INTRODUCTION:

Natural products obtained from the plants are enriched source used as effective and prevailing drugs in order to treat diseases. In many countries, plant was found to be one of the significant source in medicine that have been used as popular folk medicines.[1] Preliminary standardization Methods are used to select the plant extracts for its medicinal properties to further proceed for pharmacological studies.[2] Modern medical science applies the knowledge of using natural products and its secondary metabolites for treating various diseases like cancer, inflammation, fever etc.,.[3,4,5,6,7]. To analyse the phytoconstituents present in the herbal extract, HPTLC fingerprints [8] was found to be one of the analytical tool. Gas Chromatography Mass Spectroscopy (GCMS) was used for the identification and quantification [9] of unknown compounds present in the extract that can be analysed by matching with reference spectra.

Recent research involves identification of phytoconstituents from natural source which have medical importance and isolation of those constituents for treating various diseases [10,11].

Alstonia scholaris belongs to the family Apocynaceae and it is a large evergreen tree up to 17 to 20 m in height about 110cm in diameter. Its bark is grayish brown, rough, abounding in bitter, white milky latex; leaves 4-7 in a whorl, coriaceous, whitish beneath, obovate or elliptic or oblong, obtuse acuminate, 30-60pairs of horizontal veins joining an intramarginal one; cymes peuncled or sessile, umbellately branches; flowers aromatic, greenish white, small greenish white numerous in umbellate panicles, corolla tube short, very strongly scented; fruit follicles, 30-60 cm long; seeds papillose with brownish hair at each end [12,13]

The present study was performed to investigate the high performance thin layer chromatography (HPTLC) fingerprinting analysis and gas chromatography mass spectrometry (GC-MS) of aqueous extract of Alstonia scholaris bark.

MATERIALS AND METHODS:

Plant Collection:

Alstonia scholaris were collected from Tripathi, Andhra Pradesh, India. It was identified and authenticated by Dr. Jayaraman, Plant Botanist, Thambaram, Chennai. The plant material (bark) were cleaned thoroughly with tap water, dried in the shade and coarsely powdered. The powdered material of the plant was macerated with distilled water for a minimum period of 48hrs. Then the extract was collected and evaporated to dryness under reduced pressure. The final residue obtained was subjected to HPTLC and GC MS analysis.

HPTLC fingerprint profile from aqueous extract of A. scholaris:

TLC conditions:

TLC plate consists of 20X10cm, precoated with silica gel 60 F254 TLC plates (E. Merck) of uniform thickness 0.2mm with aluminium sheet support. The spotting device used was CAMAG Linomat V Automatic Sample Spotter. The Syringe, 100 ml (from Hamilton), the developing chamber was a CAMAG glass twin trough chamber (20X10 cm). The Densitometer consists of a CAMAG TLC Scanner 3 linked to WINCATS software. Stationary phase: Silica Gel 60 F254, Mobile phase: n-butanol: water: acetic acid (7:2:1) [14].

HPTLC analysis:

The test solution was applied on TLC plate and the plate was developed in n-butanol: water: acetic acid (7:2:1) solvent system to a distance of 8cm. The plates were dried at room temperature in air. The plate was scanned densitometrically at 254nm using TLC scanner 3 and the plate was observed under UV light at 254 nm, 310 nm & 366nm using CAMAG REPROSTAR 3. The Rf values and colour of the resolved bands were noted [14].

GC-MS Analysis of Aqueous extract of Alstonia scholaris:

Gas Chromatograph:

A Shimadzu GC-2010 Plus gas chromatograph was equipped with a straight deactivated 2 mm direct injector liner and a 15m Alltech EC-5 column (250μ I.D., 0.25μ film thickness). A split injection was used for sample introduction and the split ratio was set to 10:1. The oven temperature program was programmed to start at 35°C, hold for 2minutes, then ramp at 20°C per minute to 450°C and hold for 5 minutes. The helium carrier gas was set to 2 ml/minute flow rate (constant flow mode) [15].

