Separation and Identification of Furocoumarin in Fruits of Heracleum candicans DC . by HPTLC

Heracleum candicansWall. ex DC. (Apiaceae) is widely used in Indian system of medicines as an aphrodisiac, nerve tonic and also in the treatment of skin diseases with reports of being rich in furocoumarins which are extensively used in pharmaceutical industry for their photosensitizing activity on human skin. A simple high performance thin layer chromatography (HPTLC) method has been developed for the simultaneous determination of psoralen (1) and heraclenol (2) in the fruits of H. candicans. The analytes were separated on silica gel F 254 plates with toluene : diethy lether : acetic acid (6 : 4 : 1) and scanned densitometrically at 350 nm.The method was validated in terms of precision, repeatability, and accuracy. The linear range for psoralen and heraclenol was found to be 1–5μg spot and 1–4μg spot with correlation coefficient of 0.973 and 0.964, respectively.The two compounds were quantified in fruits ofH. candicans and were found to be present in the range of 0.021–0.036% and 0.029–0.043%w/w.Themethod was found to be very simple, accurate, precise, and economical and can be used for routine quality control.

Furocoumarins are polyphenolic compounds, synthesized from L-phenylalanine, which may occur in a linear form with the furan ring attached to the 6, 7 position of the benzo-2-pyrene nucleus.These are biologically active natural compounds found in many plants particularly in members of the Umbelliferae and Rutaceae families [9,10].They are typically phototoxic compounds leading to photodermatitis upon exposure to UV light [11].In humans and experimental animals, chronic furocoumarin treatment, in combination with UV light, is used in the therapy of psoriasis [12].On the molecular level furocoumarins bind to cellular constituents such as proteins and lipids, can damage lysosomes, lead to the formation of reactive oxygen species, and can contribute to the formation of novel antigens by covalent modification of proteins [13].Furocoumarins of H. candicans are extensively used in pharmaceutical industry for their photosensitizing activity in human skin [14].There has already been an attempt made at the spectrophotometric estimation of the total furocoumarin bitter principles from roots of H. candicans [15], but this seems to be a preliminary report on indiscriminate estimation of total furocoumarins along with a report of the root of H. candicans [16].
HPLC as a tool for the quantitative estimation of furocoumarins in fruits of H. candicans has been reported [17], but HPTLC is a cheaper and faster technique with similar resolution.HPTLC of the H. candicans root for the estimation of the heraclenin and heraclenol has also been reported [18].Thus keeping in view the importance of furocoumarins, quantitative estimation of these compounds in H. candicans has been carried out using HPTLC.We have made an attempt to qualitatively evaluate H. candicans fruit taking two furocoumarins, namely, psoralen and heraclenol as bioactive marker.An attempt has also been made to validate the developed method in terms of precision, repeatability, LOD, LOQ, and accuracy.

Plant Material and Reagents.
Fruits of H. candicans DC. were collected from Nainital (Uttarakhand), India, during April-May 2004.The samples were authenticated and herbarium of the same was deposited in the NBRI, Lucknow, India (voucher specimen no.217680).

Chromatographic Conditions.
A CAMAG (Muttenz, Switzerland) HPTLC system, comprising of a Linomat 5 automatic applicator, a twin trough plate development chamber, Camag TLC scanner 3, and win-CATs software, was used.Precoated silica gel 60 F 254 (20 × 10 cm, 0.2 mm thickness, Merck, Darmstadt, Germany) glass plates were used as stationary phase.Conditions for the HPTLC were ambient temperature with 40% relative humidity.The mobile phase consisting of petroleum ether/hexane/toluene : diethyl ether : acetic acid (6 : 4 : 1) and so forth was used for solvent optimization.Solutions of sample and standard compounds 1, 2, and 3 of known concentrations were applied on TLC F 254 in band form as 6 mm wide and 10 mm from the bottom.The scanner conditions used were slit width 5 × 0.45 mm, wavelength 350 mm, and absorption reflection scan mode.These parameters were kept constant throughout.

Preparation of Standard Solutions of Psoralen and Heraclenol.
Stock solutions of psoralen (1) and heraclenol (2) (1.0 mg mL −1 ) were prepared by dissolving 10 mg of accurately weighed 1 and 2 in methanol and making up the volume of the solutions to 10 mL each with methanol.Aliquots (200, 400, 800, 1600, 3200, and 4000 L) of stock solutions were transferred to 5.0 mL volumetric flask and the volume of each one was adjusted to 5.0 mL with methanol to obtain standard solutions containing 40, 80, 160, 320, 640, and 800 gmL −1 of psoralen and heraclenol, respectively.

