Int J Pharm Pharm Sci, Vol 9, Issue 11, 218-224Original Article


A COMPARATIVE STUDY OF CHEMICAL COMPOSITION OF ACACIA SEYAL STEM, STEM WOOD AND STEM BARK DRY DISTILLATES USED BY SUDANESES WOMEN AS COSMETIC AND MEDICINE

IKRAM MOHAMED ELTAYEB1*, ITMAD AWAD ELHASSAN2, JIHAD HASAB ELRASOUL1, EIMAN SALAH ELDIN1

1Department of Pharmacognosy, Faculty of Pharmacy, University of Medical Sciences and Technology, Khartoum, Sudan, 2Pharmaceutical Industries Department, Industrial Research and Consultancy Centre, Ministry of Science and Technology, Khartoum, Sudan
Email: kramela_07@yahoo.com

Received: 03 Aug 2017 Revised and Accepted: 21 Sep 2017


ABSTRACT

Objective: The content and chemical composition of dry distillates of the stem, stem wood and stem bark of Acacia seyal were investigated. The distillates are fumigants of A. seyal locally known in Sudan as Dokhan and widely used for its cosmetic, aromatic and medicinal value for the treatment of candidiasis, genital yeast infection, urinary tract infection, diarrhoea, respiratory tract infection, skin infection and with potent, antioxidant and antimicrobial activities.

Methods: The dry distillates were prepared by dry distillation method from the Acacia seyal stem, stem wood and stem bark and investigated chemically by GC-MS analysis.

Results: The percentage yield of dry distillates (Dokhan) wer found to be 4.0 %, 4.8%, 1.4.1% v/w for stem, stem wood and stem bark respectively. GC-MS analysis revealed the presence of one hundred and twenty three constituents in the stem distillate with major constituents of solerone (7.27%), furfural (7.15%), catechol (7.11%), syringol (5.56%), allo-inositol (4.86%), mequinol (4.81%), furfuralcohol (3.35%), 3-methyl-1,2-cyclopentanedione (3.24%), phenol (2.73%), homovanillyl alcohol (2.56%) and 3-cresol (2.11%). the wood distillate show detection of eighty compounds, with main compounds: 1, 3-dimethyl-5-methoxypyrazol (10.61%), syringol (6.75%), furfuralcohol (5.24%), mequinol (4.49%), 1,2-anhydro-3,4,5,6-alloinositol (4.26%), 3-methyl-1,2-cyclopentanedione (3.42%), catechol (3.37%), 3-methoxycatechol (3.22%), homovanillyl alcohol (2.78%), homosyringic acid (2.40%), 3-cresol (2.18%), 3-methyl-2-cyclopentenone (2.44%) and 1,2-cyclopentanedione (2.03%). Sixty six compounds were detected in the distillate bark and the main compounds were found to be hexadecanoic (62.83%), catechol (3.38%), tetrapentacontane (3.18%), phenol (2.72%), mequinol (2.30%) and 2-ethylfurane (2.22%).

Conclusion: The result concludes that the medicinal Sudanese Dokhan have good potential as sources of different bioactive compounds and antioxidants.

Keywords: Chemical composition, Dry distillates, Acacia, seyal, Stem, Stem wood, Stem bark


INTRODUCTION

Acacia seyal (Fabaceae) is a small to a medium important tree in Sudan, locally known as Talih. The pleasantly fragrant fumigate of the stem or stem wood, known as Dokhan is widely used by Sudanese women as an aromatic and medicinal plant for cleanness and perfume purposes. Dokhan from the stem or stem wood was known for its potent antimicrobial and antioxidant activity]. The aromatic oil from the plant traditionally used by Sudanese women showed preservative and therapeutic properties in addition to its pleasant aroma [1-7].