Mass Spectrum:

A Direct connection with capillary column metal quadupole mass filter prerod mass spectrometer operating in electron ionization (EI) mode with software GCMS solution ver. 2.6 was used for all analyses. Low-resolution mass spectra were acquired at a resolving power of 1000 (20% height definition) and scanning from m/z 25 to m/z 1000 at 0.3 seconds per scan with a 0.2 second inter-scan delay. High resolution mass spectra were acquired at a resolving power of 5000 (20% height definition) and scanning the magnet from m/z 65 to m/z 1000 at 1 second per scan.

RESULTS AND DISCUSSION:

High Performance Thin Layer Chromatography:

Black Spot present in the TLC plate derivatized with Vanillin and H₂SO₄ indicates the presence of glycosides. Absence of orange spot in the TLC plate derivatized with Dragendorff’s Reagent indicated the absence of alkaloids (Fig 1). HPTLC Fingerprinting analysis was performed by using CAMAG HPTLC Sanner III and bands were observed on the HPTLC plates (Figure 2,3). Rf values were calculated which is obtained from TLC (Table 1,2)

Fig 1: TLC Analysis of aqueous extract of Alstonia scholaris

Fig 2: Chromatogram of aqueous bark extract of A. scholaris at 254 nm.

Table 1: Chromatogram Data at 254nm

Peak	Start Rf	Start Height	Max Rf	Max Height	Max %	End Rf	End Height	Area	Area%	Assigned substance
1	0.14	3.4	0.17	12.4	1.53	0.19	4.9	240.3	0.58	unknown*
2	0.39	9.8	0.45	35.7	4.39	0.46	35	1108.7	2.66	unknown*
3	0.46	34.1	0.52	86.8	10.67	0.53	79	2892.6	6.94	unknown*
4	0.53	79.9	0.59	304.3	37.41	0.63	34.4	11908.7	28.59	unknown*
5	0.64	35.1	0.67	59	7.25	0.7	41.7	1994	4.79	unknown*
6	0.7	41.9	0.81	291.9	35.88	0.96	15.3	23112.5	55.49	unknown*
7	0.96	15.5	0.98	23.4	2.87	1	2	398.3	0.96	unknown*

Fig 3: Chromatogram of aqueous bark extract of A. scholaris at 366 nm.

Table 2: Chromatogram Data at 366 nm

Peak	Start Rf	Start Height	Max Rf	Max Height	Max %	End Rf	End Height	Area	Area%	Assigned substance
1	0.13	2.6	0.17	16.0	8.28	0.20	5.8	484.4	6.22	unknown*
2	0.25	3.9	0.26	16.4	8.49	0.28	1.1	239.6	3.07	unknown*
3	0.46	6.0	0.51	48.0	24.87	0.54	21.3	1481.0	19.00	unknown*
4	0.59	16.9	0.62	25.2	13.05	0.63	22.9	619.3	7.95	unknown*
5	0.73	29.0	0.80	87.5	45.30	0.86	32.5	4969.0	63.76	unknown*

GC-MS Analysis:

Identification of the components by GC-MS was matching their recorded spectra with the data bank mass spectra (Fig 4) of NIST library V 11 provided by the instruments software.

GC/MS metabolomics Database was used for the similarity search with retention index.

