TY - JOUR
AU1 - Shah,, Sonal
AU2 - Dhanani,, Tushar
AU3 - Sharma,, Sonu
AU4 - Singh,, Raghuraj
AU5 - Kumar,, Satyanshu
AU6 - Kumar,, Bhanu
AU7 - Srivastava,, Sharad
AU8 - Ghosh,, Srikant
AU9 - Kumar,, Rajesh
AU1 - Juliet,, Sanis
AB - Abstract Background Ageratum conyzoides is an aromatic plant. It is considered as an invasive and cosmopolite weed, widely spread in tropical and subtropical regions. Phytochemicals such as benzopyrenes, flavonoids, and terpenoids are reported from A. conyzoides. Objective Development and validation of a reversed-phase HPLC-photodiode array (PDA) detection method for simultaneous identification and quantification of coumarin, precocene-I, β-caryophyllene oxide, α-humulene, and β-caryophyllene in extracts of A. conyzoides and essential oils was carried out. Methods Separation of analytes was achieved on a RP-18 (250 mm × 4.6 mm, 5 µm) column using a solvent system comprising of a mixture of acetonitrile and water with 0.05% trifluoroacetic acid in gradient elution mode at ambient temperature with flow rate of 1 mL/min. Results The retention time of coumarin, precocene-I, β-caryophyllene oxide, α-humulene, and β-caryophyllene was 4.38, 12.86, 20.10, 33.34, and 35.11 min, respectively. Limits of detection for coumarin, precocene-I, β-caryophyllene oxide, α-humulene, and β-caryophyllene were 2.5, 2.5, 2.5, 0.025, and 2.5 µg/mL, respectively. Similarly, LOQ were 10, 10, 10, 0.10, and 10 µg/mL for coumarin, precocene-I, β-caryophyllene oxide, α-humulene, and β- caryophyllene, respectively. Repeatabilities (RSD, %) values for intraday and interday precision for coumarin, precocene-I, β-caryophyllene oxide, α-humulene, and β-caryophyllene was 0.765–2.086 and 0.886–2.128; 0.879–1.672 and 0.979–1.825; 0.696–2.418 and 0.768–2.592; 1.728–2.362 and 1.965–2.378; 1.615–2.897 and 1.658–2.906, respectively. Conclusions The separation of five analytes was achieved within 50 min. The developed and validated HPLC-PDA method was successfully applied for identification and quantification of above five analytes in A. conyzoides extracts and essential oils. The method could be used for meeting the characterization criteria of phytoformulations. Ageratum conyzoides L., belongs to the family of Asteraceae, tribe Eupatoriae. It is an invasive weed distributed in many tropical and subtropical countries due to its adaptability to a wide range of climatic conditions. It is an annual herb of 30–90 cm height and found as one of the most common weeds in India. The whole plant of A. conyzoides is widely utilized in traditional systems of medicine in different countries. In India, this plant is used for its bactericidal, antidysentric, and antilithic properties by traditional communities. Use of A. conyzoides as a purgative, febrifuge, and for the treatment of opthalmia, colic, ulcers, and wounds have also been reported (1–4). Also, acaricidal properties of A. conyzoides extracts against Rhipicephalus (Boophilus) microplus, acaricides resistant ticks, infesting cattle and buffaloes have been reported (5, 6). Chromenes, coumarins (CMs), and sesquiterpenes are the major constituent (>90%) of A. conyzoides essential oil (ACO, 7). In addition to known precocenes (PRCs) I and II, four new compounds, namely an isodihydroeuparin derivative, a chromene, chromone, and a chromanone were reported from the essential oil of A. conyzoides (8). Chromenes also occur in marine organisms and fungi. The ability of chromenes to interact with different molecular targets makes them highly valuable templates for modification or synthesis of new pharmacologically active molecules (9–12). CM (1, 2 benzopyrone, Figure 1A) occurs naturally in the essential oils of a number of plants (13). CM has been used as an additive in food and cosmetics. It also exhibits biological activities such as inhibition of microbes, antiplatelet coagulation, induction of cell differentiation, and protection of the liver against hepatic injury and steatosis (14, 15). Figure 1. Open in new tabDownload slide Chemical structure of (A) CM, (B) PRC-I, (C) CAO, (D) HUM, and (E) CAR. Figure 1. Open in new tabDownload slide Chemical structure of (A) CM, (B) PRC-I, (C) CAO, (D) HUM, and (E) CAR. PRC-I (7 methoxy-2, 2-dimethylchromene, Figure 1B) and PRC-II (6, 7-dimethoxy-2, 2-dimethylchromene) are chromene derivatives of plant origin. A. conyzoides is an abundant source of PRC-I and II. PRC-I and II are well known for their insecticidal and anti-juvenile hormone activities (16). These features, together with its alleolopathic effects confer agricultural potential to A. conyzoides as a natural pesticide and herbicide safer for the environment and human health (1, 17–20). Sesquiterpenes are the main components of plant-based essential oils. Several sesquiterpenes possess interesting biological activities. β-caryophyllene oxide (CAO, Figure 1C), α-humulene (HUM, Figure 1D), and β-caryophyllene (CAR, Figure 1E) are