TGA) (Q600 SDT, TA Instruments, Brussels, Bel- gium) analysis of the HNT and HNT loaded with the DR extract was conducted using Pyris 1 TGA equipment. The analysis was conducted in N2 atmosphere at a heating rate of 20 ◦C/min. 2.2.3. Culturing and Cyclic Voltammetry Analysis of MCF-7 Breast Cancer Cell Lines MCF-7 cells (HTB-22) were grown and maintained in DMEM-F12 supplemented media. Media was changed every 2–3 days, and cells were passaged at 65–80% confluence. The cells were harvested after complete rinsing with 0.25% (w/v) Trypsin and 0.53 mM Ethylenediamine tetra acetic acid (EDTA) solution to remove all traces of fetal bovine serum, which contains trypsin inhibitor. A volume of 2.0 to 3.0 mL of Trypsin-EDTA solution was added to the flask, and the cells were observed under an inverted microscope. Separations 2021, 8, 26 4 of 14 A volume of 6.0 to 8.0 mL of complete growth medium was used to aspirate the cells, and the suspension was centrifuged at 125 mg for 5 to 10 min. After re-suspension, cell density of 5.6 × 106 was obtained. The inhibitory effects (expressed as Percentage Activity, (PA)) of the metabolites concentration of 2 mg/mL on the cells was determined using Equation (2). Mo is the initial concentration of the extracts, Mph is the concentration of the extracts at particular pH. Mo − MphPA = × 100 (2) Mo A stock solution of 2 mg/mL of the metabolites from pH ~5.2 was prepared for elec- trochemical detection studies using cyclic voltammetry under steady-state conditions. The electrochemical detection was carried out using A CheapStat potentiostat device (IO Rodeo, Pasadena, CA, USA) connected to interdigitated gold electrodes (IDEs)/Microelectrodes (Metrohm, DropSens Llanera, Asturias, Spain). A volume of 5µL of cells of cell density of 2.3 × 106 cells/well was suspended in 0.1 mM Phosphate Buffer Saline (PBS), and the metabolite was also dispensed in 0.002 mM dimethyl sulfoxide (DMSO). The samples were deposited on the active electrode for cyclic voltammetry measurements. The voltam- mograms were obtained using a potential range from 690 to 970 mV at a scan rate of 10 mVs−1. Cell viability studies was conducted using trypan blue assay and confirmed by MTT assay. The MTT assay protocol is based on the conversion of water soluble MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) compound to an insoluble formazan product by the viable cells. [36–40]. 3. Results 3.1. Characterization of HNT and loaded HNT using XRD, TGA, and FTIR In an effort to determine the presence of trapped compounds in the HNT, preparations were subjected to analysis using XRD, TGA, and FTIR techniques. In Figure 1a (red), the characteristic 2◦ peak positions of HNT occur at 11.7, 20.5, 24.8, 37.5, and 62.2, representing (001), (110), (011), (131), and (331) crystallographic planes, respectively. After immobi- lization of the DR extracts on the nanotubes, there was dramatic reduction of the peak intensities at the same 2◦ positions, indicating that the resulting structure of the composite material (HNT with DR) is more amorphous than that of HNT. This is expected since the metabolites from the DR are mostly amorphous in nature. The absence of additional peaks aside the characteristic peaks of HNTs after loading with DR depicts the amount of DR loaded was less than 5% by weight of the HNTs. Since the metabolite may be amorphous, it is likely that the peaks of the HNTs overshadowed the amorphous peaks of the extract, and, as a result, no clearly defined XRD peaks were found. The bioactive con- stituents were eluted with 70% ethanol and resulted in HNTs signature peaks, as shown in Figure 1a (blue). The characteristic peak intensities reverted to those observed in the control, implying that the reduced intensities were due to the plant extract. The changes in the signatures peak intensities