1. INTRODUCTION:

The term chromatography refers to a number of highly efficient techniques for the separation of a wide variety of substances ranging from simple ions to biopolymers^1,. All methods of chromatography basically aim to separate two or more substances.² The separation is carried out, by distributing the component mixtures between a fixed stationary and a moving mobile phase³.

At the end of 50’s thin layer chromatography (TLC) was introduced to the laboratory as a rapid technique for simple qualitative analysis⁴. In this capacity the method is still widely used. In the middle of 60’s with the development of efficient desitometers⁵, TLC found acceptance as a quantitative analysis method⁶. A further step forward was the introduction of high performance plate materials⁷. The high performance thin layer chromatography (HPTLC) is a major advancement of TLC principal requiring shorter time and better resolution. Now a day it is regarded as a most useful technique for qualitative and quantitative analysis.⁸ TLC densitometry is a method for the standardization of crude medicinal plant material⁹. Results obtained from a number of medicinal plants studied in the laboratory show that in most cases¹⁰. The TLC densitometry is a more convenient method than high performance liquid chromatography (HPLC) and gas liquid chromatography (GLC). The prepurification steps essential to the application of HPLC and GLC are considerably simplified or can be avoided¹¹. Moreover the chromatograms obtained during quantitative analysis are useful both for extract characterization on an overall fingerprint and for the detection of the possible adulteration or degradation of the drugs¹². In order to ensure adequate therapeutic efficacy of herbal preparations it is necessary that the herbs be characterized and standardized for their active chemical constituents¹³. With the advent of sophisticated modern analytical techniques particularly GC, HPLC and HPTLC it is now possible to test and control the quality of the herbal drugs^14,15. It is thus desirable that methods using such instrumental techniques be developed for evaluation and standardization of cruces herbal drugs for their chemical constituents¹⁶. Modern HPTLC is comparable to HPLC results in terms of sensitivity accuracy, precision etc.¹⁷

2. EXPERIMENTAL WORK:

2.1 Thin Layer Chromatography of formulations:

Thin layer chromatographic studies of formulations and authentic drugs were performed in various solvent systems.

a) Preparation of extract:

One gram of each powder of root and leaf accurately weighed was taken and macerated in 20 ml of methanol for 3 hours with occasional shaking. It was filtered through whatsmann filter paper and residue was washed with small quantity of methanol. Finally volume of filtrate was made upto 20ml with methanol.

b)Preparation of thin layer chromatographic plates:

Slurry of Silica gel was prepared by mixing approximately 30 gm of Silica gel G in 75ml of distilled water and triturated uniformly in a glass pestle mortar.The clean and dry glass plates were uniformly coated with slurry using a glass rod. The plates were allowed to dry at room temp. and then activated at 120 degree centigrade in a oven for 30 min.

c) Preparation of solvent systems (Mobile Phase):

The solvent system consisting of various solvents of different polarity were prepared by mixing and shaking the solvent in a conical flask. The solvent system was kept aside for 15 min. In case the solvents were not miscible, they were separated in a separating funnel and upper or lower layer of mobile phase was used as per requirement. The following mobile phase was prepared: n-Butanol :Acetic acid : Water (4: 1: 5)

d)Preparation of detecting reagents:

· Vanillin in sulphuric Acid: 0.5 gmof vanillin was dissolved in 100 ml mixture of sulphuric acid :Ethanol (40 : 10). After spraying, the plates were heated at 120 degree centigrade until maximum colour intensity of spot reached.

· Ferric chloride solution : Five gram of ferric chloride was dissolved in 0.5 N hydrochloric acid and made up the volume upto 100ml.

· Iodine vapours : The plates were placed in a dense atmosphere of Iodine vapours for 5 minute. The excess of iodine evaporated on standing in air.

e) Chromatographic procedure:

The solvent system was poured to a depth of 1.5 cm in a rectangular chromatographic glass chamber of 25x25x6 cm. The chamber was lined with a piece of whatman filter paper to ensure adequate equilibrium. The spots of extract of formulations and drugs were applied on a silica get G plate with the help of capillary tube. The distance between two spots was kept 2.0 cm. The coloured spots visible on the plate were marked and the Rf value of each separated component was calculated using the following formula.