structurally related sesquiterpenes (21). CAR is one of the ubiquitous sesquiterpene hydrocarbons in the plant kingdom. It has been found, often together with its isomer HUM, in many well-known aromatic plants such as cloves, cannabis, pine trees, tobacco, basil, oregano, sage, menthe, and ginger. Many interesting biological activities of CAR have been reported, with its anticancer and analgesic properties having been summarized in a recent review (22). In addition to that, CAR has antioxidant, antimicrobial, and chemo-protective effects (23, 24). HUM is also widespread in nature. Hops represent the best-known source of HUM. Many biological activities of HUM beneficial to human health have been reported. It inhibits cell growth in breast and colon cancer cell lines (25). HUM also induces cancer cell apoptosis and concomitantly decreases cellular glutathione content as well as producing an increase in ROS production. CAO is a derivative of CAR in which the olefin of CAR has become an epoxide. It is present in lemon balm, caraway, cloves, hops, basil, oregano, lavender, rosemary, cinnamon, etc. Strong antifungal and insecticidal activity of CAO has been reported (26). CAO has also been indicated as a potential candidate for both prevention and treatment of cancer (20). CAO exhibits anti-cancer effects in MG-63 human osteosarcoma cells by inhibiting cell migration, generation of reactive oxygen species, and induction of apoptosis (27). It interferes with multiple signaling cascades involved in tumorigenesis. Treatment with CAO reduces the expression of procancer genes/proteins, while increasing the levels of those with pro apoptotic properties (22, 28). In studies carried out on laboratory animals, CAO was found to be a significant central as well as peripheral analgesic along with possessing anti-inflammatory activity (29). The main compounds from A. conyzoides are volatile and the gas chromatography (GC)-mass spectrometry (MS) technique is widely used for their characterization. However, the GC-MS technique cannot be used directly for qualitative and quantitative analysis of CM, PRC-I, CAO, HUM, and CAR in finished herbal products/phytopharmaceuticals as they contain aqueous constituents. Additional steps are required for extraction/partitioning using an immiscible solvent followed by removal of the solvent before carrying out GC-MS analysis of formulations containing water. Therefore, the present investigation was carried out to develop and validate a HPLC-PDA method for simultaneous identification and quantification of five analytes – CM, PRC-I, CAO, HUM, and CAR – in A. conyzoides extracts and essential oil from A. conyzoides as well as from other plants namely Syzygium aromaticum, Murraya koenigii, Psidium guajava, and Aegle marmelos. METHOD Validation Materials and Chemicals The air-dried herbage of A. conyzoides (Indian Veterinary Research Institute, Bareilly, India) was ground to a fine powder (80–100 mesh size) using an electric grinder (ANJO plus, Food Processing Machinery, Vasai, India). Reference compounds of the high purity grade CM, PRC-I, CAO, HUM, and CAR were purchased from Sigma Aldrich, St. Louis, MO (CM: CAS-91-64-5, MW: 146.14, assay ≥ 99% (HPLC); PRC-I: CAS-17598-02-6, MW: 190.24, assay 99%; HUM: CAS-6753-98-6, MW: 204.35; CAR: CAS-87-44-5, MW: 204.36, assay ≥ 80%; CAO: CAS-1139-30-6, MW: 220.35, assay 95%. HPLC grade methanol, acetonitrile, and analytical grade trifluroacetic acid (TFA) were purchased from Merck, Mumbai, India. Milli Q grade water used throughout the experiment was prepared using a Millipore purification system (Millipore, Milli Q gradient A10, France). Preparation of Extracts and Calibration Standards The extracts were prepared by refluxing ground plant material (5 g) with ethanol (95%, hydroalcoholic) and hexane, separately, for 5 h in a water bath. The test portion to solvent ratio was 1: 20. After extraction, the flask was cooled at room temperature and the contents were filtered to collect supernatant using vacuum filtration. The collected supernatant was concentrated under reduced pressure at 60 °C using a rotary evaporator and the dried extract was kept under refrigeration for further use. As CM, PRC-I, CAO, HUM, and CAR have been traditionally obtained from the essential oils, the plant material was also subjected to hydro distillation by Clevanger apparatus for 6 h to get ACO. S. aromaticum (Clove) essential oil (SAO) was also obtained from bud through hydro distillation (6 h) by Clevanger apparatus. Essential oils from leaves of M. koenigii (MKO), P. guajava (PGO), and A. marmelos (AMO) were also obtained by the method described above. Methanolic and hydroalcoholic (20% methanol-water) extract of P. guajava leaves were also prepared by reflux method. Supercritical fluid extraction (SFE) of A. conyzoides herbage was carried out using a SFE system (Model: SFE-5000 M1-2 FMC 50, Thar Technologies, Inc., Pittsburgh, PA) consisting