of the halloysite nanotubes (HNTs) was monitored using FTIR spectroscopy, as shown in Figure 1b. The FTIR spectra revealed all the functional groups present in the empty HNTs (black). Six major peaks were identified in the HNT. The inner Al-OH and outer Si-OH groups have characteristic stretching peaks at 3622 and 3694 cm−1, respectively. Bending vibrations of Al-OH and Si-OH revealed absorption peaks at 902 cm−1. In addition, the uneven stretching vibrations of the Si-OH bond gave a strong absorption peak at 995 and 1118 cm−1. In addition, the deformation vibration of the interlayer water molecules of the HNT was observed at 1647 cm−1. There was a significant increase of the transmission peak intensities after immobilization of the DR extracts on the HNTs (blue). Four distinct additional peaks were observed in the FTIR spectra of the halloysite nanotubes loaded with the DR extract, as can be seen in Figure 1b. These peaks were also present in the FTIR spectra of the DR extract. The peak observed at 1744 cm−1 is due to the C = O stretching, while the peak observed at 1460 cm−1 represents the symmetric C–H vibration. The symmetric and asymmetric vibrations of CH2 were represented by the peaks observed at 2923 and 2854 cm−1 [41]. Since the peaks present Separations 2021, 8, x FOR PEER REVIEW 5 of 14 Si-OH bond gave a strong absorption peak at 995 and 1118 cm−1. In addition, the defor- mation vibration of the interlayer water molecules of the HNT was observed at 1647 cm−1. There was a significant increase of the transmission peak intensities after immobilization of the DR extracts on the HNTs (blue). Four distinct additional peaks were observed in the FTIR spectra of the halloysite nanotubes loaded with the DR extract, as can be seen in Figure 1b. These peaks were also present in the FTIR spectra of the DR extract. The peak observed at 1744 cm−1 is due to the C = O stretching, while the peak observed at 1460 cm−1 Separations 2021, 8, 26 represents the symmetric C–H vibration. The symmetric and asymmetric vibrations of 5 of 14 CH2 were represented by the peaks observed at 2923 and 2854 cm−1 [41]. Since the peaks present in the HNT and DR extract were observed in the HNT loaded with DR extract sample, it depicts that the HNT was indeed loaded with the DR extract. To fiunrtthheerH cNonTfiarnmd tDhaRt tehxetr DacRt ewxetrraecot bwsearsv leodadinedth inetHo NthTe HloNadTe,d TwGAith anDaRlyesxistr oafc tthsea mple, it raw HNdTes paincdts HthNatTt lhoeaHdeNdT wwitahs thined DeeRd elxotardacetd wwaist hcotnhdeuDcRtedex. tTrhacist. analysis also helped in estimatingT othfeu ratmheorucnotn ofifr mDRth eaxtttrhaectD loRaedxetdra icnttwo atshelo HadNedTsi.n Ttohteh TeGHAN Tre, sTuGltAs iasn palryes-is of the sented inra FwigHuNreT 1sca.n TdwHoN dTisltoinadcte ddewcoitmh tphoesDitiRonesx twraecrtew oabssceornvdedu catet da.pTphroisxaimnaaltyesliys a6l0s o°Ch elped in and 450 e°sCti.m Tahtein wg ethigehatm loosusnest oofbDseRrveexdtr aactt 6lo0 a°dCe danindt o45th0 e°CH NcaTns .bTeh aetTtrGibAutreedsu tlots tis presented in◦he de- ◦ composiFtiiognu roef 1thc.eT wwaotedri mstionlcetcduelecso ambpsoosrbitions were observe◦ed onto the ◦HNT s duraftaacep panrodx timhea dteelhyy6d0roCxyalna-d 450 C. tion of thTeh He NwTeisg, hretslpoesscetisvoelbys.e Crvoendsiadter6i0ngC thaen HdN45T0 loaCdceadn wbietha tthtreib DuRte edxttoratcht,e ad seigconmif-position icant maosfs tlhoessw wataesr ombsoelervculesHNTs, respectively.eCd obe atbwsorbed ontnsideeerinn g25t–h8e5H o°Cth. eTHNT lhoi N asd m T asuedss r flaocses and the dehydrwith th ceaDn Rbee xattrtaricbtu, ate odx tyolation of thesigni fithcea nt mass decompolossistiowna sofo bthsee r7v0e%d beethtwaneoenl s2o5l–u8t5io◦nC u. sTehdis inm tahses lloosasdcinang bperoactetrsisb.u Ttehde tHoNthTe ldoeacdoemdp osition with theo fDtRh eex7t0r%acte cthoanntaoilnesodl