Abstract
Pinus merkusii Jungh & De Vries. has become increasingly gathered more attention from researchers because the plant has a range of folk medicinal uses. Heartwood plant is the major source of dehydroabietic acid (DHAA) and abietic acid (AA), which possesses several medicinal properties, such as antiviral, antimicrobial, antiobesity and anti-inflammatory. The research proposed herein a low-cost, fast, specific, uncomplicated, sensitive, precise reverse-phase high-performance liquid chromatography (RP-HPLC). This method was conducted and validated for evaluating an amount of DHAA and AA in ethanol extract and oral spray containing P. merkusii heartwood extract. Additionally, stability and antimicrobial activities against clinically isolated Streptococcus mutans of the oral spray were determined. The separation was achieved on Pursuit 200Å PFP column, 150 × 4.6 mm, particles of 3 µm with a flow rate of 1.0 mL min−1. Methanol and water (70:30 v/v) were used as eluent with an isocratic mode and sample analysis volume was set at 10 µL, at a detection wavelength of 210 and 245 nm. The developed HPLC method for analysis of DHAA and AA showed good linearity with correlation coefficients equal to 1. Moreover, other validation parameters, comprised of accuracy, precision, specificity and detection and quantitation limits of this method displayed excellent reliability, validity and sensitivity. This method could be an interesting alternative for quantitative measurement of P. merkusii heartwood extract, oral spray formulation and other P. merkusii heartwood extract preparations. The result from antibacterial tests suggested that the oral spray containing P. merkusii heartwood extract is able to inhibit the oral pathogens causing dental caries. The oral spray decreased S. mutans population size by about 0.5–2 Log CFU mL−1 at 1–4 h and complete elimination of all bacteria strains within 24 h. This study provides validity for using P. merkusii heartwood extract as an alternative for preventing and treating oral infectious diseases.
Introduction
Pinus merkusii Jungh. & de Vriese, species in the Pinaceae family, are present in India, Myanmar, Thailand and Indonesia. It is mainly found in the mountains at 50–800 m above sea level in the north, east and northeast of Thailand. P. merkusii wood is used as raw material for furniture structure, building construction, paper and varnish oil [1]. Several studies report the antibacterial activities of P. merkusii. The previous study of antibacterial activity against Streptococcus mutans, which is the virulent microbe of the oral disease, of 22 ethanolic plant extracts revealed that three plant extracts including Dracaena lourieri, P. merkusii and Senna garrettiana demonstrated the antibacterial activities against all of S. mutans strains. Nevertheless, the most potent was observed with P. merkusii heartwood extract [2]. The phytochemical constituents include phenolic compounds, flavonoids, terpenoids and resin acid [3, 4]. Thus, P. merkusii could be a new natural product source of anti-S. mutans applied in pharmaceutical or cosmetic products such as oral health care sprays. Dehydroabietic acid (DHAA) and abietic acid (AA) are the major compounds of P. merkusii rosin. Rosin, extracted from the wood of pines appears as transparent yellowish to brownish is a solid resin. Major important chemical constituents of rosin include resin acids. The general chemical formula is C20H30O2 which represents free acids or dimers or anhydrides [5]. DHAA and AA are tricyclic diterpene carboxylic acid, AA is the main constituent of resin acid and abietane diterpenoid that is abieta-7,13-diene substituted by a carboxyl group at position 18. It has a role as a plant metabolite. It is an abietane diterpenoid and a monocarboxylic acid. It is a conjugate acid of an abietate while DHAA is an abietane diterpenoid that is abieta-8,11,13-triene substituted at position 18 by a carboxyl group. It has a role as a metabolite and an allergen. It is an abietane diterpenoid, a monocarboxylic acid and a carbotricyclic compound. It derives from an abietic acid. It is a conjugate acid of a dehydroabietate.
There are several analytical methods that have been reported in the current literature for the quantitative analysis of DHAA and AA. Analyses have been carried out mainly by gas chromatography (GC) and HPLC. The derivatization of these compounds was required before analysis with GC instrument [6].