of an extraction vessel (1 L), high pressure pumps (P-50A, P-200A; Thar Technologies, Inc.), recycler, and chiller (Accel 500 LC; Thermo Scientific, Waltham, MA). The extraction conditions were optimized using SuperChrom software (SFC suit v5.9). The optimized conditions for SFE for A. conyzoides were as follows: automated back pressure (150 bar), carbon dioxide flow (40 g/min), methanol as modifier (4 g/min), extraction time (2 h), and temperature of extraction vessel (40 °C). The collected SFE extract was further concentrated using a rotary evaporator under reduced pressure at 60 °C. Stock solutions of different extracts and essential oils were prepared by dissolving extract in methanol and filtering it through a 0.45 µm membrane filter. Stock solutions of CM, PRC-I, CAO, HUM, and CAR were prepared in HPLC grade methanol (1.0 mg/mL, each). Working solutions of lower concentration were prepared by appropriate dilution of the stock solutions. Solutions of extract and standards were stored at 4 ± 1 ºC and it was brought to room temperature before use. Chromatographic Conditions and Method Validation Chromatographic separation was achieved using an HPLC (Waters, USA) system consisting of quaternary pumps, an in-line vacuum degasser, and a PDA detector. Mobile phase consisted of a mixture of the following solvents: water with TFA (0.05%, solvent A) and acetonitrile with TFA (0.05%, solvent B). Separation was achieved in a linear gradient elution mode on a reversed phase C18 column (250 × 4.6 mm, 5 µm, Sunfire, Waters). Gradient programming was as follows: 0 min 40% A, 10 min 25% A, 15 min 15% A, 25 min 5% A, 30 min 5% A, 35 min 40% A, then at 36 min re-establishing the primary condition to 41 min. Total run time was 50 min. The peaks obtained in the chromatogram of extracts were monitored in the wavelength range of 200–350 nm using PDA detector. However, wavelength selected for quantitative analysis of five analytes in plant extracts was 215 nm. Calibration Curves of CM, PRC-I, CAO, HUM, and CAR The calibration curves for CM (10–50 µg/mL), PRC-I (10–50 µg/mL), CAO (10-100 µg/mL), HUM (0.10–0.50 µg/mL), and CAR (10–100 µg/mL) were prepared by plotting the graph between peak area and concentration. Linearity and Concentration Ranges The linearity of each analyte was evaluated by analyzing different concentrations of five analytes by establishing a relationship with concentration and peak area. Limit of Detection (LOD) and Limit of Quantification (LOQ) The LOD and LOQ of individual analytes were determined on the basis of signal-noise (S/N) ratio in accordance to International Conference on Harmonization (ICH) guidelines (30). LOD was defined as the concentration of the analyte, which had S/N ratio of 3:1 and for LOQ, S/N ratio was 10:1. For determination of LOD and LOQ of CM, PRC-I, CAO, HUM, and CAR a series of their concentrations were injected and S/N ratios as well as areas of individual concentration were recorded. The minimum concentration of standard solution of CM, PRC-I, CAO, HUM, and CAR which had S/N ratios of 3 was fixed as LOD of the respective analyte. Similarly, the minimum concentration of the individual five analytes which had S/N ratios >10 was fixed as LOQ of the respective analyte. LOD for CM, PRC-I, CAO, HUM, and CAR were 2.5, 2.5, 2.5, 0.025, and 2.5 µg/mL, respectively. Similarly, LOQ values were 10, 10, 10, 0.1, and 10 µg/mL for CM, PRC-I, CAO, HUM, and CAR, respectively. The values of LOQ of CM, PRC-I, CAO, HUM, and CAR are in accordance with ICH guidelines. For fixing the concentration of test solutions of extract and essential oils, the minimum concentration of test solutions which had areas corresponding to LOQ values of individual analytes i.e., CM, PRC-I, CAO, HUM, and CAR, was selected. For this, stock solutions of test solutions were diluted to get a minimum concentration for HPLC-PDA analysis. Precision and Accuracy Within-day precision (intraday) and accuracy for the developed method were studied at three different concentration levels for each of the five analytes using three replicates. Similarly, between-day precision (interday) and accuracy were evaluated by analyzing the same three concentrations using three replicates repeated on 3 days. The repeatability of peak areas was expressed in terms of relative standard deviation (RSD). The accuracy was determined based on the recovery of analytes. Two different concentrations of individual analytes was spiked in a blank extract as well as in plant samples (extracts and essential oils) to find analytical recovery. The recovery percentage was calculated by using the formula: Recovery(%)=[(amount found – original amount)/(spiked amount)]×100 Robustness The robustness of the method is defined as its capacity to remain unaffected by minuscule changes in method conditions. The robustness was evaluated by deliberate changes in composition of mobile phase and flow