Abstract
Orthosiphon stamineus extract was fractionated by C18 reversed phase absorbent to recovery high yield of rosmarinic acid. The mixture of chloroform-ethyl acetate (70:30) was chosen as the solvent system in this fractionation because rosmarinic acid gave the lowest solvation free energy in that solvent system based on the computational solubility prediction. The content of rosmarinic acid was increased from 4.0 %w/w to 6.7 %w/w after fractionation. The radical scavenging activity of rosmarinic acid rich fraction (IC50 = 38.3 μg/mL) was higher than the crude extract (IC50 = 58.85) based on the DPPH assay. Several phytochemicals were also identified based on the detection of fragment ions of target compounds. Fraction 1 to 3 could be combined to be a rosmarinic acid rich fraction. Simultaneously, the combination of fraction 4 to 6 could obtain plant fraction rich in rosmarinic acid, sinensetin and eupatorin, whereas fractions 7 to 9 could be combined as a sinensetin rich fraction. The preparation of known phytochemical profile of O. stamineus fraction is highly required for further investigation in value added product formulation and pharmacological studies, particularly for anti-diabetes and treatment for kidney diseases which had been previously reported for this herbal plant.
Keywords: Orthosiphon stamineus, radical scavenging activity, solvation free energy, fragment ions, solid phase extraction
Introduction
Orthosiphon stamineus is a member in the Lamiaceae family and in the genus Orthosiphon. It is broadly used as traditional medicine in South East Asia countries for health improvement as well as treating kidney diseases, gout, diabetes and menstrual disorder (Adam et al. 2009). The pharmacological properties of the plant are attributed by the presence of different class of phytochemicals such as terpenoids, polyphenols and sterols (Banskota et al. 2003). The phytochemicals of O. stamineus are mostly from the class of polyphenols. There are 20 phenolic compounds including 9 lipophilic flavones, 2 flavonol glycosides, and 9 caffeic acid derivatives have been identified (Olah et al. 2003).
Phytochemicals are also known as plant secondary metabolites which are used as chemical messengers and defensive chemicals in plants and possess important biological activity, including antioxidant activity (Liu 2011). The phenolic compounds with antioxidant property can scavenge free radicals such as hydroxyl and superoxide anion, as well as chelate metal ions (Brewer 2011). The natural antioxidants from plants show the potential in human health protection, as well as food preservatives and additives (Brewer 2011). Rosmarinic acid is one of the antioxidative plant phenolics which can be abundantly found in Lamiaceae species (Brewer 2011). O. stamineus was reported to have rosmarinic acid up to 53 mg/g extract (Akowuah et al. 2004).
Owing to the importance of rosmarinic acid in many pharmacological applications, O. stamineus extract was fractionated using solid phase extraction in order to obtain a rosmarinic acid rich fraction with high antioxidant activity. The solvent system of fractionation was chosen based on the previous studies and computational solubility prediction for high yield of rosmarinic acid recovery. The chemical characteristics of this rosmarinic acid rich fraction were also evaluated in terms of the total phenolic content, total flavonoid content and antioxidant capacity compared to the crude extract of O. stamineus. Several target phytochemicals were also identified using high sensitivity analytical tool of LC-MS/MS. This technique is highly reliable and accurate without the use of the standard chemical in compound identification.
Materials and methods
Plant material
The dried whole plant of O. stamineus (white flower species) was supplied by Fidea Resources (Selangor, Malaysia). The stems and leaves of the plant were ground into fine powder (<1 mm) prior to extraction.
Reflux extraction of Orthosiphon stamineus
The dried plant sample (273.5 g) was extracted with 70% ethanol (2.5 L) at 56.5 °C for 3 hours. The crude extract was then filtered and concentrated by a rotary evaporator (Heidolph, Laborota 4000, Germany) at 60 °C. The crude extract was kept in an oven at 50 °C until completely dry and stored at -4°C until further analysis.
Prediction of rosmarinic acid solubility by solvation free energy
The solubility of rosmarinic acid was predicted in term of solvation free energy to select the appropriate solvent system for the fractionation processes. Several types of mobile phases, namely, ethyl acetate, chloroform, methanol, acetic acid and formic acid were used for the pre-screening process based on the lowest solvation free energy. The solvents were tried based on the solvent selection of previous researchers who carried out fractionation of rosmarinic acid by SPE from different plant species (Arafat et al. 2008; Ly et al. 2006; Maleš and Medić-Šarić 2001; Vundać et al. 2005). The calculation of solvation free energy for rosmarinic acid in different solvents was performed computationally by Forcite Package in Material Studio 7.0 (Accelrys Inc., San Diego, CA).
