Improved antioxidant, antimicrobial and anticancer activity of naringenin on conjugation with pectin

Abstract

The purpose of the present study was to improve the aqueous solubility of naringenin by conjugating with water-soluble polysaccharide carrier, pectin. The pectin–naringenin conjugate was synthesized employing dicyclohexylcarbodiimide and dimethylaminopyridine. The conjugation was confirmed by various physicochemical characterizations. The results of differential scanning calorimetry, X-ray diffraction and morphological analyses revealed semi-crystalline nature of the conjugate. The chromatographic analysis showed 37.069 µg naringenin/mg of conjugate. The conjugate was determined to have molecular weight of 6.22 × 10⁴ kDa by static light scattering. In silico molecular mechanistic simulations performed for pectin and naringenin revealed the energetic and geometrical stability within the polysaccharide-polyphenol conjugate. The critical aggregation concentration was in the range of 44.67–56.23 μg/mL as determined by dynamic light scattering and fluorescence spectroscopy. On in vitro release, 99.4% (pH 1.2) and 57.62% (pH 7.4) of naringenin were found to be released over a period of 30 h and 48 h, respectively. Further, the release of naringenin followed Higuchi’s square-root kinetics with diffusion as the possible release mechanism. A comparative evaluation for antioxidant activity revealed a significantly higher radical scavenging activity of conjugate over the naringenin. Further, the conjugate exhibited significantly higher antimicrobial action against Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa while a comparable antimicrobial activity was observed against Escherichia coli and Bacillus subtilis. The cytotoxicity studies of the synthesized conjugate showed anti-cancer activity against NIH: OVCAR-5 cells. In conclusion, the pectin-naringenin conjugate presented hydrocolloidal properties with improved therapeutic efficacy and delivery over the native polyphenol.

Electronic supplementary material

The online version of this article (10.1007/s13205-019-1835-0) contains supplementary material, which is available to authorized users.

Introduction

Flavonoids are polyphenolic phytochemicals existing in a wide variety of fruits and vegetables. They are categorized as flavanones, flavones, flavonols and isoflavonols. Flavonoids are present in almost all foods of plant origin (Kyle and Duthei 2006). These natural compounds have gained interest because of their extensive pharmacological effects on biological systems such as anti-oxidant (Saleh et al. 2017), anti-inflammatory, anti-microbial (Tripathi et al. 2019), anti-allergic and anti-cancer activities. Naringin is a flavonoid glycoside, found mainly in grapefruit. Naringenin (5,7-dihydroxy-2-(4-hydroxyphenyl)-chroman-4-one) is an aglycone of naringin (Erlund 2004). Naringenin also exhibits a broad spectrum of pharmacological activity yet it has not been fully explored as a therapeutic agent due to its unfavourable pharmacokinetics. The major problem associated with the usage of naringenin and naringin as nutraceuticals is their low solubility and bioavailability. To address these issues of low solubility and poor bioavailability, various attempts were made in the past using nanoparticles (Ji et al. 2016), complexation with β-cyclodextrin (Semalty et al. 2014), solid dispersions (Khan et al. 2014), liposomes (Tsai et al. 2015) and lipid carriers such as self nanoemulsifying drug delivery systems (Khan et al. 2013).

The conjugation of a drug with natural polysaccharide has been reported to improve the physicochemical and pharmacological properties of the attached moiety (Pasut and Veronese 2007). Polymer-drug conjugation of a variety of biopolymers such as sodium alginate (Dey and Sreenivasan 2014; Sarika et al. 2016), pectin (Bai et al. 2017; Mundlia et al. 2018), hyaluronic acid (Manju and Sreenivasan 2011), gum arabic (Sarika et al. 2015) have been reported for various biomedical applications. The conjugation not only protects the drug from degradation but also imparts enhanced bioavailability and water solubility. During earlier studies, the conjugates of poorly water-soluble bioactives such as curcumin with hydrocolloids (Bai et al. 2017) have already been reported. This aroused our interest to synthesize hydrocolloid conjugate of naringenin which has not been reported sofar.

Pectins are a family of hydrocolloids consisting of partially esterified α-(1–4)-d-galacturonic acid residues. It is an attractive natural polysaccharide due to its biocompatibility and biodegradability and thus, finds application in food, cosmetic and pharmaceutical industry (Sriamornsak et al. 2008). The special structure of pectin with numerous carboxyl and hydroxyl groups present along the backbone makes it a vulnerable polymer for modification and as a hydrophilic carrier for various drug molecules (Chen et al. 2015). Developing drugs as hydrophilic conjugates with pectin may prove to be a suitable strategy to improve drugs’ stability and bioavailability. Pectin may carry the covalently attached naringenin molecules to the desired site thereby increasing its concentration inside the cell before the biodegradable ester bond gets hydrolysed/breaks to release the active moiety.

