Keywords

HPTLC, Xylopia Aethiopica, Fingerprint

Introduction

Xylopia aethiopica is an evergreen, aromatic tree, of the annonaceae family that can grow up to 20m high. It is widely and abundantly available and native to the lowland rainforest and moist fringe forests in the savannah zones of Africa. The dried fruits of xylopia aethiopica (Grains of Selim, Africa pepper) is one of the most commonest and economically important indigenous versatile wonder nutritional and medicinal plant that has been in use extensively in recent time in the form of food supplements in traditional system of medicine and reputedly either alone or in polyherbal formulations as a remedy for treatment of various diseases. Included are: lowering of cholesterol or reduced heart disease-high blood pressure, heart problems and obesity; postpartum tonic to relieve pains and inflammation; heal after birth wounds, improve the immune system and enhance mood; terminate of unwanted pregnancy (obstetric potential); antimalarial, antifungal, antiviral and antimicrobial agent; treatment of gastrointestinal problems and other medical conditions, including anaemia, common cold, diabetes, corneal ulcers, the list is much longer. Though it has been suggested that xylopia aethiopica holds a place as an effective potential therapeutic agent against cancer [1][2] development and maintenance of reproduction functions in males [3][4][5][6][7] and females [8][9][10] as analgesic and anti-inflammatory substance [11][12] maintenance of blood pressure and haemorheological parameters [13][14] and antimicrobial [15], there is paucity of literature about specific biological markers to distinguish or that can be directly correlated with the acclaimed therapeutic activities.

Preliminary comparative phytochemical analysis of extractive phytocompounds of Xylopia aethiopica based on local methods of dispensing finished medicinal products and practices [16][17] assessed in accordance with the methods for medical plant materials of World Health Organization (18) showed variations and significant differences in phytochemical constituents. It was suggested that hydro-methanolic plant extract yielded higher percentage extractive value of phytocompounds and could provide better information regarding various types of biomarker or fingerprint compounds in tissues and consequently, may be useful for quantitative and qualitative phytochemical profile in identifying characteristics of medicinal plants [16][17]. The use of HPTLC profile to identify characteristics and bioactive principles of herbal materials of medical importance is well recognized [19]. Therefore, an attempt has been made to identify the appropriate biomarker(s) in quality assessment of xylopia aethiopica using hydromethanol chloroform fractionation based on their HPTLC fingerprint profile [19].

Materials and Methods

HPTLC finger printing profiling; HPTLC [20] isolation and characterization of the bioactive constituent of pure isolates of xylopia aethiopica was carried out at the Department of Pure and Applied Chemistry, Usmanu Danfodiyo University, Sokoto, Nigeria on chloroform fraction of hydromethanolic plant extract [16][17]. Chloroform fractionation of plant extracts has been suggested to potentiate higher extractive values of phytocompounds; and as well, can give a better result compared with butanol, ethyl acetate or Nhexane extractive solvents [21][22].

Briefly, forty-five (45) gram of hydromethanolic plant extract was fractionated successfully with 100mls of the solvent on 200 ml boiling water until the various components at each stage were exhaustively extracted and the various fractions obtained were concentrated to dryness in-vacuo at 40oC taking cognizance that elevated temperatures of extraction can affect the stability of major phytocompounds [22]

Results and Discussion

Based on the results of the preliminary comparative phytochemical studies of plant products based on the local methods of dispensing finished medicinal products and practices [16][17], chloroform fraction [21][22] of hydro-methanolic xylopia aethiopica extract was quantified by HPTLC technique for isolation, identification and characterization of physio-chemicals present in the plant material. In this study, HPTLC analysis revealed the presence of about eighty-one (81) phytotherapeutic chemicals in xylopia aethiopica (Table 1-5) which constituted phytosteroid (γ-Sitosterol, Clionasterol 22,23- Dihydroporiferasterol, 1% ), carbohydrate (1.3%), alkaloids (22.2%), flavonoids (31%) terpenes (44.5%) , and in addition, some fatty acids ( unsaturated, 54.3% and saturated 22.2%) and others (protein/amino acids etc., 23.5%). The study revealed that xylopia aethiopica contains both medicinal and non-medicinal properties suggesting that it could be administered at an appropriate therapeutic dose when necessary but with caution. Figure 1-5 and Table 1-5 show typical HPTLC fingerprint profile / chromatogram / peak purity spectra or biomarkers of hydromethanolic chloroform fraction of xylopia aethiopica responsible for various therapeutic activities documented (for review see Nworah 2013[21])

