Pharmacognostical Standardization, Chromatograhic and Spectral Analysis of Methanolic Extract of Echinops echinatus Linn. Roots and Fractions

Department of Pharmacognosy, Faculty of Pharmacy, The Islamia University of Bahawalpur, Bahawalpur, Pakistan. University College of Pharmacy, University of the Punjab, Lahore, Pakistan. Department of Pharmacognosy, Faculty of Pharmacy and Pharmaceutical Scienes, University of Karachi, Karachi, Pakistan. Department of Pharmaceutics, Faculty of Pharmacy, The Islamia University of Bahawalpur, Bahawalpur, Pakistan. 5 Department of Pharmacognosy, Faculty of Pharmacy Federal Urdu University of Arts, Science and Technology, Karachi, Pakistan. University College of Conventional Medicine, The Islamia University of Bahawalpur, Bahawalpur, Pakistan. Department of Pharmacy, BBS University, Lyari, Karachi, Pakistan. 8 Islam College of Pharmacy, Sialkot, Pakistan. Colistan Institute of Desert Studies, The Islamia University of Bahawalpur, Bahawalpur, Pakistan. Department of A& E, Bahawal Victoria Hospital, Bahawalpur, Pakistan.


Reagents and Equipments
All chemicals, solvents and reagents used were of analytical grade and were purchased from Merck and Sigma-Aldrich. Methanol

Macroscopic evaluation
Macroscopic evaluation of the root was performed as per standard procedures [6,7].

Powder microscopy
Binocular microscope was used to observe various cells. The so observed microscopic structures were identified by comparing with the standard work [8,9].

Fluorescence Analysis
E.echinatus dried powder of the roots was studied after treating with water, NaOH, HCl, HNO 3 , H 2 SO 4 , picric acid, acetic acid, methanol and ethanol, using ordinary and UV.

Physico-chemical Analysis
Percentage of moisture content or loss on drying, total ash value, acid insoluble and water soluble ash value, extractive values and swelling and foaming index of E. echinatus (powder) were examined [6].

Extraction and Fractionation
Plants were well washed to remove all the external dirt and unwanted material. 1 kg of powdered material was macerated in 2 L of methanol for 72 h at room temperature. The soaked material was filtered three times for coarse filtration. The filtrate was filtered through Whatman Grade-1filter paper. The filtrate was evaporated under controlled pressure and temperature (-760mm Hg at 45-50 o C) on the rotary evaporator. The filtered extract was made free from solvent. A dark brownish green gummy extract was placed in oven, and percentage yield was calculated. Moreover, successive solvent extraction was used as previously described by Tiwari et al. [10]. Extracts were dried, weighed, labelled and placed at 4°C. Methanolic crude extract and two fractions so obtained were named as follows: EME = Methanolic crude extract; EPE= Petroleum ether fraction; ECE= Chloroform fraction.

Column Chromatography
A glass column of 55x 4.5 cm. was used. Chloroform was used for packing the column. 12 g of EME was adsorbed on 10 g of silica gel. The column was first run with chloroform then the polarity of the system was changed [12]. 20 x 5 cm glass plates by applying 30 g silica gel were used for this purpose [13,24] 3.9 Ultraviolet Visible Absorption EME was analyzed in UV-Visible range between 200-800 nm.

Infra-Red Spectroscopy
IR spectra of EME were scanned over the range from 4000-400 cm-1.

Macroscopic evaluation
E. echinatus is an erect, 1-3 ft. high low growing much branched herb with white cottony stems. The fresh leaves are simple and sessile, 3-5 inch long, pinnatifid with lobes ending in spines up to 20 mm long; undersurface white tomentose. Flower heads are 1 flowered, numerous, aggregated into a white ball, 2.2 -2.4 cm in diameter, subtented by stout spines; and flowers are tubular with narrow lobes. Achens are 1/6 inch long, densely silky, surrounded by the connate hardened inner involucral bracts [1].

