Pharmacognostical-physico-chemical Evaluation and Development of HPTLC Finger print for Cichorium intybus L . fruits

Introduction: Many herbal medicines are lacking pharmacognostical, phytochemical, pharmacological and toxicological data even though used widely for medicinal purposes. Cichorium intybus L. (Asteraceae) – chicory is an ancient folklore medicine. Various parts of these plants are in use for a wide range of ailments including those affecting liver and kidney. The aim of the current study is to standardize the fruit of C. intybus for macroscopy, microscopy, physicochemical parameters, TLC photo documentation along with development of HPTLC fingerprint profiles. Methods: Following standard pharmacopoeial procedures, detailed macro-microscopic characterization along with preliminary phytochemical features of the drug has been recorded from the current study. Results: Macro-microscopic study has revealed the authenticity of this medicinal achene type fruit. Physico-chemical and HPTLC studies revealed constants for identification and authentication of fruits of C. intybus. Conclusion: The current study will serve as a reference tool for quality maintenance, authentication as well as scientific validation of chicory fruits.

clinical trial reports, cultivation and sustainable uses of various anatomical parts of C. intybus has been reviewed by earlier researchers. 9Alcoholic extract of seeds of C. intybus has been reported to exhibit a potent antihepatotoxic activity on CCl 4 induced liver damage in albino rats. 10The histopathological study has shown the normalization of the fatty accumulation of the tissues and resolvation of necrosis. 7nvestigation of the hepatoprotective activity of aqueous methanolic extract of C. intybus seed resulted in a significant reduction in death rate, serum levels of alkaline phosphatase, glutamyl oxaloacetate transaminase and glutamyl pyruvate transaminase against acetaminophen and CCl 4 induced hepatic damage in mice. 11The aqueous extract of C. intibus seed showed significant antimicrobial activity against Staphylococcus aureus but not against Candida albicans.Ethyl acetate extract showed activity against Pseudomonas aeruginosa and Staphylococcus aureus. 12The present study is aimed at standardization the fruit of C. intybus and development of HPTLC finger profile for quality maintenance, authentication as well as scientific validation of this commercially important medicament.

Material
Cichorium intybus fruits were procured from local market of Mettur and was authenticated by Dr. M. Padma Sorna Subraminian, Research Officer (Botany), Siddha Medicinal Plants Garden, Mettur, Tamil Nadu.

Macro-microscopy and powder microscopy
The external features of the test sample were documented using Nikon COOLPIX 5400 digital camera followed by Zeiss Discovery.V8.For microscopy the sample was left in FAA (Formalin-5 ml + Acetic acid-5 ml + 70% Ethyl alcohol-90 ml) for more than 48 hr.The preserved specimens were hand cut into thin transverse section using 7 O'Clock platinum blades and were stained with safranine.Microsccopic features were photographed using Nikon ECLIPSE E200 trinocular microscope attached with Zeiss AxioCam ERc5s digital camera under bright field.The powdered sample was sieved through #80 mesh and a pinch of it was mounted on a microscopic slide with a drop of glycerin-water.Characters were observed using Nikon ECLIPSE E200 trinocular microscope attached with Zeiss AxioCam ERc5s digital camera under bright field.Diagnostic characters were captured, and photomicrographs were documented.Magnifications of the figures are indicated using pre-calibrated scale-bars.

Physico-chemical analysis
All the physico-chemical parameters were performed according to the method mentioned in standard books. 13mple preparation for HPTLC Exactly 2 g of coarse powder was taken in a Soxhlet apparatus and extracted with 100 ml each of n-hexane, chloroform and ethanol successively.These extracts were filtered, concentrated over water bath and made up to 10 ml with the corresponding solvents in standard flasks.The extracts were filled in separate sample vials of ATS 4 (CAMAG, Switzerland) for application.

Chemicals, solvents and materials
AR grade solvents n-hexane, chloroform, ethyl acetate, ethanol and formic acid were purchased from Merck.For visualizing purpose vanillin (1 g) sulphuric acid in ethanol (5%) solution (VSA) was used.For HPTLC, automatic sampler 4, twin trough chamber 10 × 10 cm, TLC visualizer, TLC scanner 4, TLC plate heater (all from CAMAG, Switzerland) were used.15 and 20 µl of each extract were spotted using ATS 4 on silica gel 60F 254 coated aluminium plates (6 × 10 cm) as 8 mm band width with 15 mm inter band distance.First application position was 15 mm and distance from the bottom of the plate was 10 mm.Plates were developed using solvent systems with n-hexane -ethyl acetate -formic acid (7.5:2.5:0.5, v/v/v), toluene -ethyl acetate -formic acid (6.5:4.0:0.5, v/v/v) and chloroform -methanol -formic acid (9:1: 0.8, v/v/v) for n-hexane, chloroform and ethanol extract respectively.The twin trough chamber was previously saturated for 15 min with the above mobile phases prior to development.The developed plates were dried, and photographs were taken by the visualizer under short UV and long UV.Scanning were performed by TLC Scanner 4 (Scanner_210441) under λ 254 and λ 366 nm in absorbance mode (D 2 lamp) and fluorescence mode (Hg lamp) respectively with a slit dimension 6×0.45 mm with scanning speed 20 mm/s.The scanned plates were dipped in VSA and heated over TLC plate heater at 105º C until the appearance of the coloured spots.Photographs were taken immediately at white light mode followed by scanning at λ 520 nm at absorption mode (W lamp).

