3.1. Prediction of Rosmarinic Acid Solubility by Simulation
Solubility is one of the dominant physiochemical properties to explain the performance of target phytochemical separation from extraction process. According to Savjani et al. [20
], solubility is defined as the capacity of a solute to dissolve in a solvent to form a homogeneous solution. The solubility of a solute is affected by the properties of both solute and solvent, as well as environmental conditions such as pressure and temperature [20
]. Thus, solubility can be applied to determine the appropriate solvent system for extraction and fractionation process. Previously, the solubility was estimated by ‘like dissolve like” rule, at which certain amount of solute is dissolved in solvent. The mutual solubility between solute and solvent should be determined by their intermolecular interactions. Ideal dissolution tends to achieve when the attraction forces of solute–solvent overcomes the attraction forces of solute–solute and solvent–solvent [21
]. Hence, the solubility of a solute in a solvent can be predicted numerically from molecular structure. In this study, the magnitude of solubility between rosmarinic acid and solvents was estimated by solvation free energy calculation. The determination of solvation free energy is one of the computational approaches to estimate solubility of solute. It is widely applied in the prediction of solute aqueous solubility, as well as drug solubility. The solvation free energy is determined based on the interaction force between solvent and solute, as well as the entropy associated with creation of cavity in solvent and disruption of solvent structure. The more negative the value of solvation free energy indicates better solubility [22
In this study, the solvation free energy of rosmarinic acid in mobile phase for fractionation was calculated by Forcite Package in Material Studio 7.0 (Accelrys Inc., San Diego, CA, USA). The solvation free energy is the summation of the free energy of the charge removal in vacuum (ideal free energy), the free energy of addition of a neutralized molecule in the solvent (van der Waals) and the free energy of addition of charges on the solute (electrostatic). Figure 1
shows the solvation free energy of rosmarinic acid in six difference binary solvent systems. Rosmarinic acid showed to have the lowest solvation free energy in the mixture of chloroform–ethyl acetate (30:70), followed by ethyl acetate–methanol–formic acid–water (100:13.5:2.5:10), ethyl acetate–acetic acid–formic acid–water (100:11:11:26), ethyl acetate–methanol–water (77:13:10), ethyl acetate–formic acid–water (80:10:10), and methanol–water (10:90). Rosmarinic acid is an intermediate polar molecule, hence, it is more preferable in chloroform–ethyl acetate. Only this mixture of solvent system exhibited negative value of solvation free energy. Therefore, the solvent system of chloroform–ethyl acetate was selected as the mobile phase for the fractionation of rosmarinic acid due to its low solvation free energy.
3.2. Relationship of Rosmarinic Acid and Scavenging Activity
The total phenolic content (TPC) and total flavonoid content (TFC) of crude extract and its fractions are illustrated in Figure 2
. The figure clearly shows that TPC was about 2 to 10 times higher than TFC for all fractions. The TPC of O. stamineus
fractions varied from 1.97 to 3.06 mg GAE/100 g extract, whereas the TFC ranged from 0.23 to 1.62 mg RE/100 g extract in a bell shape curve from fractions 1 to 9.
The ratio of TPC to TFC is presented in a “S” shape curve from fractions 1 to 9 in line with the concentration of rosmarinic acid (Figure 3
). Rosmarinic acid seems to be the most abundant phenolic acid in O. stamineus.
A sudden increase of rosmarinic acid content in fraction 9 was also increased the TPC/TFC ratio. This was because the remaining rosmarinic acid and other phenolic acids were rinsed out from the SPE cartridge by strong solvent, methanol.