rate. Results and Discussion Extraction Yield The extraction yields (%) of A. conyzoides for hydroalcoholic solvent, hexane, and SFE were 9.76 ± 0.49, 2.71 ± 0.12, and 2.56 ± 0.14, respectively. Hydrodistillation gave 0.13 ± 0.02% ACO. The yields (%) of MKO, PGO, and AMO were 0.56, 0.80, and 0.37, respectively. Extract yields (%) of methanolic and hydroalcoholic extract of P. guajava leaves were 24.78 and 24.37. HPLC Method Validation Both isocratic and gradient elution modes with water and acetonitrile with TFA (0.05%) were attempted to get the separation of CM, PRC-I, CAO, HUM, and CAR. However, gradient elution mode provided sharp peaks and better resolution of the five analytes. Hence, gradient elution mode was selected for validation of the developed method. Selected mobile phase was safe for use as the concentration of TFA in mobile phase was very low. The peaks of CM, PRC-I, CAO, HUM, and CAR were obtained at mean retention time of 4.38, 12.86, 20.10, 33.34, and 35.11 min, respectively, and baseline separation of analytes was obtained at 215 nm (Figure 2A). Chromatographic peaks in the test solutions were identified by matching their retention time and UV absorption spectra with the peaks in the chromatogram of mixed standards (Figure 2B–D). External standard calibration method was used for quantification of all analytes in test solutions. The linear equation between the injected concentration of the standard analytes CM, PRC-I, CAO, HUM. and CAR and their peak areas were expressed as y = mx + c, where y is the peak area, x is the concentration of the standard, m and c are constants. Using the equation for individual analytes, concentration (x) of respective analytes in the extracts were calculated by putting the value of integrated peak areas (y) of the individual analytes in the calibration equation prepared for corresponding standards (Table 1). Figure 2. Open in new tabDownload slide HPLC-PDA chromatogram of (A) standard mixture of CM, PRC-I, CAO, HUM, and CAR, (B) hydroalcoholic extract, (C) SFE extracts of A. conyzoides and (D) on-line recorded PDA spectra (210 nm) for mixture of CM, PRCI, CAO, HUM, and CAR. Figure 2. Open in new tabDownload slide HPLC-PDA chromatogram of (A) standard mixture of CM, PRC-I, CAO, HUM, and CAR, (B) hydroalcoholic extract, (C) SFE extracts of A. conyzoides and (D) on-line recorded PDA spectra (210 nm) for mixture of CM, PRCI, CAO, HUM, and CAR. Table 1. Retention time, equation for calibration curve, linear range, LOD, and LOQ for CM, PRC-I, CAO, HUM, and CAR using the developed HPLC-PDA method (n = 5) Analyte . Retention time (min), mean . Regression equation (y = a.x + b) . r2 . Linear range, µg/mL . LOD, µg/mL . LOQ, µg/mL . CM 4.38 y = 98 900.x - 2230 0.999 10–50 2.5 10 PRC-I 12.86 y = 110 000.x - 99 100 0.999 10–50 2.5 10 CAO 20.1 y = 10 300.x - 1530 0.999 10–100 2.5 10 HUM 33.34 y =4 770 000.x - 98 600 0.990 0.10–0.50 0.025 0.10 CAR 35.11 y = 20 500.x + 4700 0.999 10-100 2.5 10 Analyte . Retention time (min), mean . Regression equation (y = a.x + b) . r2 . Linear range, µg/mL . LOD, µg/mL . LOQ, µg/mL . CM 4.38 y = 98 900.x - 2230 0.999 10–50 2.5 10 PRC-I 12.86 y = 110 000.x - 99 100 0.999 10–50 2.5 10 CAO 20.1 y = 10 300.x - 1530 0.999 10–100 2.5 10 HUM 33.34 y =4 770 000.x - 98 600 0.990 0.10–0.50 0.025 0.10 CAR 35.11 y = 20 500.x + 4700 0.999 10-100 2.5 10 Open in new tab Table 1. Retention time, equation for calibration curve, linear range, LOD, and LOQ for CM, PRC-I, CAO, HUM, and CAR using the developed HPLC-PDA method (n = 5) Analyte . Retention time (min), mean . Regression equation (y = a.x + b) . r2 . Linear range, µg/mL . LOD, µg/mL . LOQ, µg/mL . CM 4.38 y = 98 900.x - 2230 0.999 10–50 2.5 10 PRC-I 12.86 y = 110 000.x - 99 100 0.999 10–50 2.5 10 CAO 20.1 y = 10 300.x - 1530 0.999 10–100 2.5 10 HUM 33.34 y =4 770 000.x - 98 600 0.990 0.10–0.50 0.025 0.10 CAR 35.11 y = 20 500.x + 4700 0.999 10-100 2.5 10 Analyte . Retention time (min), mean . Regression equation (y = a.x + b) . r2 . Linear range, µg/mL . LOD, µg/mL . LOQ, µg/mL . CM 4.38 y = 98 900.x - 2230 0.999 10–50 2.5 10 PRC-I 12.86 y = 110 000.x - 99 100 0.999 10–50 2.5 10 CAO 20.1 y = 10 300.x - 1530 0.999 10–100 2.5 10 HUM 33.34 y =4 770 000.x - 98 600 0.990 0.10–0.50 0.025 0.10 CAR 35.11 y = 20 500.x + 4700 0.999 10-100 2.5 10 Open in new tab Dose Response Curve, LOD, and LOQ The solutions of five different concentrations of CM, PRC-I, CAO, HUM, and CAR were used for the construction of calibration curves. The detailed description of calibration curves and limit of sensitivity are depicted in Table 1. The residual values in terms of the actual and predicted concentration of the analytes used for the preparation of calibration curves were also checked for individual concentrations of the analytes. RSD (%) and standard error (SE) of the residual values for concentrations used for calibration curves of individual analytes are described in Table 