Distance travelled by the component

Rf = -----------------------------------------------

Distance travelled by the solvent front

Table 2.1: Rf value of each separated component

S. No.

Solvent System

Solvent front

Detecting agent

Rf value

n-Butanol:Acetic acid: Water(4:1:5)

10.5 cm

Visible light

0.57

0.77

n-Butanol:Acetic acid: Water(4:1:5)

10.5 cm

U.V Light

0.57

0.77

2.2 High Performance Thin Layer Chromatography of Formulations;

· Sample : Barleria prionitis

· Marker constituents: Beta-sitosterol, Gallic acid and Saponins

· Preparation of sample :Fresh leaves and roots were dried under shade and coarsely powdered. The powdered mass was defatted with petroleum ether and followed by extraction with alcohol(95%).

· Sample of whole plant powder was also prepared using the above method.

· HPTLC Plates

The precoated and preactivated TLC plates (E.Merck No. 5548) of silica gel 60F with the support of aluminium sheets having thickness of 200 um and size 20x20 cm were cut into smaller size plates according to required dimensions.

a) Preparation of solvent systems (Mobile Phase)

b) The various components of solvent system were measured separately and placed in the conical flask and shaken thoroughly. The solvent systems were set aside for 15 minutes. Every time a fresh solvent system was prepared.

The following mobile phases were prepared for HPTLC analysis of various formulations

Toluene : Ethyl acetate : Formic acid (10:3:1)

Toluene : Ethylacetate : Formic acid (7:3:0.5)

Toluene : Ethyl acetate : Diethylamine (90:10:5)

Chloroform: Glacial acetic acid: Methanol: Water (6.4:3.2:1.2:0.8)

Ethyl acetate: Formic acid:Glacial acetic acid(10:1.1:1.1:2.6)

c) Chromatographic procedure:

Ten ml of freshly prepared mobile phase consisting of specific solvents was poured in a 10x20 cm twin through glass chamber. The extract was applied on the plate using Camag Linomat IV Sample applicator) The linomat sprayed extract samples in the form of bands. The length of band was 6.0 mm and a distance of 8mm was kept between two bands. The plates were dried at room temperature.

Table 2.2 HPTLC of the whole plant sample in comparison with the Barleria prionitis in the monograph.

S. No.	Rf value of whole plant	Rf value of B.prionitis in monograph (Ayur. Pharmacopoeia)
1.	0.09	0.18
2.	0.34	0.43
3.	0.40	0.57
4.	0.49	0.77
6.	0.57	0.91
7.	0.65	0.94
8.	0.73
9.	0.80

Fig 2.1 After Iodination

Fig 2.2 Image @ 254 nm

Fig 2.3 Image @ 366 nm

Table 2.3 HPTLC of Leaf ext. , root ext., and standard beta sitosterol.

S. No.	Leaf ext.		Root ext.		Beta-sitosterol
S. No.	Rf	%area	Rf	%area	Rf	%area
1)	0.02	5.05	0.02	8.66	0.49	4.82
2)	0.06	2.92	0.10	3.03	0.58	95.18
3)	0.23	5.57	0.14	0.74
4)	0.29	1.93	0.23	7.93
5)	0.32	3.22	0.30	7.15
6)	0.40	12.10	0.36	4.20
7)	0.58	13.61	0.50	7.78
8)	0.63	34.74	0.56	22.68
9)	0.76	19.39	0.66	27.32
10)	0.86	0.20	0.74	7.99
11)	0.87	0.22	0.90	0.87
12)	0.93	1.04	0.93	1.63

Solvent system: Toluene: Ethyl acetate: Formic acid (10:3:1)

FOR β-SITOSTEROL:

Fig 2.4 Image @ 254 nm

Fig 2.5 Image @ 366 nm

Fig 2.6

U1 : Leaf; U3 : Root; U4 : Whole plant; S1 : β-Sitosterol standard

Beta Sitosterol:

Fig 2.7 Leaf 10 ul

Fig 2.8 Root 10 ul

Fig 2.9 Standard Beta Sitosterol

Table 2.4 HPTLC of Leaf extract Root extract and standard Gallic acid.

