Pharmacognostical, physico-chemical and phyto-chemical standardization of Cichorium intybus L. seed

P. Bigoniya¹*, C. S. Singh¹ and B. Shrivastava²

¹Radharaman College of Pharmacy, Fatehpur Dobra, Ratibad, Bhopal, Madhya Pradesh, India.

²School of Pharmaceutical Sciences, Jaipur National University, Jaipur, Rajasthan, India.

ABSTRACT:

Alternative and traditional medicines, largely herbal in nature, are now regarded as important but underutilized tools against disease. WHO recognized this fact and encouraged governments to effectively utilize local knowledge of herbal medicines for disease prevention and health promotion. Herbal medicines, however, suffer from a range of shortcomings. These include insufficient and unacceptable evidences of safety, efficacy, standardization and inconsistent production practices. There is a growing concern for documentation of research work carried out on traditional medicines needed for regulatory control. With this backdrop, it becomes extremely important to make an effort towards standardization of plant material used for therapeutic purposes. The process of standardization can be achieved by stepwise pharmacognostical, phyto- and physico-chemical studies and minimizing the inherent variation of natural product composition through quality assurance practices. WHO recommends various physico-chemical and phyto-chemical evaluation parameters for standardization and quality control of herbal medicinal plants. In view of ethnopharmacological importance of Cichorium intybus Linn, preliminary phyto-chemical screening and quantitative estimation of seed were performed along with morphology and microscopy to establish the salient diagnostic characters. The characteristics evaluated will be helpful for establishing quantitative and qualitative standardization of herbal preparations containing Cichorium intybus seed.

KEYWORDS: Cichorium intybus seed, pharmacognostical, physico-chemical, phyto-chemical studies.

INTRODUCTION:

Despite the promise that plant derived medicine exhibited activity, the one major obstacle in using plant-based drugs is lack of reproducibility. In olden times, the traditional medicine used to be a personalized one, with the healers preparing the medicines on an individual basis, where the quality of the medicine and hence the safety and efficacy were taken care of completely. Large scale production of herbal drugs has only started in the last 100 years or so. Now due to globalization and fast paced economic growth, the onus of maintaining quality of herbal medicine falls to a large extent on the scientists and to a certain extent on the manufacturers and consumers. In many countries, the herbal market is poorly regulated and plant derived medicines are increasingly being sought out without label claim as healthcare products through the health food shops and pharmacies over the counter as self medication.

Among consumers, there is a widespread misconception that “natural” always means “safe”, and a common belief that remedies from natural origin are harmless and carry no risk. However, some medicinal plants/formulations are inherently toxic that may attributable to the poor quality and noncompliance of standardization parameters. The herbal raw material is prone to a lot of variation due to several factors, the important ones being the identity of the plants, seasonal, ecotypic, genotypic and chemotypic variations, drying and storage conditions and the presence of xenobiotics¹.

In this scenario, the assurance of standardization of medicinal plant in term of safety, quality and efficacy has become an important issue. The National Center for Complementary and Alternative Medicine and the WHO stress on the importance of the qualitative and quantitative methods for characterizing the samples, quantification of the bioactive and the fingerprint profiles². The advancements in application of sophisticated analytical techniques have made it possible to fulfill many of these requirements of regulatory authorities. Still, the development of suitable biomarker for herbal drugs requires an innovative and creative approach, different from the routine methods. Starting from collection of the raw material, standardization of crude, preparation of the extracts, formulation of the extracts into suitable dosage form, the problems vary with each part/plant that is being used. At each and every step, phytochemical profiles have to be generated along with development of multiple marker based standardization strategy to maintain batch to batch consistency and confirmation of correct amount of extract per dosage unit³.

Considering the above mentioned problems, an attempt has been made to standardize the ethnopharmacologically useful seed of Cichorium intybus Linn. (C. intybus) on the basis of pharmacognostical, physico-chemical and phyto-chemical characteristics.

The plant, C. intybus (Family: Compositae) commonly known as chicory or kasni has a long history of herbal use and is especially of great value for its tonic effects upon the liver and digestive tract. The root and the leaves are appetizer, cholagogue, depurative, digestive, diuretic, hypoglycemic, laxative and tonic. Root decoctions are being used in the treatment of jaundice, liver enlargement, gout and rheumatism⁴. The tonic of chicory was considered to be beneficial in the treatment of enlarged spleen and diarrhea. Studies have shown that chicory root callus and seed possessed potent anti-hepatotoxic activity⁵.