Powder microscopy (root)
Fine powder revealed that root contains annular vessels showing pits and spiral vessels with parenchyma cells. Reddish brown Cork cells are also present.

Physico-chemical analysis
Percentage of moisture content or loss on drying, total ash value, acid insoluble and water soluble ash value, extractive values and swelling and foaming index are shown in Table 6.

Fluorescence analysis
The results of fluorescence analysis are shown in Table 7.

Column chromatography
Results of pooled fraction of EME by column chromatography are shown in Table 5.

Thin layer chromatography
Results of TLC of EME, EPE, ECE are shown in Table 2,3,4.

Ultraviolet visible absorption
Results of UV-Visible spectra of the five compounds (Ee1 to Ee 5) isolated from fractions of E.echanitus are shown in Fig. 1,3,5,7 and 9.

Infra-Red spectroscopy (IR)
Results of IR spectra of of the five compounds (Ee1 to Ee 5) isolated from fractions of E. echanitus are depicted in Fig. 2,4,6,8 and 10.

DISCUSSION
Despite different sophisticated modern research techniques and tools, macroscopic and microscopic methods are still the simplest, reliable, precise and economical methods for correct identity of the plant source. As per WHO [6], the macroscopic and microscopic description is first criterion for identity and purity of material. Organoleptic standardization is a qualitative test based on the study of macroscopical characters. In current study, research was conducted on roots of a medicinal plant E. echinatus. The microscopic studies of the powder showed different histological structures. Different stains differentiate different cells on the basis of their chemical nature [14,15,16]. Fluorescence is an important phenomenon for purity and quality of the sample and their chemical constituents [17,18]. Powder, qualitative and fluorescence standards provide valuable information for authentication. Preliminary phytochemical screening showed the presence of various phytoconstituents in the plant which may have diversified therapeutic value for curing ailments; for example, saponins, flavonoids, tannins, alkaloids and phenols have anti-inflammatory activities whereas flavonoids, alkaloids, tannins and phenols have hypoglycemic and liver protective potential [19]. Water soluble extractive value indicates sugars, inorganic compounds and acids; and alcohol soluble extractive value shows polar components like flavonoids, steroids and phenols etc. To prevent chemical decomposition and microbial contamination low moisture content is needed. Due to presence of mucilage swelling index was in range of 5 ml; while foaming index was less than 100, i.e., insignificant. By estimating ash value quality and also the purity of powdered sample can be determined. Total ash determines that how much care is required in preparation of a crude drug [20]. Ash value is also signifies adulterant added for adulteration [21]. Ash value usually represents inorganic salts which are present in the drug sample [6]. Total ash value indicates the inorganic composition or earthy materials presence [19].
Ten pooled fractions were obtained from EME based on TLC analysis. Five major compounds (Ee1 to Ee 5) were isolated and purified from EME by silica gel column and TLC (Tables 1 and  2). Compound Ee-1 isolated from the first column fraction, showed (Fig. 1) absorption maximum in UV as: λmax=213 nm and λmax=269 nm (Fig. 1). The strong absorption at λmax=213 nm was probably due to presence of open chain diene; while at λmax=269 nm was due to substituted ring [22]. IR spectrum of Ee-1 compound (Fig. 2) showed abroad intermolecular hydrogen bonding around 3437 cm-1 due to -OH showing presence of some alcoholic/phenolioc hydroxyl group. The band at 2078 cm-1 showed stretching vibration present in alkane. The presence and the number of -CH3=CH2 and ≡CH groups in the molecule were further indicated by the peaks in the finger print region at 1500, 1200 and 1000 cm-1. A strong peak at 1637 cm-1 indicated a coupled C=C-C=C conjugated diene (alkene) with aromatic ring [23,22,19].