HPLC method for the quantitative determination of DHAA and AA in ethanol extract and the oral spray formulation has been conducted, thoroughly validated and subjected to analyse P. merkusii oral spray formulation. The HPLC method on a C-18 column with on‐line spectrophotometric and fluorimetric detection for measurement of AA and DHAA in propolis was published previously, the isocratic mode was eluted with a solvent system composed of methanol and water (87:13 v/v) containing 0.05% formic acid with a run time of 20 min. AA was detected with a UV detection wavelength at 238 nm and DHAA was detected with a fluorescent detector with excitation and emission wavelength at 225 and 285 nm, respectively [7]. Moreover, DHAA and AA were evaluated using isocratic elution, eluted with a mobile phase consisting of methanol and water (87:13 v/v) containing 0.02% phosphoric acid. DHAA and AA were detected at a wavelength of 200 and 239 nm with a UV detector. Despite this, the fluorescent detection with an excitation and emission wavelength of 225 and 285 nm, provided more selectivity for the determination of DHAA [8, 9]. Although fluorescence detector has high selectivity and sensitivity, they are high-cost, due to their limited availability and compatibility issues with some solvents and columns. Additionally, not all analytical compounds are fluorescent or have the same fluorescence intensity.
The present work aims to develop and validate a simple RP-HPLC/DAD method to separate DHAA and AA, allowing a short analysis time and good separation followed by a measurement of the quality and quantity of DHAA and AA in the oral spray containing P. merkusii heartwood extract which is important for reducing cost and understanding the stability of P. merkusii products. In addition, antimicrobial activities against clinically isolated S. mutans of the oral spray were evaluated.
Experimental
Chemicals and reagents
The plant was collected from Phu Kradueng district of Loei Province, Thailand. The voucher herbarium specimen was approved by the Department of Pharmacognosy, College of Pharmacy, Rangsit University, Pathumthani, Thailand. P. merkusii heartwood was dried in a hot air oven at 50 ºC for overnight and screened through the 40-mesh stainless steel sieve to coarse powders. The dried powder was extracted with ethanol by sonication method for 30 min, then filtered through filter paper (Whatman no.1) and concentrated under vacuum in the rotary evaporator at 45 °C (Rotavapor® R-100, USA). The reference standard of DHAA and AA were obtained from Sigma-Aldrich (USA). Ultrapure water for HPLC was obtained from Direct-Q® 3 UV water purification system. The solvent in chromatography (HPLC grade) and analytical grade solvents were purchased from Burdick and Jackson®, SK chemicals (Korea). All chemicals for the preparation of oral spray were food (TCFF, Thailand) and pharmaceutical grade (Namsiang, Thailand).
Instrumental and chromatographic conditions
HPLC was employed for determining a quantitative measurement of DHAA and AA, and it was achieved on Agilent 1260 MODEL equipped with a 1260 VL quaternary pump, 1260 TCC autosampler and 1260 DAD diode array detector. The separation was done in Pursuit 200Å PFP column 150 × 4.6 mm (3 µm). The mobile phase consisting of methanol and water in a portion of 70:30 v/v was used. DAD detector was set at the wavelength of 210 and 245 nm. The injection volume was 10 µL and the flow rate was set at 1 mL min−1 with an isocratic system. HPLC condition was carried out at 25 ± 0.5 °C with a run time of 25 min. Data acquisition was performed on Ezechrom® software.
Preparation of oral spray containing P. merkusii heartwood extract
The oral spray containing P. merkusii heartwood extract (5 mg mL−1) was prepared by the cosolvent method as previously published [10]. Table 1 shows the ingredients of the oral spray formulation.