usotimone umsoedistiunret hwehleona dtihneg TpGroAc easnsa. lyTshise wHaNs Tbelionagd ceodnw- ith the ducted. DInR adexdtirtaiocnt ctoo nthtaiisn oebdsesrovmede mdeociosmtupreoswithioenn, athneotThGerA graandaulyasl ids ewcoams bpeoisnigtiocno nwdausc ted. In observeda dfrdoimtio anptportohxiismoabtseelyrv 2e4d0–d3e7c0o m°Cp.o Tshitiiso ngr, aadnuoathl edregcoramdpuoaslidtieocno cmanp obsei taiottnriwbuatseodb served to the exftrroamct. aTphpisr oimxipmliaetse ltyha2t4 t0h–e3 H70N◦TC l.oTadheisd gwraitdhu tahle dDeRco emxtpraocsti tiinodneceadn cobnetaaitntreidb uthteed to the DR extraecxtt.r Tahcte. aTmhiosuinmt polfi ethseth loatadtheed HDNR Texlotraadcte dwwasi tehsttihmeaDteRd etox tbrae catpinpdroexeidmcaotenltya i5n.1ed8 the DR wt.%. extract. The amount of the loaded DR extract was estimated to be approximately 5.18 wt.%. 300 HNT loaded DR HNT 250 Eluted HNT 20.5 200 11.7 150 24.8 100 37.5 62.2 50 0 10 20 30 40 50 60 70 80 2θ / degree (a) Figure 1. Cont. Intensity Separations 2021, 8, x FOR PEER REVIEW 6 of 14 Separations 2021, 8, 26 6 of 14 1647 2854 3622 995 1118 1460 1744 2923 3694 HNT DR Extract 902 HNT loaded with DR Extract 0 500 1000 1500 2000 2500 3000 3500 4000 4500 Wavenumber (cm-1) (b) 100 80 60 HNT HNT with DR Extract DR Extract 40 100 200 300 400 500 600 700 800 Temperature (Degree C) (c) Figure 1.F (iag)u Xr-era1y. (dai)fXfr-arcatyomdieftfrrya c(tXoRmDe)t rsyp(eXctRraD o) fs pheaclltoryasoitfehs anllaonyostiutebsesn a(HnoNtuTb) easn(dH HNNTT) alonaddHedN T loaded with the wDiitohcltehae RDeifolecxleaa (DRRefl) eexxatr(aDctR. ()be)x Ftroaucrti.e(rb t)raFnosuforiremr tirnafnrasfroerdm (FiTnIfRra)r sepde(cFtrTaI Rof) HspNecTtsr aanodf HNTs and loaded HlNoaTdse wd iHthN pTlsanwt imtheptalabnotlimteest. a(bc)o TliGteAs. o(cf) HTNGAT aonfdH HNNTTa nlodaHdeNdT wloitahd DedRw exitthraDctR. extract. 3.1.1. Optimization of Parameters to Increase Metabolites Immobilization onto HNTs Concentration dependent analysis of the bioactive compounds entrapped on the HNT using varied concentrations of the nanotubes is displayed in Figure 2a. It was observed that the maximum extractable bioactive compounds at the loading range from 720 to 1080 mg Weight % Transmittance (%) Separations 2021, 8, x FOR PEER REVIEW 7 of 14 3.1.1. Optimization of Parameters to Increase Metabolites Immobilization onto HNTs Concentration dependent analysis of the bioactive compounds entrapped on the HNT using varied concentrations of the nanotubes is displayed in Figure 2a. It was ob- served that the maximum extractable bioactive compounds at the loading range from 720 to 1080 mg of HNTs using DR concentration of 0.2 g/mL in a total volume of 30 mL of crude extract solution was about 14–19%. Separations 2021, 8, 26 Figure 2 showed the inhibitory effects of 2 mg/mL of the entrapped metabolites on 7 of 14 breast (MCF-7) cancer cell lines at cell density 6.3 × 106. It was observed that the crude extracts from the HNTs lumen did not show significant increase in percent antiprolifera- tive effectso ofnH tNheT sMuCsFin-7g cDeRllsc, oansc ienndtircaattiiovne obfy0 t.2heg /stmatListiincaalltyo tianlsvigonluifmicaenotf v3a0rimatLioonfsc irnu de extract cell viabilitsyo. lu tion was about 14–19%. 55 (a) (c) 60 50 50 40 45 30 20 40 10 35 40 (b) (d) 70 35 60 30 50 25 40 20 15 30 10 20 5 10 0 0 120 240 360 480 600 720 840 960 1080 1200 1320 4.1 5.2 6.4 7.5 8.6 9.7 Concentration (mg/mL) pH Figure 2. OptimFiziagtuiorne 2o.f Othpetcimonizdai tiions offo rthDeR coendtriatpiomnse nfot ru sDinRg eHntNraTps.m(ae)nTt huesirnegla HtivNeTasm. (oau) nTthien rpeelracteivneta agme ouf ennt trapped bioactive compoiunn pdesr(cmengt/agmeL o)fu esnintrgap5pgerdam bsiobaecdtiveo lcuomepofuHndNsT (sm. Tgh/me Lre)l euassinegw 5a sgrcaomndsu bcetded vfoolrum2 dea oyfs HtoNeTnss.u re all the bound moleculeTshwe erreelecaosme pwleatse