Quantitative Estimation of Phytoconstituents in Hydroalcoholic Extract of Pancratium triflorum Roxb. by RP-HPLC Method

Quality is important in every product and services, but it is vital in medicine as it involves life. Quality of a drug is determined by using various analytical methods chemistry. Traditional medicine remains a primary source of healthcare for over 80% of the world's population. Approximately 40% of modern pharmaceuticals are derived from natural products or traditional leads. However, the global demand for herbal remedies necessitates scientific evidence for quality control and standardization.

Pancratium triflorum Roxb

Pancratium triflorum Roxb. is traditionally valued for its therapeutic potential, including antioxidant, anti-inflammatory, and antimicrobial activities ^[1]. Quercetin, a prominent flavonoid found in various plants, is known for its potent biological activities, such as inhibiting pro-inflammatory enzymes like COX-2 and LOX ^[2].

High-Performance Liquid Chromatography (HPLC) is the gold standard for separating and quantifying components in complex herbal mixtures. Among its modes, Reverse-Phase HPLC (RP-HPLC) is the most widely used due to its versatility and reproducibility. RP-HPLC operates on the principle of hydrophobic interactions. It utilizes a non-polar stationary phase typically a C18 column and a polar mobile phase such as water and methanol. In this system, molecules are separated based on their polarity ^[3]. Polar compounds elute faster, while non-polar compounds like Quercetin are retained longer. This research focuses on establishing a measurable benchmark for P. triflorum by developing a validated RP-HPLC method for Quercetin.

MATERIALS AND METHODS

Materials

Quercetin reference substance was purchased from Kanton Laboratories (batch no: KL4239); Methanol and Phosphoric acid employed here were HPLC grade reagents. Distilled water and Ethanol were used in the experiment. The plant Pancratium triflorum is collected from Malappuram and identified as the same by Dr. Pramod C, University of Calicut. The plant is dried and coarse powdered.

METHODS

?Preparation of stock and working standard solution

A stock solution of Quercetin was prepared in HPLC-grade methanol. 10 mg Quercetin was accurately weighed and put into 10ml volumetric flask containing 5ml of methanol and sonicated for 10 minutes. Then the volume was adjusted with methanol up to the mark. Standard solution of different concentration (62.5, 125, 250, 500 and 1000ppm) was prepared from above solution and diluted with methanol and filtered through 0.22µ millipore membrane filters and injected in HPLC system.

Extraction of plant sample

The plant is extracted by using water and alcohol in Soxhlet extractor. The extract is then concentrated by evaporation. The 10µL of P. triflorum extract was taken in 10 ml volumetric flask and was dissolved in methanol up to the mark, sonicated for 15 minutes, and filtered through a 0.22µ millipore membrane filter.

RP-HPLC Method Development

?The analysis was performed on a Shimadzu HPLC system equipped with an SPD-M40 PDA detector and a C18 column (4.6 x 150 mm, 5 µm) with an autosampler and a LabSolutions software. The mobile phase consisted of Methanol: O-Phosphoric acid in a 60:40 ratio. The flow rate was maintained at 1 ml/min, with an injection volume of 20 µl and detection at 370 nm.

Instrumentation and Optimized chromatographic Conditions

Table 1. Instrumentation of HPLC

Table 2. Optimized chromatographic condition

METHOD VALIDATION

1. Linearity:

The linearity of analytical method is its ability to elicit test results that are directly proportional to the concentration of analyte in sample within a given range. The range of analytical method is the interval between the upper and lower levels of analyte that have been demonstrated to be determined within a suitable level of precision, accuracy and linearity.

A calibration curve was plotted over a concentration range of 62.5 to 1000 µg/L for Quercetin. Accurately measured working stock solution of drug were transferred to separate series of 10ml volumetric flask and diluted up to the mark with methanol. Filtered through 0.2 µm membrane filter and injected for HPLC analysis The Linearity was constructed by plotting concentration against area from each reading.

2. Limit of Detection and Limit of Quantification

The detection limit of an individual analytical procedure is the lowest amount of analyte in the sample which can be detected but not necessarily quantitated as an exact value. The Quantitation limit of an individual analytical procedure is the lowest amount of analyte in the sample which can be quantitatively determined with suitable precision and accuracy. The LOD and LOQ of the proposed method were determined by using calibration curve:

LOD=3.3σS

LOQ=10σS

Where σ is the standard deviation of response and S is the slope of the calibration curve.

