**3. Results**

The stepwise procedure in this work probes the mechanism of action of standard drugs for treating microbial infections and the results compared to plant bioactive metabolites and chitosan nanocomposites. Two classes of standard drugs were used for comparison; anti-microbial drugs that include amphotericin B, rifampicin and fluconazole as well as cancer drugs which included curcumin and gossypol. Whereas the plant metabolites were derived from *Dioclea reflexa (DR)*. The therapeutic effect of nanocomposites were also tested. Chitosan nanocomposites were synthesized using chitosan as the base material and Tetraethyl orthosilicate (TEOS) as well as acetic acid as modifiers. Our hypothesis stipulated that drug candidate target membrane environment of cells leading to polarization or depolarization. The outcome in this case would enhance ionic mobility across membranes which could be captured through electrochemical detection as shown in **Figure 1**.

The various standardized drugs, plant metabolites as well as the synthesized nanocomposites used to investigate the electrochemical behaviour of the cells are displayed in **Table 1**. The cell lines and their sources are also indicated in **Table 2**. First, the electrochemical behaviour of *S. cerevisiae* cells was investigated using the standard antimicrobial drugs, amphotericin B, fluconazole and rifampicin. As shown in **Figure 2**, fluconazole and rifampicin exhibited very limited changes in the anodic peak potential (**Figure 2A** and **B**) compared to the amphotericin B doped *S. cerevisiae* cell lines (**Figure 2C**). The results obtained confirmed membrane polarization/depolarization behaviour of the *S. cerevisiae* due to the

#### **Figure 1.**

*Schematic illustration of the mechanism of drug interaction with biological membranes and how its electrochemical response (using a miniature electrode) correlates to cell viability as captured by the a cell counting device (Cellometer) [33].*


*\* Extracted or synthesized and unpurified.*

*1 The following composites were tested: CT; chitosan modified by TEOS followed by acetic acid, CC; chitosan modified with TEOS/acetic acid mixture, CA; chitosan modified with acetic acid, and CT, chitosan modified with TEOS. 2 Plant metabolites obtained from pH ~4.1–9.6.*

#### **Table 1.**

*List of compounds used for the study.*

presence of amphotericin B leading to increase ionic mobility. Fluconazole and rifampicin which are not directly linked to membrane destabilization as the former are mainly responsible for RNA synthesis inhibition and rifampicin being responsible for inhibition of ergosterol synthesis.

A correlation between cell death and electrochemical response was established using *S. cerevisiae* cell lines and MTT assay detection. Although the result indicated cell death in the presence of the antimicrobial drugs, Amphotericin B exhibited


#### **Table 2.**

*Cell lines and Detection methods used for Investigating membrane mediated effects.*

enhanced cell death probably due to the high degree of membrane permeability of ions as indicated in the voltammograms in **Figure 2C**.

To test the electrochemical behaviour and redox activity of the *Dioclea reflexa* extracts, cyclic voltammetry analysis was conducted using interdigitated gold electrodes (IDEs), (Metrohm, DropSens). The current from the quasi-reversible oxidation curve was plotted against concentration of the plant metabolites. **Figure 3A** (*control, black*) indicated insignificant redox mechanism, however, the extracts showed quasi-reversible oxidation values ranging from 0.25 to 0.70 mA at a scan rate of 10 mV/s. The water extract (SWE) had higher redox potential compared to those of the ethanol extract (SEE). The water extract (SWE) *(red)* demonstrated the most cell death followed by methanol extract (SME) (*purple*) and ethanol extract (SEE) (*blue*) respectively with the control cells *(black*) exhibiting the least cell death at higher concentrations and extended incubation time periods. *SWE* (*red*) revealed cell death of about 57%, whereas *SME (purple), SEE (blue*) recorded about 31 and 22% respectively at the same concentration. It was concluded that the extracts caused membrane porosity to initiate reactive oxygen species release leading to cell death.

Another important strategy in plant phytochemical studies is to develop local immobilization materials that can capture bioactive plant metabolites and release the cargo steadily onto diseased cells. Optimization parameters were developed to capture plant metabolites from *Dioclea Reflexa (DR)* seed extracts on halloysites nanotubes (HNTs). An encapsulating capacity of 13% was obtained when approximately 5 g of *DR* extracts was immobilized onto about 1 g of HNTs. Evidence of plant metabolites entrapment was monitored with FTIR and X-ray diffraction methods. As shown in **Figure 4A**, changes in the FTIR signatures peak intensities of the halloysite nanotubes (HNTs) revealed all the functional groups present in the empty halloysites nanotubes (black). The inner Al-OH and outer Si-OH groups have characteristic stretching peaks at 3624 and 3691 cm−1, respectively. Bending vibrations of Al-OH and Si-O revealed absorption peaks at 907 cm−1. In addition, the uneven stretching vibrations of the Si-O bond gives a strong absorption peak at 1005 cm−1. There was a significant reduction of the transmission peak after immobilization of the *DR* extracts on the halloysites nanotubes (blue) and this indicated a modification of the nanotubes with the plant metabolites. Following the release of the bioactive compounds from the nanotubes, the transmission peaks reverted to the original peaks of the empty nanotubes (red). Similarly, **Figure 4B**, showed the characteristics 2θ ° peak positions of the nanotubes which occurs at 11.7, 20.5, 24.8, 37.5, 43.3 and 64.4° (red). After immobilization of the *DR* extract on the nanotubes, there was dramatic reduction of peak intensities at the same 2θ ° positions and that indicated chemical modifications of the nanotubes by the bioactive constituents in *DR* extract (black). The bioactive constituents were eluted with 70% ethanol and resulted in the reversal of the nanotubes peaks as shown in **Figure 4B** (blue). The characteristic peak intensities reverted to those observed in the control.