Leaf extract Root extract Gallic acid

S. No	Rf value	%area	Rf value	%area	Rf value	%area
1)	0.00	28.32	0.01	35.15	0.01	5.27
2)	0.17	4.51	0.09	6.11	0.08	51.99
3)	0.19	3.67	0.19	16.53	0.18	9.74
4)	0.27	5.36	0.29	19.97	0.38	11.96
5)	0.32	12.24	0.53	17.96	0.42	14.09
6)	0.47	4.14	0.93	4.28	0.53	3.50
7)	0.55	9.84			0.59	2.97
8)	0.63	3.42			0.70	0.48
9)	0.67	1.60
10)	0.70	3.38
11)	0.75	5.48
12)	0.79	5.86
13)	0.88	9.99
14)	0.92	2.19

Solvent system: Toluene: Ethyl acetate: Formic acid (7:3:0.5)

Tannins:

Fig 2.13 Leaf 10 ul

Fig 2.14 Root 10 ul

Fig 2.15 Standard gallic acid

Table 2.5- HPTLC of Leaf extract Root extract and standard saponin

S. No.	Leaf extract		Root extract		Saponins
1.	Rf value	%area	Rf value	%area	Rf value	%area
2.	o.oo	5.17	0.00	6.52	0.12	100
3.	o.06	2.54	0.04	8.41
4.	0.09	19.31	0.09	5.88
5.	0.20	3.33	0.14	18.96
6.	0.27	3.69	0.21	18.17
7.	0.30	1.23	0.27	8.29
8.	0.33	2.55	0.36	1.08
9.	0.46	27.02	0.45	14.46
10.	0.63	27.66	0.61	14.84
11.	0.74	2.27	0.70	2.44
12.	0.79	5.23	0.77	0.95

Solvent system: Chloroform: glacial acetic acid: methanol : water (6.4:3.2:1.2:0.8)

Fig 2.16 Image @ 254 nm Fig 2.17 Image @ 366 nm Fig 2.18 Anchrom

Fig 2.19 Leaf 10 ul

Fig 2.20 Root 10 ul

Fig 2.21 Standard Saponin

Table 2.6: Chemical Components in the samples of leaf and root

Sample	Beta-sitosterol	Tannins	Saponins
.Root	0.006%	0.023%	0.07%
Leaf	0.0121%	0.045%	0.78%

3. RESULTS AND DISCUSSION:

The seperation of compounds on silica gel requires a solvent system. Mixtures of two or three solvents produce a satisfactory seperation. The criterion for good seperation is Rf value and compactness of the spots on the plate.

Initially, the alcoholic extract of roots and leaves extract was subjected to thin layer chromatography. The solvent system used was as per the monograph in the Ayurvedic Pharmacopoeia Vol-III i.e. n-butanol : acetic acid : water (4:1:5). But, it did not give a clear resolution. Rf value calculated are given in table 5.1. Therefore the extracts were subjected to HPTLC.

The qualitative analysis of the samples of the root and leaf (alcoholic extract) was carried out earlier and this showed the presence of saponins, tanins and flavanoids, but alkaloids were found absent in both the samples (Wagner 1984).

Fingerprinting analysis was carried of alcoholic extract, of the whole plant by HPTLC.By using the solvent system. n-butanol : acetic acid : water (4:1:5). (Ayurvedic Pharmacopoeia Vol-III) separation at Rf values 0.09, 0.14, 0.18, 0.36, 0.41, 0.49, 0.57, 0.64. was observed (table 2.2) . 0.18, 0.41and 0.57 were the Rf values (table 5.2) of the samples which matched with the Rf value of Barleria prionitis as given in the monograph (Ayurvedic Pharmacopoeia, Vol-III) .This may be due to the fact that plants obtained from different regions show a major variation in the herbal ingredients present even in the same country (Mukherjee, 2002).Absence of Alkaloids in the samples in the present study can be attributed to this fact.

HPTLC of the alcoholic extract of both roots and leaves were also carried out using the solvent system and method as given for whole plant in Ayurvedic Pharmacopoeia (Vol. III). Common spots of roots, leaves and whole plant were observed at Rf- 0.36, 0.57 and 0.64. (table-2.2).

