Chemical constituents and nitric oxide inhibitory activity of supercritical carbon dioxide extracts from Mitragyna speciosa leaves

2.1 Plant material

Fresh leaves of M. speciosa were collected from the forest of Sungai Setong, Kelantan, Malaysia, in November 2006. The plant materials were identified by the herbarium authority at the Department of Chemistry, University of Malaya, where the plant specimen was deposited for future reference (Voucher specimen no. KL5321). The leaves were dried under the shade, assisted by electric light bulbs as a heat source of 35–40 °C for a period of one week. The shed dried leaves were ground by an electrical blender to produce coarse powder of approximately 5 mm size particle.

2.2 Reagents

Carbon dioxide (99.995% purity, SFE grade), contained in a cylinder with an eductor tube, was purchased from MOX (Selangor, Malaysia). Analytical grade solvents, chemical reagents and 95% ethanol were purchased from Merck. The standard mixture of C₇–C₂₄ and other homologous series of n-alkanes used in the gas chromatography analysis were obtained from SIGMA Chemical Co., USA. Antibiotic (5000 U/mL penicillin and 5000 μg/mL streptomycin) and Dulbecco’s Modified Eagle’s Media (DMEM) were obtained commercially from Flowlab™, Australia; foetal bovine serum (FBS) was obtained from Mycoplex™ (PAA Lab. GmbH, Austria); recombinant mouse interferon gamma (IFN-γ) was obtained from BD Pharming, USA; lipopolysaccharide (LPS) was from Escherichia coli (strain 0111:B4), and sulphanilamide and naphtylethylenediamine were obtained from SIGMA Chemical Co., USA. Dimethyl sulfoxide (DMSO) was purchased from Fisher Scientific, USA, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MMT) from Fluka Chemie GmbH, Switzerland.

2.3 Supercritical fluid extraction (SFE)

Approximately 25 g of the dried ground leaves was loaded into a 100 mL stainless steel extraction vessel of the analytical-scale SFT 100 XW SFE system (illustrated in Fig. 1). An orthogonal test design L₉ (3)³ was employed in order to determine a suitable extraction condition in a wide range with a minimum number of trials, whereby the temperature, pressure and the percentage of modifier (ethanol) were considered to be three major factors for effective extraction. The combinations of three different levels of each factor are listed in Table 1. In each test, two-step extractions were performed. First, the plant material was pre-extracted with pure CO₂ in Step 1 (S1) and afterwards the residue was re-extracted with CO₂ modified with aqueous ethanol solution in Step 2 (S2) as depicted in Table 2. CO₂ was delivered at the rate of 24 mL/min by the SFT-10 Constant Pressure pump. For those studies involving modifier, ethanol solution was added directly to the plant material in the extraction vessel using an additional High Performance Liquid Chromatography (HPLC) pump (Series II Isocratic HPLC pump) prior to the extraction by supercritical CO₂.

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Figure 1

The flow sheet of the analytical-scale of SFT 100 XW SFE system.

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In both steps, after 60 minutes of static extraction (no CO₂ flow), the sample material was subjected to dynamic extraction by flowing the CO₂ liquid (depressurized). The extraction process was terminated when it was visually observed that there was no more analyte being trapped in the collection vial (exhaustive extraction). The extracts obtained were evaporated to dryness under reduced pressure at 40 °C (yielding dark green residue). Then their final mass was weighed (Table 2) and later checked by thin layer chromatography (TLC) for their individual profile.

TLC separations were performed using aluminium sheets pre-coated with 0.20 mm thick Silica gel 60 F₂₅₄ (20 cm × 20 cm) and developed with a mobile phase dichloromethane:methanol (96:4 v/v). The extracts were dissolved in dichloromethane and applied to silica gel plates. TLC plates were then visualized under UV light (254 and 365 nm) and further sprayed with Dragendorff’s reagent to detect the presence of alkaloidal compounds, which was indicated, by the presence of orange colour spots.

2.4 Nitric oxide inhibitory activity

2.5 MTT cell viability assay

After the removal of culture media, the wells in micro titre plate were topped up with 100 μL of DMEM followed by the addition of 20 μL of MTT (5 mg/mL). The cells were incubated under 5% CO₂ at 37 °C for 4 h. The formed formazan crystals were dissolved in DMSO and the absorbance was recorded at 570 nm on a micro plate reader (SpectraMax, Plus 384, Molecular Devices, Inc., USA). The optical density (OD) of the samples was compared to that of negative control cells (untreated stimulated cells and contained 0.1% DMSO) to obtain the percentage viability using the formula stated below (Syahida et al., 2006): $$\text{Cell viability}(\%)=\frac{{\text{OD}}_{\text{sample}}}{{\text{OD}}_{\text{control}}}\times 100\%$$

2.6 Gas chromatographic analyses

The analyses were performed on the SFE extract that possesses the strongest anti-inflammatory activity with no toxicity effect.