clyonredluecatseedd fforro m2 dthayesl utom eennsuorfet haell HthNe Tb.o(ubn)Md TmToalescsuaylesfo wr eMreC cFo-7mcpelleltveliyab rielilteyasaenda lysis of the entrapped bfirooamct itvhee cluommpeno uonf dthsef rHoNmTt.h (ebv)MarTioTu asscsrauyd feore xMtrCaFct-s7 ucesliln vgia2bmiligt/y manLa.l(ycs)isT ohfe tehfefe ecntstroafpbpuedff ebriopaHc-on the release of the imtmivoeb ciloizmepdobuionadcst ifvroemco tmhpe ovuanridosu.s( dcr)uTdhee erxeltaraticvtes aucstiinvgit y2 omfgp/HmLd.e (pce) nTdheen et fbfeiocatsc toivf ebucoffmerp pouHn odns othneM CF-7 release of the immobilized bioactive compounds. (d) The relative activity of pH dependent bioac- cell viability usintigve2 cmomg/pmouLncdosn ocenn MtraCtFio-n7 sc.ell viability using 2 mg/mL concentrations. Figure 2 showed the inhibitory effects of 2 mg/mL of the entrapped metabolites on 3.1.2. PH-Dberpeeanstd(eMntC EFl-u7t)iocnan ocfe trhcee Mll elitnaebsoalittecse lflrodmen HsiNtyT6s. 3 × 106. It was observed that the crude The pHex-tdraecptesnfdroemnt trheeleHasNeT osfl uthme ebniodaicdtinvoet csohmowposuignndifis cisa ndtisinpclareyaesde iin pFiegrucerne t2a. nTthiper oliferative results reveafflecdt spHon eftfheectM onC Fth-7e rcellelsa,sea sofi nthdeic baitoivaectibvye tchoemsptoautinstdicsa flrloymin HsiNgnTisfi wcaanst tvhaer iations in same basedc eolnl vthiaeb pilritoyf.ile exhibited in Figure 2. To investigate the inhibitory effects of the pH eluted samples, a 2 mg/mL was prepared from all the samples and tested on the cancer cells, as sh3o.w1.n2 .iPnH F-iDguerpee n2d.e nAtllE tlhueti osnamofptlehse tMesetteadb odleitmesonfrsotmratHedN sTosme level of percent inhibition with Tthe epxHtr-adcetp fernomde nptHr(e~le5a.2s eanofdt h~9e.6b)i,o raecvtievaelicnogm tphoe uhnigdhseistd ainspdl athye dloinwFesigt ure 2. The degree of prerscuelntst raenvteipalreodlifpeHrateifvfec etfofenctsh eofr e7l4e aasnedo 3f 6th, erebsipoeaccttiivveelyc.o Tmhpeo IuCnd sinfr oTmabHleN 1T s was the of the watesra manedb eatsheadnonl ethxetrpacrotsfi, laese wxheilbl iatesd thinosFei gouf rteh2e. pTHo idnevpeestnigdaetnet tehleutinedh isbaitmorpyleesf,f ects of the pH eluted samples, a 2 mg/mL was prepared from all the samples and tested on the cancer cells, as shown in Figure 2d. All the samples tested demonstrated some level of percent inhibition with the extract from pH(~5.2 and ~9.6), revealing the highest and the lowest degree of percent antiproliferative effects of 74 and 36, respectively. The IC50 in Table 1 of the water and ethanol extracts, as well as those of the pH dependent eluted samples, were determined to confirm the trend of inhibition demonstrated in Figure 2. The inhibitory effects of the bioactive compounds eluted at acidic pH had much lower IC50 of 0.8–1.6, with pH~5.2 revealing the lowest IC50, which indicated the metabolites from that fraction are more potent to the cells, as shown in Table 1. Antiproliferate effects (%) Loading capacity (%) Antiproliferate effects (%) Entrapment efficiency (%) Separations 2021, 8, x FOR PEER REVIEW 8 of 14 were determined to confirm the trend o f inhibition demonstrated in Figure 2. The inhibi-tory effects of the bioactive compounds eluted at acidic pH had much lower IC50 of 0.8–Separations 2021, 8, 26 1.6, with pH~5.2 revealing the lowest IC , which indicated the metabolites from that 8 of 14 fraction are more potent to the cells, as shown in Table 1. Table 1. IC50 values of the extracts eluted at different pH conditions, all measured in milligram, and quaTnatbitliees1 .oIfC th50e vseaelude esxotfrathcte. extracts eluted at different pH conditions, all measured in milligram, and quantitiTesreoaf tthmeesneet d extract. Normalized Sample/TDrerayt mEexntrtact IC50 (mg) NormaRli zseqduared Value SamWplaet/eDrr y Extract I3C3.3 50 (mg) 0R.95S9q uared Value EthaWnaotle r 1.363 .3 0.991 0.959 pHE t4h.a1n