Novel formulation for co-delivery of cinnamon- and cumin-loaded polymeric nanoparticles to enhance their oral bioavailability

Nanobiotechnology has been an encouraging approach to improving the efficacy of hydrophobic bioactive compounds. The biologically active constituents present in herbal extracts are poorly absorbed, resulting in loss of bioavailability and efficacy. Hence, herbal medicine and nanotechnology are combined to overcome these limitations. The surface-to-volume ratio of nanoparticles is high and as the size is small, the functional properties are enhanced. The present study reports the synthesis of cinnamon and cumin (Ci–Cu) dual drug-loaded poly (D, L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) to overcome the limitations of oral bioavailability and extend the effect of these drugs for alleviating health problems. The solvent evaporation method was adopted for the synthesis, and the as-prepared nanoparticles were characterized by Scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, Transmission electron microscopy (TEM) and X-ray diffraction (XRD). The average size of the formed spherical Ci-Cu nanoparticles ranged between 90 and 120 nm. The encapsulation efficiency of the drug was found to be 79% ± 4.5%. XRD analysis demonstrated that cinnamon and cumin were amorphously scattered in the PLGA matrix. The FTIR bands showed no evident changes suggesting the no direct molecular interactions between the drug and the polymer. At pH 6.9, the release studies in vitro exhibited a burst initially followed by a tendency to obtain a slower steady release. The results indicated that the Cu-Ci dual drug-loaded polymeric NPs has drug release at a slower rate. The time taken for 25% release of drug in Ci-Cu-loaded PLGA NPs was twice as compared to cumin-loaded PLGA Nps, and three times compared to cinnamon-loaded PLGA NPs.


Introduction
Spices are aromatic substances found in dried plant parts, mostly seeds, leaves, fruits, bark, roots, or other plants with a distinct smell. As all spices are obtained from plants, they have been generally recognized as safe (GRAS). Plant leaves, fruits, seeds and bark have polyphenols due to which they are added to increase flavor, preserve food or add color. However, spices can be used for functions more than just these in the foods to which they are added. Spices have usage as traditional medicine in various countries and play a great role because of their antioxidant, anti-inflammatory, antimicrobial, antimutagenic, antidiabetic and anti-cancerous activities (Yashin et al. 2017;Paswan 2021).
Nowadays, poly(D, L-lactide-co-glycolide) (PLGA)based nanotechnology has gained immense attention in medical applications as is easily broken down by hydrolysis into lactic acid and glycolic acid. The monomers are also biocompatible. It owes unique properties like bioavailability, biocompatibility, changing the kinetics of degradation, stability, increased drug loading and prolonged release of drug in comparison with other carriers. Also, these systems protect sensitive ingredients from unwanted reactions that lead to structural changes (Campos et al. 2016). These new drug delivery approaches, including polymeric nanoparticles (NPs), are projected to transform the view of pharmaceutical companies.
The present study emphasizes on the preparation of cinnamon and cumin (Cu-Ci) dual drug-loaded PLGA NPs with simple and cost-effective solution evaporation method. Till now, no attempts have been made to co-encapsulate these drugs and to study their combined potency. Based on the studies, the drug encapsulation efficiency was calculated to be 79% ± 4.5%. Moreover, the Ci-Cu-loaded polymeric NPs exhibited nearly two and three-times slower drug release rate compared to cinnamon-loaded and cumin-loaded polymeric NPs, respectively.

Materials
PLGA [poly (D, L-lactide-co-glycolide)] with a 50:50 copolymeric ratio of DL-lactide to glycolide (Mw = 24,000 to 38,000 Da) was obtained from Sigma-Aldrich. Pluronic F-68 used as the surfactant was taken from Sigma-Aldrich. Analytical grade methanol and acetonitrile organic solvents were used. Disodium hydrogen phosphate (Na 2 HPO 4 ), sodium dihydrogen phosphate (NaH 2 PO 4 ), and trisodium phosphate (Na 3 PO 4 ) used in this work were also analytical grade. For in vitro release studies, dialysis