2.7 Statistical analysis

Results for NO inhibition and cell viability are expressed as mean ± standard deviation (SD). Data were statistically assessed using Graph pad prism version 5.0 for one-way analysis of variance (ANOVA) with Dunnett’s test. Differences were considered statistically significant at p < 0.05.

3.1 Supercritical fluid extraction (SFE)

Optimization of the experimental conditions signifies a crucial step in the development of an efficient SFE method through the adjustment of certain parameters that potentially affect the extraction process. Optimization of the method can be performed step-by-step or by using an experimental design. Table 2 exhibits the results of the SFE of M. speciosa carried out under different conditions. Pressure and temperature of the fluid and percentage of the modifier were the selected factors assessed using a three-level orthogonal array design with L₉ (3)³ matrix. Consequently, a complete evaluation of the three factors will require three level tests, i.e., 27 tests. In order to reduce the number of tests, an L₉ (3)³ orthogonal design graph was employed, which reduced the total number of tests to 9 (Table 1). A two-step extraction process was designed for this study that included pre-extraction with pure CO₂, makes it a total of 18 tests to be executed.

In general, results depicted in Table 2 indicate the percentage of total yield (S1 + S2) improved with the increment in pressure. At the static pressure of 3000 psi, the yield raised as the temperature was increased from 40 to 60 °C but dropped slightly when the temperature was further increased from 60 to 80 °C. As at 1300 psi and 5000 psi, a rise in temperature resulted in lower yield.

The yield of extraction by pure CO₂ also trailed the same trend of increased yield for an increase in the pressure. A rise in the pressure can improve the density of the fluid and thereby resulting in an increased solvating power-giving rise to more yields. At static pressure of 1500 psi, the yield gradually reduced as the temperature increased. Within the pressure range of 3000–5000 psi, the yield increased between the temperatures of 40–60 °C and somehow decreased from 60 to 80 °C. Supercritical CO₂ has a low polarity, making the extraction of polar analytes difficult. This limitation can usually be overcome by adding small amounts of polar entrainer, such as ethanol, to the supercritical CO₂ in order to raise its solubilizing power and thus increase the extraction efficacy. In our experiment, the second step of each test in which the entrainer was added in succession was also meant to be the exhaustive process for completing the recovery of the high-molecular mass components. Anyhow, the results revealed that the addition of entrainer (as modifier) negligibly improved the yield than that of pure CO₂. The increments in the yield were only by 0.01–0.27% except in M7S2, where it was 0.48%.

Basically, in Step-1 (extraction by pure CO₂) and Step-2 (extraction by CO₂ + modifier) process, when the pressure factor was set to 1300 psi, the yields were low and the total yields were not more than 0.3%. A raise in the pressure from 3000 psi to 5000 psi resulted in a satisfactory improvement in the yield (>0.9%), except for M4S1 (0.69%) for Step-1 process. Overall, the total yields at 3000 psi and 5000 psi, were significant (>0.9%).

3.2 TLC profile of the extracts

Each crude was examined to point out possible interesting differences in the chemical composition obtained by different extraction conditions.

TLC sprayed with Dragendorff’s reagent revealed several orange spots for all crudes extracted at 5000 psi but not for those extracted at 1300 and 3000 psi. This indicated that alkaloidal compounds can only be furnished at relatively high pressure and the addition of a polar solvent as modifier did not give any effect in this aspect as shown by crudes extracted at 1300 and 3000 psi. Nevertheless, the orange colour spots of the alkaloid were more intense in M7S2, M8S2 and M9S2 compared to M7S1, M8S1 and M9S1 respectively, consequently suggesting that the addition of modifier might enhance the yield of the alkaloid components.

