The plant, Lagenaria siceraria (Family: Cucurbitaceae), known as bottle gourd, Calabash, Doodhi, and Lauki, is a common fruit vegetable used throughout the India [1,2]. Lagenaria siceraria is mentioned medicinal plant in Ayurvedic Pharmacopoeia of India. L. siceraria fruits (Figure 1) are traditionally used as a nutritive agent having cardioprotective, cardiotonic, general tonic, diuretic, aphrodisiac, antidote to certain poisons and scorpion stings, alternative purgative, and cooling effects [3,4]. Literature survey also revealed that no spectrophotometric or chromatographic methods have been reported for standardization of Lagenaria siceraria fruit or its extract and or market formulations [5]. Hence the reported HPTLC method is only suitable for analysis of Lagenaria siceraria fruit. In present study attempt was made to isolate and estimate common phytoconstituent from crude extract of Lagenaria siceraria fruit and marketed powder formulation. HPTLC has been widely used for the phytochemical evaluation of the herbal drugs, due to its simplicity and minimum sample clean up requirement. Hence a densitometric HPTLC method has been developed for quantification of quercetin. The HPTLC method proposed in this paper is useful for simple, specific, rapid, precise and accurate analysis of Lagenaria siceraria fruits and its formulations by both peak-area and peak-height estimation method.

Figure 1: Lagenaria siceraria Fruit.

Plant material

The fruits of Lagenaria siceraria (Molina) Standley, were collected from local market of Amravati, and were authenticated by Principal investigator, Department of Botany, Govt. Vidarbha institute of science and Humanities, Amravati.

Chemicals and reagents

Toluene, ethyl acetate and formic acid were procured from Merck, India all of analytical grade reagent and silica gel F²⁵⁴ precoated TLC aluminum plates (E-Merck). Standard Quercetin was procured from Total Herb Solution, Mumbai. Market formulation in the form of powder was purchased in local market of Amravati.

Extraction and isolation

The fresh and semi ripe fruit were sliced using a home slicer and the slices obtained were shade-dried, pulverized and passed through 20 mesh sieve and stored in airtight container at room temperature (30 ± 20°C). About 100 gm of dried, coarsely powdered plant material was extracted with 99% ethanol using Soxhlet apparatus. The solvent recovered by distillation, evaporated under vacuum to give semisolid mass (20% w/w) which further dried and used for isolation of phytochemicals. The ethanolic extract was suspended in small portion of water, extracted with ethyl acetate and then resulting solution were concentrated to provide ethyl acetate soluble parts. These parts were subjected to silica gel (60-120 mesh) column chromatography for the isolation of individual phytoconstituents. Ethyl acetate soluble part was eluted gradiently using chloroform, chloroform: ethyl acetate (50:50), ethyl acetate, ethyl acetate: methanol (90:10) and methanol to give a compound which shown a single spot on TLC plate developed in Tolune: ethyl acetate: formic acid (6:2:0.8) and sprayed with Aluminium chloride reagent. This isolated compound further confirmed as quercetin using UV and FTIR spectroscopy (Figure 2). Sonicated ethanolic extract of market formulation of fruits was prepared and used for HPTLC estimation.

Figure 2: UV and FTIR spectrum of isolated compound-quercetin.

Instrumentation and chromatographic conditions

Chromatography was performed on 10 cm×10 cm HPTLC plates coated with 0.2 mm layers of silica gel 60 F²⁵⁴ (Merck, Darmstadt, Germany). Before use the plates were washed with AR grade methanol and activated at 115°C for 30 min. Samples were applied as bands 4 mm wide and 4 mm apart by use of a CAMAG Linomat IV sample applicator (Muttenz, Switzerland; supplied by Anchrom Technologists, Mumbai) equipped with a 100-μL syringe (Hamilton, Nevada, USA). A constant application rate of 6s μL^-1 was used. After several trials of ethyl acetate and toluene in different proportions, ultimately the mobile phase selected was toluene: ethyl acetate: formic acid, 6: 2: 0.8 (v/v/v). Linear ascending development was performed in a CAMAG 15 cm×15 cm glass twin-trough chamber. Before insertion of the plate into the mobile phase, the chamber was saturated with mobile phase vapor for 25 min at room temperature (25 ± 2°C). The development distance was 80 mm. After development the plates were dried by hot air spray drier. Densitometric scanning at 575 nm was performed with a CAMAG TLC scanner III in reflectance–absorbance mode controlled by CATS 4 software (Version 1.4.1; CAMAG) resident in the system. The amounts of the compounds chromatographed were determined from the intensity of diffusely reflected light. The method was further validated according to the ICH guidelines.