Preparation of cinnamon and cumin extract
The bark of cinnamon and seeds of cumin was purchased from the market. The plant samples were further submitted to Raw Material Herbarium and Museum, Delhi (RHMD) for certification. Authentication for both the plants in the study was done as follows: After that, the impurities were removed, and both spices were air-dried at room temperature and grounded in a mixer to a fine powder. The powder was sieved and stored in air-tight bottles to get an even size range. Extraction was done using methanol. Powdered samples (8 gm) were soaked for 72 h in 100 ml methanol in a sterile beaker covered with aluminum foil to avoid evaporation, and then it was filtered using filter paper (Whatman No. 1). The solvent from the filtrate was evaporated under room temperature conditions. Finally, the dried-up extract was kept in bottles till further usage.

Formulation of PLGA nanoparticles loaded with cinnamon and cumin
Solvent evaporation method was adopted to formulate polymeric NPs with or without methanolic extract of cinnamon and cumin. Scheme 1 shows the schematic of PLGA nanoparticles loaded with cinnamon and cumin. To prepare the organic phase, 10 ml acetonitrile was added to 5 mg methanolic extracts of cinnamon and cumin, along with 50 mg of PLGA. The organic phase was added dropwise to an aqueous solution (25 ml of 0.1% Pluronic F-68). Further the mixture was sonicated in an ultrasonicator (Hielscher Ultrasonics, Germany) for 3 min. The organic solvent was evaporated from the resulting dispersion of nanoparticles by continuous stirring (400 rpm) at room temperature for 4 h followed by centrifugation (REMI, INDIA) at 15,000 rpm for 25 min. The obtained nanoparticles were washed several times with double distilled water followed by lyophilization. After that, the NPs were stored at 4 °C.

Size determination
Nanoparticle size was determined using Dynamic Light Scattering (Zeatasizer Malvern Instruments, Worcestershire, UK). For measurements at scattering angle 90° and temperature 25 °C, 100 μl suspension of the nanoparticles was dispersed in 1 ml of distilled water and then sonicated for 1 min. The average diameter and the standard deviation were calculated for ten determinations of each sample.

SEM studies
The nanoparticle's surface morphology and shape were analyzed using scanning electron microscopy (SEM-Zeiss EVOMA10. Metal stubs were covered with double-sided adhesive tape, and a suitable nanoparticles sample was spread on the sticky surface. Vacuum dried samples were coated with a palladium layer through an ion sputter. Samples were observed after palladium coating of 30 nm at an accelerating voltage of 20 kV and 10 Pa.

TEM studies
The morphology of nanoparticles was further studied and compared employing transmission electron microscopy (TEM-JEOL-2100F, Japan). After dispersing the polymeric nanoparticles in distilled water, they were sonicated. The sample was prepared by placing a drop on a copper grid so that a thin liquid film was formed, which was allowed to dry before the examination, using a TEM without being stained.

XRD studies
PANalytical X-ray diffractometer at 40 mA anodic current was used to record the XRD patterns of cinnamon, cumin and cinnamon-cumin-loaded polymeric nanoparticles using an accelerating voltage of 45 kV. XRD patterns was obtained at a scanning speed 2° per minute were done at diffraction angles (2θ) from 5° to 90°.

FTIR studies
Fourier-transform infrared (FTIR) spectroscopy was used for sample analysis in the range 400-4000 cm −1 to validate the compatibility among polymeric, cinnamon-loaded polymeric nanoparticles, cumin-loaded polymeric nanoparticles and cinnamon-cumin-loaded polymeric nanoparticles. KBr was used to disperse the powdered samples and compress them into pellets.

Determination of Ci-Cu entrapment efficiency
Drug encapsulation efficiency was calculated using UV-Visible spectroscopy. The supernatant collected after centrifugation was analyzed to check the amount of available drug in the nanoparticles. The absorbance of the drug was checked, and the presence was calculated using the standard curve of the drug prepared in water. The calibration curve for the quantification of cinnamon and cumin was linear over the range of standard concentrations from 0.1250 mg to 10 mg with a correlation coefficient of R 2 = 0.998.