3. Precision

The term precision is defined by the ISO International Vocabulary of Basic and General Terms in Metrology (ISO-VIM) and ICH as the closeness of agreement between quantity values obtained by replicate measurements of a quantity under specified conditions. Assessing the precision implies expressing numerically the random error or the degree of dispersion of a set of individual measurements by means of the standard deviation, the variance or the coefficient of variation.

Repeatability is the concordance of a series of measurements of the same quantity when the experiments are conducted under same conditions (analyst, apparatus, instrument, and day) in a rapid succession. For this experiment, standard solution of Quercetin (62.5 μg/L) was prepared and analysed three times as per the proposed method.

4. Accuracy

Accuracy was determined by means of recovery experiments, by the determination of % mean recovery of sample at three different levels (80-120%). At each level, two determinations were performed. The accepted limits of recovery are 90% - 120%.

RESULTS AND DISCUSSIONS

Identification of marker compound

The solution of plant extract sample was chromatographed and obtained the concentration of quercetin of plant extract by using the regression equation. In the research, the HPLC process is used to study and quantify the quercetin in the Pancratium triflorum.

Preparation of the calibration curve of the quercetin

For the preparation of calibration curve, taken standard solutions (quercetin) and injected into the column and calculate the mean of peak area of standard drug and plotted the graph against different concentration (62.5, 125, 250, 500 and 1000ppm) of the drug by using methanol as solvent system. The regression equations were figured out on the basis of the curve. The calibration curve of the standard solution was used for comparing the peak of plant extract.

Table 3: Preparation of Calibration curve

Figure 1: Calibration Curve of the Quercetin

The calibration curve of flavonoids was observed linear relationship over the range of 62.5, 125, 250, 500, 1000µg/L (R²=0.99) concentration of standard. The linear regression equation for the curve was Y = 98946.6 x + 1038940, where y is the ratio of peak area of flavonoids and x is the flavonoid concentration (µg/L).

Identification of quercetin in plant extract

The qualitative and quantitative study of quercetin of selected Phytoextracts was performed by the HPLC method. The HPLC chromatogram of ethanol extracts of bulb of Pancratium triflorum revealed their highest peaks area for the quercetin as secondary metabolites component. The resulting HPLC chromatogram of standard quercetin and plant extracts sample were recorded and presented in below (Table and Figures). The qualitative analysis of phytochemicals was supportive of the quantitative analysis of its. The ongoing study confirmed the presence of flavonoids (quercetin) in medicinal plant as secondary metabolites.

Table 4: Characteristics of the analytical method derived from the standard calibration curve

Figure 2: Chromatogram of standard Quercetin

Figure 3: Chromatogram of ethanolic bulb extract of Pancratium triflorum

Chromatographic analysis of ethanolic bulb extracts of plant performed by using methanol and ortho-phosphoric acid 60:40 (V/V) solutions as mobile phase and used a flow rate of 1ml per min and absorbance at 370 nm. It gave good separation of quercetin at RT 2.25 min.

The peak of quercetin in the chromatogram of plant extract was compared with the RT values with those obtained by the chromatogram of the standard quercetin under the similar conditions.

Standard quercetin gave a strong peak area at the RT 2.24 min and HPLC of extracts of the ethanolic bulb extract of plant gave different peaks area at the same retention times as shown in the above figures. Results of the peaks of plant extracts did show more differences with standard quercetin, which further confirms the occurrence of quercetin in the ethanolic bulb extracts of Pancratium triflorum. Recently, this technique is achieving popularity within numerous analytical techniques as the primary choice for the study of fingerprinting and for quality control method of herbal plants characterization of secondary metabolites of plant extracts.

Quantitative analysis of quercetin in plant extract

This research stated the presence of a large quantity of total flavonoids content in Pancratium triflorum. Flavonoid is mainly occurred in various parts of the plant as glycosides and quercetin and is known to be imparting luxuriant colour to the leafy parts, fruits, flowers, etc. It is stated to reveal anti-cancer, anti-histamine, and anti-inflammatory activities which mostly followed its antioxidant features. Quantitative estimation of quercetin in hydroalcoholic extracts of Pancratium triflorum were calculated in the given table.

Table 3: Quantitative estimation of Quercetin in extract of Pancratium triflorum