GC-MS profiling of aqueous extract of Alstonia Scholaris revealed the presence of 27 compounds (Table 3)such as L-Isoleucine, N-acetyl- Isoleucine, N-acetyl-, L-N-Acetyl-L-isoleucine N-Acetylisoleucine, beta.-d-Glucofuranosiduronic acid, methyl gamma.-lactone, Valeraldehyde, 2,2-dimethyl-, semicarbazone (1E)-2,2-Dimethylpentanal semicarbazone, D-Streptamine, O-2-amino-2-deoxy-.alpha.-D-glucopyranosyl-(14)-O-[O-2,6-diamino-2,6-dideoxy-.beta.-L-idopyranosyl-(13), alpha.-Ketostearic acid $$ 2-Oxooctadecanoic acid, 6. Citronellic acid, trimethylsilyl ester, 5-Chlorovaleric acid, nonyl ester Nonyl 5-chloropentanoate, 3-tert-Butyl-5-chloro-2-hydroxybenzophenone $$ (3-tert-Butyl-5-chloro-2-hydroxyphenyl)(phenyl)methanone, 3-Ethyl-3-methylheptane $$ Heptane, 3-ethyl-3-methyl, Allantoin $$ Urea, (2,5-dioxo-4-imidazolidinyl)- Allantol AVC/ Dienestrolcream Cordianine Glyoxyldiureid, aS-Triazine-3,5(2H,4H)-dione, 6-(dimethylamino)- 5-Dimethylamino-6-azauracil 6-(Dimethylamino)-as-triazine-3,5(2H, 4-(2-Piperidin-1-yl-ethyl)-pyridine 1-[2-(4-Pyridinyl)ethyl]piperazine, 4-(2-Piperidin-1-yl-ethyl)-pyridine 1-[2-(4-Pyridinyl) ethyl] piperazine, aS-Triazine-3,5(2H,4H)-dione, 6-(dimethylamino)- 5-Dimethylamino-6-azauracil 6-(Dimethylamino)-as-triazine-3,5(2H), dl-Threitol/1,2,3,4-Butanetetrol, Nanofin Piperidine, 2,6-dimethyl- 2,6-Lupetidine Lupetidin/Lupetidine/Naniopinum/Nanophin/ Nanophine, Isoquinoline, decahydro- Decahydroisoquinoline Perhydroisoquinoline Perhydroisoquinoline, Furazan-3-amine, 4-(hydroximino)(triazirinyl)methyl, N-dl-Alanylglycine/Glycine, N-DL-alanyl- [(2-Aminopropanoyl) amino]acetic acid 19. Glycerin 1,2,3-Propanetriol/ Glycerol/ Glycerine /Glyceritol/Glycyl alcohol / Glyrol / Glysanin /Osmoglyn, n-Propyl Nonyl Ether, Cedrene-V6, 2-Formylhistamine, Quinoline-5,8-dione-6-ol, 7-[[(4-cyclohexylbutyl)amino]methyl], Furazan-3-amine, 4-(hydroximino) (triazirinyl)methyl, Benzimidazol-2-amine, 1-[2-(1-piperidyl)ethyl]- 1-[2-(1-Piperidinyl)ethyl]-1H-benzimidazol-2-ylamine, l-Alanine, N-(m-anisoyl)-, heptadecyl ester, Piperidine, 3-methyl- 3-Pipecoline /beta.-Methylpiperidine /beta.-Pipecoline/ 3-Methylpiperidine.