ol 1.41 .6 0.967 0.991 pHp 5H.24 .1 0.81 .4 0.998 0.967 pHp 6H.45 .2 1.60 .8 0.975 0.998 pHp7H.46 .4 1.91 .6 0.983 0.975 pH7.4 1.9 0.983 pHp 8H.18 .1 2.32 .3 0.948 0.948 pHp 9H.69 .6 3.13 .1 0.994 0.994 3.1.3. C3V.1 R.3e.sCpVonRsees opfo tnhsee MofCt-h7e CMelCls- 7inC tehlels PinretsheencPer eosfe tnhcee MofetthaeboMlietetas b olites The infTluheenicnefl oufe tnhcee mofettahbeomlietetas bfroolimte spfHro~m 5.2p Hon~ b5r.e2aosnt (bMreCaFst–(7M) cCaFn–c7e)r ccaenllcse vriaceblillsitvyi ability was mownaistomreodn iwtoirthed cywciltihc cvyoclltiacmvmolteatmrym (CetVry). (FCigVu).rFe i3g ushreo3wsehdo twheed otxhideaotxioidna ptieoankp peoatkepno- tential tial anda ncudrrceunrtr eflnutcfltuuacttiuoantsi oans sthaes rtehseporensspeo vnasreiavbalerisa wblheesnw thhee ncetlhlse wceelrles twreearteedtr weaittehd thwei th the metabomlietetas baot lditieffseartendti fpfeHre. nInt pFHig.uIrne F3iAgu, trhee3 eAm, pthtey eemlecpttryoedlee cdtirdo dneotd eidxhniobtite exlhecibtritoeclheecmtro- chemi- ical rescpaolnrsees,p aosn isned, iacsatinedd ibcyat ae dhobryizaohnotarli zloinnet,a wl lhineere, awsh tehree tarsetahteedt rceealtles dsacmelplslessa mshpolwesedsh owed a quasia-rqeuvaerssi-irbelve eCrsVib pleroCfiVle.p Trohfiel ev.oTlthaegev–oplHta gaet– tphHe saatmthee csoanmceenctorantcieont roaft imonetoabf omlietetasb olites describdeeds carnib iendvearnsei ncvoerrrseelactioornre blaettiwoneebne tmweteanbomlieteta cboonlictenctorantcioen traantdio noxaidnadtioxni dpaetaiokn peak potentipaol,t reenvtieaall,irnegv ae aPlienagrsaonP ecaorrsroenlactioornr ecloaetifofinciceonetf (fiRc2ie) n9t8(.6R42,) a9s8 i.n6 4F,iagsuirne F3Big. uHreow3Be.vHero, wever, the peatkh ecupreraeknct ucrorrernetlactoiorrne leaxtihoinbietexdh iab itreidanagturilanr gwualavrew pavtteerpna tatse ran fausnactfiuonc otifo mn oetfamboe-tabolite lite conccoencteranttiroanti,o ans, sahsoswhonw inn FinigFuirgeu 3rCe .3 C . MCF-7 cells with plant extracts 400 Empty electrodes (A) 200 0 -200 -400 -600 -400 -200 0 200 400 600 800 1000 V/mV Figure 3. Cont. I/μA Separations 2021, 8, x FOR PEER REVIEW 9 of 14 Separations 2021, 8, 26 9 of 14 0.1 0.0 (B) -0.1 -0.2 100 (C) 95 90 85 80 4 5 6 7 8 9 10 pH Figure F3.i g(Aur)e E3ff.e(cAts) oEff ftehcets boiof athcteivbeio caocmtivpeouconmdsp ofruonmd sthfreo pmHt h~e5.p2H on~ t5h.2e odnepthoeladriezpaotliaorni zpaotitoenntpiaolt ential of of the MthCeFM-7C cFe-ll7s.c e(Bll,sC. )( BT,hCe) iTnhfleuiennflcuee onfc tehoef vtohletavgoel toange cuonrrceunrt roefn tthoef MthCe FM-7C cFe-l7lsc ealsl saa fsuancfutinonct ioofn of pH. pH. TheT hcyecclyicc vlioclvtaomltammomgroagmr ameamsuearesmurenmtse ncotsncdoitniodnitsi ownesrwe: eSrcea:nSncianngn firnogmf r6o9m0 m69V0 tmo V97t0o m97V0 mV at a at a scasnc raanter aotfe 1o0f m10Vm sV−1. sM−1C.FM-7C cFa-n7ccearn cceellr vcieallbviliatyb isltiutydsietus doife tshoef btihoeabctiiovaec tciovme pc omunpdosu enxd-s extracted at tracted paHt p5H.2 5a.t2 caetl lcecolln ccoenncternattiroantionf 1of× 1 1×0 160c6e cllesl/lsw/welell.l. 3.1.4. F3lo.1w.4 .CFyltoowmCetyrtyo AmneatrlyysAisn oafl ythsies AofcttihveeA McteitvaeboMlietetasb oonli tCeeslol nBeChealvl iBoerh avior Flow cFyltoowmectyrtyo maneatlryysiasn (aBlyDs iLsS(RBDFoLrtSeRssFao rXt-e2s0s,a BXD-2 B0,ioBsDcieBnicoessc,i eLnec ePso,nLt edPe oCnltadixe, Claix, FranceF) rwanacse c)owndasucctoendd tuoc tiendvetsotiignavtees tthigea mteetchheamnisemch aonf iisnmhiobfitiinonh iboift itohne obfretahset b(MreaCs-t7)( MC-7) cancer ccaenllcse rbyce tlhlseb my etthaebomlietteasb ooblittaeisnoedb tafrionmed pfHro~m5.2p Han~d5 .2thaen rdestuhletsr ecsoumltpsacroemd pwairtehd aw ith a commecrocmiamllye racviaalillaybalev adirlaubgl e(Cdurrucgum(Ciunr),c ausm sihno)w, ans isnh Foiwgunrien 4F. iTghuer equ4a. dTrhaenqtsu raedferrarnetds troe ferred the cellt ocothnedicteiollnc, oans ddiitsipolna,yaesdd inis pthlaey