2. Table 2. Residual values (RSD, %) of standard concentration used for preparation of calibration curves (CM, PRC-I, CAO, HUM, and CAR) with standard error (SE) Analyte . Concentration, μg/mL . RSD, % . SE . CM 10 3.536 0.105 15 3.624 0.031 25 3.746 0.036 30 3.454 0.008 50 3.077 0.009 PRC-I 10 4.245 0.013 15 3.724 0.023 30 3.652 0.040 40 3.660 0.020 50 4.791 0.001 CAO 10 4.220 0.085 15 3.295 0.049 40 3.133 0.002 60 4.141 0.001 100 4.941 0.001 HUM 0.10 2.231 0.047 0.15 1.372 0.057 0.30 1.428 0.058 0.40 1.615 0.024 0.50 1.441 0.028 CAR 10 3.626 0.034 20 3.121 0.046 40 2.899 0.004 80 3.181 0.022 100 2.598 0.011 Analyte . Concentration, μg/mL . RSD, % . SE . CM 10 3.536 0.105 15 3.624 0.031 25 3.746 0.036 30 3.454 0.008 50 3.077 0.009 PRC-I 10 4.245 0.013 15 3.724 0.023 30 3.652 0.040 40 3.660 0.020 50 4.791 0.001 CAO 10 4.220 0.085 15 3.295 0.049 40 3.133 0.002 60 4.141 0.001 100 4.941 0.001 HUM 0.10 2.231 0.047 0.15 1.372 0.057 0.30 1.428 0.058 0.40 1.615 0.024 0.50 1.441 0.028 CAR 10 3.626 0.034 20 3.121 0.046 40 2.899 0.004 80 3.181 0.022 100 2.598 0.011 Open in new tab Table 2. Residual values (RSD, %) of standard concentration used for preparation of calibration curves (CM, PRC-I, CAO, HUM, and CAR) with standard error (SE) Analyte . Concentration, μg/mL . RSD, % . SE . CM 10 3.536 0.105 15 3.624 0.031 25 3.746 0.036 30 3.454 0.008 50 3.077 0.009 PRC-I 10 4.245 0.013 15 3.724 0.023 30 3.652 0.040 40 3.660 0.020 50 4.791 0.001 CAO 10 4.220 0.085 15 3.295 0.049 40 3.133 0.002 60 4.141 0.001 100 4.941 0.001 HUM 0.10 2.231 0.047 0.15 1.372 0.057 0.30 1.428 0.058 0.40 1.615 0.024 0.50 1.441 0.028 CAR 10 3.626 0.034 20 3.121 0.046 40 2.899 0.004 80 3.181 0.022 100 2.598 0.011 Analyte . Concentration, μg/mL . RSD, % . SE . CM 10 3.536 0.105 15 3.624 0.031 25 3.746 0.036 30 3.454 0.008 50 3.077 0.009 PRC-I 10 4.245 0.013 15 3.724 0.023 30 3.652 0.040 40 3.660 0.020 50 4.791 0.001 CAO 10 4.220 0.085 15 3.295 0.049 40 3.133 0.002 60 4.141 0.001 100 4.941 0.001 HUM 0.10 2.231 0.047 0.15 1.372 0.057 0.30 1.428 0.058 0.40 1.615 0.024 0.50 1.441 0.028 CAR 10 3.626 0.034 20 3.121 0.046 40 2.899 0.004 80 3.181 0.022 100 2.598 0.011 Open in new tab Accuracy and Precision Repeatability is the degree of variability in results when experimental conditions are as described in the method protocol. Fresh dilutions were prepared from stock solutions of standards each day for measurement of intraday and interday precision. The experiments were repeated on three successive days. Repeatability of the developed HPLC method was evaluated at three different concentrations of CM, PRC-I, CAO, HUM, and CAR. Repeatability (RSD, %) values for intraday and interday precision for CM, PRC-I, HUM, CAR, and CAO was 0.765–2.086 and 0.886–2.128; 0.879–1.672 and 0.979–1.825; 0.696–2.418 and 0.768–2.592; 1.728–2.362 and 1.965–2.378; 1.615–2.897 and 1.658–2.906, respectively (Table 3). Table 3. Precision (RSD, %) of the developed HPLC-PDA method for three different concentrations of CM, PRC-I, CAO, HUM, and CAR Analyte . Concentration, µg/mL . RSD, % . Interday Intraday CM 10 2.128 2.086 25 1.562 1.239 50 0.886 0.765 PRC-I 10 1. 825 1.672 30 1.012 1.001 50 0.979 0.879 CAO 10 2.592 2.418 40 1.783 1.472 100 0.768 0.696 HUM 0.10 2.378 2.362 0.30 2.219 2.121 0.50 1.965 1.728 CAR 10 2.906 2.897 40 2.009 2.108 100 1.658 1.615 Analyte . Concentration, µg/mL . RSD, % . Interday Intraday CM 10 2.128 2.086 25 1.562 1.239 50 0.886 0.765 PRC-I 10 1. 825 1.672 30 1.012 1.001 50 0.979 0.879 CAO 10 2.592 2.418 40 1.783 1.472 100 0.768 0.696 HUM 0.10 2.378 2.362 0.30 2.219 2.121 0.50 1.965 1.728 CAR 10 2.906 2.897 40 2.009 2.108 100 1.658 1.615 Open in new tab Table 3. Precision (RSD, %) of the developed HPLC-PDA method for three different concentrations of CM, PRC-I, CAO, HUM, and CAR Analyte . Concentration, µg/mL . RSD, % . Interday Intraday CM 10 2.128 2.086 25 1.562 1.239 50 0.886 0.765 PRC-I 10 1. 825 1.672 30 1.012 1.001 50 0.979 0.879 CAO 10 2.592 2.418 40 1.783 1.472 100 0.768 0.696 HUM 0.10 2.378 2.362 0.30 2.219 2.121 0.50 1.965 1.728 CAR 10 2.906 2.897 40 2.009 2.108 100 1.658 1.615 Analyte . Concentration, µg/mL . RSD, % . Interday Intraday CM 10 2.128 2.086 25 1.562 1.239 50 0.886 0.765 PRC-I 10 1. 825 1.672 30 1.012 1.001 50 0.979 0.879 CAO 10 2.592 2.418 40 1.783 1.472 100 0.768 0.696 HUM 0.10 2.378 2.362 0.30 2.219 2.121 0.50 1.965 1.728 CAR 10 2.906 2.897 40 2.009 2.108 100 1.658 1.615 Open in new tab The recovery percentage was calculated for two different added concentrations of CM, PRC-I, CAO, HUM, and CAR (Table 4). Table 4. Recovery data for CM, PRC-I, CAO, HUM, and CAR in test solutions of extracts and essential oils Extracta . Analyte . Added concentration, µg/mL . Recovery, % . AC 95% EtOH CM 15 97.15 30 99.76 MKO CM 15 101.66 30 106.48 MeOH extract PGL CM 15 100.18 30 105.23 HA extract PGL CM 15 103.78 30 105.19 AC 95% EtOH PRC-I 15 97.85 40 99.37 MKO PRC-I 15 100.79 40 102.26 MeOH extract PGL PRC-I 15 102.86 40 106.92 HA extract PGL PRC-I 15 104.54 40 107. 