Ingredients of oral spray base and oral spray containing P. merkusii heartwood extract
Ingredients | Amount (g) | Role of Ingredients | |
Control | Test formulation | ||
Purified water | 35.0 | 35.0 | Co-solvent |
Ethanol | 10.0 | 10.0 | Co-solvent |
PG | 15.0 | 15.0 | Co-solvent |
PEG400 | 40.0 | 40.0 | Co-solvent |
Menthol | 0.3 | 0.3 | Soothing agent |
Xylitab® | 4.0 | 4.0 | Sweetening agent |
Peppermint oil | 0.1 | 0.1 | Flavouring agent |
Paraben conc | 1.0 | 1.0 | Preservative |
P. merkusii heartwood extract | 0 | 0.2 | Active ingredient |
Method validation
Validation of the analytical methodology process was performed through linearity and range, accuracy, precision, limits of detection and quantification studies (LOD and LOQ).
Linearity and range
Linear calibration curves of DHAA and AA were obtained at six points of the concentration range. Linearity is determined by following six different concentrations in the range of 0.0045–0.4400 mg mL−1 for DHAA and 0.0313–1.000 mg mL−1 for AA. The two reference standard solutions were dissolved in methanol. Linear calibration curves were plotted between six different concentrations versus peak area. Calculation of a regression line by the least squares method.
Accuracy
Recovery was used to determine the accuracy of an analytical method. The method was tested through spiking experiments by adding three different known amounts of standards, analysing in triplicate and then calculating the recovery percentage. The test concentration ranges of DHAA and AA were 0.028–0.176 and 0.125–0.500 mg mL−1, respectively.
Precision
This parameter was evaluated through intra-day and inter-day precision. Each sample was analyzed six times within a day (intra-day) and three sequential days (inter-day). The precision was calculated as percent relative standard deviation (%RSD).
LOD and LOQ
LOD is the smallest quantity of an analyte in a sample that can be detected under the specific conditions of the test [11]. It is expressed as a concentration at a specified signal-to-noise ratio of 3. LOD was calculated following the equation as LOD = 3.3(σ/S). LOQ is the lowest concentration of a component in a sample that can be quantitatively determined with acceptable precision and accuracy [12]. This limit is defined as the signal-to-noise ratio of 10. LOQ was calculated following the equation as LOQ = 10(σ/S). Where “σ” is the standard deviation of the blank and “S” is the slope of the calibration curve.
Application to oral spray
An oral spray containing P. merkusii heartwood extract stock solution was prepared in 10 mL volumetric flask for quantitative analysis. Oral spray (50 mg) was dissolved in methanol, mixed and adjusted the volume to 10 mL. Then, a stock solution of the reference standard (DHAA and AA), oral spray base (placebo) and P. merkusii extract was prepared by weighing each sample accurately at 50 mg, dissolved in methanol, mixed and adjusted the volume to 10 mL in a volumetric flask. All solutions were filtered through a 0.45 µm Nylon filter into an HPLC vial, and 10 µL of each sample was injected for analysis.
Stability testing of DHAA and AA in ethanol extract the oral spray
The oral spray and ethanol extract of P. merkusii heartwood kept in closed amber glass containers were exposed to three different storage conditions consisting of 4 ± 2 °C, room temperature (30 ± 2 °C) and accelerated condition (45 ± 2 °C, 75% humidity) for 4 months. An aliquot of each sample was taken at 0, 7, 14, 30, 60, 90, and 120 days for analyzing the percent remaining DHAA and AA contents by HPLC.
Microorganisms and culture conditions
The oral pathogens causing dental caries used in this study, S. mutans ATCC12175 and three clinically isolated S. mutans (NPRC001, NPRC002 and NPRC003) were obtained from the Department of Microbiology, Faculty of Science, Prince of Songkla University, Songkhla, Thailand. All microorganisms were cultured onto brain heart infusion (BHI) agar (Becton, Dickinson and Company, USA) at 37 °C under anaerobic conditions for 24 h. The bacterial cultures were stored in BHI broth containing 20% glycerol at −80 °C until use.
