|Year : 2013 | Volume
| Issue : 4 | Page : 227-233
Phytochemical analysis of ethanolic extract of Dichrostachys Cinerea W and Arn leaves by a thin layer chromatography, high performance thin layer chromatography and column chromatography
M Vijayalakshmi1, K Periyanayagam2, K Kavitha1, K Akilandeshwari1
1 Department of Pharmaceutical Technology, Anna University, Bharathidasan Institute of Technology, Tiruchirappalli, India
2 Department of Pharmacognosy, Madurai Medical College, Madurai, India
|Date of Web Publication||6-May-2014|
Department of Pharmaceutical Technology, Anna University, Bharathidasan Institute of Technology, Tiruchirappalli - 620 024, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Background: The leaves of Dichrostachys cinerea are used as laxative, diuretic, painkiller. It is also used in the treatment of gonorrhoea, boils, oedema, gout, veneral diseases and nasopharyngeal affections, etc.
Materials and Methods: The Phytochemical investigation of ethanolic extract of D. cinerea leaves were performed by standard chemical tests, thin layer chromatography (TLC) by using various solvent systems, and by high performance liquid chromatography (HPTLC). Two compounds were isolated by column chromatography and one of the compounds was identified by various spectral studies.
Result : Preliminary phytochemical screening of ethanolic extract of D. cinerea leaves showed the presence of Carbohydrates, proteins, Glycosides, Saponins, Tannins, Aminoacids and Terpenoids. The TLC and HPTLC fingerprint of ethanolic extract were studied and various fractions were isolated by column chromatography and one of the fraction contain β-amyrin glucoside which was confirmed by Infra Red[IR] Spectroscopy, 1 H-Nuclear Magnetic Resonance (NMR), C- 13 NMR and Mass spectroscopic (MS) studies.
Keywords: Column chromatography, Dichrostachys cinerea, high performance thin layer chromatography, leaves extract, phytochemical evaluation
|How to cite this article:|
Vijayalakshmi M, Periyanayagam K, Kavitha K, Akilandeshwari K. Phytochemical analysis of ethanolic extract of Dichrostachys Cinerea W and Arn leaves by a thin layer chromatography, high performance thin layer chromatography and column chromatography. Ancient Sci Life 2013;32:227-33
|How to cite this URL:|
Vijayalakshmi M, Periyanayagam K, Kavitha K, Akilandeshwari K. Phytochemical analysis of ethanolic extract of Dichrostachys Cinerea W and Arn leaves by a thin layer chromatography, high performance thin layer chromatography and column chromatography. Ancient Sci Life [serial online] 2013 [cited 2019 Nov 19];32:227-33. Available from: http://www.ancientscienceoflife.org/text.asp?2013/32/4/227/131978
| Introduction|| |
Dichrostachys cinerea W and Arn, which belongs to the family of mimosaceae is a perennial much - branched thorny shrub sometimes a small tree up to 2 m in height, bark light colored furrowed branchlets ending in spines, indigenous to North West - India, central India, Rajasthan, N. Australia. Leaflets are minute, sessile 12-20 pairs, close, linear or strap shaped oblique, subacute. The leaves of D. cinerea are laxative and used to treat gonorrhea and boils. It is also used as a fodder. Powder from leaves is used in the massage of fractures. The leaf also used as pain killer, in conditions like edema, gout, veneral diseases, swellings, and also naso-pharyngeal infections. It is also used as a diuretic. , Brushed young shoots which are astringent are used in the treatment of ophthalmia, rheumatism and urinary calculi.
This study elaborates the phytochemical characteristics of D. cinerea W and Arn by qualitative chemical tests, thin layer chromatography (TLC), high performance thin layer chromatography (HPTLC) and phytoconstituents were isolated by column chromatography. The ethanolic extract of leaves of D. cinerea W and Arn with coconut oil as carrier possessed significant antilice activity (P < 0.05). 
| Materials and Methods|| |
The plant specimens were collected from forest of Perambalur District, Tamil Nadu during August month and identified and authenticated by Botanist. A voucher specimen of leaves (PCG DC 005) has been deposited in the Department of Pharmacognosy herbarium Madurai Medical College, Madurai.
Preparation of plant extract
The healthy leaves of D. cinerea was collected during August 2007 at Perambalur, Tamil Nadu, washed and dried in the shade. The drug material was powdered sieved, (powder mesh size 60). The powder was defatted with petroleum ether by continuous hot percolation using soxhlet apparatus then extracted with ethanol (99%) for 6 h. , The solvent was removed under reduced pressure. The residue (2.75%) was semisolid dark green in color; viscous in consistency. The obtained extract was stored at 4°C until experiments.
The ethanolic extracts were subjected to preliminary Phytocthemical tests, TLC with various solvent system by using TLC Aluminium sheet precoated with silica gel 60 F254- Merck and HPTLC were performed using CAMAG HPTLC system. ,, All the solvents used for extraction and isolation was of GR grade and purchased from Merck.
