INTRODUCTION:

Medicinal plants are traditionally used around the world to cure different diseases with natural resources the demand for medicinal plants is increasing both in developing and developed countries¹. The development by means of biological products has greater than before worldwide and the dynamic plant extracts are commonly screened as part of innovative strategy for new drug discovery². Cichorium intybus Linn. (Compositae) is a potent anti-hepatotoxic plant and is a major component of indigenous drugs such as Geriforate, Acilvan, Livex³. In traditional System of Medicine, chicory whole plant are used to promote diuresis, lower body temperature, prevented gastrointestinal diseases, blood cleansing, in treatment and prevention of constipation⁴.

The plant found in the various state of India like Punjab, Kashmir, Andhra Pradesh, Karnataka and Maharashtra and it can easily grows almost on all types of soil⁵. As per Ayurveda medicinal system, cichory have the property to balance pitta and used to cure gall bladder stones by excreting them out of the body. In Ayurveda, Unani and Siddha medicinal system plant is known to treat hepatobiliary, liver disorders, jaundice, hepatitis and renal disorders⁶. Cichorium intybus Linn., is reported to have anti-diabetic, gastroprotective, cardiovascular, anthelmintic, antimicrobial, analgesic, anti-inflammatory, hepatoprotective and free-radical scavenging effects^7,8,9,10,11. C. intybus plant contains several chemical compounds like Sonchuside A, Cichoriolide, Chlorogenic acid, 3,5- Dicaffeolyquinic acid, 4,5 Dicaffeolyquinic acid, Crepidiaside A, Cichoralexin, Malic acid, Caffeic acid, 3-Caffeoylquinic acid, 5- Caffeoylquinic acid, 4-Caffeoylquinic acid, Dicaffeoyltartaric acid, (chicoric acid), Cyanidin, Glucoside are identified in chicory plant^{12,13,14,15,16,17,18,19.}

MATERIAL AND METHOD:

Chemicals:

All chemicals used were of analytical grade purchased from Sigma Chemical Co. and Merck.

Plant material:

The plant specimen for the study was obtained from the Herbal-garden of Jamia Hamdard, New Delhi and identified by taxonomist (voucher specimen No. PRL/JH/05/28), Department of Botany, Faculty of Science, Jamia Hamdard (Hamdard University), New Delhi.

Plant part: Stem

Botanical Name: Cichorium intybus Linn.

Family: Compositae

Macroscopical Evaluation:

The macroscopical evaluation was done by observing the stem under simple microscope and with naked eyes and taking note of the colour, odour, taste, shape, size and other diagnostic parameters. Different macroscopic features of stem were noted.²⁰

Microscopy:

Fresh green, fully-grown and healthy chicory plant stem was collected from Herbal garden of Jamia Hamdard. It was washed in pure water to remove all the impurities. Microscopy of the sample was done as per the method described by O Brien et al²¹. Photograph of anatomical features of the stem was taken with the help of Nicon laptop 2 microscopic units. The polarized light was used for the study of crystals, starch grains and lignified cell²².

Powder Microscopy:

The coarsely stem powdered was studied under the microscope and it was soaked in chloral hydrate regent, after that treat it with phloroglucinol and iodine reagents independently. After that stained powder were mounted on a slide with glycerine, observed various distinct characters under microscope and took the photos of the particular cell structures.

Physico-chemical studies:

Different physico-chemical values for instance ash value, extractive values, loss on drying, total resin and pH value²⁰ were determined.

Preliminary Phytochemical Analysis:

The successive extraction was carried out by using nonpolar and polar solvents such as petroleum ether, chloroform, acetone, ethanol and water, through soxhlet apparatus from dried stem coarse powder (10g), extract thus obtained dried and weighed. This extract was used for the detection of secondary metabolites for examples alkaloids, glycosides, sterols, carbohydrates, phenolics, saponin and flavanoids etc, through chemical tests as per method described by Peach and Tracy²³.

Fluorescence analysis study of powdered drug material with different reagents performed for observation of the color reactions²⁴. WHO protocols follows for the noting the bitterness value, swelling index and foaming index respectively²⁵.