Correct identification and quality assurance of the starting materials is prerequisite to ensure reproducible quality, which will intern ensure safety and efficacy of herbal medicine. This study was undertaken to generate standardized data on various pharmacognostical, physico- and phyto-chemical characteristics of the seed. The outcome of the present study will be helpful in identification, authentication and quality control of the plant material.

MATERIALS AND METHODS:

Chemicals:

FAA solution (95% ethyl alcohol: glacial acetic acid: formalin: water in 50:5:10:35), hemalum, safranin, hydrochloric acid, phloroglucinol and other chemicals used in the study were of analytical grade.

Collection and identification of plant material:

The seeds of C. intybus were collected from local market of Bhopal, M.P., India. The seeds were identified by Dr. H. B. Singh, Scientist F and Head, Raw Material Herbarium and Museum, NISCAIR, New Delhi, India. A voucher specimen (NISCAIR/RHMD/Consult/-2009-10/1234/47) of the seed has been retained in the department for reference purpose. The collected seed were cleaned, shade dried and pulverized with mechanical pulverizer for size reduction. The size pulverized seed powder was passed through mesh 40-60 and used for determination of physiochemical parameters and preparation of different solvent extracts. The fresh seed samples were used for macroscopic and microscopic studies.

Macroscopic and microscopic analysis:

The macroscopy and microscopy of the seed were studied to the method described by Brain and Turner, (1975)⁶. In microscopy, the desired part of fresh seeds were cut into pieces of 2-5 mm without compression and immediately transferred into FAA solution for one day to kill and fix the tissues. The pieces were embedded with paraffin wax. The paraffin embedded specimens were sectioned with the help of rotary microtome having thickness of 10-12 μm. Dewaxing of the sections was performed by customary procedure⁷. The sections were stained with hemalum and safranin. A drop of HCl and phloroglucinol were used to detect lignified cell in the cut sections⁸. The microphotographs were captured using trinocular microscope with digital Olympus camera.

Preparation of extracts:

Coarse powder (25 gm) of seed was defatted with sufficient quantity (500 ml) of petroleum ether (40-60°C) with the aid of Soxhlet apparatus for 24 hr. The defatted seed cakes (5 gm each) were then extracted separately with 100 ml each of ethyl acetate, chloroform methanol, ethanol and water for 48 hr by maceration and then filtered to obtain respective extracts. The petroleum ether fraction obtained after defatting was recovered as petroleum ether extract after filtration. The extracts in different solvent were collected separately and volume reduced under low pressure. Twenty five ml of the each extract was used to determine the percentage extractive values of seeds in different solvents. The remaining extract was stored in air tight glass container at 4-8°C for fluorescence analysis.

Physico-chemical studies:

The percentage of foreign matter, loss on drying and ash were determined according to the method described in WHO guidelines on Quality Control Methods for Medicinal Plant Materials⁹. The dried seed powders were subjected to fluorescence analysis, as it is and also after treating separately with water, 1 N of HCl, HNO₃, H₂SO₄, NaOH, KOH, alcoholic NaOH, alcoholic KOH and ammonia against normal and ultra-violet light (254 nm). Color reaction of petroleum ether, ethyl acetate, chloroform and methanol extract was also observed in normal light and UV light (254 nm)¹⁰.

Preliminary phytochemical screening:

Preliminary phytochemical screening of the seed extracts in different solvents has been performed to detect the phytoconstituents like; alkaloid, amino acid, carbohydrate, glycoside, inulin, mucilage, tannin, starch, saponin, steroid, triterpenoid and flavonoid^{11, 12}.

Quantitative estimation of phytoconstituents:

Alkaloid estimation: Alkaloid estimation was performed according to the method described by Obdoni and Ochuko, (2001)¹³.

Flavonoid estimation: Aluminium chloride colorimetric technique was used for flavonoids estimation¹⁴.

Saponin estimation: Saponin estimation was performed according to the method described by Obdoni and Ochuko, (2001)¹³.

Estimation of total phenols: The total phenols of the extracts were measured at 765 nm by Folin Ciocalteu reagent method¹⁴.

RESULTS:

Macroscopic evaluation of seed:

Seeds are brown in color which is approximately 5 mm long, 1-1.5 mm wide and 0.7-1 mm thick with 4 or 5 ridges near the base. Pappy containing 28-45 imbricate scales which are about 0.2-0.3 mm in length (Figure 1).