Extraction of Plant Materials.
Air dried (40-50 ∘ C), powdered fruits (2.0 g each) of H. candicans were extracted with methanol (3 × 20 mL) by stirring for 30 min on shaker.The combined extracts were concentrated under vacuum.The extracts were obtained in a yield of 20.22%.Solutions of 10 mg mL −1 concentration were prepared by dissolving the extracts in methanol for analysis.
2.5.Calibration.10 L of each of the standard solutions (0.4, 0.8, 1.6, 3.2, 6.4, and 8.0 g spot −1 ) of psorelen and 8 L of the standard solutions (0.32, 0.64, 1.28, 2.56, 5.12, and 6.4 g spot −1 ) of heraclenol were applied in triplicate onto silica gel 60 F 254 HPTLC plates using a Linomat 5 automatic sample applicator.The plates were developed with a solvent system of toluene : diethy lether : acetic acid (6 : 4 : 1 v/v) in a CAMAG glass twin trough chamber, up to a distance of 8.0 cm.After development, the plates were air dried and scanned at 350 nm.The peak areas were recorded.Calibration curves were prepared by plotting peak areas versus concentration.

Quantification of Psoralen and Heraclenol in Methanolic
Extract of H. candicans Fruits.10 L of sample solution was applied in triplicate on a silica gel 60 F 254 HPTLC plate with the CAMAG automatic sample applicator.The plate was developed and scanned as described above.The peak areas were recorded.The amount of 1 and 2 in the sample was calculated using the calibration curves.Peak identification was achieved in comparison of both   and UV absorption spectrum with those obtained for standards.

Method Validation.
Precision was checked by repeated scanning of the same spots of 1 (3.2 g spot −1 ) and 2 (2.56 g spot −1 ) six times and was expressed in terms of relative standard deviation (% RSD).The repeatability of the method was done by analyzing the spots of standard solutions of 1 and 2 after application on the HPTLC plate ( = 5) and expressed in terms of % RSD.Variability of the method was studied by analyzing aliquots of standard solutions of 1 (0.4, 0.8, 1.6, 3.2, 6.4, and 8.0 g spot −1 ) and 2 (0.32, 0.64, 1.28, 2.56, 5.12, and 6.4 g spot −1 ) on the same day (intraday precision) and on different days (interday precision) and the results were expressed in terms of % RSD.The accuracy of the method was determined by recovery studies.Plant samples spiked with three different amounts of 1 (1.0, 2.0, and 3.0 g) and 2 (400, 600, and 800 g) were analysed and the percent recovery was calculated.Different dilutions of standard solutions of 1 and 2 in methanol were applied.Limit of detection (LOD) and limit of quantification (LOQ) were determined on the basis of-signal to-noise ratios of 3 : 1 and 10 : 1.

Results and Discussion
Earlier a simple TLC method has been reported for the simultaneous determination of only two compounds from the roots of Heracleum candicans [18].The quantitative estimation of furocoumarins in fruits was also reported by HPLC only [17].In the present study different compositions of the mobile phase consisting of petroleum ether/hexane/toluene : diethyl ether : acetic acid (6 : 4 : 1) and so forth were tested for solvent optimization.In systems other than toluene: Diethyl ether: Acetic acid either the resolution of the spots was not very clear or there was interference from the matrix.Best resolution and reproducible peaks were obtained only by using toluene : diethyl ether : acetic acid (6 : 4 : 1) as mobile phase (1:   = 0.61, 2:   = 0.16).Linearity was observed in the range of 1 to 5 g per spot for 1 and 1 to 4 g per spot for 2. The specificity of the method was ascertained by analyzing standards and the

Conclusion
The method for the quantification of psoralen (1) and heraclenol (2) in the fruits of H. candicans was found to be simple, accurate, precise, and suitable for rapid screening of a large number of samples without any special sample pretreatment.The method can be used for the routine quality control of H. candicans as well as for the quantification of psoralen and heraclenol in other plant materials as well as in compound herbal formulations containing these furocoumarins.

Table 1 :
Method validation parameters for the estimation of psoralen and heraclenol by HPTLC densitometry.

Table 2 :
Percent of psoralen and heraclenol in H. candicans fruits.