High Performance Thin Layer Chromatography Fingerprints:

HPTLC fingerprint profile done by referring Stahl methods²⁶. The weighed quantity of dried stem powder was used for successive extraction by nonpolar and polar solvents like petroleum ether, chloroform, acetone and ethanol. These extracts were concentrated and used for the determination of fingerprint profile of chemical components which were present in respective extracts through HPTLC method. Individual extract sample applied (5µl each) on TLC plate (precoated silica gel G60 F_254,aluminium sheets, 10cm × 10cm) in triplicate with band width of 8mm using CAMAG Linomat V applicating device on separate plates. TLC plates were allowed to develop in the presaturated twin trough chamber and scanning of developed TLC plate was done using CAMAG TLC Scanner III and maximum numbers of compounds were determined via deuterium and tunguston lamp at best suitable wavelength.

RESULT:

Macroscopic characters:

Macroscopy investigation showed small the stems were 2-3 feet in height, angled or grooved, with spreading branches, given off at a very considerable angle from the central stem. Stems were green in colour outside and white inside. Shape: cylindrical, straight; branching: fibrous (Figure 1).

Microscopic evaluation:

The stem was circular with even, smooth surface with broad central pith canal, stem portion from surface to the pith canal was 1.5mm thick. The stem had a thin epidermal layer of narrow rectangular cells followed by two or three layers of collenchyma. The cortex was fairly wide and parenchymatous. The vascular strands were collateral with massive sclerenchyma caps, inner phloem and wedge-shaped radial, dense xylem files. Vascular strands were numerous, unequal in size and in circular shape. The xylem elements were circular and fairly thicken wide wall. The metaxylem elements were 40 μm wide. The outer pith consists of large, thin walled and compact parenchyma cells (Figure. 2, 3 and 4).

Figure 1 Stems of C. intybus.

Figure 2 T.S. of stem showing portion from surface to pith canal (5x)

Figure 3: T.S. of small stem (100 x). BCF: bundle cap fibres; Co: cortex; Ep: epidermis; OP: outer pith; PC: pith canal; Ph: phloem, X: xylem

Figure 4: T.S. of stem showing enlarged xylem and outer pith (10 x 2.5). MX: metaxylem; Pi: pith, PX: protoxylem.

a b

c d

Figure 5: Powder microscopy of C. intybus stems

a. Spiral vessel and fragment of cortex (40 x); b. yellow matter (40 x).

c. fibres (Fi) and vessel element (VE) (10 x 2.5); d. fibres (40 x).

Powder study of stem:

The stem powder exhibited fibres and vessel elements, fibres were long, thick walled, had narrow lumen and tapering ends. The fibres were 500 - 550 μm long. The vessel elements were narrow, long and cylindrical; they were 400 – 900 μm long and 30 – 40 μm wide. The lateral wall pits were minute and dot like, densely distributed on the walls (Figure 5).

Physico-chemical values:

The total ash, acid insoluble ash, water soluble ash and mean values of different solvent extractives have been evaluated in Table 1. Presence and absence of different phytoconstituents were detected in Table 2. Result of fluorescence analysis study was tabulated in Table 3. The foaming index estimated less than 100; successive extractives were calculated; quantitative estimation of bitterness value, swelling factor, total resin, total phenolic and flavonoid content were also study and was tabulated in Table 4.

Table 1: Percentage of loss on drying, ash and extractive value of C. intybus stem

S.No.	Parameters	*C.intybus* (mean^a S.D)
1	Loss on drying	3.78 ± 0.08
2	Total ash	2.8 ± 0.01
3	Water soluble ash	2.80 ± 0.01
4	Acid insoluble ash	0.02 ± 0.00
5	Water soluble extractive	15.07 ±0.19
6	Alcohol soluble extractive	11.08 ± 0.47

HPTLC fingerprints of C. intybus stem extracts:

Petroleum ether, chloroform, acetone and methanol successive extracts investigated by HPTLC for development of fingerprints. The chromatograms obtained after development in different solvent system followed by scanning at 254nm in absorbance mode depicted presence of number of substances in the extracts. Petroleum ether, chloroform, acetone and ethanol showed presence of 2,13,16,17 spots respectively, with different R_fvalues as given in Table 5.