The aim of the present investigation is to conjugate pectin, a water-soluble polysaccharide to naringenin, a hydrophobhic drug to enhance its therapeutic efficacy. To study its stability, molecular modeling via static lattice atomistic simulations has also been carried out. Additionally, the synthesized conjugate was characterized for physicochemical characteristics (NMR, FTIR, DSC and XRD), morphological properties (scanning electron microscopy) and colloidal properties (dynamic light scattering and fluorescence spectroscopy). The release of naringenin from the conjugate was investigated and various release kinetic profiles were generated. The preliminary anti-cancer potential of the polysaccharide-polyphenol conjugate was also studied.

Experimental

Materials

Naringenin (MW = 272.26, purity > 95%) was procured from HiMedia Lab. Pvt. Ltd. (Mumbai, India). Pectin (GENU^®pectin (citrus) type USP/100, CP Kelco US, Inc.) was procured as an ex-gratia sample from Burzin and Leons Agenturen Pvt. Ltd. (Mumbai, India). N, N′-dicyclohexylcarbodiimide (DCC), 4-Dimethylaminopyridine (DMAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH) and dialysis tubing cellulose membrane (MWCO 3500 Da) were purchased from Sigma-Aldrich (Bangalore, India). Buffer salts and solvents were supplied by Thermo Fischer Scientific India Pvt. Ltd. (Mumbai, India). The silica plates were precoated and were purchased from Macherey–Nagel GmbH & Co. KG (Germany). NIH: OVCAR -5 cells were collected from Dr. Tom Hamilton, Fox Chase Cancer Institute, PA, USA. All the reagent grade chemicals were used as received.

Esterification of pectin with naringenin

Naringenin was conjugated to pectin by an esterification reaction previously reported elsewhere by the researchers and was based on the method proposed by Sarika et al. (2015) with slight modifications (Sarika et al. 2015). Briefly, pectin (500 mg) was added to DMSO (50 mL) to form dispersion and was kept for stirring at 25 °C. The carboxylic groups of polymer were activated using DCC (80 mg) while DMAP (30 mg) was added as a coupling agent to the pectin dispersion, and stirred for another 2 h at 25 °C. A 10 mL solution of naringenin in DMSO (25 mg/mL) was slowly added under nitrogen atmosphere to the activated polymer dispersion and stirred for 6 h at 60–65 °C. The cream coloured solution so obtained was subjected to dialysis against DMSO (for 12 h) to remove the unreacted molecules followed by dialysis against distilled water (for 72 h) to remove the solvent. The dialyzed polymer-drug conjugate mixture was frozen at − 75 °C for 4 h prior to its lyophilisation (Alpha 2–4 LD Plus, Martin Christ, Germany) at 0.0010 mbar and − 76 °C for 24 h. The pectin-naringenin (Pec-Nrg) conjugate so obtained was kept in dessicator for future use.

Characterization of Pec-Nrg Conjugate

Ultraviolet visible (UV) absorption spectroscopy

The UV absorption spectra of naringenin and Pec-Nrg conjugate was recorded using UV–Vis-NIR spectrophotometer (Cary 5000, Varian, Netherland). The solutions of naringenin and Pec-Nrg conjugate (1 mg/mL) prepared in water: DMSO (50: 50 v/v) were scanned in the range of 200–600 nm to study any shift in the absorption maxima.

Fourier transform infrared (FTIR) analysis

FTIR investigations of naringenin, pectin and Pec-Nrg conjugate were accomplished using Fourier–transform infrared spectrophotometer (IR Affinity-I, Shimadzu, Japan). The spectrum was obtained using diffuse reflection method. Background measurement is first performed on KBr. The sample powder is diluted to 1% in KBr and then placed into sample plate to carry out the infrared spectrum measurement over a wave number region of 400–4000 cm⁻¹.

¹H nuclear magnetic resonance (NMR) analysis

To investigate the bonding/conjugation mode of naringenin with pectin, ¹H NMR analyses of naringenin and Pec-Nrg conjugate were studied in DMSO-d₆ using 400 MHz spectrometer (Bruker Ascend 400, Bruker Corporation, Faellanden, Switzerland). The typical parameters set for NMR analysis were: 20 scans; 1 s relaxation delay; 297.9 K pulse degree. The chemical signals were determined in terms of parts per million (ppm).

Differential scanning calorimetry (DSC) analysis

Thermal characterization of naringenin, pectin and Pec-Nrg conjugate was performed using differential scanning calorimeter (TA instruments, DSC25, California, USA) and analysed using TRIOS V4.1 software. A 5 mg of sample sealed securely in a standard aluminium pan was heated at a temperature range of 40–300 °C with a heating rate maintained at 10 °C/min under constant N₂ flow and the cell/base purge were kept at 50/350 mL/min, respectively.

X-ray diffraction (XRD) analysis

Study of crystal structures and atomic spacing of pectin, naringenin and Pec-Nrg conjugate was performed using a Benchtop X-ray diffractometer (Miniflex 600, Rigaku Corporation, Tokyo, Japan) maintained at 40 kV and 15 mA and were subjected to measurements from 3° to 90° at a scan speed of 10°/min.

