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

Mandal, Ramachandran, Divya, Rubeena, Sunil Kumar, and Sathiyarajeswaran: Pharmacognostical-physico-chemical Evaluation and Development of HPTLC Finger print for Cichorium intybus L. fruits

Authors

INTRODUCTION

Traditional system of medicines is the major backbone of primary health care systems throughout the whole world even in this century. The genus Cichorium comprises of six species majorly distributed in Asia and Europe.1,2 C. intybus L. is an erect perennial herb with a fleshy taproot up to 75 cm.2,3 It has a very pronounced history in folklore medicine as a very familiar plant in traditional system of medicine in Europe and Asia. Despite this fact, this plant is not described in European pharmacopoeia.4 Chicory contains many medicinally important phytochemicals which belong to the categories, viz., carbohydrates, alkaloids, flavonoids, triterpenoids, tannins, fatty acids, volatile oils, etc.5 The seed (botanically achene type of indehiscent fruit) contains triterpenoids, cichoridiol and intybusoloid along with 11 known compounds namely lupeol, fridelin, β-sitosterol, stigmasterol, betulinic acid, betunaldehyde, syringic acid, vanilic acid, betulin, 6,7-dihydroxycoumarin and methyl-α-D-galactopyranoside.6 Chicory fruits are one of the main ingredients of jigerine, a commercial product of India which is used for the treatment of various diseases of liver.7,8 Traditional uses, chemical constituents, pharmacological activities, toxicological studies, clinical trial reports, cultivation and sustainable uses of various anatomical parts of C. intybus has been reviewed by earlier researchers.9 Alcoholic extract of seeds of C. intybus has been reported to exhibit a potent antihepatotoxic activity on CCl4 induced liver damage in albino rats.10 The histopathological study has shown the normalization of the fatty accumulation of the tissues and resolvation of necrosis.7 Investigation 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 CCl4 induced hepatic damage in mice.11 The 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.12 The 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.

MATERIALS AND METHODS

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.13

Sample 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 60F254 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 (D2 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).

TLC Photo documentation

TLC finger print profile of n-hexane extract of Cichorium intybus L. fruits revealed 6 bands with Rf 0.18, 0.29, 0.37, 0.49, 0.58 and 0.75 (green) under short UV; 6 spots with Rf 0.23, 0.29, 0.36 (red), 0.42 (fluorescent blue), 0.55 and 0.63 (blue) under long UV; 8 spots with Rf 0.06, 0.14 (violet), 0.17 (ash), 0.32 (violet), 0.45, 0.53, 0.75 and 0.82 (purple) under white light (post derivatization) (Figure 1). Successive chloroform extract showed 6 bands with Rf 0.10, 0.21, 0.32, 0.51, 0.73 and 0.79 (green) under short UV; 7 bands with Rf 0.14, 0.21, 0.28 (blue), 0.32 (red), 0.53 (violet), 0.64 and 0.73 (red) under long UV; 10 spots with Rf 0.07 , 0.09 (violet), 0.14 (grey), 0.16, 0.37 , 0.54 (purple), 0.61, 0.70, 0.75 and 0.81 (violet) under white light (post derivatization) (Figure 2). In case of successive ethanol extract of 6 bands with Rf 0.04, 0.11, 0.22, 0.37, 0.53, 0.85 (green) were found under short UV, 6 spots with Rf 0.11, 0.34 (blue), 0.40 (fluorescent blue), 0.58 (blue), 0.82 and 0.90 (red) were seen under long UV and 9 bands with Rf 0.18 (grey), 0.21, 0.36 (violet), 0.49 (brown), 0.53, 0.66, 0.74, 0.79 and 0.93 (violet) were found under white light (post derivatization) (Figure 3). The Rf values and corresponding colours of the bands are enlisted in for n-hexane, chloroform and ethanol extract respectively (Table 2).

HPTLC denistometric scan

The fingerprint profile of n-hexane extract under λ 254 nm revealed that the major peak is Rf 0.58 with an area of 18.52% followed by the peaks at Rf 0.29 (17.38 %), Rf 0.37 (9.39%) and 9 more minor peaks (Figure 4.1); under λ 366 nm, the major peak appeared at Rf 0.36 with an area 32.01%, second major peak at Rf 0.42 with an area 23.77% followed by Rf 0.29 with an area of 13.72% along with 8 more peaks (Figure 4.2); under white light after derivatization, the peak at Rf 0.75 with an area 25.22% came out as the major followed by the peak at Rf 0.53 (24.19%) and Rf 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 Rf 0.32 with an area of 30.28% followed by Rf 0.21 (16.71%), Rf 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 Rf 0.73 with an area 30.21% followed by the peak at Rf 0.21 with an area 30.03% along with 9 more peaks (Figure 5.2); under white light the fingerprint profile showed the major peak at Rf 0.81 with an area 25.13%, whereas the peak having Rf 0.61 with an area 13.25% was second major and the peak having Rf 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 Rf 0.85 with an area of 15.67% followed by Rf 0.90 (12.67%), Rf 0.42 (12.03%) along with 10 more minor peaks (Figure 6.1); under λ 366 nm, the major peak appeared at Rf 0.40 with an area 53.75% followed by the peak at Rf 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 Rf 0.93 with an area 26.87%, whereas the peak having Rf 0.74 with an area 15.00 % was second major and the peak having Rf 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, physico-chemical 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.

CONFLICT OF INTEREST

We declare no conflict interest with any.

ABBREVIATIONS

AG: Aleurone grains; Ec: Epicarp; Eco: Epidermis of cotyledon; Enc: Endocarp; IMe: Inner mesocarp; Mec: Mesocarp; OMe: Outer mesocarp; Scl: Sclerenchyma; Te: Testa; T: Trichome; VB: Vascular bundle.