felodwin cythtoemfleotwry cryetsoumltse,t rayfterer sau 4lt8s ,ha ifntecrubaa4t8iohn itnimcueb ation frame. tTimhee sfyrammbeo.lsT hQe1s, yQm2,b Qol3s, Qan1d, Q Q24, oQn3 ,thaen dgrQap4ho nwtehree ugrsaepdh tow reerperuesseendt tdoerberpisr,e dseenatdd ebris, cells, lidveea dcecllesl,l sa,nldiv eapcoelplsto, tainc dcealplso, prteostpiceccteivllesl,yr.e Tspheec tciovnedlyi.tiTonhse ocof nthdeit icoenllss obfetfhoerec ealnlsdb efore after traenadtmaeftnetr wtriethat mtheen dtrwugit,h asth weedllr uags ,tahse wmeeltlaabsotlihteesm, aentadb tohleit erse,suanltds cthome rpeasrueldts wcoitmh pared the conwtritohl tihnediccoantetrdo lthinadt itchaete cdutrhcuatmthine schuorcwuemdi ndrsahmowateicd cderlal mdeaatitchc aetll cdoenactehnatrtactoionnce onft ration 0.02 mogf/m0.0L2 wmigth/omuLt gwoiitnhgo utthgroouinggh thsirgonuigfihcasnigt naipfiocapntotsaips.o Cptoonsvise.rCseolny,v ethrsee lmy,etthaebomlietteasb olites showedsh soowmeed lseovmel eolfe cveelll odfecaethll bdueta twhibthu tsiwgnitihficsaignnt iafipcoanpttoatpico peftfoetcictse. f fects. I/μA E/μV Separations 2021, 8, x FOR PEER REVIEW 10 of 14 Separations 2021, 8, 26 10 of 14 (a) (b) (c) Figure 4. FFliugourrees c4e. nFclueoarcetsivceantecde acectllivsaotretdin cge(llF AsoCrtSin) gan (aFlAysCiSs)o afnthaleyisnish oibfi tthorey inehffiebcittsoroyf ebfrfeeacstts (oMf bCrFe-a7s)t cancer cells using (MCF-7) cancer cells using bioactive extracts at pH 5.2 (best IC50 concentration). The results were bioactive ecxotmrapctasreadt p tHo t5h.e2 i(nbhesibt iItCo5ry0 ceoffneccetsn torfa tai ocno)m. Tmheercrieaslulylt-sawvaeirlaebcloem capnacreedr dtoruthge, ciunchuibrimtoirny. (eaff)e Uctns-of a commercially- available ctarneacteerdd creulgls, ,c (ubc)u cremllisn t.r(eaa)teUdn wtreitaht eedxtcrealclst,, (abn)dc (ecll)s cterlelsa tterdeawteidth weixtthr accut,rcaunmd i(nc), acelll last tcreelalt ceodnwceitnhtrcau-rcumin, all at cell concentitora 6 nt ioofn 1 o×f 110×6 c1e0lls/cwelellsl/. well. 4. Discussion 4. Discussion The current worTkh eseceukrsr eton tpwrooprokssee aelkusmtoinporsoilpicoastee amluinmeirnaolss,i lHicNatTesm, tion ebrea lus,seHdN toT s, to be used to entrap plant meetanbtroalpitepsl afonrt mbioetmabeodliictaels afporplbicioamtioendsi.c aTlhaep bpiloicmatimionetsi.c Tmhaetberioiaml ihmase tbiceemna terial has been used for variousu hseeadltfho ravpaprliiocautsiohnesa ldthuea ptop iltics autinoinqsued uneattuoriatls duensiiqgune cnoantsuisrtailndge osfi genmcpotnys isting of empty cylinders with wciydlitnhds eorfs awboituht w10id0 tnhasnoofmabeoteurts.1 T00hen aXnRoDm, eFtTerIRs., Tahned XTRGDA, FreTsIuRl,tsa ncldeaTrGlyA results clearly imply that the HiNmTpsl ywtahsa ltotahdeeHdN wTisthw thase leoxatdraecdtewdi tchrutdhee eDxRtr.a Tchteed recdruudcetioDnR o. fT XhRe Dre dpueackti on of XRD peak intensities implyi nthteen csrityisetsailmlinpiltyy tohfe thcrey lsotaadlleindi tHyNofTt hise rleodaudceeddH dNueT tois trheed iumcemdodbuileiztaotitohne immobilization of bioactive comopfobuionadcst iovne cthoem HpoNuTn dansdo nthtihse oHbsNerTvaatniodnt hisis ino bcsoenrfvoartmiointyi stoin licteornaftourrme ity to literature which indicates wthhaitc hcriynsdtaicllainteistyt hoaf tHcrNyTst adlelicnrietaysoesf HwNheTn dbeicoraecatsivees wcohmenpobuionadcst icvoemcpomlexp ounds complex with the materiawl i[t4h2]t.h Tehme aDteRr ieaxl t[r4a2c]t. iTsh eexDpeRcteexdt rtaoc bt eis aemxpoerpctheodutso, bheenacme,o trhpeh oobusse,rhveendc e, the observed reduction in the rpeedaukc tiinotneninsittihees poef athkei nHteNnTsi ltoieasdoefdt hweitHh NthTe lDoaRd eexdtrwacitth. Tthhee DFTRIRex stpraecctt.raT he FTIR spectra transmission fintgrearnpsrministssi oonf tfihneg HerNprTin atsndof HthNeTH lNoaTdaendd wHiNthT DloRa dexedtrawcitt hcoDnRfiremxterda ctthcaotn firmed that the the HNTs was lHoaNdTeds wwaisthl otahdee HdNwTitSh. tThheeHreN