Introduction: The present study sought to stabilize the nanosuspension of potent antioxidant such as Vitexin, Petunidin as well as Tricosane from the root related to Cynodon dactylon by utilizing the RESSAS process. These compounds include low efficacy and bioavailability since are rarely soluble in aqueous media. Thus, producing stable nanosuspensions can solve the related problem by decreasing the size of the particle. In addition, central composite design (CCD) was used for analyzing the impact of oven temperature, pressure, CO2 flow rate, and modifier volume on the antioxidant activity index (AAI) in the outcome of RESSAS process. Further, DLS, FE-SEM, and LC-MS techniques were implemented for evaluating the features of nanosuspension. Based on the results, the behavior of the particles after forming particle was emphasized, which indicated that the RESSAS process results in decreasing the agglomeration of the particle and enhancing AAI for the extract. Thus, the bioavailability of the products related to herbal medicinal can be dramatically increased in biological media.
Methods: Traditional herbal medicines have been extensively used all over the world as the oldest source of drugs and has attracted a lot of attention in today’s medical practice. Most of the natural compounds used in plants consist of some forms of biological activities which are implemented to treat different diseases and can positively affect human health. In addition, during the last decades, the natural bioactive compounds have less been emphasized after the creation of molecular biology and combinatorial chemistry, which plays a role in discovering new drugs and structures. However, natural compounds and their role have been supported more as a formidable skeleton for producing drugs (Pollio et al., 2016). Further, some of the phyto-compounds and nutrients in foods are normally insoluble or include insignificant solubility in biological media. Therefore, a decrease occurs in using these kinds of drugs due to their poor solubility features, which results in creating a low bioavailability, along with its threshold concentration of toxicity which is approximately near the therapeutic dosage (Müller et al., 1999; Xu and Luo, 2014). In this respect, nanoparticles manufacture can be regarded as a good choice for increasing the dissolution rate of such bioactive compounds, along with their related bioavailability (Chai et al., 2020; Huang et al., 2010; Momenkiaei and Raofie, 2019). Nano dispersions formulation is considered as an appropriate method for solving the problems related to bioavailability since an increase in the accessible surface areas can lead to an increase in the volume dissolution related to low soluble compounds available in water (Ahire et al., 2018; Merisko-Liversidge and Liversidge, 2008). Furthermore, the process of rapid expansion of supercritical carbon dioxide solution results in creating strong solvating force of the supercritical fluid (Eckert et al., 1996) and fragmenting the heat-sensitive compounds without producing any damage and creating particles less than 500 nm in diameter (Türk, 2009; Young et al., 2000). In addition, high rapidity of supersaturation leads to the speedy precipitation of the extracted substance as micro or nanoparticles because of reduction in pressure related to the expansion chamber (Karimi and Raofie, 2019; Pessi et al., 2016; Türk, 1999).
The main challenging issue is related to the behavior of the particles after it is created based on nanoparticle reports. Nanoparticle may be improved through condensating, agglomerating, and coagulating in the period of residence in the collection vessel. However, it is worth noting that dispersing most of the systems again is approximately inoperative because theoretical framework of expanding supercritical solution process can predict the formation of primary particles with a diameter of smaller than 50 nanometers (Weber and Thies, 2007). Regarding water-insoluble substances, spraying RESSAS outcome straightly into an aqueous media encompassing a surfactant via heating the capillary jet is regarded as a satisfactory method for obtaining the fine particle (Sane and Thies, 2005; Uquiche and Martínez, 2016). RESSAS technique has been used in some studies in the pharmaceutical field and the results represented the preparation of successful submicron particle with stabilizing factors and various pharmaceutical compounds (Lee et al., 2018; Pathak et al., 2006; Sane and Limtrakul, 2009; Türk and Lietzow, 2004). Manipulation on particle size, size distribution, and possible polymorph can be achieved through altering the operating conditions in RESSAS process. Based on the results of the previous studies, an aqueous solution encompassing emulsifier at the expansion chamber avoids growing particle, coagulating, and agglomerating of the herbal extract (Liu et al., 2006; Ya-Ping et al., 2005).
Results: 3.1. Optimization of RESSAS process for the antioxidant activity
In this study, pressure (A), oven temperature (B), modifier volume (C), and CO2 flow rate (D) were taken into consideration. In addition, a design by five levels (rotatable CCD, α = 1.68) was utilized for optimizing the factors playing a role in the antioxidant capacity of bioactive compounds in the Cynodon dactylon root. Based on the results, DPPH tests represented a decrease in intensity of absorption at 517 nm in the visible region to measure the AAI related to the extracted Cynodon dactylon root by focusing on RESSAS process at 200 µg/mL concentration in every sample (DPPH 0.2 mM). Table 1 indicated the plan matrix of the parameters related to the CCD. To this aim, 29 spaced runs (24 + (2 ×4) + 5) were used for performing the CCD for A, B, C, and D factors, including a (24) factorial plot enhanced with (2 ×4) star and 5 central points. This plan makes the reply possible for modelling by inserting a second-order polynomial as shown in Eq. (4):
AAI = -4.4675 + 0.0150917 A + 0.0952222 B + 0.0106875 C + 0.742083 D - 0.000008125 A2 - 0.000065 AB - 0.0000096875 AC - 0.0006375 AD - 0.000827778 B2 + 0.0000354167 BC 0.000916667 BD - 0.0000275391C2 - 0.000609375 CD - 0.0753125 D2 (4)
Based on the ANOVA results, the model had a well proficiency in R2 regulated 0.82 (Table S2). As indicated in Table 1, the superlative AAI is 2.61 and maximum extracted yield of Cynodon dactylon root flour is 41.12 mg oil/g Cynodon dactylon root and as the Pareto chart is shown in Fig. 2, the pressure and modifier volume had a positive effect on this outcome. Further, a wide gain of the extract is not often related to a wide gain with respect to antioxidant capacity.