Figure 1: HPTLC finger print profile /chromatogram of hydro-methanolic extract of X. aethiopica under

Table 1

Corresponding biomarkers

S/No Names R.T. (min) Peak height Corr. Area Corr. % max % of total
1 1,4:3,6-Dianhydro-α-d-glucopyranose 6.918 14607 66552 15.36 1.52
2 2,3,5-Trimethylanizole 8.817 21986 78649 18.16 1.796
3 Bicyclo[2.2.1]heptan-2-ol,4,7,7-trimethyl- 9.452 22316 75381 17.4 1.721
4 2-Hexenal,2-ethyl- 9.672 22472 85253 19.68 1.947
5 1H-Imidazole-4-methanol,5-methyl- 9.785 34543 122133 28.19 2.789
6 1,2-Cyclohexanediol,1-methyl-4-(1-methylethenyl)- 10.283 15640 66941 15.45 1.529
7 4(axial)-n-Propyl-trans-3-oxabicyclo[4.4.0]decane 11.108 21623 95413 22.02 2.179
8 2-(2-Methyl-propenyl)-cyclohexanone 11.34 30787 128008 29.55 2.923
9 Bicyclo[2.2.1]heptan-2-ol,1,3,3-trimethyl- 11.731 27285 100629 23.23 2.298
10 7-Oxabicyclo[4.1.0]heptan-2-one,6-Methyl-3-(1-methylethyl)- 12.557 14868 81589 18.83 1.863
11 2,3-Dioxabicyclo[2.2.2]oct-5-ene,1-methyl-4-(1-methylethyl)- 13.15 73023 330517 76.3 7.548
12 Bicyclo(3.1.0)hexane-6menthanol,2-hydroxy-1,4,4-trimethyl 13.803 41986 161850 37.36 3.696
13 Bicyclo[3.1.1]hept-2-ene-2-methanol,6,6-dimethyl- 14.034 45004 160421 37.03 3.663
14 3,7-Dimethyl-6-nonen-1-ol acetate 14.195 19677 82374 19.01 1.881
15 1-Methyl-4-ethylaminocytosine 14.42 21910 75363 17.4 1.721
16 4-Methoxy-2,2,5-trimethylcyclopent-4-ene-1,3-dione 15.874 17403 79061 18.25 1.805
17 2-Cyclohexen-1-one,3-(hydroxymethyl)-6-(1-methylethyl)- 16.112 47611 192061 44.33 4.386
18 Cyclohexanone,2-(hydroxymethylene)-3-methyl-6-(1-methyethyl)- 16.533 16855 75066 17.33 1.714
19 (1,5,5,8-Tetramethyl-bicyclo[4.2.1]non-9-yl)acetic acid 21.786 38696 140684 32.47 3.213
20 Spiro[2.5]octane,3,3-dimethyl-2-(1-buten-3-on-1-yl)- 22.593 18778 96826 22.35 2.211
21 1H-Cycloprop[e]azulen-7-ol,decahydro-1,1,7-trimethyl-4-methylene-,[1ar-(1aα,4aα,7β,7aβ,7bα)]- 22.747 33736 118326 27.31 2.702
22 2,21 -Dichlorostilbene 23.537 15638 120949 27.92 2.762
23 Isoaromadendrene epoxide 23.881 31495 127903 29.52 2.921
24 2-Naphthalenemethanol,decahydro-α,α,4a-trimethyl-8-methylene-,[2R-(2 α,4a α,8aβ)]- 24.16 33064 132808 30.66 3.033
25 Cyclopentaneacetic acid,3-oxo-2-(2-pentynyl)-,methyl ester 24.397 24992 149842 34.59 3.422
26 1-Hexadecanol 25.062 20526 88772 20.49 2.027
27 Platambin 25.448 94917 433208 100 9.893
28 7,9-Di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione 25.935 20720 91860 21.2 2.098
29 Platambin 26.131 20146 91490 21.12 2.089
30 n-Hexadecanoic acid 26.896 36006 174144 40.2 3.977
31 1,2-Benzenedicarboxylic acid,butyloctyl ester 27.092 23926 94634 21.84 2.161
32 2H-Bisoxireno[2,3:8,8a]azuleno[4,5-b]furan-7(3aH)-one,octahydro-3a,8c-dimethyl-6-methylene- 28 16411 76089 17.56 1.738
33 5H-Benzol[b]pyran-8-ol,2,3,3,3,8a-pentamethyl-6,7,8,8a-tetrahydro- 29.092 22585 81556 18.83 1.862
34 Acetic acid, chloro-, octadecyl ester 30.125 27386 113462 26.19 2.591
35 Cyclotetrasiloxane,octamethyl- 32.078 14569 77840 17.97 1.778
36 Octasiloxane,1,1,3,3,5,5,7,7,9,9,11,11,13,13,15,15-hexadecamethyl- 40.138 26784 111418 25.72 2.544