In vitro release studies
At pH 6.9, phosphate-buffered saline (PBS) solution was used to carry out release studies of the drug up to 25 h from Ci-Cu-loaded polymeric NPs. About 10 mg each of cinnamon, cumin, cinnamon-loaded polymeric NPs, cuminloaded polymeric NPs and Ci-Cu-loaded NPs were put in a dialysis membrane. Further dialysis membrane was then put into a beaker having 20 ml PBS. This setup was kept under magnetic stirring (100 rpm) at room temperature. At predetermined time gaps (30 min, 1 h, 2 h, 4 h, 8 h, 24 h) withdrawal of 3 ml solution was done. Shimadzu UV-Vis spectrophotometer was used to record absorbance for analyzing the drug release at a wavelength of 275.5 nm. The cumulative release from various formulations was also drawn as a function of time. Figure 2(a-c) shows the morphology of cinnamon-loaded, cumin-loaded, and Ci-Cu-loaded nanoparticles. SEM studies indicated that distinctive spherical nanoparticles were formed. The TEM images of cinnamon-loaded, cuminloaded and Ci-Cu-loaded polymer nanoparticles is shown in Fig. 3(a-c). TEM analysis also revealed the spherical shape of the synthesized nanoparticles. It was noticed that the size of the nanoparticles significantly decreased from 100 to 270 nm to 100-180 nm in cinnamon-and cuminloaded PLGA nanoparticles. Whereas a size distribution of 90-120 nm was observed in the Cu-Ci dual drug-loaded polymeric NPs. It is expected that Ci-Cu-loaded NPs' decreased size can contributes majorly to slow drug release. Entrapment efficiency is an important parameter, as inappropriate entrapment would lead to an initial burst release of the drug, which deters its sustained-release property. Based on the studies, the drug encapsulation efficiency was calculated to be 79% ± 4.5%.

XRD Studies
The XRD patterns for Ci-Cu-loaded PLGA nanoparticles is shown in Fig. 4. For the drugs entrapped in polymeric nanoparticles, no characteristic peak was shown, possibly owing to the amorphous nature of the PLGA.

In vitro release studies
The release profiles under in vitro conditions were studied of cinnamon, cumin, cinnamon-loaded polymeric NPs, cuminloaded polymeric NPs and Ci-Cu-loaded polymeric Nps by UV-Vis spectroscopy (see Fig. 6). A biphasic release profile was shown by the drug-loaded PLGA nanoparticulate formulation. Various studies have reported the exponential release pattern for PLGA nanoparticles (Saxena et al. 2004;Brandt et al. 2021). The release profile of all formulations of PLGA nanoparticles exhibited a faster exponential release in the initial phase followed by a slower release phase. The results indicated that the Cu-Ci dual drug-loaded polymeric NPs has drug release at a slower rate. Figure 7 indicates that the time taken for 25% drug release from Ci-Cu-loaded polymeric NPs increases significantly as compared to cinnamon-loaded polymeric nanoparticles and cumin-loaded polymeric nanoparticles. The time taken for 25% release of drug in Ci-Cu-loaded PLGA nanoparticles is twice as compared to cumin-loaded PLGA nanoparticles, and three times compared to cinnamon-loaded PLGA nanoparticles.

Conclusion
Cinnamon and cumin (Ci-Cu) dual drug-loaded PLGA polymeric nanoparticles were successfully synthesized by solvent evaporation method. The spherical nanoparticles prepared have size distribution of 90-120 nm with a good encapsulation efficiency of 79% ± 4.5%. The in vitro drug release studies showed the controlled biphasic release of drugs from Ci-Cu-loaded polymeric nanoparticles. The Ci-Cu-loaded polymeric nanoparticles exhibited nearly two-and three-times slower drug release rate compared to cinnamon-loaded and cumin-loaded polymeric NPs, respectively. The reported results provided a simple method for synthesizing polymeric nanoparticles loaded with cinnamon and cumin to cure numerous ailments.
Funding Open Access funding provided by the Qatar National Library. This study was funded by the Qatar University, Kishor Kumar Sadasivuni Grant no: QUCG-CAM-21/22-1.

Conflict of interest The authors declare no conflict of interest.
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