Anti-S. mutans activity of oral spray
The minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were evaluated by the broth microdilution method according to the National Committee for Clinical Laboratory Standards (NCCLS, 2002). The tested samples were dissolved with 5%DMSO and sequential two-fold dilution with BHI broth to a concentration ranging from 1 to 0.0005 mg mL−1. Gentamycin and DMSO were used as the positive and negative control, respectively. S. mutans suspension was prepared from the overnight broth culture and adjusted turbidity with 0.85% sterile normal saline solution to 0.5 McFarland standard (1.5 × 108 CFU mL−1) and then diluted with BHI broth (1:100 v/v) to contain 1.5 × 106 CFU mL−1. From this suspension, 50 µL was mixed with 10 µL of test sample solution into each well. The mixtures were then incubated under proper conditions. The MIC was recorded as the lowest concentration of the test sample that prevents visible S. mutans growth.
The MBC was determined by the streakes of the mixture suspensions from the wells without any growth of S. mutans during MIC assays in BHI agar plate and incubating at 37 °C under anaerobic conditions overnight. The lowest concentration of mixture suspensions that did not show any growth of S. mutans was taken as the MBC.
Time-kill assay
Time-kill assays were determined as described by Foerster et al. [13] with slight modifications. The bacteria cultures (24 h) in BHI broth were standardized (105 CFU mL−1) and then 10 mL of the bacterial suspension was mixed with the oral spray containing P. merkusii heartwood extract (5 mg mL−1) (40 mL) and incubated at 37 °C under anaerobic conditions. The controls were observed with gentamycin and without the extract (spray base). A 50 µL aliquot was withdrawn from the culture medium at 0, 0.5, 1, 2, 4, 8, 16 and 24 h for the determination of bacterial survival (CFU mL−1) by plate count technique.
Results and discussion
There are several analytical procedures reported in the current pharmaceutical published works for the quantitative analysis of DHAA and AA in P. merkusii, including HPLC. The analytical method was carried out on a Pursuit 200Å PFP column (150 mm × 4.6 mm, 3 µm) using the mobile phase consisting of methanol and water in the ratio of 70:30 v/v. The separation was carried out with an isocratic system, which is the simplest method for evaluation. The percentage of methanol in the mobile phase systems was investigated varying from 60 to 80%. The result indicated that the mobile phase consisting of methanol 70% provides the best separation and a sharp symmetrical shape without tailing. Some of the mobile phases that have been previously described showed a good separation was achieved with containing acidic such as formic acid and phosphoric acid. However, in this study, a non-acidic mobile phase was observed. The developed solvent system proved to be linear with a correlation coefficient (R2) of DHAA and AA greater than 0.9999. Therefore, the retention time as well as the peak shape of DHAA and AA was not influenced by the pH of the mobile phase.
The HPLC conditions were operated to achieve a good separation of DHAA and AA. The retention time of DHAA and AA reference standards were shown in the peak signal at 13 and 21 min, respectively with 25 min runtime (Fig. 1A and B). The UV absorption spectrum in the wavelength of 190–400 nm was determined for DHAA and AA. The UV λmax of DHAA was 210 nm and AA was 245 nm, these wavelengths were selected due to the suitable molar absorptivity for the detection of DHAA and AA. However, interferences in HPLC chromatograms have possibly presented problems at lower wavelengths such as 210 nm. Therefore, the chromatogram of the oral spray base solution (placebo) was compared.
The chromatogram of DHAA and AA (A) DHAA standard solution, (B) AA standard solution, (C) DHAA of P. merkusii extract solution, (D) AA of P. merkusii extract solution, (E) oral spray base solution (placebo) at 210 nm, (F) oral spray base solution (placebo) at 245 nm, (G) DHAA of P. merkusii oral spray solution and (H) AA of P. merkusii oral spray solution
Citation: Acta Chromatographica 36, 3; 10.1556/1326.2023.01149
A column is an important part of HPLC because it is responsible for the separation of the sample components. The HPLC methods on a C-18 column for measurement of DHAA and AA were published previously. The initial study of the column was focused on C-18 column, performed in different C-18 columns including Zorbax C-18 column (100 mm × 4.6 mm, 3.5 µm) and ACE C-18 column (250 mm × 4.6 mm, 5 µm) column. The results showed that C-18 column was not suitable for the measurement of DHAA and AA in P. merkusii with this chromatographic condition. Therefore, Pursuit pentafluorophenyl (PFP) column (150 mm × 4.6 mm, 3 µm) is being tested for analysis. It was found that Pursuit PFP column provides a good separation. PFP phases have shown multiple mechanisms for the separation of many compounds by an enhanced dipole-dipole, π-π interaction, charge transfer, and ion-exchange interactions when compared to C-18 phase. DHAA and AA were highly hydrophobic and soluble only in organic solvents such as acetone, ethanol, methanol and ethyl acetate. Both DHAA and AA contain carbon atoms in their skeleton with cyclic structures and unsaturated π-bonds.