Microscopical evaluation of seed:

The longitudinal section of seed showed presence of embryo, testa, tegmen, alleurone layer, cotyledons and endosperm. Triangle shaped embryo were attached with both cotyledons mouth. The cells of the embryo were small and polygonal in shape. Testa was thick, 1-2 layered and appeared as pinkish white in color whereas, tegmen layer was attached to inner side of testa layer and appeared as singled layer. The major bulk of seed was endosperm and both the cotyledons were well separated by alleurone layer (Figure 2)

Physico-chemical evaluation:

The seed powder was evaluated for its physico-chemical parameters like foreign matter, loss on drying, total ash, acid insoluble ash and different extractive values. The results showed that only 0.92 ± 0.08% of foreign matter were present in C. intybus seed. The results of loss on drying turned out to be 7.34 ± 0.06%. Content of total ash in crude was found to be 9.33 ± 0.08% whereas, acid insoluble ash showed low content of ash i.e. 1.97 ± 0.01%.

The results suggest that seed has high water (23.92 ± 0.05%) and alcohol (16.80 ± 0.09%) soluble extractive value in comparison to the petroleum ether (1.47 ± 0.02%), chloroform (1.16 ± 0.03%), ethyl acetate (1.18 ± 0.03%) and methanol (3.90 ± 0.04%) soluble extractive values.

Table 1: Fluorescence analysis of C. intybus seed powder

Seed	Observation Under	Powder + Solvents
Seed	Observation Under	Dry Powder	Powder + Water	Powder + HCl	Powder + HNO₃	Powder + H₂SO₄	Powder + NaOH	Powder + KOH	Powder + Alc. NaOH	Powder + Alc. KOH	Powder + Ammonia
C. intybus	Normal light	Saddle brown	Maroon	Maroon	Golden rod	Maroon	Black	Golden rod	Maroon	Golden rod	Maroon
C. intybus	U.V. light	Saddle brown	Dark brown	Dark brown	Greenish yellow	Dark brown	Dark brown	Greenish yellow	Dark brown	Greenish yellow	Dark brown

Table 2: Fluorescence analysis of C. intybus seed with different solvents

Seed	Extracts	Normal light	U. V. light
C. intybus	Petroleum ether	Transparent	Transparent
	Ethyl acetate	Light yellow	Light yellow
	Chloroform	Medium forest green	Light green
	Methanol	Light yellow	Light yellow

Preliminary phytochemical test for seed extract:

Preliminary phytochemical investigation was undertaken for the identification of different type of chemical constituents present in the plant material. Results of preliminary phytochemical screening are compiled in Table 3. Petroleum ether extract showed presence of carbohydrate and triterpenoid in C. intybus. Chloroform extracts showed the presence of steroid along with inulin. Screening of ethyl acetate extract indicated the presence of steroid only in seed. Methanol and ethanol extract of crude seed showed the positive results for carbohydrate, mucilage, tannin and steroid along with flavonoid. Screening of aqueous extract showed the presence of carbohydrate, glycoside, mucilage, tannin and flavonoid. Alkaloid, amino acid, starch and saponin were absent in all the extract of seed sample.

Table 3: Preliminary phyto-chemical screening of C. intybus seed with different solvents

Phytoconstituents

Solvent

Alkaloid

Amino acid

Carbohydrate

Glycoside

Inulin

Mucilage

Tannin

Starch

Saponin

Steroid

Triterpenoid

Flavonoid

Petroleum ether

Chloroform

Ethyl acetate

Methanol

Ethanol

Water

“+” = Presence of constituent, “-” = Absence of constituents

Quantitative estimation of phytoconstituents:

Quantitative estimation of phytoconstituent like alkaloid, flavonoid, saponin and total phenol in crude were performed. The result indicated that chicory seed have higher amount of flavonoid (215 mg/gm) and total phenol (383 mg/gm), whereas the alkaloid and saponin was found to be absent.

DISCUSSION:

To ensure the batch to batch consistency, homogeneity and reproducible quality of herbal products, proper control of starting material is utmost essential. Thus, in recent years, there has been an emphasis on standardization of medicinal plants of therapeutic potential. Despite the advances in the hyphenated techniques, identification and characterization of plant drugs by macroscopic and microscopic evaluation is still more reliable, accurate and inexpensive¹⁵. The present study was focused on the structural features of C. intybus including macro and microscopic features, physico-chemical and phyto-chemical properties of grinded seeds. According to WHO guidelines for Quality Control Methods for Medicinal Plant Materials, herbal materials are usually categorized on the basis of their sensory, macroscopic and microscopic characteristics. An examination to determine these characteristics should be the ﬁrst step towards establishing the identity and the degree of purity of plant materials and should be carried out before any further tests are undertaken in consideration¹⁶.