Scanning electron microscopy (SEM) analysis

The surface morphology of the native polymer, drug and conjugate were studied using FEI Nova Nanolab 600 SEM (FEI, Hillsboro, Oregon, USA). The photomicrographs of the samples were captured at various magnifications after sputter coating with gold/palladium. The scans were further processed for brightness and contrast using Paint.net (freeware raster graphics editor program).

High-performance liquid chromatography (HPLC) analysis

A previously reported and validated HPLC method was used to determine the naringenin content in Pec-Nrg conjugate (Wang et al. 2017). The resolution of naringenin was carried out using a HPLC system (1260 Infinity series, Agilent Technologies, Germany) comprising of the manual injector (G1328C, 1260 Agilent Technologies, Germany) and a photodiode array detector (1260 DAD-VL, Agilent Technologies, Germany). Freshly prepared aqueous solutions of conjugate were first filtered through nylon syringe filter (0.22 μm) and then injected (20 μL) to pass through C18 (Zorbax SB USA) column of pore size 5 μm and dimensions 4.6 × 150 mm. The mobile phase used was methanol: water (0.1% o-phosphoric acid) (50: 50) at a flow rate of 1 mL/min, with signal being detected at 290 nm. The time for the analysis was 10 min. The retention time of naringenin was 7.5 min. The naringenin concentration was calculated using a calibration curve of standard naringenin (2–40 μg/mL), with regression equation of the plot, $y=2037x+5491$ (R² = 0.996).

Estimation of critical aggregation concentration (CAC)

To assess the self-aggregation behavior of the conjugate, critical aggregation concentration (CAC) was determined using fluorescence spectroscopy and dynamic light scattering (DLS) techniques. Briefly, pyrene solution (50 μL) of concentration 0.5 μg/mL in acetone was placed in vials and evaporated. Aqueous solutions (10 mL) of Pec-Nrg conjugate (0.05–1000 μg/mL) were added to the pyrene vials followed by sonication in bath sonicator (Powersonic 405, Hwashin Technology, Seoul, Korea) for 40 min and further left undisturbed overnight. The conjugate solutions were analysed for the emission spectra in spectrofluorophotometer (RF-5301PC, Shimadzu, Japan) at room temperature from 350 to 500 nm (excitation wavelength = 334 nm: slit widths = 5 nm. The intensity ratio (I₃₇₃/I₄₇₅) of fluorescence bands of pyrene was plotted on ordinate against the logarithm of the conjugate concentration on the abscissa. The CAC was obtained by plot extrapolation on abscissa and determining the point of inflection (Guo et al. 2013).

The determination of critical aggregation concentration by DLS was carried out by measuring average particle size. Different concentrations of Pec-Nrg conjugate from 0.05 μg/mL to 1000 μg/mL prepared in HPLC grade water were analyzed at 25 °C in a particle sizer (Zetasizer, Nano ZS90, Malvern Instruments, UK). One milliliter of the conjugate dispersion was scanned (n = 3) at an angle of 90° with an equilibration time of 120 s (disposable sizing cuvettes DTS0012).

Molecular weight studies by static light scattering

The average molecular weight of pectin and Pec-Nrg conjugate was determined using a Malvern Zetasizer Nano ZS90 instrument (Malvern Instruments, UK) by static light scattering technique (Puskas et al. 2013). Various graded concentrations of pectin and Pec-Nrg conjugate were prepared in HPLC grade water (0.05–1000 μg/mL) and stirred at 25 °C in bath sonicator for 15 min. One milliliter of the sample solution was scanned in automated mode using toluene as a reference with standard refractive index of 1.496 and standard Rayleigh ratio of 1.35 × 10⁻⁵ at 25 °C (glass cuvette PCS1115).

In silico molecular profiling of Pec-Nrg conjugate

In silico molecular simulations were employed to elucidate and confirm the potential sites of ester formation and conjugation between pectin and naringenin. Briefly, static lattice atomistic simulations (SLAS) were carried out using default molecular mechanics algorithm and geometrical/energetic minimizations (HyperChem™ 8.0.8 Molecular Modeling Software, Hypercube Inc., Gainesville, FL, USA). The structures of polysaccharide (pectin) and naringenin was developed using the saccharide building module and natural bond angles, respectively. The individual structures were optimized with respect to energy and geometry employing MM + Force Field algorithm followed by AMBER 3 (Assisted Model Building and Energy Refinements) Force Field algorithm. The lowest energy conformers were then employed to develop the final molecular complex of Pec-Nrg conjugate by parallel disposition technique followed by energy-minimization. The Polak–Ribiere Conjugate Gradient method was employed to carry out full geometrical optimization until an RMS gradient of 0.001 kcal/mol was achieved (Kumar et al. 2012).