Determination of the specificity, the stock solution of standards DHAA and AA, oral spray base solution, P. merkusii extract solution and P. merkusii oral spray solution were dissolved in methanol. All sample solutions were completely dissolved and filtered through a 0.45 µm Nylon filter. Ten microliters of each sample were analyzed, and the peak areas were measured. The chromatograms of P. merkusii extract (Fig. 1C and D), oral spray base (Fig. 1E and F) and P. merkusii oral spray (Fig. 1G and H) were compared with the chromatographic standards of DHAA and AA (Fig. 1A and B). As a result, the chromatogram of the oral spray base did not show the peak signals at 13 and 21 min, respectively. Thus, the ingredients in the oral spray from P. merkusii extract did not interfere with the process.
Validation method of HPLC analysis
Linearity
The linearity was determined at six different concentration levels. The different concentrations of DHAA and AA were plotted against their respective peak areas. The results represented the linearity with a correlation coefficient (R2) equal to 1 in the concentration range of 0.0045–0.4400 mg mL−1 and 0.0313–1.0000 mg mL−1, respectively as presented in Figs 2 and 3. The linear regression equations of DHAA and AA were obtained as y = 1,245,960,943.01x + 4,669,126.70 and y = 539,209,002.21x + 5,030,711.82, respectively.
Linearity of DHAA determined at 210 nm
Citation: Acta Chromatographica 36, 3; 10.1556/1326.2023.01149
Linearity of AA determined at 245 nm
Citation: Acta Chromatographica 36, 3; 10.1556/1326.2023.01149
Accuracy
The accuracy of an analytical method was evaluated by spiking a known amount of standard DHAA and AA at three concentration levels to quantify DHAA and AA in samples and calculation for accuracy as %recovery. The mean values of the %recoveries were 98.7 and 98.6% for DHAA and AA, respectively. The average percent recovery of DHAA and AA acquiesced with the requirement of AOAC Guidelines, which is acceptable in the range of 92–105%. The results obtained are presented in Table 2.
Validation results of the analytical method
Samples | Linearity (R2) | Accuracy (%recovery) | Precision (%RSD) | LOD (µg mL−1) | LOQ (µg mL−1) | |
Intra-day | Inter-day | |||||
DHAA | 1 | 98.7 ± 1.23 | 0.66 | 1.95 | 0.0079 | 0.0240 |
AA | 1 | 98.6 ± 0.89 | 0.98 | 1.63 | 0.0109 | 0.0332 |
Precision
The intra-day precision of the assay was evaluated with six replicates of DHAA and AA test samples on the same day. For inter-day precision, three different concentrations of samples were analysed on three consecutive days. These percentage relative standard deviation values of DHAA and AA were within limits of <2% (Table 2). Thus, the results obtained guaranteed the precision of this method.
Limits of detection and quantification
LOD and LOQ of the analytical method were calculated as described in ICH Guidelines based on the standard deviation of the response and slope of the calibration curve. LOD and LOQ of DHAA and AA were 0.0079, 0.0109 mg mL−1 and 0.0240, 0.0332 mg mL−1, respectively. The results are shown in Table 2 and indicated the sensitivity of this method. The developed HPLC method is suitable for the quantitative analysis of DHAA and AA in P. merkusii extract and P. merkusii oral spray formulation.