Solid phase extraction to prepare plant fractions
Solid phase extraction (SPE) was applied for the fractionation of O. stamineus crude extract using Chromabond C18ec cartridges (6 mL, 500 mg, Macherey-Nagel, Düren, Germany). The SPE cartridge was first pre-conditioned with methanol (12 mL), and then equilibrated by water acidified with 0.5% formic acid (6 mL). 0.15 g of crude extract was dissolved in 6 mL of methanol (60 %v/v), and only 1 mL of the crude extract solution was loaded onto the SPE cartridge. A number of 10 fractions were collected from the SPE cartridge, where the fractions 1-8 were collected by eluting 0.2 mL of chloroform-ethyl acetate mixture in a ratio of 30:70, and the rest of the fractions were collected by eluting 1.0 mL of methanol for washing purpose. The collected fractions were dried by using an IR concentrator coupled with cold trap system (Micro-Cenvac NB 503CIR, N-BIOTEK Co. Ltd., Korea) at 40 °C.
Determination of total phenolic content and total flavonoid content
The total phenolic content (TPC) of crude extract and its fractions were determined by the Folin–Ciocalteu colorimetric method according to the International Organization for Standardization (ISO) 14502-1 (ISO 2005). 1 mL sample (1 mg/ml) was mixed with 10 % Folin–Ciocalteu reagent (5 mL) and incubated for 5 minutes at room temperature. Then, 4 mL of sodium carbonate solution (7.5 %w/v ) was added to the mixture. The mixture was incubated at room temperature for 60 minutes before the absorbance was measured at 765 nm. Blank was prepared by replacing the sample solution with 1 mL distilled water. The TPC was expressed as gallic acid equivalent (GAE) in mg per 100 g dry extract. The standard curve of gallic acid was plotted at the concentration ranged from 10 to 50 µg/mL. All experiments were performed in triplicate and averaged for the mean of absorbance.
The total flavonoid content (TFC) of crude extract and its fractions were determined according to the method suggested by Chua et al. with some modification (Chua et al. 2011). A 1 mL sample (1 mg/mL) was mixed with 2 mL 2 % aluminium chloride methanolic solution. The mixture was incubated for 15 minutes at room temperature before the measurement of absorbance at 430 nm. The TFC was expressed as rutin equivalent (RE) in mg per 100 g dry extract. The standard curve of rutin was prepared at the concentration ranged from 10 to 60 µg/mL.
Determination of free radical scavenging activity
The free radical scavenging activity of crude extract and its fractions from O. stamineus were evaluated by DPPH assay. The extracts were prepared at different concentrations ranging from 10-250 ppm. A 0.5 mL sample solution was mixed with 3.5 mL DPPH methanolic solution (0.1 mM). The samples were then incubated in a dark for 30 minutes at room temperature. Control was prepared by replacing the extract solution with 0.5 mL methanol. The absorbance was measured at 517 nm. Ascorbic acid, rosmarinic acid and rutin were used as positive control. The antioxidant activity of the sample was expressed in term of IC50, which is estimated at 50 % inhibitory activity from the plot of inhibition against sample concentration.
(Eq. 1)
Chromatographic fingerprinting by liquid chromatography
An analytical liquid chromatography (Ultimate 3000) system coupled with a diode array detector (Dionex, Thermo Scientific; MA, USA) and a C18 reversed phase XSelect High Strength Silica (HSS) column (2.1×100 mm, 2.5µm, Waters; Milford, MA), was applied to analyse the phytochemicals profile in the crude extract and fractions of O. stamineus. The mobile phase consisted of 0.1% formic acid in water (A) and acetonitrile (B) (Chua et al. 2011). The separation was performed in a gradient program with the following condition; 0-10 min, 10%B; 10–25 min, 10–80%B; 25–30 min, 80%B; 30–35 min, 10%B at the flow rate of 150 µL/min. The samples were filtered by using syringe filters with 0.22µm pore size (Membrane Solutions, Dallas, TX, USA) before injection.
Identification of targeted phytochemicals by liquid chromatography tandem mass spectrometry
The targeted phytochemicals were identified by an ultra-performance liquid chromatography (UPLC, Waters Acquity; Milford, MA) system coupled with a triple quadrupole-linear ion trap tandem mass spectrometer (Applied Biosystems 4000 QTRAP; Life Technologies Corporation, Carlsbad, CA) and a C18 reversed phase Acquity column (2.1×150 mm, 1.7 µm). Both positive and negative modes of multiple reaction monitoring with the published transition ions were used for compound identification (Table 1). The standard solution of rosmarinic acid was prepared in a serial concentration from 0.2 – 1.0 ppm for quantitation.
The mixture of 0.1% formic acid in water (A) and acetonitrile (B) was used as mobile phases (Chua et al. 2011). The separation was performed in a gradient elution with the following condition; 0-10 min, 10% B; 10–12 min, 10-90%B; 12–14 min, 90%B; 14–15 min, 10%B at the flow rate of 0.2 mL/min and the injection volume of 5 µL. All samples were filtered prior to injection.