3.3 Nitric oxide inhibitory activity

A total of 15 plant extracts were screened for their inhibitory effect of NO release in RAW 264.7 cells. The overall efficacy of all plant extracts on NO₂⁻ production in IFN-γ/LPS-activated macrophage and cell viability on RAW 264.7 cells are presented in Table 3. The plant extracts were considered as having a strong, moderate or weak activity, if the inhibitory of NO was more than 90%, between 50% and 89%, or less than 50%, respectively, as compared to the control. In addition, the viability of RAW 264.7 cells assessed by MTT method must be above 85% to prove that the decrease in NO production was not a result of cell death (Mosmann, 1983). The results revealed that the extracts obtained at 3000 and 5000 psi, using pure CO₂ exhibited stronger NO inhibitory activity (lower production of NO) than those extracted in the presence of modifier (Table 3). Hence suggesting that extraction with 100% CO₂ alone is sufficient enough to enrich extract with compound/s of potent anti-inflammatory property. The results showed that six of the plant extracts demonstrated 51–64%, and nine exhibited 16–48% inhibitory activity towards NO production when tested at a final concentration of 100 μg/mL. Meanwhile, NOS inhibitor, N-nitro-arginine methyl ester (L-NAME), used as the positive control in this assay strongly (86.65%) inhibited the secretion of NO without any cytotoxic effect at the concentration of 250 μM (0.674 μg/mL). Among those samples that moderately reduced NO production, M5S1 showed the highest NO inhibitory activity at 60.08 ± 10.02% without any cytotoxicity effect (cell viability, 91.98 ± 5.58%). Thus, the optimal conditions determined from these tests to furnish an extract that possesses the strongest anti-inflammatory activity with no cytotoxicity effect were extraction conducted using pure CO₂ at 3000 psi of pressure and temperature at 60 °C. The constituents of this extract (M5S1), were identified via GC and GC–MS analyses.

3.4 GC and GC–MS analyses of M5S1

Fig. 2 depicts the GC–MS profile of the SFE extract that possesses the strongest NO inhibitory activity without showing toxicity effect towards the test cells (M5S1). Table 4 lists the compounds identified in M5S1, the relative GC peak areas of these compounds and their experimental KI. RSD values less than 10% were considered good precision for a method. The gas chromatographic profile shows 15 identifiable components that represented 93.53% of the total peak area. The prominent constituents were fatty acids, accumulated to 39.01% of the total yield. The rest of the crude was made up of four hydrocarbons (36.67%), three phytosterols (9.32%), two esters (3.45%), two alcohols (2.30%) and last but not least, a tocochromanol (2.78%). Palmitic acid, at 34.90% was the most abundant constituent in the crude matrix followed by heptacosane (18.56%) and nonacosane (11.00%).

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Figure 2

The GCMS total ion chromatogram of M. speciosa extract obtained by SFE that possesses the strongest nitric oxide inhibitory activity without cytotoxicity effect, M5S1.

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Fatty acids have been identified to involve in the formation of healthy cell membranes, proper development and functioning of the brain and nervous system, regulating blood pressure and viscosity as well as immune and inflammatory responses (Eastwood, 1997; Sánchez-Vicente et al., 2009). This may explain the NO inhibition exhibited by M5S1, as it is rich in palmitic acid that has been communicated for its anti-inflammatory activity. Ester bond hydrolysis of membrane phospholipids by phospholipase A₂ and consequent release of fatty acids are the initiating steps of inflammation. Therefore, phospholipase A₂ inhibitory is one of the ways to control inflammation. The structural and kinetics studies have revealed that palmitic acid is an inhibitor of phospholipase A₂, and hence, is an anti-inflammatory compound (Aparna et al., 2012).

Furthermore, the appearance of phytosterols may also enhance the NO inhibition activity exhibited by M5S1 as they have been reported to possess anti-inflammatory (Park et al., 2001), antioxidant (Yoshida and Niki, 2003) and angiogenic (Choi et al., 2002) activities. β-Sitosterol, stigmasterol and campesterol were detected at an appreciable amount, i.e., 6.67%, 0.99% and 1.66%, respectively. Along with these phytosterols, the presence of tocopherol that constituted 2.78% of M5S1, although present at a low concentration, might contribute towards the observed NO inhibition activity through its antioxidant properties, especially against lipid peroxidation in biological membranes (Gast et al., 2005).

Interest has long been focused on the organic solvent extracts of M. speciosa obtained through cool percolation or hot extraction (Avula et al., 2015; Beckett et al., 1965; Kitajima et al., 2006; Seaton et al., 1960; Shellard et al., 1978). Almost all of the previous biological studies have been conducted on the alkaloidal fractions and its major alkaloid constituents (Idayu et al., 2011; Kong et al., 2011; Parthasarathy et al., 2009; Sabetghadam et al., 2013; Shaik Mossadeq et al., 2009; Utar et al., 2011). There is lack of studies on the non-alkaloidal crude. In this current investigation, studies have been done on the non-organic solvent crudes that consist of not only the alkaloids but also the non-alkaloidal fractions. It highlighted the advantage of the SFE technique in affording the non-alkaloid anti-inflammatory compounds such as campesterol, stigmasterol, sitosterol and palmitic acid. This is the first report revealing non-alkaloidal extract of M. speciosa exhibited NO inhibition activity, which may be of important potential in anti-inflammatory treatment.