Application to P. merkusii oral spray
HPLC chromatogram of oral spray containing P. merkusii extract (Fig. 1G and H) was compared with the reference standard, P. merkusii extract and oral spray base chromatograms. As a result, the chromatogram of the oral spray base did not show any peak signals at 13 and 21 min, respectively as shown in Fig. 1E and F. Oral spray from P. merkusii extract formulation was composed of DHAA and AA, the major compounds in P. merkusii extract. The chromatogram was shown peak DHAA and AA at 13 and 21 min, respectively. The spray preparation contained more than 10 ingredients but all ingredients were not interrupted in the process of analysis.
Stability test
The effect of temperature (4 °C and 30 °C) and accelerated conditions on the stability of DHAA and AA in ethanolic extract and the oral spray were examined. The percentage remaining contents of DHAA and AA in ethanolic extract and oral spray stored at three different storage conditions for 4 months were shown in Tables 3 and 4. The result demonstrated that the percentage remaining contents of DHAA and AA in the ethanolic extract and oral spray did not change even when stored under conditions of 4°C. At room temperature, AA in the ethanolic extract and oral spray slightly decomposed, but DHAA showed more stability than AA through the period of 4 months. Under the accelerated condition, a marked decrement of AA in both samples was observed after 2 weeks of storage, while the percentage of remaining contents of DHAA was increased. This result is related to the previous report which indicates that the thermal treatment of AA is disproportionated to DA and dihydroabietic acid homologues which are more resistant to oxidation [14]. This result indicates that AA is not stable at room temperature and accelerated conditions. Thus, P. merkusii heartwood extract exposure should be avoided at high temperatures.
The remaining percentage of DHAA and AA in ethanolic extract under 4 °C, room temperature and accelerated storage conditions for 4 months
Time (days) | Ethanol extract | |||||
% remaining content | ||||||
DHAA | AA | |||||
4 °C | Room temperature | Accelerated condition | 4 °C | Room temperature | Accelerated condition | |
0 | 100 | 100 | 100 | 100 | 100 | 100 |
7 | 101.15 ± 0.21 | 100.12 ± 0.13 | 101.31 ± 0.24 | 100.09 ± 0.42 | 102.41 ± 0.43 | 98.10 ± 0.11 |
14 | 100.01 ± 0.12 | 101.42 ± 0.41 | 112.33 ± 0.32 | 101.48 ± 0.51 | 100.11 ± 0.44 | 97.49 ± 0.51 |
30 | 101.16 ± 0.11 | 104.28 ± 0.34 | 119.59 ± 0.21 | 99.89 ± 0.25 | 98.21 ± 0.31 | 61.82 ± 0.67 |
60 | 101.28 ± 0.23 | 103.54 ± 0.13 | 129.07 ± 0.13 | 102.17 ± 0.53 | 96.22 ± 0.41 | 49.31 ± 0.65 |
90 | 101.10 ± 0.10 | 100.22 ± 0.36 | 135.67 ± 0.23 | 101.41 ± 0.41 | 93.52 ± 0.12 | 43.13 ± 0.46 |
120 | 100.11 ± 0.15 | 102.49 ± 0.52 | 151.25 ± 0.21 | 101.62 ± 0.55 | 91.34 ± 0.41 | 39.53 ± 0.19 |
The remaining percentage of DHAA and AA in the oral spray containing P. merkusii heartwood extract under 4 °C, room temperature and accelerated storage conditions for 4 months
Time (days) | Oral spray | |||||
% remaining content | ||||||
DHAA | AA | |||||
4 °C | Room temperature | Accelerated condition | 4 °C | Room temperature | Accelerated condition | |
0 | 100 | 100 | 100 | 100 | 100 | 100 |
7 | 101.65 ± 0.63 | 100.29 ± 0.33 | 103.33 ± 0.24 | 102.07 ± 0.13 | 104.13 ± 0.71 | 95.19 ± 0.81 |