Figure 2: HPTLC finger print profile /chromatogram of hydro-ŵethaŶolic extract of X. aethiopica (γ-Sitosterol)

Table 2

Corresponding biomarkers

S/No Names R.T. (min) Peak height Corr. Area Corr. % max % of total
1 γ-Sitosterol 45.07 168784 1857390 100 100

Figure 3: HPTLC finger print profile /chromatogram of hydro-methanolic extract of X. aethiopica

Table 3

Corresponding biomarkers

S/No Names R.T. (min) Peak height Corr. Area Corr. % max % of total
1 4(axial)-n-Propyl-trans-3-oxabicyclo[4.4.0]decane 9.577 17005 53517 13.87 2.233
2 Bicyclo(3.1.1)heptane-2,3-diol,2,6,6-trimethyl- 9.66 23391 75158 19.48 3.136
3 1,1-Di(prop-2-enyl)-1-silacyclobutane 9.779 25327 80287 20.81 3.35
4 4-Ethyl-trans-3-oxabicyclo[4,4,0]decane 9.903 22509 86679 22.47 3.616
5 1,2-Cyclohexanediol,1-methyl-4-(1-methylethenyl)- 10.271 16535 52165 13.52 2.176
6 4(axial)-n-Propyl-trans-3-oxabicyclo[4.4.0]decane 11.102 112261 385807 100 16.097
7 2-(2-Methyl-propenyl)-cyclohexanone 11.334 46351 161150 41.77 6.724
8 9-Ethylbicyclo(3.3.1)nonan-9-ol 11.447 81168 273632 70.92 11.417
9 Bicyclo(3.1.0)hexane-6-menthanol,2-hydroxy-1,4,4-trimethyl 11.726 22261 75356 19.53 3.144
10 Benzeneethanol,4-hydroxy- 12.165 13688 50900 13.19 2.124
11 1-Cyclohexyl-2-methyl-prop-2-en-1-one 12.527 20294 145618 37.74 6.076
12 2,3-Dioxabicyclo[2.2.2]oct-5-ene,1-methyl-4-(1-methylethyl)- 13.138 49967 204370 52.97 8.527
13 2-Cyclopenten-1-one,4-hydroxy-3-methyl-2-(2-propenyl)- 13.779 15438 84722 21.96 3.535
14 1,3-Cyclohexadiene-1-methanol;4-(1-methylethyl)- 14.04 27645 98004 25.4 4.089
15 2,2,7-Trimethyl-octa-5,6-die-3-one 14.189 13007 52556 13.62 2.193
16 1-Decalone(cis-trans) 14.29 19286 64719 16.77 2.7
17 Bicyclo(3.1.0)hexane-6-menthanol,2-hydroxy-1,4,4-trimethyl 14.432 69088 268127 69.5 11.187
18 4,5,9-Trihydroxy-dodeca-1,11-diene 14.563 10286 43513 11.28 1.815
19 2-Cyclohexen-1-one,3-(hydroxymethyl)-6-(1-methylethyl)- 16.118 37207 140492 36.42 5.862

Figure 4: HPTLC finger print profile /chromatogram of hydro-methanolic extract of X. aethiopica