87 AC 95% EtOH CAO 15 96.76 60 100.31 MKO CAO 15 102.92 60 104.48 MeOH extract PGL CAO 15 101.26 60 103.33 HA extract PGL CAO 15 97.99 60 100.98 AC 95% EtOH HUM 0.15 101.95 0.40 103.86 MKO HUM 0.15 106.34 0.40 108.68 MeOH extract PGL HUM 0.15 100.89 0.40 102.78 HA extract PGL HUM 0.15 102.85 0.40 103.35 AC 95% EtOH CAR 20 98.85 80 101.46 MKO CAR 20 105.35 80 107.61 MeOH extract PGL CAR 20 103.85 80 105.89 HA extract PGL CAR 20 100.06 80 102.38 Extracta . Analyte . Added concentration, µg/mL . Recovery, % . AC 95% EtOH CM 15 97.15 30 99.76 MKO CM 15 101.66 30 106.48 MeOH extract PGL CM 15 100.18 30 105.23 HA extract PGL CM 15 103.78 30 105.19 AC 95% EtOH PRC-I 15 97.85 40 99.37 MKO PRC-I 15 100.79 40 102.26 MeOH extract PGL PRC-I 15 102.86 40 106.92 HA extract PGL PRC-I 15 104.54 40 107. 87 AC 95% EtOH CAO 15 96.76 60 100.31 MKO CAO 15 102.92 60 104.48 MeOH extract PGL CAO 15 101.26 60 103.33 HA extract PGL CAO 15 97.99 60 100.98 AC 95% EtOH HUM 0.15 101.95 0.40 103.86 MKO HUM 0.15 106.34 0.40 108.68 MeOH extract PGL HUM 0.15 100.89 0.40 102.78 HA extract PGL HUM 0.15 102.85 0.40 103.35 AC 95% EtOH CAR 20 98.85 80 101.46 MKO CAR 20 105.35 80 107.61 MeOH extract PGL CAR 20 103.85 80 105.89 HA extract PGL CAR 20 100.06 80 102.38 a AC 95% EtOH= hydroalcoholic extract of A. conyzoides, MKO= M. koenigii leaf EO, MeOH extract PGL= methanolic extract of P. guajava leaf, HA extract PGL= hydroalcoholic extract of P. guajava leaf. Open in new tab Table 4. Recovery data for CM, PRC-I, CAO, HUM, and CAR in test solutions of extracts and essential oils Extracta . Analyte . Added concentration, µg/mL . Recovery, % . AC 95% EtOH CM 15 97.15 30 99.76 MKO CM 15 101.66 30 106.48 MeOH extract PGL CM 15 100.18 30 105.23 HA extract PGL CM 15 103.78 30 105.19 AC 95% EtOH PRC-I 15 97.85 40 99.37 MKO PRC-I 15 100.79 40 102.26 MeOH extract PGL PRC-I 15 102.86 40 106.92 HA extract PGL PRC-I 15 104.54 40 107. 87 AC 95% EtOH CAO 15 96.76 60 100.31 MKO CAO 15 102.92 60 104.48 MeOH extract PGL CAO 15 101.26 60 103.33 HA extract PGL CAO 15 97.99 60 100.98 AC 95% EtOH HUM 0.15 101.95 0.40 103.86 MKO HUM 0.15 106.34 0.40 108.68 MeOH extract PGL HUM 0.15 100.89 0.40 102.78 HA extract PGL HUM 0.15 102.85 0.40 103.35 AC 95% EtOH CAR 20 98.85 80 101.46 MKO CAR 20 105.35 80 107.61 MeOH extract PGL CAR 20 103.85 80 105.89 HA extract PGL CAR 20 100.06 80 102.38 Extracta . Analyte . Added concentration, µg/mL . Recovery, % . AC 95% EtOH CM 15 97.15 30 99.76 MKO CM 15 101.66 30 106.48 MeOH extract PGL CM 15 100.18 30 105.23 HA extract PGL CM 15 103.78 30 105.19 AC 95% EtOH PRC-I 15 97.85 40 99.37 MKO PRC-I 15 100.79 40 102.26 MeOH extract PGL PRC-I 15 102.86 40 106.92 HA extract PGL PRC-I 15 104.54 40 107. 87 AC 95% EtOH CAO 15 96.76 60 100.31 MKO CAO 15 102.92 60 104.48 MeOH extract PGL CAO 15 101.26 60 103.33 HA extract PGL CAO 15 97.99 60 100.98 AC 95% EtOH HUM 0.15 101.95 0.40 103.86 MKO HUM 0.15 106.34 0.40 108.68 MeOH extract PGL HUM 0.15 100.89 0.40 102.78 HA extract PGL HUM 0.15 102.85 0.40 103.35 AC 95% EtOH CAR 20 98.85 80 101.46 MKO CAR 20 105.35 80 107.61 MeOH extract PGL CAR 20 103.85 80 105.89 HA extract PGL CAR 20 100.06 80 102.38 a AC 95% EtOH= hydroalcoholic extract of A. conyzoides, MKO= M. koenigii leaf EO, MeOH extract PGL= methanolic extract of P. guajava leaf, HA extract PGL= hydroalcoholic extract of P. guajava leaf. Open in new tab The purity of the peaks in chromatographic analysis for standard analytes, CM, PRC-I, CAO, HUM, and CAR in plant extracts as well as in reference was checked by comparing their peak purity angle and purity threshold values using Empower Software. For the peaks of these five analytes in standard as well as in plant extracts, purity angles were lesser than the purity threshold values, thereby confirming that eluted peaks were pure and no peak mixing was there due to co-elution. Therefore, the developed method was specific for determination of CM, PRC-I, CAO, HUM, and CAR as their peak purity values established that peaks were pure and had no co-eluting peaks. Robustness To test the robustness of the developed HPLC-PDA method, chromatographic conditions which could affect the performance of the method were deliberately changed. In the present study, TFA content in mobile phase (0.05, 0.08, and 0.1%), flow rate (0.75, 1.25, and 1.35 mL/min) and wavelength of detection (±5 nm) were changed. The RSD of retention time and peak areas of all analytes were calculated for changes in each parameter. The results demonstrated that the developed HPLC-PDA method was insensitive to minor changes (RSD, %  < 2). Quantification of CM, PRC-I, CAO, HUM, and CAR in Extracts and Essential Oil The developed and validated HPLC-PDA method was applied for the quantification of CM, PRC-I, CAO, HUM, and CAR in A. conyzoides extracts namely AC 95% EtOH, SFE and AC Hexane. CM, PRC-I, CAO, HUM, and CAR were