14 | 101.04 ± 0.22 | 102.79 ± 0.61 | 117.43 ± 0.54 | 99.98 ± 0.32 | 101.78 ± 0.24 | 95.58 ± 0.33 |
30 | 101.14 ± 0.13 | 105.83 ± 0.64 | 121.59 ± 0.11 | 97.89 ± 0.92 | 97.23 ± 0.86 | 54.28 ± 0.45 |
60 | 102.39 ± 0.41 | 104.48 ± 0.53 | 135.07 ± 0.08 | 101.38 ± 0.43 | 96.72 ± 0.95 | 46.52 ± 0.53 |
90 | 101.11 ± 0.12 | 100.43 ± 0.07 | 148.56 ± 0.85 | 102.20 ± 0.61 | 92.56 ± 0.23 | 42.63 ± 0.76 |
120 | 101.14 ± 0.05 | 99.09 ± 0.72 | 162.05 ± 0.68 | 101.81 ± 0.66 | 90.34 ± 0.82 | 38.75 ± 0.14 |
Antibacterial activity of oral spray
The oral spray containing P. merkusii heartwood extract (5 mg mL−1) displayed antimicrobial activity against reference and three clinically isolated strains of S. mutans with the MIC and MBC values range of 31.2–250.0 and 125.0–500.0 μg mL−1, respectively (Table 5). The results from the time-kill assay of all S. mutans strains are shown in Fig. 4A–D. As a result, a graph of Log CFU mL−1 was plotted against incubation time, the oral spray decreased the amount of viable S. mutans in all strains by about 0.5–2 Log CFU mL−1 at 1–4 h and the complete elimination was achieved within 24 h.
MIC and MBC of oral spray containing P. merkusii heartwood extract (5 mg mL−1) and positive control against standard and clinically isolated S. mutans strains
Sample | S. matans strains | |||||||
MIC (µg mL−1) | MBC (µg mL−1) | |||||||
ATCC 12175 | NPRC 001 | NPRC 002 | NPRC 003 | ATCC 12175 | NPRC 001 | NPRC 002 | NPRC 001 | |
Oral spray | 62.5 | 125.0 | 250.0 | 31.2 | 125.0 | 250.0 | 500.0 | 125.0 |
Base spray | ND | ND | ND | ND | ND | ND | ND | ND |
DHAA | 31.2 | 62.5 | 62.5 | 62.5 | 31.2 | 125.0 | 125.0 | 125.0 |
AA | 7.8 | 15.6 | 15.6 | 15.6 | 15.6 | 31.2 | 31.2 | 31.2 |
Gentamycin | 0.5 | 7.8 | 7.8 | 7.8 | 7.8 | 15.6 | 15.6 | 15.6 |
ND = not detected.
Time-kill curves of (A) S. mutans ATCC12175 and clinically isolated S. mutans ((B) NPRC001, (C) NPRC002 and (D) NPRC003) after being treated with the oral spray containing P merkusii heartwood extract (5 mg mL−1), gentamycin (0.2 mg mL−1), spray base and control at different times
Citation: Acta Chromatographica 36, 3; 10.1556/1326.2023.01149
Conclusions
The analytical RP-HPLC method for analysing the amount of DHAA and AA was developed and validated carefully. This method was an interesting alternative and proved to be suitable for the detection and quantification of DHAA and AA. Although, several HPLC methods described in previous studies, none of them relate an isocratic with a non-acidic mobile phase. The developed method was cost-efficient, simple, fast and sensitive which could be occupied for quantitation of P. merkusii preparation and applied for quality control process in further study. Additionally, the oral sprays containing P. merkusii heartwood extract has antibacterial activity against clinically isolated S. mutans. Thus, P. merkusii heartwood extract may be a new potent natural product source for the prevention of oral pathogens causing dental caries. Clinical efficacy and safety studies should be further conducted.
Acknowledgements
The authors are grateful to grant number 12/2560 from the Research Institute of Rangsit University for financial support and providing language assistance and proofreading the article.
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