Chromatography
Deore et al., J Chromatograph Separat Techniq 2013, 4:6 http://dx.doi.org/10.4172/2157-7064.1000191
Separation Techniques Research Article Research Article
Open OpenAccess Access
Isolation and Quantitative Estimation of Quercetin in Lagenaria siceraria Fruit Sharada L Deore1*, Kumudini Nikole1, Bhushan A Baviskar1 and Somshekhar S Khadabadi2 1 2
Department of Pharmacognosy and Phytochemistry, Govt. College of Pharmacy, Amravati-444 604, Maharashtra, India Department of Pharmacognosy and Phytochemistry, Govt. College of Pharmacy, Aurangabad-431 305, Maharashtra, India
Abstract Lagenaria siceraria is an important medicinal plant belonging to family Cucurbitaceae. Now a day its use in Juice form is very popular. In present article, quercetin has been isolated from ethyl acetate fraction of fruit extract and confirmed by Co-TLC, UV and FTIR analysis. In addition, a simple, rapid, precise, and accurate high-performance thin-layer chromatographic (HPTLC) method has been established, validated for flavonoid quercetin. The linearity of the method was investigated in the range of 1-3.5 ng mL-1. Percentage recoveries for quercetin found to be 99.79%. The method was validated for precision, accuracy, specificity, and ruggedness. Densitometric quantification was performed at 575 nm after spraying with aluminum chloride reagent.
Keywords: Lagenaria siceraria; Flavonoids; Quercetin; HPTLC Introduction 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.
Matrrials and Methods 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
*Corresponding author: Sharada L Deore, Department of Pharmacognosy and Phytochemistry, Govt. College of Pharmacy, Amravati-444 604, Maharashtra, India, E-mail:
[email protected] Received June 27, 2013; Accepted July 22, 2013; Published July 25, 2013 Citation: Deore SL, Nikole K, Baviskar BA, Khadabadi SS (2013) Isolation and Quantitative Estimation of Quercetin in Lagenaria siceraria Fruit. J Chromatograph Separat Techniq 4: 191. doi:10.4172/2157-7064.1000191
Figure 1: Lagenaria siceraria Fruit.
J Chromat Separation Techniq ISSN:2157-7064 JCGST, an open access journal
Copyright: © 2013 Deore SL, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Volume 4 • Issue 6 • 1000191
Citation: Deore SL, Nikole K, Baviskar BA, Khadabadi SS (2013) Isolation and Quantitative Estimation of Quercetin in Lagenaria siceraria Fruit. J Chromatograph Separat Techniq 4: 191. doi:10.4172/2157-7064.1000191
Page 2 of 4 2.325 2.000
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Kbr
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Figure 2: UV and FTIR spectrum of isolated compound-quercetin.
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.
ethanolic extract of market formulation of fruits was prepared and used for HPTLC estimation.
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 6 s 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 J Chromat Separation Techniq ISSN:2157-7064 JCGST, an open access journal
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.
Results and Discussion 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
Volume 4 • Issue 6 • 1000191
Citation: Deore SL, Nikole K, Baviskar BA, Khadabadi SS (2013) Isolation and Quantitative Estimation of Quercetin in Lagenaria siceraria Fruit. J Chromatograph Separat Techniq 4: 191. doi:10.4172/2157-7064.1000191
Page 3 of 4
Figure 4: Typical HPTLC densitogram of standard Quercetin and sample showing Quercetin.
Method property
Value
Linearity range (ng)
500-2500
Standard deviation
2.21
Correlation coefficient (r)
0.99
Limit of Dectection (ng)
150
Limit of Quantification (ng)
50
Instrumental precision RSD {%} n=3
0.879
RSD=relative standard deviation Table 1: Method validation parameters for HPTLC quantification of quercetin. Sr. No.
% of Quercetin
1
1.09 ± 0.002
2
0.65 ± 0.012
3
0.89 ± 0.04
Mean ± SD
0.876 ± 0.051
% RSD
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).
1.81 Table 2: Precision study.
Sample
Amount Amount Present (ng) Added (ng) 1000 ng
Amount Recovered
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. (