The Fabaceae family produces more nitrogen-containing secondary metabolites than other plant families such as quinolizidine, pyrrolizidine, indolizidine, piperidine, pyridine, pyrrolidine and many other nitrogenous compounds [8]. Acacia genus was reported to have many secondary metabolites such as amines, alkaloids, cyanogenic glycosides, cyclitols, fatty acids and seed oils, fluoroacetate, amino acids, essential oils, diterpenes, phytosterol, triterpenes, saponins and hydrolyzable tannins. The most evident and best known are polysaccharides (gums) and complex phenolic substances (condensed tannins) [9]. A. seyal is highly nitrogen-fixing and moderately salt tolerant species and characterized by high content of proteins, phenols and flavonoids [10].

The present paper represents the first attempt to investigate and compare of the content and composition of the dry distillates (Dokhan) of A. seyal stem, stem wood and stem bark with potent antioxidant and antimicrobial activities [1] which is traditionally used by Sudanese women for cosmetic and therapeutic purposes mainly for the treatment of candidiasis, genital yeast infection, urinary tract infection, severe stomach cramps, diarrhea, vomiting, respiratory tract disease, cold and throat infection, wound and skin infection and toothache [1, 11].

MATERIALS AND METHODS

Plant materials collection and preparation

The stem of A. seyal was collected from Omdurman local market, Sudan, and then it was authenticated by taxonomist at the Department of Silviculture, Faculty of Forestry, University of Khartoum. The voucher specimen; IKR4, December-2015 was deposited at the Department of Pharmacognosy, Faculty of Pharmacy, University of Medical Science and Technology. The collected material was cleaned, dried and separated into two parts: one part of the plant material was chopped into small pieces and the other was separated into stem bark and stem wood and they were preserved separately for further studies.

Phytochemical screening

The phytochemical constituents of the plant material were detected using a standard procedure described by Farhat et al., et al., [12], Prashant et al., [13] and Mosa et al., [14]. The physiochemical parameters of the plant materials were detected according to the methods described by the WHO [(15].

Distillates preparation and determination of physiochemical properties

The stem, stem wood and stem bark distillates were prepared from the samples by dry distillation technique described by lewandowki and Milchert [16] with a minor modification. The percentage yield was determined in (v/w) and (w/w) with reference to the dried sample weight.

Solubility, specific gravity, refractive index, acid value, ester value and a saponification value of the prepared distillates were determined according to the British Pharmacopoeia, (2002) [17, 18].

GC-MS analysis

The gas chromatography-Mass spectrometry analysis was carried out on gas chromatograph coupled to a mass spectrometer (GC-MS QP). The temperature was programmed at 180 °C for 2 min. at a rate of 10c/min, and then increased to 289 °C for 1 min. at a rate of 15c/min and the dry distillate was injected with split injection mode. The identification of different components was achieved from their mass spectra, retention time (RT), compared to those in NIST library [19]. The fragmentation mode of major constituents was carried out and their m/z value was compared with those obtained in the Mass spectrometry spectra.

RESULTS

Phytochemical screening

The phytochemical screening of A. seyal stem, stem wood and stem bark (table 1) revealed the presence of tannuns, terpenoids, cardiac glycoside and reducing sugar in the three assessed materials. Flavonoids, alkaloids and steroids are detected in the stem and stem wood, whereas, they are absence in the stem bark. Saponnins are not detected in any of the three plant materials.

Percentage yields and physiochemical properties of the distillates

The oily dry distillates of A. seyal stem, stem wood and stem bark were found to be slightly different in their physiochemical properties. The physiochemical property of stem bark is more differ from that of stem and stem wood (table 2).