wTaSs. Tnoh eorbesweravsendo sohbifste irnv ethdes hpiefatkins tohf ethpee aks of the HNT HNT after loadinafgt.e Tr hloisa diminpgl.ieTsh tihsaitm thpeli feusntchtaiot nthael gfuronucptiso nparel sgernotu ipns thpere DseRn texintrathcte mDaRye xtract may not not have interachteadv echinetmericaacltleyd wchitehm thicea lfluynwctiitohntahl egfruonucptsio pnraelsgenrot uinp sHpNreTsse.n Itf itnheHreN wTsa.sI f there was any any chemical intcehreamctiicoanl, itnhteenra ictt iiosn l,iktheleyn tihteis inlitkeerlaycttihoeni nwtearsa nctoito nprwonasounnoctepdr oennoouungche dtoe nough to cause cause any signifaincaynsti/gonbisfiecrvanabt/leo bssheifrtv ainb ltehseh pifetaiknst.h Tehpee arekvs.erTshael oref vtehres apleoafkt hinetpeneasiktiienst ensities back to back to that of thteh aetmopfttyh etuebmupletsy itnudbiucaletessi nthdaict athtees btihoaatctthiveeb cioomacptiovuencdosm epnotruanpdpseedn itnra tphpe ed in the lumen lumen of the HNofTt hareeH trNanTsaierentt raannds ireenvtearnsdiblree.v Terhsuibsl, et.hTeh pulsa,ntth me petlaabnot lmiteesta cbaonli teeassiclayn beea sily be released released using buusffienrg wbiuthff edrifwfeirtehndt ipffHer veanltupeHs, avsa wluaess ,raefslewcatesdr einfl ethctee FdTiInRt ahnedF TXIRRDa snpdeXc-RD spectra. The TGA results further confirmed that the HNT was indeed loaded with the DR extract. The decomposition of the DR extract in the HNT loaded DR was observed between 240–370 ◦C, Separations 2021, 8, 26 11 of 14 and the amount of the DR extract loaded into the HNT was estimated to be approximately 5.1 wt.%. The captured bioactive compounds fail to show significant bioactivity when the crude extracts from the optimization parameters are tested on model cancer cell lines. Though literature reveals DR contains flavonoids, phenolics compounds, alkaloids, and antioxidants, as confirmed by UV studies and other analytical characterizations, our studies show the cells are not compromised in their cell viability in the presence of the metabolites [6,26,43]. However, further studies are required to carry out careful analysis on the extracts to confirm the presence of these metabolites. Nonetheless, the rationale of the current study is to develop a simple procedure to capture metabolites mixtures for further characterization. We used pH-dependent elution of the bioactive compounds from the HNTs to further validate the activity of the captured metabolites on cell death. HNTs have SiOH and AlOH groups, which are found on the outer surface and the inner surface, making the outer and inner surfaces have different charges, respectively. The charge disparity has drawn interest from the research community, whereby overall negatively charged proteins taken above their isoelectric points are mostly loaded into the positively charged tube’s lumen [16]. Thus, depending on the pH conditions, aluminol (AlOH) and the siloxane (SiOH) groups can either be protonated or deprotonated, leading to different affinities towards certain macromolecules and organic compounds. Our hypothesis here was that partially positive metabolites will be weakly attracted to the SiOH groups, whereas negatively charged metabolites will prefer the latter. The pH dependent release of the metabolites from the HNTs are not statistically different after determining the amount in milligram quantities and expressing the entrapment efficiency as a percentage value. However, when tested against the breast (MCF-7) cancer cell lines, the acidic pH elution demonstrates significant anti-proliferative activity against the cancer cell lines compared to the basic pH metabo- lites. The most profound activity is found in the pH~5.2, which is supported by IC50 calculated values. Polarization and depolarization are attributes associated with mitochondrial dysfunc- tion in most cancer cells and can be used to