INTRODUCTION:
India is the land of most of the medicinal plants and is hence called as “Botanical Garden of the world”. According to the World Health Organization (WHO) estimates, more than 80 % of the people in developing countries depend on traditional medicine for their primary health needs1. Medicinal plants constitute a source of raw materials for both traditional systems of medicines (e.g. Ayurvedic, Chinese, Unani, Homeopathy and Siddha) and modern medicine. Plant materials are widely used as home remedies over the years as we have our strong base of knowledge about traditional systems of medicine as such, they represent a substantial proportion of the global drug market2.
Medicinal plants have been known for their therapeutic potency for in the world of natural therapeutics. For treatment of various diseases, maintenance of good health and conditions; multiple medicinal plants have been used3.
Trachyspermum ammi (T. ammi), commonly known as ‘Ajwain’, is a plant indigenous to Egypt and widely distributed throughout India. It is commonly used plant in kitchen for variety of purposes. Ajwain is known for multiple historic applications in traditional medicine as broad spectrum of ailments including flatulence, atonic dyspepsia, diarrhea, abdominal tumors, abdominal pains, piles, bronchial problems, lack of appetite, galactagogue, asthma, and amenorrhea4. Such diverse therapeutic uses have garnered significant interest in exploring the phytochemical composition of T. ammi. Constituents like fiber (11.9%), carbohydrates (24.6%), tannins, glycosides, moisture (8.9%), protein (17.1%), fat (21.1%), saponins, flavones and other components (7.1%) involving calcium, phosphorous, iron, cobalt, copper, iodine, manganese, thiamine, riboflavin and nicotinic acid are from proximate evaluation of Ajwain. Alcoholic extracts of the plant have shown considerable amount of saponins5-7.
Thymol, is one of the major phytoconstituent present in T. ammi and it plays a pivotal role in conferring the medicinal attributes. This compound exhibits a wide array of pharmacological properties including antioxidant, anti-inflammatory, analgesic, antispasmodic, antibacterial, antiseptic, and anticancer activities8,9. Consequently, it has become the focus of numerous studies aiming to elucidate its mechanisms of action and therapeutic potential.
Although a similar literature detailing the analysis of thymol using High Performance Liquid Chromatography (HPLC)was done with a mobile phase of Acetonitrile: water (50:50) in T. vulgaris, the primary objective of this study is to introduce a novel method and mobile phase for the analysis and quantification of thymol in Trachyspermum ammi leaves. Previous methods were not efficient in extracting and quantifying thymol from leaves Trachyspermum ammi10,11.
Standardization of method of extraction, optimization of chromatographic conditions and validation is a main approach of the current work. Validation of a method signifies that method will be suitable for its intended use. Different parameters of validation by HPLC have been evaluated as per ICH guidelines. This validated method assures consistent, reliable and accuracy of the data for detection, separation and analysis of thymol. Analytical method validation includes the determination of accuracy, precision, LOD, LOQ, linearity and range. The results from method validation can be used to moderate the quality, reliability and consistency of analytical results, which is an integral part of any good analytical practice. Validation of analytical methods is also required by most regulations and quality standards that impact laboratories12.
This validation process ensures the robustness and reproducibility of this analytical approach, thereby providing a reliable tool for the quantification of thymol in T. ammi samples.
MATERIALS AND METHODS:
Collection of plant material:
The fresh leaves of Trachyspermum ammi (Ajwain) were plucked from the Ajwain plant procured from Matunga market. All the solvents used were of chromatographic grade (Merck) such as Methanol, water, Acetonitrile, Glacial Acetic Acid, thymol standard (purity 99%).
Preparation of sample:
Sample was prepared by accelerated maceration technique for Trachyspermum ammi (ajwain). Freshly collected and identified leaves were washed thoroughly with water and pat dry to remove dirt, dust and excess moisture. Leaves were ground and approximately 10 grams of leaf material was weighed and introduced to 20 ml of extracting solvent, Methanol in a conical flask for 24hours. Accelerated maceration was performed using Rotary Shaker cum Incubator. After extraction the sample was filtered using filter paper to collect the liquid extract, which was then analysed using HPLC.
Preparation of standard:
1000 ug/ml Stock solution was prepared in methanol. Working standard used for further dilution was 100 ug/ml.
Instrument details:
High Performance Liquid Chromatograph Prominence i LC2030-3D plus manufactured by Shimadzu Analytical India Pvt. Ltd.