Preparation of standard and sample stock solution

Standard quercetin 10 mg was accurately weighed into a 10 mL volumetric flask, dissolved in 5 mL ethanol and the solution was made up to 10 mL with the same solvent (1 mg/mL or 1000 μg/1000/μl). From this stock solution, further dilutions were made, in such a way that of concentrations ranging from 0.5 to 3.5 μg/μl spot. Different concentration 1000, 1500, 2000, 2500, 3000, and 3500 ng/spot of quercetin were spotted on TLC plate. Accurately weighed 10 mg of dried alcoholic extract of fruit L. siceraria and market formulation was transferred to a 10 ml volumetric flask dissolved in 10 ml of ethanol separately. It was then sonicated for 10 minutes and the contents of the flask were filtered through Whatman No. 1 paper (Merck, Mumbai, India). The final volume of the solution was made up to 10 mL with ethanol to get stock solution containing 1 mg/ mL.

HPTLC method development and optimization

Normal phase HPTLC on silica gel 60 F²⁵⁴ with Tolune: ethyl acetate: formic acid (6:2:0.8 v/v/v) as mobile phase enabled good separation of quercetin at 3.6 R_f (Figure 3). The well defined peaks were obtained only when the chamber was saturated with the mobile phase for 25 min at a controlled temperature before plate development. Recovery close to 100% indicates that the proposed method is free from interference from the additives added.

Figure 3: HPTLC Photograph of various sample [Quercetin standards (first five spots), isolated quercetin, alcoholic extract, and marketed formulation] and Calibration curve of standard Quercetin.

Method validation

As recommended by ICH guidelines [6] the all validation was performed during the development of the procedure. A typical HPTLC densitogram is shown in Figure 4. The proposed method was validated for linearity, limits of detection (LOD) and quantification (LOQ), precision, accuracy, specificity, and ruggedness.

Figure 4: Typical HPTLC densitogram of standard Quercetin and sample showing Quercetin.

Linearity: Linearity was established by least squares linear regression analysis of the calibration curve. The constructed calibration plots were linear over the concentration ranges 1-4 ng (Figure 3). Correlation coefficient was 0.99 for standard quercetin (Table 1).

Table 1: Method validation parameters for HPTLC quantification of quercetin.

Precision: Instrumental precision, intra-assay precision, and intermediate precision of the method were determined. Instrumental precision was measured by replicate (n=3) application of the same quercetin standard solution (concentration 3.5 μL/per spot). Intra assay precision was evaluated by analysis of three replicate applications of freshly prepared standard solutions of same concentration, on the same day. Intermediate precision was evaluated by analysis of three replicate applications of standard solution of same concentration on three different days. The measurement of the peak area at three different concentration levels showed low values of % R.S.D. (<2%) for interand intra-day variation, which suggested an excellent precision of the method (Table 2).

Table 2: Precision study.

Limits of detection and limit of quantification: The limits of detection (LOD) and (LOQ) were determined from the standard deviation and the slope of line of calibration curve. The limits of detection (LOD) and quantification (LOQ) are 50 and 150 ng/μL respectively indicates the adequate sensitivity of the method (Table 1).

Recovery studies: Accuracy data for the assay of the compounds of interest are summarized in Table 3. The accuracy of the method was established by performing recovery experiments at three different levels using the standard addition method. In 3.5 μL of sample, and known amounts of quercetin standard (1000 ng per spot) were added by spiking. Results from recovery studies are within acceptable limits (98.0 to 102.0%), indicating the accuracy of the method was good.

Table 3: Recovery study.

Specificity: The specificity of the method was ascertained by analyzing the standard drug and extract. The spot for quercetin in the sample was confirmed by comparing the R_f values and spectra of the spot with that of the standard. The peak purity of the quercetin was assessed by comparing the spectra at three different levels, viz. peak start, peak apex and peak end positions of the spot.

Robustness and ruggedness: By introducing small changes in the mobile phase composition, mobile phase volume and duration of mobile phase saturation, the effects on the results were examined. Robustness of the method was done in triplicate at a concentration level of 3.5 μL/spot and the % R.S.D peak area was calculated (Table 4).

Table 4: Ruggedness and robustness studies.

A rapid, simple, accurate and specific HPTLC method for quantitative estimation of quercetin present in the fruit of Lagenaria siceraria has been developed and validated. The data could be used as a QC standard. The method used in this work resulted in good peak shape and enabled good resolution of quercetin from other constituents of the plant material. Because recovery (99.79%) was close to 100%, there was no interference with the quercetin peak from other constituents present in the plant. Quercetin belongs to the group of flavonoids that are largely effective as anti-inflammatory, antioxidant, anticancer agents. Most important achievement of the present work is that Quercetin can be used as a marker for Lagenaria siceraria standardization by HPTLC.

We are grateful to Dr V. K Mourya, Principal, Government College of Pharmacy, Amravati for providing laboratory facilities.