GRAPHICAL ABSTRACT

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SUMMARY

  • 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, physico-chemical standardisation, TLC finger print profiles will help to check the quality of the drug prior to its use in traditional medicine.

Table 1

Physico-chemical constants of Cichorium intybus fruits.

ParameterResult
Loss on drying at 1050 C3.43 %
Total ash9.98 %
Water soluble ash1.93 %
Acid insoluble ash1.60 %
Water soluble extractive9.85 %
Ethanol soluble extractive11.25 %
pH5.86
Successive Extraction
n-Hexane1.70 %
Chloroform1.25 %
Ethanol3.25 %
Table 2

Rf values and color of spots of extracts of Cichorium intybus fruits.

Name of the extractUV 254 nmUV 366 nmWhite light after derivatizationRfColourRfColourRfColour
n-HexaneGreen0.18Blue0.06Violet0.06
Green0.27Blue0.09Violet0.11
Green0.31Red0.22Violet0.13
Green0.35Red0.28Grey0.17
Green0.48Red0.35Violet0.30
Green0.60F Blue0.41Purple0.44
Green0.73Blue0.53Purple0.54
Blue0.62Purple0.74
Purple0.83
ChloroformGreen0.10Blue0.13Violet0.06
Green0.21Blue0.21Violet0.09
Green0.32Blue0.28Grey0.14
Green0.51Red0.32Grey0.16
Green0.73Violet0.52Purple0.37
Green0.80Red0.64Purple0.53
Red0.74Violet0.62
Violet0.69
Violet0.75
Violet0.82
EthanolGreen0.05Blue0.11Grey0.19
Green0.12Blue0.34Violet0.22
Green0.20F Blue0.42Violet0.36
Green0.36Blue0.60Brown0.50
Green0.55Red0.68Violet0.53
Green0.86Red0.85Violet0.66
Green0.93Red0.92Violet0.74
Violet0.80
Violet0.94

F - Fluorescent

Plate 1

Macroscopy Cichorium intybus L. fruit

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Plate 2

Microscopy Cichorium intybus fruit. AG - Aleurone grains; Ec - Epicarp; Eco - Epidermis of cotyledon; Enc - Endocarp; IMe - Inner mesocarp; Mec - Mesocarp; OMe - Outer mesocarp; Scl - Sclerenchyma; Te - Testa; T - Trichome; VB - Vascular bundle.

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Plate 3

Microscopy of powder of Cichorium intybus fruit

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Figure 1

Photo documentation of n-hexane extract of Cichorium intybus fruit.

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Figure 2

Photo documentation of chloroform extract of Cichorium intybus L. fruit.

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Figure 3

Photo documentation of ethanol extract of Cichorium intybus L. fruit.

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Figure 4

Densitometric scan of n-hexane extract of Cichorium intybus fruit.

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Figure 5

Densitometric scan of chloroform extract of Cichorium intybus fruit.

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Figure 6

Densitometric scan of ethanol extract of Cichorium intybus fruit.

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References

1 

Quality Standards of Indian Medicinal Plants. New Delhi: Indian Council of Medical Research; 2005. 3. p. 126

2 

Bias HP, Ravishankar GA, authors. Cichorium intybus L.- cultivation, processing, utility, value adiition and biotechnology, with an emphasis on current status and future prospects. Journal of the Science of Food and Agriculture. 2001;(81)5:467–84

3 

Vanwyk BE, Van Oudtshoorn B, Gericke N, authors. Medicinal Plants of South Africa. Briza Publications; Pretoria, South Africa: 1997

4 

European Medicines Agency. “Assessment report on Cichorium intybus L., radix,”. EMA/HMPC/113041/2010. 2013

5 

Nandagopal S, Ranjithakumari BD, authors. Phytochemical and antibacterial studies of Chicory (Cichorium intibus) a multipurpose medicinal plant. Advances in Biological Research. 2007;(1-2):17–21

6 

Rahman A, Zareen S, Choudary MI, Akhtar MN, Khan SN, authors. Alpha-glucosidase inhibitory activity of triterpenoids from Cichorium intybus. J Nat Prod. 2008;(71)5:910–13

7 

Ahmed B, Al-Howiriny TA, Siddiqui AB, authors. Antihepatotoxic activity of seeds of Cichorium intybus. J Ethnopharmacol. 2003;2-3:237–40

8 

Najmi AK, Pillai KK, Pal SN, Aqil M, authors. Free radical scavenging and hepatoprotective activity of jigrine against galactosamine induced heptatopathy in rats. J Ethanopharmacol. 2005;(97)3:521–5

9 

Street RA, Sidan J, Prinsloo G, authors. Cichorium intybus: Traditional uses, phytochemistry, pharmacology and toxicology. Evidence-Based Complementary and Alternative Medicine. 2013;13. pages. http://dx/doi.org/10.1155/2013/579319.

10 

Gadgoli C, Mishra SH, authors. Antihepatotoxic activity of Cichorium intybus. J Ethanopharmacol. 1997;(58)2:131–4

11 

Gilani AH, Janbaz KH, authors. Evaluation of the liver protective potential of Cichorium intybus seed extract on acetaminophen and CCl4 – induced damange. Phytomedicine. 1994;(1)3:193–7

12 

Shaikh T, Rub Ra, Sasikumar S, authors. Antimicrobial screening of Cichorium intibus seed extracts. Arabian Journal of Chemistry. 2016;9:S1569–S73

13 

Anonymous. Quality control Methods for medicinal plant materials. World Health Organizing; Geneva.: 1998