The alcoholic extract of both roots and leaves taken for the present study were then subjected to HPTLC for specific groups of compounds i.e. alkaloids, tannins, beta-sitosterol and saponins.

The analysis of the alcoholic extracts, for alkaloids, using toluene: ethylacetate: diethylamine (7:2:1) as the solvent system did not show any fluorescence of blue or blue green colour which is normally shown by the alkaloids. This further confirmed the absence of alkaloids. Hence it is reported absent in present studies.

Toluene: Ethylacetate: Formic acid (10:3:1) was used as the solvent system for the analysis of beta-sitosterols. The samples showed quenching at U.V 365 nm and further spraying with Anisaldehyde confirmed the presence of beta-sitosterol. A standard of beta-sitosterol (sigma) was subjected to HPTLC studies simultaneously. The Rf- value 0.58 in the leaf sample and 0.56 in the root sample showed the presence of beta-sitosterol on comparing with the Rf-value of the standard beta-sitosterol which was found to be 0.58 (table 2.3).

Maximum area in the densitogram was also seen at this Rf value and the percentage of the beta-sitosterol in root and leaf was calculated using the formula:

Sample area weight of standard

---------------X ----------------------- X 100

Standard area weight of sample

0.006% in the roots and 0.0121% in leaf was observed. (Table-2.6)

Toluene: Ethylacetate: Formic acid (7:3:0.5) was used as the solvent system for estimation of tannins in both the samples used for the present studies.

Both the samples showed quenching at U.V wavelength 254nm and 365nm. Gallic acid was run simultaneously as standard for quantitative estimation. Presence of Rf- 0.0, 00.19, 0.55, and 0.7 in the leaf sample coincided with the Rf-value 0.01, 0.18, 0.53, and 0.70 of gallic acid so, the presence of tannins in reference with the gallic acid (standard) was confirmed. The Rf-value 0.01, 0.19, 0.53 of the root sample confirmed the presence of tannins in the root sample on comparing it with the Rf values-0.01, 0.18, and0.53. of the standard (Table 2.4).

On quantitative estimation 0.045% tannins in leaf and 0.023% tannins in root were observed (Table 2.6).

Chloroform: glacial acetic acid: methanol : water (6.4:3.2:1.2:0.8) was used as solvent system for thr HPTLC of saponins. Normally saponins are not deflectable by exposure to toluene: ethylacetate:formic acid (7:3:0.5) solvent system at 254nm and 366nm except for glycyrrhetic acid or glycyyrrhizin (Wagner 1984). Quenching was observed at 610nm. Anisaldehyde was used as the spray reagent. The Rf-value of 0.14 of root and 0.09 of leaf confirmed the presence of saponins (Table 2.5). On quantitative estimation by running a standard simultaneously, 0.78% saponins in leaf and 0.07% saponins in root were observed (Table 2.6).

Ethyl acetate: Formic acid: Glacial acetic acid: Water (10:1.1:1.1:2.6) was used as the solvent system for estimation of flavanoids. The samples used in the present study showed quenching at UV 254 nm nd 366nm. Blue fast reagent was used as the spray reagent and this indicated the presence of flavanoids. Standard for flavanoids could not be made available, hence quantitative estimation was not carried out.

4. CONCLUSION:

Chloroform: glacial acetic acid: methanol : water (6.4:3.2:1.2:0.8) was used as solvent system for thr HPTLC of saponins. Normally saponins are not deflectable by exposure to toluene:ethylacetate:formic acid (7:3:0.5) solvent system at 254nm and 366nm except for glycyrrhetic acid or glycyrrhizin. The samples showed quenching at U.V 365 nm and further spraying with Anisaldehyde confirmed the presence of beta-sitosterol. On chromatographic analysis of the alcoholic leaf and alcoholic root, it was observed that the leaf had a higher percentage of beta-sitosterol, tannins and saponnins as compared to root sample. For estimation of flavanoids. The samples used in the present study showed quenching at UV 254 nm and 366nm.

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Received on 14.03.2019 Modified on 17.04.2019

Research J. Pharm. and Tech 2019; 12(8):3679-3686.

DOI: 10.5958/0974-360X.2019.00628.0