RESULT AND DISCUSSION
Macroscopy Macroscopically, the fruit of C. intybus is creamish -brown in colour, measures up to 3 to 3.5 mm in length and about 1.25 mm in breadth; oblong in shape pointed towards base and broader towards upper side; topped with scaly pappus derived from accrescent calyx; surface smooth and shining and comprises of about 8 ridges (Plate 1); odour and taste nil.

Microscopy
The pericarp of fruit consists of a single epidermal layer which bears randomly distributed groups of unicellular papillose trichomes; the outer mesocarp shows 3 to 4 layers of sclerenchymatous cells arranged beneath the epidermal cells while bulk of sclerenchymatous cells arranged under elevated areas giving the appearance of ridges and furrows; the inner mesocarp is made up of large compactly arranged columnar cells followed by thin layer compressed cells forming endocarp enclosing two cotyledons.Endocarp and testa are fused and cannot be differentiated as the fruit is an achene.The cotyledon has a thin walled parenchymatous epidermis enclosing parenchyma cells containing globoid aluerone grains (Plate 2).

Powder microscopy
Powder shows polygonal epicarp cells in surface and oblique views; pitted as well as normal parenchyma; parenchyma containing oil globules; cells of cotyledons containing aleurone grains; bundle of fibre and pitted vessels (Plate 3); odor and taste nil.

Physico-chemical analysis
Moisture plays animportant role in deterioration of a drug due to microbial load leading to decline in shelf life.The loss on drying value which indicates the presence of moisture content of air dried sample was found to be 3.43 %.Total ash which indicate the total inorganic content including physiological and non-physiological salts of the drug was estimated to be as 9.98 %.Water soluble ash which predicts the water soluble inorganic salts was calculated as 1.93 %.The acid insoluble ash, majorly composed of silica in a plant drug, was found to be 1.60 %.The water and ethanol soluble extractive value which indicate the solubility of the active ingredients of the plant in water and ethanol respectively were estimated to be 9.85 % and 11.25 %.The pH of the drug was determined as 5.86 which indicates the acidic nature of the plant (Table 1).

HPTLC denistometric scan
The fingerprint profile of n-hexane extract under λ 254 nm revealed that the major peak is R f 0.58 with an area of 18.52% followed by the peaks at R f 0.29 (17.38 %), R f 0.37 (9.39%) and 9 more minor peaks (Figure 4.1); under λ 366 nm, the major peak appeared at R f 0.36 with an area 32.01%, second major peak at R f 0.42 with an area 23.77% followed by R f 0.29 with an area of 13.72% along with 8 more peaks (    the major peak at R f 0.81 with an area 25.13%, whereas the peak having R f 0.61 with an area 13.25% was second major and the peak having R f 0.24 with an area 9.25% was the third major peak.It also showed 11 more minor peaks (Figure 5.3).The finger print profile of successive ethanol extract under λ 254 nm showed the major peak at R f 0.85 with an area of 15.67% followed by R f 0.90 (12.67%),R f 0.42 (12.03%) along with 10 more minor peaks (Figure 6.1); under λ 366 nm, the major peak appeared at R f 0.40 with an area 53.75% followed by the peak at R f 0.34 with an area 15.19% along with 7 more peaks (Figure 6.2); under white light, the fingerprint profile showed the major peak at R f 0.93 with an area 26.87%, whereas the peak having R f 0.74 with an area 15.00 % was second major and the peak having R f 0.89 with an area 11.52 % was the third major peak; it showed 12 more minor peaks (Figure 6.3).

CONCLUSION
Cichorium intybus fruit (chicory seed) is an important ingredient of several Ayurveda, Siddha, Unani and other traditional formulations for the treatment of various ailments.The current attempt of preparation of monograph including macro-microscopy, powder microscopy, physicochemical standardisation, TLC finger print profiles will help to check the quality of the drug prior to its use in traditional medicine.

ACKNOWLEDGEMENT
The authors extend their heartfelt thanks to Director General, CCRS for the support and Dr. Padma Sorna Subramanian, Research Officer (Botany), Medicinal Plant Garden, Mettur, for providing authentic samples.came out as the major followed by the peak at R f 0.53 (24.19%) and R f 0.82 15.58 (15.58%) along with 9 more minor peaks (Figure 4.3).The finger print profile of successive chloroform extract showed the major peak at R f 0.32 with an area of 30.28% followed by R f 0.21 (16.71%),R f 0.79 (9.62%) along with 7 more minor peaks (Figure 5.1) under λ 254 nm; under λ 366 nm the major peak appeared at R f 0.73 with an area 30.21% followed by the peak at R f 0.21 with an area 30.03% along with 9 more peaks (Figure 5.2); under white light the fingerprint profile showed
Figure 4.2); under white light after derivatization, the peak at R f 0.75 with an area 25.22% Pharmacognosy Journal, Vol 10, Issue 4, Jul-Aug, 2018

Figure 3 :
Figure 3: Photo documentation of ethanol extract of Cichorium intybus L. fruit.