Table 1: Optimised Chromatographic Conditions
|
Parameter |
Description |
|
Instrument |
HPLC- Prominence-i, LC-2030C 3D Plus Liquid chromatograph |
|
Pump |
LC 2030 pump |
|
Injector |
LC 2030, Autosampler |
|
Injection volume |
30uL |
|
Column oven |
LC 2030 oven,400C |
|
Column |
Shimadzu, C18 250mm X 4.6 mm, 5µm |
|
Mobile phase |
Acetonitrile (99.9%): water along with 0.1%GAA (80:20v/v) |
|
Flow rate |
1mL/min |
|
Detector |
LC 2030 PDA |
|
Detection wavelength |
274nm |
RESULT:
Figure 1: HPLC chromatogram of standard Thymol
Figure 2: HPLC chromatogram of Trachyspermum ammi leaf extract
Figure 3: HPLC chromatogram of mobile phase
Figure 4: HPLC chromatogram of blank diluent (Methanol)
Figure 5: (A)
Figure 5: (B)
Figure 5: (A) Spectrum of standard Thymol; (B) Spectrum of Thymol from sample
Table 2: Summery of validation
|
Parameter |
Thymol |
|
System Suitability(%RSD) |
Area=414650, Rt=4.49, RSD=0.094642 |
|
Specificity and Robustness |
Specific and Robust |
|
Precision(%RSD) Intraday, 5μg/ml |
0.389574 |
|
Precision(%RSD) Interday, 5ug/ml |
0.245099 |
|
LOD |
0.1ug/ml |
|
LOQ |
0.3ug/ml |
|
Linearity |
2-10ug/ml |
|
Assay |
1.32% |
|
Recovery |
93.84% |
DISCUSSION:
Current research work focusses on standardizing a suitable method of extraction and analysis of Thymol from the used plant leaves. Accelerated maceration of plant leaves extract has shown consistent good extraction efficiency and hence is selected further. In the initial phases of optimization of chromatographic parameters, various mobile phases and their concentrations were used to achieve resolution of the peak of interest. CAN (99.9%): H2O along with 0.1% GAA (80:20) in an isocratic mode was selected amongst them with the flow rate of 1ml/min gave the best possible result.
Optimization and Method Development:
The mobile phase was optimized after several trials with ACN and water (spiked with few acids like OPA, formic acid, Glacial acetic acid, etc.). Finally, a mobile phase consisting of acetonitrile and glacial acetic acid were chosen for further analysis as it gave good results. Flow rates were kept between 0.3ml/min to 1.0ml/min.
The UV spectrum of Thymol showed maximum absorption at 274 nm; therefore, the compounds were monitored at this wavelength using a photodiode array detector (PDA).
Method Validation:
The developed analytical method was validated in accordance with the International Conference on Harmonization (ICH) guidelines. The technique has been validated for linearity, specificity, recovery, accuracy, the limits of detection (LOD) and quantification (LOQ).
System Suitability:
Six replicates of a single standard solution showed %RSD to be less than 1% proving the system suitable for the analysis.
Selectivity:
As depicted in chromatograms in figure 3 and 4; no interference is seen at the retention time of thymol from either mobile phase or the diluent blank.
Linearity:
Linearity refers to an analytical method’s capacity to yield test findings that are directly or by well-defined mathematical transformation proportional to analyte concentration in a sample within a defined range. Linearity of this method was carried out by injecting five different concentrations of standard thymol solution ranging from 2ug/ml - 10ug/ml respectively as per the developed method. The calibration curve was constructed based on the response of peak (i.e.AUC) and concentration of thymol in standard solutions. The coefficient of correlation was shown to be 0.9991.
Precision:
Precision of the method indicates the degree of dispersion within a series to be determined of the same sample. Precision was examined by analysing the standard series (1-5ug/ml) intraday (repeatability) on the same day thrice and for different days with different analysts for inter day (intermediate) precision by three replicates of every injection.
The repeatability and intermediate precision of the method was determined by calculating the percentage relative standard deviation of the three (%RSD) The developed method was found be precise with RSD below 2%.
Sensitivity:
Sensitivity of the method was evaluated by determining the LOD (Limit of detection) and LOQ (Limit of Quantification). LOD and LOQ were measured based on the signal to noise ratio by comparing measured signals with known low concentrations of analyte in comparison to blank samples and to determine the minimum concentration at which analyte can be detected and quantified with certainty. A signal-to-noise ratio are within the acceptable range for LOD and LOQ respectively. LOD was found to be 0.1ug/ml and LOQ was found to be 0.3ug/ml.
Assay:
Assay was quantified according to the calibration curve, y = mx + c by analysing native sample using the developed method. Concentration of thymol from sample was found to be 1.32%.
Recovery:
Accuracy for this method was evaluated by spiking the standard solution of concentration in the levels of 80, 100 and 120% in the sample. As depicted in summery table of validation; the method is accurate for the purpose with the RSD of 0.76%.
CONCLUSION:
A novel HPLC-PDA detection method has been established for the efficient quantification of thymol, a crucial bioactive component, from Trachyspermum ammi leaves. This method surpasses existing techniques by offering rapidity, simplicity and robust validation following ICH guidelines. The validation confirms exceptional selectivity, precision, and accuracy making it a highly reliable tool for thymol analysis using Reverse phase HPLC. This novel approach significantly reduces analysis time compared to conventional methods, offering a practical and efficient alternative for HPLC-based studies. The enhanced accessibility and reliability provided by this method will significantly accelerate research into pharmacological properties and therapeutic potential of thymol. Furthermore, the versatility and robustness of this technique position it as a potential standard analytical tool, paving the way for advancements in the utilization of medicinal plants. Overall, this validated method represents a significant contribution to the field, providing a dependable and efficient means to quantify thymol, thereby fostering a deeper understanding of its medicinal applications.
CONFLICT OF INTEREST:
The authors have no conflicts of interest regarding this investigation.
ACKNOWLEDGMENTS:
The authors acknowledge the support received from Rashtriya Uchchatar Shiksha Abhiyan (RUSA) for the HPLC facility at the Department of Bioanalytical Sciences, Ramnarain Ruia Autonomous College.
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Received on 20.07.2024 Revised on 27.09.2024 Accepted on 01.11.2024 Published on 25.11.2024 Available online from December 27, 2024 Asian J. Research Chem.2024; 17(6):319-323. DOI: 10.52711/0974-4150.2024.00054 ©AandV Publications All Right Reserved
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