Table 1: Qualitative phytochemical screening of A. seyal stem, stem wood and stem bark

Phytochemical Result
Stem Wood Bark
Flavoniods +ve +ve -ve
Alkaloids +ve +ve -ve
Tannins +ve +ve +ve
Saponins -ve -ve -ve
Steroids +ve +ve -ve
Terpenoids +ve +ve +ve
Cardiac glycosides +ve +ve +ve
Reducing sugars +ve +ve +ve

+: presence,-: absence

Table 2: Percentage yields and physiochemical properties of dry distillates of A. seyal stem, wood and stem bark

Plant part Percentage yield Physiochemical
V/W W/W Sp. Gr. Ref. I Ac. V. Es. V Sap. V
Stem 15 % 83.8 % 0.95g 1.341 23.6 ml/g 34.8 ml/g 50.9 ml/g
Stem wood 14.1 % 48 % 0.95g 1.352 24.6 ml/g 34.8 ml/g 58.9 ml/g
Stem bark 13.6 % 53 % 0.95g 1.341 21.7 ml/g 16.1 ml/g 37.8 ml/g

V/M= volume/weight, W/W= weight/weight, Sp. Gr.= specific gravity, Ref. I = refractive index, Ac. V= acid value, Es. V. = ester value, Sap. V. = saponification value

GC-MS analysis

The results of GC-MS analysis of the distillates showed a slight difference between the chemical constituents of stem and stem wood distillates, whereas, the chemical constituent of stem bark distillate is more different from those two distillates. The analysis revealed the presence of one hundred and twenty-three, eighty and sixty-six constituents of the stem, stem wood and stem bark distillates respectively (table 3 and fig. 1, 2, 3, 4).