also identified and quantified in ACO, SAO, MKO, PGO, and AMO. The developed and validated HPLC-PDA method was also applied for identification and quantification of these five analytes in methanolic and hydroalcoholic (20% methanol-water) extract of P. guajava leaves (Tables 5 and 6). Table 5. Concentration (mean ± standard deviation) of CM, PRC-I, CAO, HUM, and CAR in test solutions of A. conyzoides extract (n = 3) Extractsa . Extract yield, % . Amount in extract, % . CM . PRC-I . CAO . HUM . CAR . ACc 95% EtOH 9.76±0.49 0.063±0.002 0.123±0.006 0.156±0.008 0.001±0.000b 0.159±0.007 SFEd 2.56±0.14 0.212±0.009 0.249±0.013 0.459±0.015 0.001±0.000b 0.530±0.037 AC hexanee 2.71±0.12 0.059±0.003 0.239±0.019 0.465±0.027 0.003±0.000b 0.512±0.029 Extractsa . Extract yield, % . Amount in extract, % . CM . PRC-I . CAO . HUM . CAR . ACc 95% EtOH 9.76±0.49 0.063±0.002 0.123±0.006 0.156±0.008 0.001±0.000b 0.159±0.007 SFEd 2.56±0.14 0.212±0.009 0.249±0.013 0.459±0.015 0.001±0.000b 0.530±0.037 AC hexanee 2.71±0.12 0.059±0.003 0.239±0.019 0.465±0.027 0.003±0.000b 0.512±0.029 a Test concentration: 20000 µg/mL. bSD restricted to three places after decimal. c AC 95% EtOH = Hydroalcoholic extract. d SFE = Supercritical fluid extract. e AC hexane = Hexane extract of A. conyzoides. Open in new tab Table 5. Concentration (mean ± standard deviation) of CM, PRC-I, CAO, HUM, and CAR in test solutions of A. conyzoides extract (n = 3) Extractsa . Extract yield, % . Amount in extract, % . CM . PRC-I . CAO . HUM . CAR . ACc 95% EtOH 9.76±0.49 0.063±0.002 0.123±0.006 0.156±0.008 0.001±0.000b 0.159±0.007 SFEd 2.56±0.14 0.212±0.009 0.249±0.013 0.459±0.015 0.001±0.000b 0.530±0.037 AC hexanee 2.71±0.12 0.059±0.003 0.239±0.019 0.465±0.027 0.003±0.000b 0.512±0.029 Extractsa . Extract yield, % . Amount in extract, % . CM . PRC-I . CAO . HUM . CAR . ACc 95% EtOH 9.76±0.49 0.063±0.002 0.123±0.006 0.156±0.008 0.001±0.000b 0.159±0.007 SFEd 2.56±0.14 0.212±0.009 0.249±0.013 0.459±0.015 0.001±0.000b 0.530±0.037 AC hexanee 2.71±0.12 0.059±0.003 0.239±0.019 0.465±0.027 0.003±0.000b 0.512±0.029 a Test concentration: 20000 µg/mL. bSD restricted to three places after decimal. c AC 95% EtOH = Hydroalcoholic extract. d SFE = Supercritical fluid extract. e AC hexane = Hexane extract of A. conyzoides. Open in new tab Table 6. The amount of CM, PRC-I, CAO, HUM, and CAR in test solutions of essential oils of M. koenigii, P. guajava, and A. marmelos leaves and extract of P. guajava Columna . Amount, % . CM . PRC-I . CAO . HUM . CAR . Essential oil ACO NDb 7.304±0.229 10.415±0.175 NQc 9.443±0.391 SAO ND ND ND 0.010±0.000d 9.998±0.465 MKO ND ND 6.790±0.280 0.018±0.001 9.548±0.334 PGO ND ND 10.085±0.216 0.017±0.000d 10.321±0.218 AMO ND ND NQ NQ 2.446±0.117 P. guajava extract MeOH extract PGL ND ND 0.135±0.000d NQ 0.493±0.027 HA extract PGL ND ND NQ NQ NQ Columna . Amount, % . CM . PRC-I . CAO . HUM . CAR . Essential oil ACO NDb 7.304±0.229 10.415±0.175 NQc 9.443±0.391 SAO ND ND ND 0.010±0.000d 9.998±0.465 MKO ND ND 6.790±0.280 0.018±0.001 9.548±0.334 PGO ND ND 10.085±0.216 0.017±0.000d 10.321±0.218 AMO ND ND NQ NQ 2.446±0.117 P. guajava extract MeOH extract PGL ND ND 0.135±0.000d NQ 0.493±0.027 HA extract PGL ND ND NQ NQ NQ a ACO = A. conyzoides essential oil (test concentration: 1000 μg/mL); SAO = S. aromaticum essential oil (test concentration: 1000 μg/mL); MKO = M. koenigii leaf essential oil (test concentration: 1000 μg/mL); PGO = P. guajava leaf essential oil (test concentration: 1000 μg/mL); AMO = A. marmelos leaf essential oil (test concentration: 1000 μg/mL); MeOH extract PGL = Methanolic extract of P. guajava leaf (test concentration: 20000 μg/mL); HA extract PGL = Hydroalcoholic extract of P. guajava leaf (test concentration: 20000 μg/mL). b ND = Not detected. c NQ = Not quantifiable. d SD restricted to three places after decimal. Open in new tab Table 6. The amount of CM, PRC-I, CAO, HUM, and CAR in test solutions of essential oils of M. koenigii, P. guajava, and A. marmelos leaves and extract of P. guajava Columna . Amount, % . CM . PRC-I . CAO . HUM . CAR . Essential oil ACO NDb 7.304±0.229 10.415±0.175 NQc 9.443±0.391 SAO ND ND ND 0.010±0.000d 9.998±0.465 MKO ND ND 6.790±0.280 0.018±0.001 9.548±0.334 PGO ND ND 10.085±0.216 0.017±0.000d 10.321±0.218 AMO ND ND NQ NQ 2.446±0.117 P. guajava extract MeOH extract PGL ND ND 0.135±0.000d NQ 0.493±0.027 HA extract PGL ND ND NQ NQ NQ Columna . Amount, % . CM . PRC-I . CAO . HUM . CAR . Essential oil ACO NDb 7.304±0.229 10.415±0.175 NQc 9.443±0.391 SAO ND ND ND 0.010±0.000d 9.998±0.465 MKO ND ND 6.790±0.280 0.018±0.001 9.548±0.334 PGO ND ND 10.085±0.216 0.017±0.000d 10.321±0.218 AMO ND ND NQ NQ 2.446±0.117 P. guajava extract MeOH extract PGL ND ND 0.135±0.000d NQ 0.493±0.027 HA extract PGL ND ND NQ NQ NQ a ACO = A. conyzoides essential oil (test concentration: 1000 μg/mL); SAO = S. aromaticum essential oil (test concentration: 1000 μg/mL); MKO = M. koenigii