HPTLC analysis of ethanolic extract of D. cinerea
The ethanolic extract of D. cinerea leaves were applied in a concentration of 10 μl using CAMAG Linomat IV sample applicator on Aluminum sheets precoated with silica gel 60 F 254 HPTLC plates of 0.2 mm thick, 5 cm × 20 cm, used as a stationary phase. The plates were developed in the mobile phase, hexane:ethyl acetate (7:3) for the ethanolic extract to a distance of 120 mm in CAMAG - Twin trough glass chamber. The track was scanned using CAMAG densitometer scanner II equipped with CAMAG software 1998 CATS 3.20", at a wavelength of 254 nm using deuterium lamp and the finger print profiles were recorded. 
Column chromatography of ethanolic extract of leaf of D. cinerea
The column was packed by wet packing and separation was started by eluting with solvent gradually with increasing order of polarity using hexane and ethyl acetate, Iso propyl alcohol. The entire fraction was collected separately pooled and solvent removed under reduced pressure using the rotary evaporator.  The fractionation of the compound was recorded in [Table 1].
Identification of isolated compound I: The compound 1 was obtained in 100% of ethyl acetate fraction (F-13). It gave single spot with terpenoid reagent. The compound purity were checked by TLC and further it was characterized by IR, NMR and Mass spectral studies. ,,,
| Results and Discussion|| |
Result of chemical tests indicate that the ethanolic extract of leaves of D. cinerea contains alkaloids, terpenoids, proteins, tannins, carbohydrates and no flavonoids. The results are shown in [Table 2].
|Table 2: Preliminary phytochemical screening for the ethanolic extract of leaf of D. cinerea|
Click here to view
TLC of ethanolic extract of D. cinerea showed the presence of terpenoids and sugar by using various solvent systems. This is shown in [Table 3].
HPTLC profile of ethanolic extract of D. cinerea leaves using Hexane - ethylacetate (7:3) showed nine peaks [Table 4], [Figure 1].
Isolation of compound was performed by column chromatography by using the various solvents of increasing order of polarity and 22 fractions were collected and TLC was performed for each fraction. The ethylacetate fraction (F-13) showed single spot in TLC and the solvent was removed by vacuum evaporator. The obtained solid compound purity was further checked by TLC and identified by 1H, C 13 NMR, Burker 500 MHz, DMSO d6 IR and mass spectroscopic techniques [Figure 2].
|Figure 1: High performance thin layer chromatography profile of ethanolic extract of Dichrostachys cinerea|
Click here to view
H 1 NMR and C 13 NMR of isolated compound I
C-1 36.69 H - 1 C-2 56.74 H - 2 1.37 (1H, m), C-3 38.78 H - 3, 1.58 (2H, m) C-4,38.78 H - 4-, C-5-23.08 H 5-1.78 (2H, m), C-6-77.79, H6-3.15 (1H, m), C-739.84, H7-, C-8 33.82 H - 81.52 (2H, m) C-9-51.06 H - 9-1.81 (1H, m) C-10-19.09 H - 10-1.79 (2H, m) C-11140.94 H - 11-, C-12-42.33 H - 12-, C-13-121.69 H - 135.18 (1H, m) C-14-23.08 H - 141.90 (2H, m) C-1531.89 H - 15-, C-16-25.34 H - 16-1.50 (2H, m) C-17-50.08 H - 17-2.50 (2H, m) C-18-25.34 H - 18-1.93 (2H, m) C-19-35.96 H - 19-1.47 (2 H, m) C-20-37.31 H - 20-1.51 (2H, m) C-21-31.80 H - 21-, C-22 45.62 H - 22 1.94 (2H, m) C-23 29.74 H - 23-0.83 (3H, s) C-2429.74 H - 240.84 (3H, s) C-2529.18 H - 25-0.89 (3H, s) C-26 19.58 H - 26 0.79 (3H, s) C-27
25.34 H - 27-0.90 (3H, s) C-28-19.41 H - 28-0.96 (3H, s) C-29-21.58 H - 29-0.99 (3H, s) C-30S21.41 H - 30-1.00 (3H, s) C-31-61.58 H - 31-3.49 (2H, m) C-32-77.39 H - 32-3.66 (1H, m) C-33-101.27 H - 33-4.86 (1H, m) C-34 73.94 H - 34-3.65 (1H, m) C - 35-77.25 H - 353.64 (1H, m) C - 36-70.58 H - 36-3.64 (1H, m) [Figure 3] and [Figure 4].