In vitro release

A comparative release profile of naringenin from naringenin (API) and Pec-Nrg conjugate was evaluated in vitro at acidic and basic pH of 1.2 and 7.4, respectively, using the dialysis method (Dey et al. 2015). The pre-activated dialysis membrane was first loaded with the dispersion of 10 mg of naringenin or conjugate (equivalent to 10 mg naringenin) in 5 mL of medium (pH 1.2 or 7.4) and the loaded sacs were then immersed in 10 mL of the corresponding buffer after tightly securing their ends with cable ties. The system was then transferred in orbital shaker-cum-incubator (Colton, Narang Scientific Works, New Delhi, India) at 37 °C and shaked at 100 rpm. At pre-determined time intervals (up to 48 h), 0.5 mL aliquots were sampled and naringenin content was analyzed by HPLC. To maintain the sink conditions, equal volume (0.5 mL) of the release media was replaced.

In vitro antioxidant activity

The antioxidant activity of naringenin and its conjugate was examined by DPPH scavenging assay (Chandrasekhar et al. 2006). Ascorbic acid was used as a reference standard. DPPH stock solution of 1 mg/mL was prepared in methanol. An aliquot of 100 µL of DPPH stock solution was added to 100 µL of each naringenin and conjugate solution (equivalent naringenin) prepared in methanol. The solutions were then thoroughly mixed and allowed to stand in the dark at room temperature for 30 min for reaction to take place. The samples were then analysed by HPLC to measure % inhibition. The chromatographic system (1260 Infinity series, Agilent Technologies, Germany) consisted of C18 (Zorbax SB USA) column of pore size 5 μm and dimensions 4.6 × 150 mm, equipped with manual injector (G1328C, 1260 Agilent Technologies, Germany) and a PDA detector (1260 DAD-VL, Agilent Technologies, Germany). The elution was carried out using methanol: water (80:20) at a flow rate of 1 mL/min, with signal being detected at 517 nm. The peak of DPPH appeared at 4.2 min. The total free radical scavenging capacity was calculated using the formula-

$$\text{Radical}\text{scavenging}\left(\%\right)=\frac{\text{PA}\text{blank}-\text{PA}\text{sample}}{\text{PA}\text{blank}}\times 100$$

where PA blank is peak area of blank, PA sample is peak area of sample.

Antibacterial activity evaluation

Minimal inhibitory concentration

The minimal inhibitory concentration of Pec-Nrg conjugate was compared with naringenin, and pectin by broth dilution assay (Nguni et al. 2015) against strains of Gram +ve bacteria—Staphylococcus aureus MTCC 7443, Staphylococcus epidermis MTCC 435, Bacillus subtilis MTCC 441 and Gram −ve strains Escherichia coli MTCC 1652, Pseudomonas aeruginosa MTCC 424. Nutrient medium was prepared by dissolving an accurately weighed amount of nutrient broth in distilled water. One mL of this nutrient medium was transferred to test tubes containing serial dilutions ranging from 3.12 to 50 µg/mL of Nrg, Pec-Nrg conjugate (equivalent naringenin) and pectin prepared in DMSO. The solutions were then incubated at 37 °C for 24 h. Minimum inhibitory concentration was then determined by the visual examination, the sample of minimum concentration which was optically clear was recorded as minimum inhibitory concentration (MIC).

Bacterial growth curve

The effect of naringenin and Pec-Nrg conjugate on the growth kinetics of Staphylococcus aureus MTCC 7443, Staphylococcus epidermis MTCC 435, Bacillus subtilis MTCC 441 and Gram −ve strains Escherichia coli MTCC 1652, Pseudomonas aeruginosa MTCC 424 were determined by culturing the bacterial suspensions in the presence of different concentrations of naringenin and Pec-Nrg conjugate (Sureshkumar et al. 2010). Briefly, 100 µL of the bacterial suspensions were added to 5 mL of the nutrient broth followed by incubation at 37 °C in an orbital shaking incubator at 100 rpm. The bacterial growths were assessed by measuring optical density at 600 nm in spectrophotometer.

In vitro cytotoxicity analysis

The potential anti-cancer activity of the Pec-Nrg conjugate was tested on NIH: OVCAR-5 cells using the MTT assay (MTT-based Sigma Cell Proliferation Kit I) as described previously by the researchers (Mundlia et al. 2018). Briefly, the cells were seeded in a DMEM: Ham’s F12 media (3:1) supplemented fetal bovine serum, glutamine, sodium bicarbonate, and an antibiotic solution and incubated at a humid, 37 °C and 5% CO₂ atmosphere. The cells at a concentration of 1.5 × 10⁴ cells/well were exposed to varying concentrations of free naringenin in medium (6.25–100 μg/mL) and the conjugate (equivalent naringenin) for 48 h. Complete medium and pectin dispersed in the medium were used as control and blank, respectively. The cell viability was quantitatively assessed by determining the absorbance at 570 nm using a multi-plate reader (VICTOR Multilabel Plate Reader, Perkin Elmer, USA) and was calculated as percentage cell viability relative to the control.

Statistical analysis

Statistical calculations were carried out employing GraphPad InStat 3 (GraphPad Software Inc., California). The results of cytotoxicity (MTT assay) on NIH: OVCAR-5 cells were subjected to one-way analysis of variance (ANOVA) followed by Tukey–Kramer multiple comparisons test. The unpaired t test was used for testing significance of free radical scavenging activity (DPPH assay). The P value < 0.0001 was considered extremely significant.