3.2. The effect of RESSAS process parameters on the antioxidant activity
As displayed in Fig. 2, Pareto diagram analyzes in the CCD related to RESSAS indicates the estimated impact of factors and their interplays on the antioxidant activity. The negative or positive mark on the plot represents a decrease or increase while crossing off the nethermost to uppermost area set for the specific parameter, respectively.
Similar studies were reported that extraction yield and antioxidative activity increased remarkably by an increment of pressure at constant other parameters, owing to an increment of the solubility. In the present study pressure plays the most effect on the results. The extraction efficiency and antioxidant activity increased by raising the pressure because increasing the extraction pressure results in decreasing intermolecular mean distance of CO2 molecule and a higher fluid (CO2) density. Thus, it causes an increment in the specific interaction between the solute and solvent molecules, which results in enhancing the solubility of the target compounds.
By increasing the temperature, the herbal compounds' vapor pressure improves the outgrowth before the optimal point. As regards, since an optimal temperature, enhancement in the temperature decreases the congestion of the supercritical CO2, which plays a negative efficacy on the outcome (Oliveira et al., 2011).
Based on the outcome, all of the antioxidant activity growth by apply ethanol for pure CO2, as a modifier. Further, extracting polar herbal compounds with pure carbon dioxide at supercritical area quantitatively cannot be successful because of the confined solubility of supercritical CO2 as a non-polar solvent (Veggi et al., 2011). Of course, the addition of ethanol to the extraction chamber affects the weight of the final product and in this way apparently increases the extraction yield (mg/g), but this residual solvent has no efficacy on the amount of absorbance and AAI. Additionally, ethanol can play a role in forming and creating stability for nanoparticles. Thus, adding a polar pharmacological modifier to supercritical CO2 can growth the extraction efficiency of natural compounds so that it cannot create disorder supercritical fluid (Bhargavi, 2011).
Based on the results, an increase in the flow rate of CO2 can positively influence the AAI in the produced extract. In fact, a high supercritical CO2 flow rate leads to a high AAI. The consequence can be normally logical as a tradeoff among a thermodynamic equilibrium state and mass transfer (Kuś et al., 2018; Yousef et al., 2001). In fact, a superior mass flow rate equals to concise residence time, which prohibit agglomeration the particles in the collection vessel, leading to the creation of smaller particles and an increase in their the absolute bioavailability in an aqueous solution. However, excessive flow rate results in decreasing the stay time more. Thus, it causes the how to deviate of equilibrium and leave the extractor by the incomplete extraction solvent.
The specimen response is conformed versus two experimental factors based on the CCD, while other factors are placed in its central surface area. Fig. 3 displays the response surface that representing the impact of (A) pressure and CO2 flow on the AAI at constant oven temperature value and modifier volume in its center value, and (B) oven temperature and modifier volume on the AAI at the fixed value of pressure and CO2 flow rate in its center value. Based on the ANOVA results in Table S2, a change taken place in the modifier volume and pressure could significantly influence extracted AAI (p <0.05).
3.3. Characterization of nanosuspension
The DLS results indicated the post-expansion conditions play a role in the RESSAS process, which prevents from growing and agglomerating nanoparticles when it is used as an appropriate surfactant at a good concentration. Thus, it caused the stable and uniform suspension about 100 nanometers. Fig. 4 displays the calculations related to the particle and distribution size after one day (A), one week (B), and one month (C).
In addition, FE-SEM technique was implemented to determine the surface of nanoparticle, along with the morphology and dimensions and of the sampled nanoparticles. Based on the FE-SEM image results in optimized mode of the RESSAS process, the initial particle size was circa 30-100 nm diameter range (Fig. 5). In other words, the process can produce nanoparticles of extratced Cynodon dactylon root.
Fig. 6 illustrates the total ion chromatogram (TIC) of the herbal extract that identified by the component LC-MS produced in optimized mode by RESSAS method. Based on the LC-MS analysis results, diagnosis of antioxidant compounds with the suitable amount in the extract from the RESSAS process was confirmed. Table 2 indicates some of the potent antioxidant compounds with their acquisition time. Finally, the extracted ion chromatogram (EIC) and mass spectra of some detected potent antioxidant compounds are shown in Fig. S1.
Conclusion: In the present study, the RESSAS process was implemented successfully for producing stable and uniform nanosuspensions of the negligible soluble herbal compound in the extracted Cynodon dactylon root. The optimization of the operative conditions with 350 bar pressure, 45 °C oven temperature, 132 μL modifier volume, and 2.6 mL/min CO2 flow rate allowed potent antioxidant nanosuspensions by a particle size about 100 nm so that it can be produced with an increased bioavailability. Tween 80, as the stabilizer, created stable nanosuspension by preventing from growing and agglomerating the particle after being sprayed into an aqueous solution. Based on the AAI results related to the processed extracts, potent antioxidant activity was observed in Cynodon dactylon root by decreasing the particle size. Thus, RESSAS process is considered as an appropriate method for producing stable nanosuspension and enhancing the bioaccessibility of insoluble or negligible soluble natural compounds related to pharmaceutical plants.