Results and discussion
Prediction of rosmarinic acid solubility by simulation
Solubility is one of the dominant physiochemical properties to explain the performance of target phytochemical separation. According to Savjani et al. (2012), 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 (Savjani et al. 2012). Thus, solubility can be applied to determine the solvent system for the extraction process, as well as fractionation process. Previously, the solubility is estimated by ‘like dissolve like" rule, at which certain amount of solute is dissolved in a solvent. The mutual solubility of 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 (Reichardt 2004). 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 the 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 the creation of the cavity in solvent and disruption of the solvent structure. The more negative the value of solvation free energy indicates better solubility (Accelrys 2011).
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). The solvation free energy is the summation of the free energy of the charge removal in the 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 the lowest solvation free energy in the mixture of chloroform-ethyl acetate (30:70), followed by ethyl acetate-methanol-formic acid-water mixture (100:13.5:2.5:10), ethyl acetate-acetic acid-formic acid-water mixture (100:11:11:26), ethyl acetate-methanol-water mixture (77:13:10), ethyl acetate-formic acid-water mixture (80:10:10), and methanol-water mixture (10:90). Rosmarinic acid is an intermediate polar molecule, hence, it is more dissolvable in chloroform-ethyl acetate mixture. Therefore, chloroform-ethyl acetate solvent system was selected as the mobile phase for the fractionation of rosmarinic acid due to its low solvation free energy.
Relationship of total phenolic, total flavonoid, scavenging activity and rosmarinic acid
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 folds higher than TFC for all samples. 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 fraction 1 to 9.
The ratio of TPC to TFC is presented in an "S" shape curve from fraction 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 change of rosmarinic acid content in fraction 9 also increased the TPC/TFC ratio. This was because the remaining rosmarinic acid was rinsed out from the SPE cartridge by strong solvent, methanol.
The TPC/TFC ratio is closely correlated to the concentration of rosmarinic acid which is also highly linked to the IC50 of the fractions 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 the lower IC50 values of fraction 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.
The antioxidant activity of O. stamineus extract and its fractions were evaluated in term of free radicals scavenging activity and expressed in IC50. This IC50 explains the concentration of sample required to exhibit 50 % of inhibition against free radicals. The phytochemicals with anti-radical 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 5 hydroxyl groups in the molecular structure which may contribute protons to scavenge free radicals of DPPH. 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.
Chromatographic fingerprinting of plant fractions
Figure 4 illustrates the chromatograms of O. stamineus crude extract and its fractions. The fraction samples show a better HPLC separation compared to the crude extract sample as the undesired impurities were removed during the fractionation process. Rosmarinic acid shows the highest peak at the retention time around 12.5 minutes in the figure. SPE fractionation produced RA rich fraction 1 to 3. Further elution was found to produce RA 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 the 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 is plotted as peak area for the relative comparison, since no standard chemicals are purchased for quantitation, except rosmarinic acid.
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 fraction 1 to 9. The highest content of rosmarinic acid was detected in fraction 1 to 3. On the other hand, the intermediate fraction 4 to 6 contained a significant amount of three major phytochemicals, namely sinensetin, rosmarinic acid and eupatorin in the descending order.
Other phytochemicals such as caffeic acid, danshensu, caftaric acid, caffeic acid derivative, salvianolic acid B, sagerinic acid, and orthosiphol A were also detected in a lower amount in the O. stamineus fractions. Caffeic acid, danshensu, caftaric acid, caffeic acid derivative, salvianolic acid B and sagerinic acid showed the 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, fraction 1 to 3 could be combined to obtain a rosmarinic acid rich extract (6.7 %). The rosmarinic acid rich fraction (fraction 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 fraction 4 to 6 could also be combined to obtain plant fraction rich in rosmarinic acid, sinensetin and eupatorin. The rest of the fractions from 7 to 9 could be combined to obtain sinensetin rich fraction.
Conclusion
The fractionation of O. stamineus crude extract was successfully performed using SPE to obtain plant fraction with high rosmarinic acid content. The fractionation process increased the rosmarinic acid content from 4.0 % in the crude extract to 6.7 % in the plant fraction using the solvent system of chloroform-ethyl acetate (70:30). The computational prediction of rosmarinic acid solubility based on the lowest solvation free energy could produce higher content of rosmarinic acid in the plant fraction (68.63 mg/g fraction) than previously reported data (53 mg/g fraction) from this herb. The increment in rosmarinic acid content also increased the scavenging capacity of the fraction (IC50 = 38.29 µg/mL). Therefore, fractionation has value-added the plant extract for further investigation.
Acknowledgement
The authors would like to thank the financial support of research grant from Universiti Teknologi Malaysia (GUP 14H24) and Ministry of Higher Education Malaysia (HiCoE 4J263).