Results and discussion

Synthesis of conjugate

DCC/DMAP coupling reaction was employed to carry out the conjugation of pectin with naringenin. The activated –COOH functionality of pectin interacted with –OH group of naringenin to form Pec-Nrg conjugate (Scheme 1). The conjugate of the polysaccharide so obtained after lyophilization was off-white film like matrix with an average naringenin content of 37.069 µg/mg of the conjugate. The successful linkage of pectin with naringenin was assured by performing thin layer chromatography showing retention factor (R_f) of 0.77 and 0.46 for naringenin and Pec-Nrg conjugate, respectively, using the mobile phase toluene: ethylacetate: formic acid:: 3:2:0.4 (ALUGRAM SIL G/UV₂₅₄ TLC plates).

Scheme 1.

Synthesis of Pec-Nrg conjugate

Characterization of Pec-Nrg conjugate

Pec-Nrg conjugate so synthesized was spectroscopically characterized by UV, FTIR, NMR, DSC, and X-ray diffraction while the morphology was assessed employing SEM. The effect of conjugation on the UV absorption of naringenin was studied by comparing the absorption spectra of naringenin and newly synthesized conjugate. Naringenin showed an absorption maximum (λ_max) at 291 nm, while the Pec-Nrg conjugate exhibited λ_max of 281 nm (Fig. S1). The blue shift in the absorption maxima can be attributed to an ester bond between the –COOH functionality of pectin with the –OH group of naringenin.

The IR spectra (Fig. S2) of naringenin exhibited characteristic absorption peaks at 3285 (–OH stretching vibration), 3055 (aromatic stretching vibration of C–H), 1629 (C=O stretching vibrations), 1601 (aromatic stretching due to C=C), 1462 (bending vibrations of CH₂), 1386 (bending vibrations of CH₃) and 1014 cm⁻¹ (C–O stretching). The FTIR spectrum of pectin showed characteristic absorption peaks at 3443 (–OH of aliphatic alcohol), 2930 (–CH₂ stretch), 1720 (–C=O stretch of –COOH) and 1103 cm⁻¹(–C–O stretch of R–OH). Pec-Nrg conjugate presented bands corresponding to both pectin and naringenin wherein 3417 cm⁻¹ represented –OH stretch of aliphatic alcohols (pectin) and 2918 cm⁻¹ due to asymmetric stretch of CH₂ (pectin and naringenin). Importantly, the appearance of 1741 cm⁻¹ confirmed the C=O stretching of ester linkage while the peak at 1637 cm⁻¹ represented the conjugated C=C and C=O of inter-ring chain.

¹H NMR (Fig. S3) analysis provided further confirmation of the synthesis of naringenin conjugate with hydrocolloid. The spectrum of naringenin shows the characteristic NMR signals at 9.7, 10.8 and 12.2 due to the hydroxyl groups. However, these NMR signals are missing in the ¹H NMR spectra of Pec-Nrg conjugate which indicate the participation of these hydroxyl groups in conjugation reaction. The low resolution of ¹H NMR spectra of pectin was in corroboration with previously reported literature and was attributed to the gelling properties of pectin (Muller-Maatsch et al. 2014).

Figure 1a shows DSC thermogram of naringenin, pectin and Pec-Nrg conjugate. Thermal curve of naringenin shows a sharp endothermic peak at 253.54 °C having an onset at 250.51 °C and endset at 259.24 °C with the enthalpy of fusion of 175.97 J/g. Thermogram of pectin presents a broad endotherm and exotherm at 101.67 °C and 241.24 °C with heat flow of 344.87 J/g and 94.397 J/g, respectively. DSC curve of Pec-Nrg conjugate exhibits two endothermic peaks at 76.97 °C and 217.45 °C with an enthapy of 108.75 J/g and 27.073 J/g, respectively. Conjugate also exhibits a small exothermic peak at 232.73 °C. Thus, due to conjugation, the characteristic endothermic peak of pectin shifts from 101.67 to 76.97 °C, while the endotherm appearing at 217.45 °C can be attributed to naringenin.

Fig. 1.

a DSC thermogram of pure naringenin, pectin and Pec-Nrg conjugate, b XRD spectrum of naringenin, c XRD spectrum of pectin and d XRD spectrum of Pec-Nrg conjugate

Figure 1b–d portrays the X-ray diffractogram of naringenin, pectin and Pec-Nrg conjugate, respectively. Naringenin showed intense diffraction peaks of crystallinity between 7° and 48° (2θ) indicating the inherent crystalline nature of the drug (Wen et al. 2010). The X-ray diffractogram of pectin indicates the amorphous nature of pectin with characteristic broad peaks appearing at 13° and 21° (2θ) (Mundlia et al. 2018). The Pec-Nrg conjugate diffractogram shows that naringenin conjugation imparted partial crystallinity to pectin with a broad peak at 20° (2θ) and crystalline peaks at 27 and 20° (2θ).