leaf essential oil (test concentration: 1000 μg/mL); PGO = P. guajava leaf essential oil (test concentration: 1000 μg/mL); AMO = A. marmelos leaf essential oil (test concentration: 1000 μg/mL); MeOH extract PGL = Methanolic extract of P. guajava leaf (test concentration: 20000 μg/mL); HA extract PGL = Hydroalcoholic extract of P. guajava leaf (test concentration: 20000 μg/mL). b ND = Not detected. c NQ = Not quantifiable. d SD restricted to three places after decimal. Open in new tab CM was quantified in hydroalcoholic (AC 95% EtOH), SFE, and hexane (AC hexane) extracts of A. conyzoides. However, it was not detected in other test solutions namely ACO, SAO, MKO, PGO, AMO, MeOH extract PGL, and HA extract PGL. PRC-I was quantified in AC 95% EtOH, SFE, and AC hexane extracts of A. conyzoides. It was also quantified in ACO. CAO was quantified in all extracts of A. conyzoides and also in ACO, MKO, PGO, AMO, MeOH extract PGL, and HA extract PGL, but it was not detected in SAO. Both HUM and CAR was quantified in all extracts including extracts of A. conyzoides, ACO, SAO, MKO, PGO, AMO, MeOH extract PGL, and HA extract PGL (Tables 5 and 6). Discussion Jaya et al. (31) reported essential oil yield (0.5% V/W) from the leaves of A. conyzoides. Tambunan et al. (32) reported extraction yield of A. conyzoides leaves with water and 70% ethanol. The extraction time was 192 h and extraction yield was 15.0% and 30.20% for 70% ethanol and water, respectively. PRCs are known to exhibit anti-juvenile effects namely antigonadotropic, ovicidal, precocious metamorphosis, and dipause induction on insects (16, 33, 34). PRC-I is the major constituent of the essential oil of A. conyzoides (35, 36). CAR is a widespread plant volatile isolated from the essential oils of various medicinal and aromatic plants such as guava (P. guajava), oregano (Origanum vulgare), cinnamon (Cinnamomum spp.), clove (S. aromaticum), black pepper (Piper nigrum), etc. (37, 38). The sesquiterpenes CAR, CAO, and HUM (formerly α -caryophyllene) are not only structural relatives, but also often occur together in the same plants. The human organism comes into frequent contact with these sesquiterpenes, as they are components of food, beverages, folk medicinal preparations, and cosmetics. Moreover, these natural compounds possess such promising biological activities that their use in pharmacotherapy should be considered in the future (39). Simultaneous identification and quantification of polymethoxyflavones, CM, and phenolic acids in A. conyzoides by UPLC-ESI-QToF-MS and UPLC-PDA was reported (40). Twenty-seven compounds, including the toxic pyrrolizidine alkaloids, phenolic acids, CM and polymethoxyflavones in the aqueous extract were identified by tandem mass spectrometry analysis. Earlier reported HPLC methods for the quantification of PRCs have long retention time (41–43). Sharma and Sharma (44) reported a reversed-phase-HPLC method for analysis of PRC-I and PRC-II in essential oils of Ageratum species. Separation of PRC-I and PRC-II was achieved with a mobile phase consisting of a mixture of acetonitrile and water (40:60) in a linear gradient elution mode. The total run time was more than 40 min and PRC-I and PRC-II were eluted within 25 min. High performance thin-layer chromatography (HPTLC) methods for quantification of PRCs were also reported. Kumar et al. (45, 46) reported a validated HPTLC method for quantification of PRC-I and PRC-II in A. conyzoides germplasms. PRC-I content was 0.0016–0.0834% on dry weight basis in A. conyzoides germplasms from Western Himalayas. PRC-II was in the range of 0.016–0.143%. In the present investigation the amount of PRC-I varied in the following order: ACO (6.034 ± 0.188, %) > SFE (0.012 ± 0.0007, %) > AC 95% EtOH (0.010 ± 0.0007, %) > AC hexane (0.0065 ± 0.0008, %). Conclusions In the present investigation, a HPLC method has been developed and validated for simultaneous determination of CM, PRC-I, CAO, HUM, and CAR in extracts and essential oils. Complete separation of the compounds was accomplished in 41 min and the method could be used for identification and quantification of the above five analytes in crude as well as in finished herbal formulations. Also, the method could be used for meeting the characterization of phytoformulations. 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TI - Development and Validation of a Reversed Phase High Performance Liquid Chromatography-Photodiode Array Detection Method for Simultaneous Identification and Quantification of Coumarin, Precocene-I, β-Caryophyllene Oxide, α-Humulene, and β-Caryophyllene in Ageratum Conyzoides Extracts and Essential Oils from Plants
JF - Journal of AOAC International
DO - 10.1093/jaoacint/qsz038
DA - 2020-06-01
UR - https://www.deepdyve.com/lp/oxford-university-press/development-and-validation-of-a-reversed-phase-high-performance-liquid-tAnMi2eFdY
SP - 857
EP - 864
VL - 103
IS - 3
DP - DeepDyve
ER -