Compound Ee-2, a light yellow oily compound, isolated from the second column fraction. The strong absorption at λmax=217 nm (Fig. 3) was probably due to ‫ﬣ‬ to ‫*ﬣ‬ transition [22,19]. IR spectrum of the compound (Fig. 4) showed absorption maximum at 3370 (medium) and 1459 (sharp) cm-1. A band at 3370 cm-1 is absorption frequency of triple bond showed alkyne, i.e., ≡C-H or -C≡C-H. Presence of a medium band at 2937 cm-1 showed C-H aliphatic asymmetric stretch.
Compound Ee-3 was a light yellow compound and chromatographically pure. The strong absorption at λmax=215 nm (Fig. 5) was probably due to ‫ﬣ‬ to ‫*ﬣ‬ transition, the compound may be α,β conjugated six-ring or acyclic ketone; while at λmax=275 nm is the positive identification of a ketone or aldehyde carbonyl group [22,13]. IR spectrum around 3419 nm (Fig.  6) emphasizes the stretching vibration of -OH with intermolecular H-bonded at OH. Two bands at 2924 and 2853 cm-1showed the presence of single bonds due to C-H stretching; or these may be saturated C-H (-CH3) and C-C in the form of 2 or 3 bonds [23,22,19].
Compound Ee-4 was a dark yellow compound and chromatographically pure. The strong absorption at λmax=207 nm (Fig. 7) was probably due to ‫ﬣ‬ to ‫*ﬣ‬ transition, which suggested that the compound may be α, β unsatured ketone or aldehyde [22,19]. IR spectrum of Ee-4 (Fig. 8) showed a band at 3387 cm-1 due to alkyne, i.e., ≡C-H or -C≡C-H. Presence of two bands at 2943 and 2881 cm-1 showed single bonds due to C-H stretching; or these may be saturated C-H (-CH3) and C-C in the form of two or three bands.  Compound Ee-5 was a dark yellow oily compound and chromatographically pure. The strong absorption at λmax=237 nm ( Fig. 9) was probably due to ‫ﬣ‬ to ‫*ﬣ‬ transition, the compound may be an acyclic diene with 2-alkyl group, one each on α and β position; while at λmax=275 nm was due to disubstituted, benzene rings and is the positive identification of a ketone or aldehyde carbonyl group, it gives rise to yellow colour of the compound [23,22,19]. IR spectrum of the compound Ee-5 (Fig. 10) showed absorption maximum at 3409 (medium) 2925 (sharp) and 1636 (sharp) cm-1. IR spectrum of Ee-5 showed a broad intermolecular hydrogen bonding around 3409cm-1 due to -OH showing some alcoholic/phenolioc hydroxyl group. A strong peak at 1636 cm-1 indicated a coupled C=C-C=C conjugated diene (alkene) with aromatic ring; it may be α,β unsaturated carbonyl compounds, usually much weaker than C=O band [23,22,19]. A band at 1378 cm-1 showed C-H bend for -CH3 symmetrical deformation; while another band at 1272 cm-1 was for -CH3 group stretch. All the five compounds Ee 1-Ee 5 contain -OH, -COOH, or ketonic group and a double bond with conjugated diene system in their molecules.  Foaming index ≤ 100 cm 7 Extractive value in water 14% 8 Extractive value in ethanol 13%

CONCLUSION
Standardized pharmacognostic evaluation for this plant has yet not been much reported in literature. The roots powder subjected for macroscopic, microscopic pharmacognostic analysis provides important information which may be helpful in the authentication of the sample and also to check adulteration for quality control of raw material. The pharmacognostic parameters observed in present study, being reported for the first time may be helpful for standardization and preparation of the crude drug's formulation and inclusion in various pharmacopoeias to be utilized as a potential therapeutic agent for treating various diseases.
The current observation may be helpful to differentiate this species from other species of family Asteraceae. UV and IR spectra may be useful for spectral analysis of the plant.

CONSENT
It's not applicable.

ETHICAL APPROVAL
It's not applicable.