Table 3: GC MS profile of Aqueous Extract of Alstonia Scholaris

Peak #	Phytocomponents	% Peak Area	Retention Time (Min)	Mol. Wt
1.	L-Isoleucine, N-acetyl- Isoleucine, N-acetyl-, L-N-Acetyl-L-isoleucine $$ N-Acetylisoleucine	32.25	5.498	173
2.	beta.-d-Glucofuranosiduronic acid, methyl, .gamma.-lactone	23.07	5.637	190
3.	Valeraldehyde, 2,2-dimethyl-, semicarbazone (1E)-2,2-Dimethylpentanal semicarbazone	0.79	6.566	171
4.	D-Streptamine, O-2-amino-2-deoxy-.alpha.-D-glucopyranosyl-(14)-O-[O-2,6-diamino-2,6-dideoxy-.beta.-L-idopyranosyl-(13)	1.43	6.633	615
5.	alpha.-Ketostearic acid $$ 2-Oxooctadecanoic acid	1.86	6.667	298
6.	Citronellic acid, trimethylsilyl ester	0.43	6.859	242
7.	5-Chlorovaleric acid, nonyl ester $$ Nonyl 5-chloropentanoate	0.49	8.908	262
8.	3-tert-Butyl-5-chloro-2-hydroxybenzophenone $$ (3-tert-Butyl-5-chloro-2-hydroxyphenyl)(phenyl)methanone	0.14	9.255	288
9.	3-Ethyl-3-methylheptane $$ Heptane, 3-ethyl-3-methyl	1.36	9.724	142
10.	Allantoin $$ Urea, (2,5-dioxo-4-imidazolidinyl)- Allantol $$ AVC/Dienestrolcream Cordianine $$ Glyoxyldiureid	0.88	9.917	158
11.	aS-Triazine-3,5(2H,4H)-dione, 6-(dimethylamino)- $$ 5-Dimethylamino-6-azauracil $$ 6-(Dimethylamino)-as-triazine-3,5(2H, 4-(2-Piperidin-1-yl-ethyl)-pyridine $$ 1-[2-(4-Pyridinyl)ethyl]piperazine	0.48	10.435	156
12.	4-(2-Piperidin-1-yl-ethyl)-pyridine $$ 1-[2-(4-Pyridinyl) ethyl]piperazine	0.14	10.549	191
13.	aS-Triazine-3,5(2H,4H)-dione, 6-(dimethylamino)- $$ 5-Dimethylamino-6-azauracil $$ 6-(Dimethylamino)-as-triazine-3,5(2H)	1.55	10.718	156
14.	dl-Threitol $$ 1,2,3,4-Butanetetrol	0.21	11.283	122
15.	Nanofin $$ Piperidine, 2,6-dimethyl- $$ 2,6-Lupetidine $$ Lupetidin $$ Lupetidine $$ Naniopinum $$ Nanophin $$ Nanophine	0.84	11.810	113
16.	Isoquinoline, decahydro- $$ Decahydroisoquinoline $$ Perhydroisoquinoline $$ Perhydroisoquinoline	1.52	12.669	139
17.	Furazan-3-amine, 4-(hydroximino)(triazirinyl)methyl	0.54	14.544	169
18.	N-dl-Alanylglycine $$ Glycine, N-DL-alanyl- $$ [(2-Aminopropanoyl) amino]acetic acid	2.93	15.304	146
19.	Glycerin $$ 1,2,3-Propanetriol $$ Glycerol $$ Glycerine $$ Glyceritol $$ Glycyl alcohol $$ Glyrol $$ Glysanin $$ Osmoglyn	0.68	15.409	92
20.	n-PROPYL NONYL ETHER	1.04	15.444	186
21.	Cedrene-V6	10.30	19.885	204
22.	2-Formylhistamine	4.36	22.258	139
23.	Quinoline-5,8-dione-6-ol, 7-[[(4-cyclohexylbutyl) amino]methyl]	1.13	23.631	342
24.	Furazan-3-amine, 4-(hydroximino)(triazirinyl)methyl	2.57	23.689	169
25.	Benzimidazol-2-amine, 1-[2-(1-piperidyl)ethyl]- $$ 1-[2-(1-Piperidinyl)ethyl]-1H-benzimidazol-2-ylamine	2.19	23.723	244
26.	l-Alanine, N-(m-anisoyl)-, heptadecyl ester	2.79	23.814	461
27.	Piperidine, 3-methyl- $$ 3-Pipecoline $$. beta.-Methylpiperidine $$ .beta.-Pipecoline $$ 3-Methylpiperidine	4.05	26.276	99

Fig 4: GC-MS Chromatogram (x-axis Retention time; y-axis % intensity/% abundance)

CONCLUSION:

Chromatographic Method is one of the analytical techniques to analyse the fingerprints obtained for the phytoconstituents present in the plant. It is used as important tool for identification of phytoconstituents from plants in order to represent chemical integrities. The present study is used to analyse the compounds present in the aqueous extract of A. scholaris bark by using HPTLC fingerprinting profile and GC-MS technique.

HPTLC profiling was performed in 254nm, 310nm and 366nm wavelength. Seven peaks were obtained in 254nm wavelength, 5peaks were obtained for 310nm wavelength and 5peaks were obtained for 366nm wavelength respectively. GC-MS analysis revealed the presence of 27 compounds in the aqueous extract of A. scholaris plant. Hence identification and isolation of these compounds will become significant in phyto pharmaceutical industry.

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Received on 05.05.2019 Modified on 01.07.2019

Research J. Pharm. and Tech. 2019; 12(12): 5706-5710.

DOI: 10.5958/0974-360X.2019.00987.9