The scanning electron micrographs of Pec-Nrg conjugate depicted a unique morphology not characteristic of either of the individual components. While naringenin existed as elongated, cuboidal crystals with sharp edges (analogous to parallelepiped geometry, Fig. 2a); pectin micrographs showed polyhedral geometry characteristic of polysaccharides with striated-folded surface morphology (Fig. 2b). Interestingly, the Pec-Nrg conjugate was obtained as a fibrous, film-like biomaterial with plain, polyhedral particles embedded in a highly porous, nanofibrous network (Fig. 2c–f). The obtained nanofibrous morphology is analogous to an amphiphilic, self-assembling peptide architecture—this is maybe due to the hydrophilic: hydrophobic composition of the pectin: naringenin conjugate (Fig. 2e, f). This is a very important finding in terms of polysaccharide-based drug conjugates wherein the novel conjugate can perform both as a drug delivery system as well as a biomaterial platform.

Fig. 2.

SEM images of a naringenin, b pectin and c–f Pec-Nrg conjugate

Conjugation of hydrophobic naringenin with hydrophilic pectin results in formation of amphiphilic conjugate, which is expected to show self assembling behavior. The critical aggregation concentration of Pec-Nrg conjugate was estimated using fluorescence spectroscopy and dynamic light scattering (DLS). In fluorescence spectroscopy, pyrene was used as a probe. Pyrene tends to reside in the close promixity of hydrophobic regions of the aggregate as a result, its photophysical characteristics are changed. The fluorescence spectral studies of conjugate solutions containing pyrene revealed the sensitivity of the ratio of intensity of pyrene probe appearing at 373 nm and 475 nm, to the concentration of conjugate (Wang et al. 2011). A plot of log concentration of conjugate with peak intensity ratio (I₃₇₃/I₄₇₅) exhibits the inflexion point at 1.75 (Fig. 3a) which correspond the conjugate concentration of 56.23 µg/mL. Figure 3b portrays the relationship between the log concentration of the Pec-Nrg conjugate and the hydrodynamic diameter of the colloidal dispersion. It can be observed that as the colloidal dispersion of Pec-Nrg conjugate is diluted from the concentration of 1000–0.05 µg/mL, the hydrodynamic diameter decreases from 748.4 to 349.7 nm. The plot reveals the inflexion point around 1.65 ($\cong$ 44.67 µg/mL). Thus, the CAC value as determined by DLS is in close agreement with fluorescence spectroscopy results.

Fig. 3.

Effect of concentration of Pec-Nrg conjugate on a fluroscence intensity ratio, b hydrodynamic diameter

Among a number of available techniques to determine the molecular weight, static light scattering also comes to a great rescue as there is no need for calibration. The molar mass (M_w) of pectin as determined by static light scattering technique comes out to be 4.31 × 10⁴ kDa (R² of Debye plot = 0.9) which is in close agreement to the molecular weight value reported in literature (Sayah et al. 2016). The molar mass for Pec-Nrg conjugate was found to be 6.22 × 10⁴ kDa (R² of Debye plot = 0.97).

Molecular mechanics assisted model building and energy refinements

A molecular mechanics energy relationship was used to provide information about the contributions of noncovalent van der Waals, noncovalent coulombic terms, and valence terms interactions for polysaccharide/naringenin interactions. The difference between the total potential energy of the complex system and the sum of the potential energies of isolated individual molecules was used to calculate the total potential energy deviation, ΔE_Total as shown below:

$$\mathrm{\Delta }{E}_{\text{Total}(A/B)}=E_{\text{Total}(A/B)}-\left[E_{\text{Total}(A)}+E_{\text{Total}(B)}\right]$$ 1

The stability of the molecule was assessed by comparison of the total potential energies of the complexed and isolated systems. The formation of complex is favored if it is more stable which is indicated by smaller potential energy of the complex in comparison to the sum total of potential energies of individual molecules in the same conformation.

The geometrical orientations of the various modes of interactions between the pectin chain and naringenin molecule is shown in Fig. 4 and the energetic profiles of the conjugate and constituent molecules are represented in Eqs. 2–4 and Table 1. It is evident from Fig. 4 that the –COOH functionality of pectin interacted with all three –OH functional groups of naringenin: 5-OH, 7-OH, and 4′-OH thereby confirming the NMR results. The conjugation of naringenin to pectin was accompanied by a −ve energy minimization (ΔE_V∑) of ≈ 18 kcal/mol confirming the favorable and energetically stable complexation between the two molecules. The energy minimized complex was stabilized primarily by the non-bonding energy terms: electrostatic interactions (ΔE_Vij = − 18.642 kcal/mol) and van der Waals forces (ΔE_Vel = − 9.680 kcal/mol). However, this energetic stabilization may have led to certain geometrical adjustments within the conjugate molecule causing a strain in the molecular structure and hence a significant destabilization of the torsional bonding energy term (ΔE_Vφ = + 9.248 kcal/mol). Given the presence of only three interacting groups in the naringenin molecule as compared to the polysaccharide and only one available for interaction in vacuo; only one intermolecular H-bond was formed between pectin and naringenin while the intramolecular H-bonds were variable in number dependent on the type of –OH group involved in conjugation. The significant stabilization of van der Waals energy terms provides a confirmation of the overlap of the respective van der Waals surfaces of the molecules with minimal repulsion.