Table 2: Preliminary phytochemical screening of C. intybus stem extracts

Extract	Alkaloids	Flavanoids	Phenol	Tannin	Saponin	Sterol	Carbohydrate	Terpenoids	Glycosides	Resins
Petroleum ether	-	-	-	-	-	+	-	-	-	-
Chloroform	-	-	-	-	-	-	-	-	-	-
Acetone	-	-	+	-	-	-	-	-	-	-
Ethanol	+	+	+	+	+	+	+	+	+	+
Methanol	+	+	+	+	+	+	+	+	+	+
Water	+	+	+	+	+	+	+	+	+	+

Table 3: Fluorescence analysis of C. intybus stems powder

S. No.	Chemical Treatment	Observations
		254 nm	366 nm
		Stem Powder
1	1N NaOH in methanol	Light Green	Brownish Green
2	1N NaOH in water	Light Yellow	Dark Green
3	50% HCl	Light Green	Blackish Green
4	50% HNO₃	Yellowish Green	Brown
5	50% H₂SO₄	Brownish Black	Dark Black
6	Hexane	Clear	Clear
7	Petroleum Ether	Green	Brown
8	Chloroform	White	Creamish White
9	Ethanol	Clear	Buff White
10	Drug as such	Creamish Yellow	Greenish Black

Table 4: Successive extractive values and other content of C. intybus stem powder

S. No.	Parameters	Values
1	Petroleum ether successive extractive value	0.52%w/w
2	Chloroform successive extractive value	0.44%w/w
3	Ethanol successive extractive value	5.68%w/w
4	Water successive extractive value	8.23%w/w
5	Loss on drying	3.82% w/w
6	Bitterness value	0
7.	Swelling index	8.56 ml
8.	ph value	8.01 (Acidic)
9.	Total phenolic content in alcoholic extract	0.100 mg/gm
10.	Total flavonolids content in alcoholic extract	3.450 mg/gm
11.	Total resin content	2.46 mg/gm
12.	Total sesquiterpene content	1.76 mg/gm

DISCUSSION:

Microscopy:

Microscopy shows normal microscopic features of stem.

Physicochemical parameters and phytochemical analysis:

Physicochemical parameters of C. intybus Linn stem were tabulated in respective sections. The shade dried stem was put through physicochemical analysis; no foreign matter was detected; total ash value, acid insoluble and water soluble ash was determined and result were tabulated. Extractive values, successive extractive values, swelling index, ph values, bitterness value, total phenolic, total flavonoids, total resin and total sesquiterpene content were also determined and tabulated. The preliminary phytochemical studies done for various extract prepare from stem powder which confirm the positive test for Alkaloids, Glycosides, Phenolics, Flavonoids and Resins.

HPTLC:

Alcohol extracts showed maximum number of peaks indicates large number of secondary metabolites in the extract.

Present investigation discloses the data generated may be handed down for determining correct identity and detection of adulterants as well.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 18.05.2021 Modified on 19.09.2021

Research J. Pharm. and Tech 2022; 15(9):4078-4082.

DOI: 10.52711/0974-360X.2022.00684

Extract	Solvent System	No.of Spots	R_f Values	Visualizing Agents
Petroleum ether extract of Stem	Toluene: Ethyl acetate: Glacial Acetic acid (9.5: 0.5: 0.2)	2	0.01, 0.26	Anisaldehyde in Sulphuric acid
Chloroform extract of Stem	Toluene: Ethyl acetate: Chloroform: Glacial Acetic acid (5.0:2.0:3.0:0.2)	13	0.00,0.06,0.09,0.20,0.22,0.35,0.45, 0.49, 0.54,0.61,0.68,0.78,0.85	Anisaldehyde in Sulphuric acid
Acetone extract of Stem	Toluene: Hexane: Glacial Acetic acid (6.0:4.0:0.1)	16	0.01,0.04,0.19,0.21,0.34,0.40,0.46,0.49,0.58,0.62,0.64, 0.73,0.76,0.78,0.86,0.88	Anisaldehyde in Sulphuric acid
Ethanol extract of Stem	Toluene: Ethyl acetate: Chloroform: Glacial Acetic acid (4.0:3.0:3.0:0.5)	17	0.04,0.08,0.16,0.20,0.25,0.27,0.30,0.33,0.35,0.38,0.43, 0.44,0.48,0.60,0.72,0.78,0.80	Anisaldehyde in Sulphuric acid