**Figure 2.** *Cyclic voltammetry response of* S. Cerevisiae *in the presence of (A) rifampicin, (B) fluconazole, and (C) amphotericin B on IDE electrode (Colour code: black, antibiotic; red, cells and blue, cell + antibiotic).*

The antiproliferative activity of the crude extracts did not reveal significant inhibitory effects on breast (MCF-7) cancer cells, however, the pH-dependent eluted 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 MTT viability test as shown in **Table 3**. pH ~ 5.2 demonstrated IC50 of 0.8 mg and a cyclic voltammetry oxidation peak potential and current of 234 mV and 0.45 μA respectively indicating membrane polarization/ depolarization of the cancer cells as shown in **Figure 5**. It was confirmed through fluorescence-activated cell sorting (FACS) studies that the plant metabolites influenced breast cancer apoptotic signaling pathways of cell death as shown in **Figure 6**. The studies proved that plant metabolites could be captured using simplified screening procedures for rapid drug discovery purposes. Such procedures, however, would require the integration of affordable analytical tools to isolate individual metabolites

**Figure 3.**

*(A) A graph of current versus concentration of plant extracts revealed the water extract generated highest current, followed by the methanol extract, ethanol extract and the control cells in that order. (B) Investigating cell death as a function time. The water extract revealed significant cell death (*red*) after 2 days of incubation, followed by the methanol extract, ethanol extract and control in that order.*

for testing. Our approach could be an important strategy to create plant metabolite database based on pH values.

Biopolymers such as chitosan, gelatin and cellulose have been used with different additives in order to modify their surfaces for biomedical application. In the

*Electrochemical Response of Cells Using Bioactive Plant Isolates DOI: http://dx.doi.org/10.5772/intechopen.95360*

#### **Figure 4.**

*(A) Characterization of HNTs and loaded HNTs with plant metabolites using FTIR techniques. Reduction in IR transmittance indicates OH on the HNTs are functionalized by at least one bioactive. (B) XRD spectra of the entrapped metabolites showing the signature peaks at two theta position of HNTs and loaded HNTs with plant metabolites. Intensity has inverse relationship to surface area. Larger area due to immobilization will cause a decrease peak intensity.*

next paragraph, a description of the synthesis of chitosan nanocomposites using tetraorthosilicate (TEOS) and acetic acid (AA) to study their influence on prostate cancer (PC3) cell lines would be described. The particles synthesized for the study include *SC*; chitosan modified by TEOS followed by acetic acid, *CC*; chitosan modified with TEOS/acetic acid mixture, CA; chitosan modified with acetic acid, and


#### **Table 3.**

*Experimental IC50 values of the extracts eluted at different pH conditions all measured in milligram quantities of the seed extract.*

*CT*, chitosan modified with TEOS. As shown in **Figure 7A** and **B**, The electrochemical response of normal cells and prostate cancer cells (PC3) when treated with the particles revealed the latter showed modest response whereas the PC3 cells anodic peak currents changed dramatically with the treatment with the nanocomposites especially the *SC* nanoparticles. The cell viability studies revealed a corresponding decrease in cell viability as measured by a cell counting device. The normal cells again showed no significant difference in cell viability after 24–48 hours of cell growth as shown in **Figure 7C** and **D**. The results were compared with a standard drug used to treat cancers, gossypol (GP).

### **4. Discussion**

In the current work, a systematic approach was undertaken to carefully investigate standard drugs, plant metabolites and chitosan nanoparticles on the electrochemical behaviour of selected cell lines and also to correlate their electrochemistry to cell viability.

It is becoming evident that taking advantage of the numerous redox mediators, scientists could develop biosensors from many biological systems. For example, *S. Cerevisiae* has several Redox centers which could be exploited using hydrophilic/ hydrophobic molecules as extensively discussed previously by *Rawson et al.* [1, 2]*.* The fact that Electrochemical behavior could be monitored using membrane targeted drugs [42–45], opens avenue for future development of a biosensor for identifying potential drug candidate from plant sources. For example, the famous antifungal drug, Amp B has been used to treat fungal infection effectively and its mechanism of action has been well characterized [46, 47]. It is established that the antifungal drug binds to ergosterol in the cell membrane to enhance leakage of ions leading to depolarization of the membrane [48]. Increase in ions leakage across membrane could ultimately increase the oxidation potential across membranes. Such source of ions can be detected through electrochemical techniques as already observed in our studies as well as studies from other groups [2].