Mass spectrum (m/e) of isolated compound I: 679 (M + 1)
The various spectrum of isolated compound I are shown in [Figure 5]:
The chemical structure of isolated compound I was elucidated and discussed in the [Table 5] and a possible structure was given below: The β-amyrin glycoside is now reported for the first time in D. cinerea leaf [Figure 6].
| Conclusion|| |
Herbal drugs are an excellent source of many constituents. TLC and HPTLC fingerprint profile are important parameters of herbal drug standardization for the proper identification of medicinal plants. In this work, the complete phytochemical investigation of ethanolic extract of D. cinerea were studied by TLC, HPTLC finger print. The compound -1 was isolated by column chromatography and identified as β-amyrin glycoside it was reported first in the leaves of D. cinerea.
| References|| |
|1.||Anomymous. The Wealth of India. Vol. III. Reprint 2006. New Delhi: CSIR Publication; 1952. p. 56. |
|2.||Varier's PS. Indian Medicinal Plants, Compendium of 500 Species. Vol. 2. Kottakkal: Arya Vaidya Sala; 1993. p. 330-3. |
|3.||Vijayalakshmi M. In vitro antilice activity of Dichrostachys Cinerea (L.) Wight and Arn. Int J Pharm Tech Res 2010;2:2210-3. |
|4.||Mukherjee PK. Quality Control of Herbal Drugs - An Approach to Evaluation of Botanicals. 1 st ed. New Delhi: Business Horizons Pharmaceutical Publishers; 2002. p. 14-24. |
|5.||Kokate CK. Practical Pharmacognosy. 4 th ed. Delhi: Vallabha Prakashan; 1994. p. 117-9, 123-5. |
|6.||Harbone JB. Phytochemical Methods, a Guide to Modern Techniques of Plant Analysis. 3 rd ed. UK: Eswar Books; 1998. p. 7. |
|7.||Bobbit JM. Thin Layer Chromatography. 2 nd ed. New York: Verlogchemise Academy Press; 1966. |
|8.||Randerath K. Thin Layer Chromatography. 2 nd ed. New York: Verlogchemise Academy Press; 1966. |
|9.||Sethi PD. High Performance Thin Layer Chromatography Quantitative Analysis of Pharmaceutical Formulation . 1 st ed. New Delhi: CBS Publishers; 1996. |
|10.||Skoog DA, West DM, Haller FJ, Crouch SR. Fundamentals of Analytical Chemistry an Introduction to Analytical Separation. 8th ed.Bangalore: Eastern Press P. (Ltd.); 2004. p. 920-1. |
|11.||Kemp W. Organic Chemistry. 3 rd ed. London: MacMillan Press Ltd.; 1994. |
|12.||Dyer JR. Application of Absorption Spectroscopy of Organic Compounds. 8 th ed. New Delhi: Eastern Economic; 1991. p. 4-17, 22-32, 58-123. |
|13.||Finar IL. Organic Chemistry. 6 th ed. London: ELBS; 1975. p. 54-60. |
|14.||Furniss BS, Hannaford AJ, Smith PW, Tatchell AC. Vogels Textbook of Organic Chemistry. 4 th ed. New Delhi: Pearson Education Publishers; 1989. p. 1412-34. |
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]
|This article has been cited by|
||Cytotoxicity of Crude Extract and Isolated Constituents of the Dichrostachys cinerea Bark towards Multifactorial Drug-Resistant Cancer Cells
| ||Armelle T. Mbaveng,Francois Damen,James D. Simo Mpetga,Maurice D. Awouafack,Pierre Tane,Victor Kuete,Thomas Efferth |
| ||Evidence-Based Complementary and Alternative Medicine. 2019; 2019: 1 |
|[Pubmed] | [DOI]|
||Wound healing potential of selected Southern African medicinal plants: a review
| ||Constance Chingwaru,Tanja Bagar,Alfred Maroyi,Petrina T. Kapewangolo,Walter Chingwaru |
| ||Journal of Herbal Medicine. 2019; : 100263 |
|[Pubmed] | [DOI]|
||Bis (Isothiocyanatomethyl) Benzene, A Plant Derived Anti-Neoplastic Compound: Purified from Moringa Oleifera Leaf Extract
| ||Samrat Paul,Piyali Basak,Namrata Maity,Chayan Guha,Nandan Kumar Jana |
| ||Anti-Cancer Agents in Medicinal Chemistry. 2019; 19(5): 677 |
|[Pubmed] | [DOI]|
||Ethnomedicinal plant species commonly used to manage arthritis in North-West Nigeria
| ||T. Salihu,J.O. Olukunle,O.T. Adenubi,C. Mbaoji,M.H. Zarma |
| ||South African Journal of Botany. 2018; 118: 33 |
|[Pubmed] | [DOI]|
||Antibacterial activity of aqueous and methanol extracts of selected species used in livestock health management
| ||Clarice P. Mudzengi,Amon Murwira,Musa Tivapasi,Chrispen Murungweni,Joan V. Burumu,Tinyiko Halimani |
| ||Pharmaceutical Biology. 2017; 55(1): 1054 |
|[Pubmed] | [DOI]|