The results of macroscopic study showed important characteristics which might be useful for distinguishing it from coffee as the chicory root is often used as an adulterant in coffee¹⁷. Microscopic evaluation allows more detailed examination of crude and use of various reagents or strains enable to identify the organized cellular structure viz; embryo, testa, tegmen, alleurone layer, cotyledons and endosperm as present in the C. intybus seed.

The physic-chemical parameters are helpful to determine the identity, purity and quality of crude drug¹⁸. The results indicated that non−considerable amount of foreign matter was present in seed. This ensuring the purity of seed and reveals the absence of organic and inorganic contaminants. Loss on drying of C. intybus was nearly seven percent. It signifies that considerable amount of moisture was present in seed. The higher moisture content may lead to hydrolysis, oxidation and rancidity of drug constituent which may result in chemical or microbial decomposition of drug constituents. So, the moisture content of drug should be controlled and minimized in order to prevent chemical/microbial decomposition and make the product of definite strength.

Controlled incineration of crude drugs resulted in ash residue which is useful for detecting low grade products, exhausted drugs and excess of sandy or earthy matter present especially in powdered drugs. Evaluation of total ash is also helpful in determining authenticity, purity and inorganic materials such as oxides of Ca, Mg, K, Na, Si, P and Fe as well as minute quantities of other elements as boron in the crude drugs¹⁹. Content of total ash in seed was found to be relatively lower which may be due to low content inorganic materials. The total ash value for a crude drug is not always reliable, since there is possibility of presence of physiological and non-physiological substances such as calcium oxide, phosphates, silicates, silica, sand and soil. So, the authentication of crude by acid insoluble ash was also performed which particularly indicate the contamination with silicious material such as earth and soil^{20, 21}. The results showed the low content of silicious material in the seed.

The results indicate that seed have high water soluble extractive value in comparison to the ethanol, petroleum ether, ethyl acetate, chloroform and methanol extractive values. The higher water soluble extractive value indicates the presence of water soluble matters such as carbohydrate, glycoside, mucilage, tannin and flavonoid in the seed. These secondary metabolites have been reported to possess promising therapeutic activities, which can be utilized to develop potential drugs for therapeutic purposes²².

Fluorescence is the phenomenon exhibited due to presence different functional groups in the plant chemical constituents. Chemical constituents may produce fluorescence either in the visible range of day light or in ultra violet light. If the substance themselves do not show fluorescence, they may often be converted into their fluorescent derivatives or decomposition products by treating with different reagents. Fluorescence analysis of seed powder in different organic and inorganic solvents showed their characteristic fluorescent color. The fluorescence character of powdered drugs plays a vital role in the determination of quality and purity of the drug material. Hence, some crude drugs are often evaluated in this way as it is an essential parameter for first line standardization of crude drug²³.

The results of preliminary phytochemical test revealed the presence of various phytochemical compounds in the seeds which are known to have multiple therapeutic importances in traditional medicine. For instance, a sesquiterpene glycoside, cichotyboside isolated from C. intybus seed has been reported to possess hepatoprotective activity²⁴. Inulin, one of the constituent of C. intybus, was reported to decrease serum triglycerides by decreasing fatty acid synthesis and reducing production of low density lipoproteins by Williams, (1999)²⁵. The present study reports not only establishes pharmacognostical, physico-chemical and phyto-chemical characterizations of seed but also microscopic and fluorescence characters of chicory seed. These characteristics will be helpful for establishing quantitative and qualitative standardization of herbal preparations containing chicory seed. Further studies are in progress in order to characterize cichotyboside by chromatographic techniques along with exploration of pharmacological activity of cichotyboside.

ACKNOWLEDGEMENT:

Authors are thankful to Tapsya Siksha Samiti, Bhopal for providing financial support and laboratory facilities to carry out the research work (Faculty Promotion/Jan-July 2012/rcp-06).

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Received on 07.04.2013

Modified on 11.04.2013

Accepted on 14.04.2013

Research Journal of Pharmacognosy and Phytochemistry. 5(3): May-June 2013, 133-138