The antioxidant activity of O. stamineus
extract and its fractions were evaluated in term of free radicals scavenging activity and expressed in IC50.
explains the concentration of sample required to exhibit 50% of inhibition against free radicals. The phytochemicals with antiradical property could scavenge free radicals by donating protons. Rosmarinic acid could act as a potent radical scavenger, most probably because of proton donator characteristics. It has five hydroxyl groups in the molecular structure which may contribute protons to scavenge free radicals of DPPH [23
The TPC/TFC ratio is closely correlated to the concentration of rosmarinic acid which is also highly linked to the IC50
of the fractions in the antioxidant assay as presented in Table 2
. Hence, the TPC/TFC ratio could also be used to explain the scavenging activity of the fractions, mainly contributed by the presence of rosmarinic acid. This can be seen from lower IC50
values of fractions 1 to 3 with higher content of rosmarinic acid in those fractions. The increase of IC50
values in other fractions was also followed by the decrease of rosmarinic acid content.
In the present study, the standard chemicals of rosmarinic acid, ascorbic acid and rutin were used as positive control (Table 2
). The results revealed that the scavenging capacity of rosmarinic acid and ascorbic acid were comparable because both standard chemicals showed almost similar IC50
values (~15 µg/mL). However, the scavenging capacity of rutin was about 3 times lower than rosmarinic acid. Therefore, rosmarinic acid was the main contributor to the high TPC/TFC ratio and high scavenging capacity of O. stamineus
fraction in a linear relationship.
3.3. Major Phytochemicals in Plant Fractions
illustrates the chromatograms of O. stamineus
crude extract and its fractions which were obtained phytochemical fingerprinting. The fraction samples show a better HPLC separation compared to the crude extract as the undesired impurities was removed during the fractionation process. Rosmarinic acid shows the highest peak at the retention time around 12.5 min in the figure. SPE fractionation produced rosmarinic acid rich fractions 1 to 3. Further elution was found to produce rosmarinic acid mixed with other less polar compounds which could be phytochemicals from the classes of flavonoids and terpenoids. They are relatively less polar, and therefore detected at the back-end of the chromatograms. The concentration of rosmarinic acid was getting less in the subsequent fractions, whereas the other less polar compounds were increased simultaneously.
Several target phytochemicals were identified in the fractions using the method of multiple reaction monitoring in liquid chromatography tandem mass spectrometer. The identification was based on the detection of characteristic ions which were previously reported in literature for O. stamineus
extracts. The parent ions and their product ions which are listed in Table 1
were used in the target phytochemical analysis. The detected phytochemicals are presented in Figure 5
and their amount are plotted as peak area for the relative comparison, since no standard chemicals are purchased for quantitation.
Rosmarinic acid, sinensetin and eupatorin are the major phytochemicals in O. stamineus
]. The presence of rosmarinic acid was detected in all fractions with decreasing concentration from fractions 1 to 9. The highest content of rosmarinic acid was detected in fractions 1 to 3. On the other hand, the intermediate fractions 4 to 6 contained significant amount of three major phytochemicals, namely sinensetin, rosmarinic acid and eupatorin in the descending order. The low rosmarinic acid in fraction 9 was not in line with the observation in Figure 3
. This phenomenon could be explained by other compounds having similar retention time with rosmarinic acid in the chromatogram of fraction 9.
Other phytochemicals such as caffeic acid, danshensu, caftaric acid, caffeic acid derivative, salvianolic acid B, sagerinic acid, and orthosiphol A were also detected in lower amount in the O. stamineus fractions. Caffeic acid, danshensu, caftaric acid, caffeic acid derivative, salvianolic acid B and sagerinic acid showed similar trend in their concentrations along the fractions. This could be due to the high similarity of chemical characteristics of the compounds.
Based on the findings of this study, fractions 1 to 3 could be combined to obtain a rosmarinic acid rich extract (6.7%). The rosmarinic acid rich extract (fractions 1 to 3) could achieve the recovery of 57% rosmarinic acid from the crude extract. SPE fractionation also improved the rosmarinic acid content from 4.0% in the crude extract to 6.7% in the plant fraction. The fractions 4 to 6 could also be combined to obtain a plant fraction rich in rosmarinic acid, sinensetin and eupatorin. The rest of the fractions from 7 to 9 could be combined to obtain a sinensetin rich fraction.