Table 4

Corresponding biomarkers

S/No Names R.T. (min) Peak height Corr. Area Corr. % max % of total
1 Bis(hexahydro-7a-methyl-3-oxo-3H-pyrrolizin-5-yl)ether 11.316 12145 43457 17.61 4.255
2 2,4-Pentanedione,1,1,1,5,5,5-hexafluoro- 11.678 10719 30088 12.19 2.946
3 1H-Pyrrole-2-carboxylic acid,5-ethyl-,ethyl ester 13.5 5835 19143 7.76 1.874
4 2-Ethoxy-3-methylpyrazine 14.901 7187 30857 12.5 3.021
5 Benzeneethanamine,4-amino-N-methyl-N-(2-phenylethyl)- 20.913 5497 36936 14.96 3.616
6 N1,N111-Ethanediylidenebis(isonicotinohydrazide) 22.908 4676 20472 8.29 2.004
7 Tetradecane,5-methyl- 23.501 19129 119644 48.47 11.713
8 Pentadecanoic acid,14-methyl-,methyl ester 24.682 32389 174378 70.65 17.072
9 1-Methyl-4-[nitromethyl]-4-piperidinol 27.145 5312 22812 9.24 2.233
10 Dodecane,2,6,10-trimethyl 28 13771 73729 29.87 7.218
11 Bicyclo[10.1.0]tridec-1-ene 31.36 9866 44534 18.04 4.36
12 9-Octadecanoic acid (Z)-,2-hydroxyethyl ester 31.591 47559 246836 100 24.166
13 Methyl 16-methyl-heptadecanoate 32.458 13913 57022 23.1 5.583
14 Oxadiazon 34.297 19904 78988 32 7.733
15 Naphthalene,1,11-(1,4-butanediyl)bis- 34.778 6171 22538 9.13 2.207

Figure 5: HPTLC finger print profile /chromatogram of hydro-methanolic chloroform fraction of X. aethiopica

Table 5

Corresponding biomarkers

S/No Names R.T. (min) Peak height Corr. Area Corr. % max % of total
1 2-Ethyl-4-methylanisole 8.883 8582 21867 28.34 6.061
2 4(axial)-n-Propyl-trans-3-oxabicyclo[4.4.0]decane 11.31 11995 35303 45.76 9.713
3 Cis,trans-4-n-Propyl-3-oxabicyclo[4.4.0]decane 11.678 7134 23592 30.58 6.491
4 3,4-Dihydroxy-5-methoxybenzaldehyde 13.494 10133 32009 41.49 8.807
5 Pentanedioic acid,(2,4-di-t-butylphenyl)mono-ester 15.103 2971 8719 11.3 2.399
6 Thioallophanicacid,N-(m-toluoyl)-,S-methyl ester 19.626 3697 13194 17.1 3.63
7 7-Acetyl-2-hydroxy-2-methyl-5-isopropylbicyclo[4.3.0]nonane 23.026 9003 33012 42.79 9.083
8 Tert-Butyl 2-(2-amino-4-methyl-5-methoxyphenyl)acetate 25.395 3952 15285 19.81 4.206
9 Benzothiazole,2-methyl- 25.703 4660 15282 19.81 4.205
10 3-(1-Phenyl-ethoxy)-3H-isobenzofuran-1-one 25.97 4348 8559 11.09 2.355
11 Platambin 27.14 21654 77150 100 21.227
12 2(1H)-Pyrimidinone,5-chloro-4,6-diphenyl- 39.538 3969 12082 15.66 3.324
13 4-Amino-2-methyl-5,6-trimethylenepyrimidine 43.687 7461 34374 44.55 9.458
14 3-(Acridin-9-ylamino)-propionic acid methyl ester 49.296 4681 14297 18.53 3.934
15 Trimethyl[4-(1,1,3,3, tetramethylbutyl) phenoxy]silane 54.104 4351 10707 13.88 2.946
16 Silicic acid,diethylbis (trimethylsilyl) ester 55.172 3704 8019 10.39 2.206

Acknowledgment

The authors are grateful to Dr. Faruq Umar Zaki of the Department of Pure and Applied Chemistry, Usmanu Danfodiyo University, Sokoto for technical assistance.