$$E_{\text{Pec}}=-25.743V_{\sum }=2.030V_{\text{b}}+16.827V_{\theta }+26.870V_{\phi }+7.085V_{\text{ij}}-3.399V_{\text{hb}}-75.157V_{\text{el}}$$ 2

$$E_{\text{Nrg}}=8.384V_{\sum }=0.532V_{\text{b}}+0.843V_{\theta }+1.080V_{\phi }+5.934V_{\text{ij}}-0.006V_{\text{hb}}+0.00V_{\text{el}}$$ 3

$$E_{\text{Pec-Nrg}}=-35.506V_{\sum }=2.805V_{\text{b}}+17.631V_{\theta }+37.198V_{\phi }+3.339V_{\text{ij}}-2.681V_{\text{hb}}-93.799V_{\text{el}}$$ 4

Fig. 4.

Representation of the geometrical preferences of the pectin molecule (tube rendering) in complexation with naringenin molecule (tube-and-ball rendering) after molecular mechanics simulations. The molecular models depict ester formation between the –COOH functionality of pectin with –OH functionality of naringenin a 5-OH; b 7-OH; and c 4′-OH [Color code for elements: C cyan, H white, O red]

Table 1.

Inherent energy attributes calculated using static lattice atomistic simulations in vacuum

Energy function	Energy value (kcal/mol)
ΔEa (V∑)b	− 18.147i
ΔE (Vb)c	0.243j
ΔE (Vθ)d	− 0.039
ΔE (Vφ)e	9.248
ΔE (Vij)f	− 9.680
ΔE (Vhb)g	0.724
ΔE (Vel)h	− 18.642

^aΔE_(A/B) = E_(A/B) − [E_(A) + E_(B)]

^bTotal steric energy for an optimized structure

^cBond stretching contributions

^dBond angle contributions

^eTorsional contribution arising from deviations from optimum dihedral angles

^fvan der Waals interactions

^gHydrogen-bond energy function

^hElectrostatic energy

ⁱValues represents the structure stabilizing contribution

^jValues represents the structure destabilizing contribution

In vitro release of conjugate

In vitro release of naringenin from naringenin (API) and Pec-Nrg conjugate was studied at 37 °C in simulated gastric (pH 1.2) and intestinal fluid (pH 7.4). The results of the study (Fig. 5a) reveal that only 36.78% and 11.84% of naringenin from naringenin (API) is released at pH 1.2 and pH 7.4, respectively, in 48 h study. However, almost all of the naringenin is released by hydrolysis within 30 h at pH 1.2 while only 57.62% of the naringenin is hydrolysed at pH 7.4 during 48 h study period from Pec-Nrg conjugate. Further, the results of the modeling kinetics of the release data of naringenin from naringenin (API) show R² value of 0.981, 0.770, 0.957 and 0.869 for zero, first, Higuchi and Korsmeyer-Peppas models, respectively, at pH 1.2 while the respective R² values at pH 7.4 were 0.986, 0.984, 0.920 and 0.824. On the other hand, data of modeling fitting of naringenin release from Pec-Nrg conjugate unveil the R² value of 0.947, 0.825, 0.997 and 0.656 for zero, first, Higuchi and Korsmeyer-Peppas models, respectively, at pH 1.2 and the respective R² values at pH 7.4 were 0.804, 0.894, 0.941 and 0.933. The values of n, the release exponent of Korsmeyer-Peppas were 0.296 (pH 1.2) and 0.161 (pH 7.4) for Pec-Nrg conjugate. Thus, the dissolution of naringenin from naringenin (API) follows zero order kinetics while the release of naringenin from the conjugate obeys Higuchi’s square root kinetics with diffusion being the primary mechanism of release.

Fig. 5.

a In vitro release profile of naringenin and Pec-Nrg conjugate at pH 1.2 and pH 7.4, b cytotoxicity analysis (MTT assay) of Pec-Nrg conjugate at varying concentrations (6.25–100.00 µg/mL) against NIH: OVCAR-5 cancer cells

In vitro antioxidant activity

The free radical scavenging activity of naringenin and Pec-Nrg conjugate was measured by DPPH assay. This method is based on the conversion of free radical form into stable form. DPPH solution is deep violet in color and when it gets reduced it becomes yellow in color in the presence of antioxidants. The total radical scavenging power was calculated by reduction in the peak area. Naringenin and Pec-Nrg conjugate showed 91.74 ± 1.2% and 96.10 ± 0.82% radical scavenging, respectively. The statistical analysis by unpaired t test of the results indicate that the Pec-Nrg conjugate has significantly higher (P < 0.05) free radical scavenging activity over the free naringenin, which may be attributed to higher aqueous solubility of the conjugate.