inform the mechanism of cell death [44]. In this work, cyclic voltammetry measurements are used to probe the extent of polarization and depolarization by relating the voltage to current surge using electrochemical detection methods. The results reveal that the metabolites exhibit quasi-reversible redox behavior and concentration dependent reduction in the applied voltage [45]. The currents also show a triangular modulation with a rise in oxidation current at lower pH, follow by another rise beyond acidic pH and further reduction in the strongly basic pH conditions. Metabolites from the pH~5.2 extract require a higher voltage application to generate the minimum amount of current in the cells, indicating that cell membrane polarization in the presence of the metabolite is achieved. The extracts from pH~7.4 and pH~8.1, even though they give higher IC50 values, the voltage required to initiate cell depolarization is at a minimum. Nonetheless, the highlights of the current studies are that the metabolites can cause cell death through a polarization/depolarization mechanism, as documented by other researchers in the literature [46]. Flow cytometer-based analysis shows that the metabolites exhibit dose-dependent apoptosis of MCF-7 cells. It is noted that exposure of 2 mg/mL concentrations of the metabolites leads to a greater than two-fold increase in apoptosis in comparison to the untreated cells. Curcumin is a well-known polyphenol obtained from Curcuma longa, and it is widely used for its anti-oxidative and anti-cancerous application. Curcumin effects on the breast cancer cells are also investigated and compared to the results from the metabolites. It is observed that curcumin improves cell death significantly, without going through the apoptotic phase, indicating the synergistic effect could be developed when both metabolites and curcumin are used to treat cancer. Separations 2021, 8, 26 12 of 14 5. Conclusions In this work, it is demonstrated that optimization of parameters for Dioclea Re- flexa (DR) extracts immobilization on HNT and subsequent releasing the cargo based on pH could find important lead metabolites for discovering druggable entities without going through complex analytical techniques. Such simplified methods will need addi- tional modified analytical tools to expediate the drug discovery pipeline. The work also intend to provide plant metabolites database with fundamental information on herbal medicine isolation and characterization to serve the scientific community in future studies of herbal medicine. Author Contributions: Conceptualization, E.K.T. and S.B.; methodology, B.O.A. and E.N.; software, P.K.A., R.A., and S.B.; validation, E.K.T., and P.K.A.; formal analysis, E.K.T.; investigation, E.K.T.; resources, P.K.A.; funding acquisition, E.K.T. and P.K.A. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Wellcome Trust, grant number 107755/Z/15/Z. Acknowledgments: We would like to thank Lily Paemka (Cancer Biology Laboratory) at the West African Center for Cell Biology of Infectious Pathogens (WACCBIP) for support in providing the facilities and technical assistants for the work. We also thank Solomon Katu and Shadrack O. Aboagye for assisting the team conducting the experiments in the Department of Biomedical engineering. Conflicts of Interest: The authors declare no conflict of interest. References 1. Saqib, Z.; Mahmood, A.; Malik, R.N.; Syed, J.H.; Ahmad, T. Indigenous knowledge of medicinal plants in Kotli Sattian, Rawalpindi district, Pakistan. J. Ethnopharmacol. 2014, 151, 820–828. [CrossRef] 2. Pal, D.; Mandal, M.; Senthilkumar, G.; Padhiari, A. Antibacterial activity of Cuscuta reflexa stem and Corchorus olitorius seed. Fitoterapia 2006, 77, 589–591. [CrossRef] 3. Bhandari, P.; Sendri, N.; Devidas, S.B. 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