The fact that Amp B and the plant extracts behave similarly on *S. cerevisiae* cell viability supported a general claim that Amp B and the plant extracts exhibited a common mechanism leading to cell death. Hence, due to the quasi-reversible oxidation process observed in the anodic response, it was concluded that membrane *Electrochemical Response of Cells Using Bioactive Plant Isolates DOI: http://dx.doi.org/10.5772/intechopen.95360*

#### **Figure 5.**

*(A) Effects of the bioactive compounds from the pH ~5.2 on the depolarization potential of the MCF-7 cells. (B) The influence of the voltage on current of the MCF-7 cells as a function of pH. The cyclic voltammogram measurements conditions were: Scanning from 690 mV to 970 mV at a scan rate of 10 mV s−1. MCF-7 cancer cell viability studies of the bioactive compounds extracted at pH ~ 5.2 at cell concentration of 1 x106 cells/well [submitted results for publication, Scientific Reports].*

polarization/depolarization leading to ionic leakage might have been the mechanism through which one or more of the organic bioactive molecules illicit their action. This correlation had highlighted an important opportunity that could be further exploited for identifying bioactive plant metabolites in the natural product field.

We used pH dependent elution of the *DR* bioactive compounds from the halloysites nanotubes to further validate the activity of the captured metabolites on electrochemical behaviour and cell death. Halloysite nanotubes have SiOH and AlOH

**Figure 6.**

*Fluorescence activated cell sorting (FACS) analysis of the inhibitory effects of breast (MCF-7) cancer cells using bioactive extracts at pH ~ 5.2 (best IC50 concentration). The results were compared to the inhibitory effects of a Commercially available cancer drug, cucurmin. (A) untreated cells, (B) cells treated with extract and (C) cells treated with curcumin all at cell concentration of 1 x106 cells/well.*

groups which are found on the outer surface and the inner surface making the outer and inner surfaces to have different charges respectively [10, 49, 50]. Thus, depending on the pH conditions, aluminol (AlOH) and the siloxane (SiOH) groups could either be protonated or deprotonated leading to different affinities towards certain macromolecules and organic compounds. Our hypothesis was that partially positive metabolites will be weakly attracted to SiOH groups whereas negatively charged metabolites would prefer the latter. The pH dependent release of the metabolites from the HNT were 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 demonstrated significant anti-proliferative activity against the cancer cell lines compared to the basic pH metabolites. The most profound activity was found in the pH ~ 5.2 which was supported by IC50 calculated values.

Depolarization is an indicator of mitochondrial dysfunction in most cancer cells and therefore investigating polarization and depolarization could inform the mechanism of cell death [51]. In this work, Cyclic voltammetry measurements were used to probe the extent of polarization and depolarization by relating the voltage to current surge using electrochemical methods. The results revealed that the metabolites exhibited quasi-reversible redox behavior and concentration dependent reduction in the applied voltage [52]. The currents also showed a triangular modulation with a rise in oxidation current at lower pH, followed by another rise beyond acidic pH and further reduction to the strongly basic pH. Metabolites from the pH ~ 5.2 extract required a higher voltage application to generate the minimum amount of current in the cells indicating cell membrane polarization in the presence of the metabolite was achieved. The extracts from pH ~ 7.4 and pH ~ 8.1, even though gave higher IC50 values, the voltage required to initiate cell depolarization

#### **Figure 7.**

*Correlation electrochemical response of the (A) normal cells and (B) prostate cancer cells (PC3) as a function of chitosan composite treatment.* SC*; chitosan modified by TEOS followed by acetic acid,* CC*; chitosan modified with TEOS/acetic acid mixture,* CA*; chitosan modified with acetic acid, and* CT*, chitosan modified with TEOS The corresponding cell viability as measured by the cell counting device. (C) normal cells and (D) cancer cells.*

in the cells was at a minimum as indicated in the maximum current generation. The results highlighted that the metabolites could cause cell death through a polarization/depolarization mechanism as documented by other researchers in the literature [53]. Flow cytometer-based analysis showed that the metabolites showed dose-dependent apoptosis of MCF-7 cells. It was noted that exposure of 2 mgmL−1 concentrations of the metabolites led to greater than two-fold increase in apoptosis in comparison to the untreated cells. Curcumin is a well-known polyphenol and widely used for its anti-oxidative and anti-cancerous application. Curcumin effects on the breast cancer cells were also investigated and compared with the result from the metabolites. It was observed that curcumin improved cell death significantly without going through the apoptotic phase indicating synergistic effect could be

developed when both metabolites and curcumin are used to treat cancer. Finally, the chitosan nanoparticles also demonstrated that membrane polarization and depolarization could also be used to monitor particle permeation into cell membrane to induce varied cell behaviour.