Antibacterial activity

The comparative results of antibacterial activity of naringenin and Pec-Nrg conjugate revealed that MIC of Pec-Nrg conjugate is lower than naringenin. It was found that Pec-Nrg conjugate exhibited MIC value of 2.5, 12, 11.5, 12.5, 12.5 µg/mL in comparison to 6.5, 24.5, 12.5, 12.5, 23.5 µg/mL of naringenin against Staphylococcus aureus (MTCC 7443), Staphylococcus epidermis (MTCC 435), Bacillus subtilis (MTCC 441), Escherichia coli (MTCC 1652), Pseudomonas aeruginosa (MTCC 424), respectively. However, in the case of pectin no inhibition of microbial growth was observed, which indicates that the antibacterial activity of Pec-Nrg conjugate is not due to the pectin. Thus, the better antibacterial activity of amphiphilic Pec-Nrg conjugate over the lipophilic naringenin may be attributed to the greater aqueous solubility and penetration of the colloidal Pec-Nrg conjugate over the hydrophobic naringenin.

The bacterial growth kinetics study was carried out for naringenin and Pec-Nrg conjugate at MIC and below MIC against Staphylococcus aureus MTCC 7443, Staphylococcus epidermis MTCC 435, Bacillus subtilis MTCC 441 and Gram −ve strains Escherichia coli MTCC 1652, Pseudomonas aeruginosa MTCC 424. The results of the growth inhibition curve study (Fig. S4a–e) correlate with the results of MIC studies, it can be easily observed that naringenin and Pec-Nrg conjugates at their respective MIC values for different strains inhibited the growth curve, while the use of naringenin and Pec-Nrg concentrations below their MIC showed a variable delay in growth ranging from 1 to 4 h.

In vitro anti-cancer activity of Pec-Nrg conjugate

The NIH: OVCAR-5 cells cytotoxicity analysis for Pec-Nrg conjugate presented a concentration-dependent decrease in cell viability with ≈ 35% viability at the highest tested concentration of 100 µg/mL (Fig. 5b). However, free naringenin dispersed/dissolved in medium shows > 80% viability over the concentration range tested and the results are very variable. The results of one-way analysis of variance indicate that as the concentration of naringenin was increased from 6.25 to 100 µg/mL no significant difference (P > 0.05) in the cell viability was observed. In case of Pec-Nrg conjugate, increasing the concentration of conjugate from 6.25 to 100 µg/mL, the cell viability decreased, and a significantly higher (P < 0.01) cytotoxicity of the conjugate was observed at the concentration of 100 µg/mL as compared to 6.25 µg/mL. Further, on comparing the effect of respective concentrations of Pec-Nrg conjugate and naringenin on cell viability, a significantly higher (P < 0.001) cytotoxic effect of the conjugate was observed. It is worth noting that the naringenin was dispersed in medium to elucidate the effect of increased naringenin aqueous solubility on the viability of the NIH:OVCAR-5 cancer cell line. Although the apparent maximum aqueous solubility of naringenin is usually in the range of 200 µg/mL (https://www.drugbank.ca/drugs/DB03467), crystal formation was observed in the upper concentration ranges. This may be the underline cause of higher viability in case of free naringenin as compared to conjugated naringenin. In case of control and pectin; the cells showed spindle formation and good adherence on the culture plate. For Pec-Nrg conjugate, the cells showed early detachment, spherical cells, and cell death was evident confirming the anti-cancer potential of the Pec-Nrg conjugate (Fig. 6). Although the results obtained with Pec-Nrg were very promising, the cellular internalization of Pec-Nrg micellar aggregates in several cancer cell lines and a 3D cell culture model may provide further information on the mechanism of action of the anti-cancer activity of the conjugate.

Fig. 6.

NIH: OVCAR-5 cancer cell morphology in the presence of a complete medium; b pectin dispersed in medium; c free naringenin; and d Pec-Nrg conjugate (error bars = ± SD; n = 5)

Conclusion

Naringenin, a hydrophobhic nutraceutical was conjugated to hydrophilic pectin employing carbodiimide coupling chemistry. The coupling was confirmed by spectral analyses. The results of in silico molecular mechanistic simulations confirmed the stability of the conjugate. Pec-Nrg conjugate was observed to be semi-crystalline with nanofibrous morphology, having self-assembling characteristics. The conjugate was found to release naringenin following Higuchi’s square root kinetics. Further, conjugation was observed to significantly improve the free radical scavenging and antibacterial activity of naringenin. The faster and higher release of naringenin at acidic pH is of therapeutic advantage for killing cancerous cells which have acidic environment. Higher cytotoxicity of conjugate over free naringenin against ovarian cancerous cells points towards its enhanced therapeutic efficacy. However, further in vivo studies in animals are required to see if the higher solubility and cytotoxicity of Pec-Nrg conjugate translates its enhanced bioavailability. In conclusion, conjugate of naringenin to pectin appears to be a promising approach to enhance its delivery.

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Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.