Table 3: GC-MS result of A. seyal stem, stem wood and stem bark dry distillates

Compound number R. T Compound name Area %
Stem Wood Bark
1 3.034 Propanal - 0.85 -
2 3.090 Propanoic acid - - 0.11
3 3.090 Butanoic acid - 0.85 0.24
4 3.090 Quinone - - 0.07
5 3.100 Pyridine,3-methyl - - 0.23
6 3.102 Furfural 7.15 0.22 -
7 3.236 Methylthiirane 0.13 - -
8 3.317 Furfuralcohol 3.35 5.24 1.47
9 3.449 Unknown 0.76 - -
10 3.581 Aniline - - 0.47
11 3.581 (S)-5-Hydroxymethyl-2-(5H)-furanone - 0.07 -
12 3.628 4-Methylhexanoic acid 0.12 - -
13 3.662 Avitrol - - 0.18
14 3.662 2,3-Pentanediol - 0.35 -
15 3.836 2-(Tetrahydrofuran-2-yloxy)-ethanol 0.11 0.52 -
16 3.915 dl-Threonine 0.64 - -
17 3.991 2-Ethylfurane 1.15 1.75 2.22
18 4.041 1, 3-Dimethyl-5-methoxypyrazol - 10.61 -
19 4.045 2-Acetylfuran 0.66 0.73 0.46
20 4.088 Dumasin 1.51 0.97 -
21 4.134 2-Cyclohexenol 0.13 - -
22 4.209 1,2-cyclopentanedione 1.28 2.03 0.15
23 4.245 β-Octalactone 0.12 0.38 -
24 4.332 3,5-Lutidine 0.14 - 0.10
25 4.334 Angelica lactone - 0.42 0.08
26 4.340 4-Methyl-2(5H)-furanone - - 0.10
27 4.350 L-Leucine,ethyl ester - 0.45 0.35
28 4.365 2-Cyclohexenone 0.09 0.16 0.79
29 4.427 6-Methyl-2,2-diphenyl-cyclohexanone 0.42 0.30 -
30 4.533 Suberone 0.23 - 0.42
31 4.640 Valerolactone 0.08 - -
32 4.694 Ethylene dipropionate 0.42 - -
33 4.740 γ-Ethoxybutyrolactone 0.16 - -
34 4.770 5-methylfurfural 0.46 - -
35 4.805 3-Methyl-2-cyclopentenone 1.03 2.44 1.07
36 4.874 Octanamide - 0.43 -
37 4.893 Hexanoic acid 0.08 1.42 -
38 4.936 Methyl 2-furoate 0.09 - 0.06
39 4.975 Phenol 2.73 1.79 2.72
40 5.088 Maleamic acid 0.12 0.72 -
41 5.137 2-Hydroxy-γbutyrolactone 0.44 - -
42 5.221 3,4-dimethylcyclopent-2-en-1-one 0.21 - 0.41
43 5.231 γ-Crotolactone - 1.50 0.28
44 5.289 Decylamide 0.82 - -
45 5.328 2,3-Dimethylpyrazine - - 0.04
46 5.329 3,5-Dimethyl-2(5H)-furanone 0.51 0.42 0.22
47 5.400 Tetrahydro, furfuryl alcohol 0.81 0.89 0.43
48 5.515 Unknown - - 0.27
49 5.515 Uridine 0.33 0.15
50 5.651 4-methyl-4-Hepten-3-ol 0.27 - -
51 5.724 3-Methyl-1,2-cyclopentanedione 3.24 3.42 0.58
52 5.864 2,3,4,5-Tetramethylfuran 0.12 - 0.06
53 5.865 Methyl 3-butenoate - - 0.17
54 5.890 Dihydro-2-methyl-3-furanone - - 0.13
55 5.905 3-Ethylpyridine - - 0.06
56 5.927 2,3-dimethyl-2-cyclopentenone 0.99 0.73 0.14
57 6.002 4-Methyl-2(5H)-furanone 0.22 0.34 -
58 6.103 Orthocresol 0.98 1.66 0.75
59 6.150 3-Ethyl-2-hydroxy-2-cyclopenten-1-one 0.30 0.85 0.36
60 6.226 2-Acetylpyrrole 0.09 - 0.06
61 6.281 2-Hexyltetrahydrofuran 0.45 - -
62 6.361 Heptanoic acid 0.21 0.12 -
63 6.414 3-Cresol 2.11 2.18 1.50
64 6.466 Thymine - 0.20 -
65 6.489 Octanal 0.32 - -
66 6.598 γ-tridecalactone 0.06 1.43 -
67 6.640 15-Amino-1-pentanol, N,O-diacetyl- 0.09 - -
68 6.707 Mequinol 4.81 4.49 2.30
69 6.783 2-Octenal 1.56 - -
70 6.876 4-Pyridinol 1.31 0.65
71 6.951 1-(2-furyl)pyrrole - 1.12 -
72 6.954 4-Octyne - - 0.19
73 6.960 2,6-Dimethylphenol 0.22 - -
74 7.064 Maltol 0.53 0.51 0.25
75 7.153 2-Isopropyl-2,5-dimethyl-cyclohexanone 0.95 - -
76 7.316 2-Ethyl-6-methylpyridine - - 0.12
77 7.382 Cyclohexane 0.23 - -
78 7.449 2-Octyne - 0.21 0.07
79 7.571 p-Xylenol 0.92 0.57 0.20
80 7.685 Diethyl azodicarboxylate 0.28 - -
81 7.758 Benzoic acid 0.12 - -
82 7.832 Octanoic acid 0.46 0.07 -
83 7.866 Meobal 0.56 - -
84 8.031 Phenylethyl Alcohol 0.08 - 0.07
85 8.088 Creosol 0.24 1.92 0.43
86 8.230 Tetrahydro-2-Furancarboxylic acid 0.92 0.44 -
87 8.332 Catechol 7.11 3.37 3.38
88 8.567 3-Hexenedioic acid, trans- 1.15 1.36 0.53
89 8.625 methyl salicylate 0.07 - -
90 8.783 Thiophene,2-propyl 0.24 - -
91 8.849 DL-Lactide 0.53 - -
92 8.945 Camphor 0.20 - -
93 9.065 Di-n-butyl diazene, - 1.70
94 9.123 Phorone 0.09 - -
95 9.172 Threitol, acetylated 0.09 - -
96 9.282 2-Methyl hydroquinone 0.57 0.71 -
97 9.356 3-Methoxycatechol 1.80 3.22 0.23
98 9.440 Hydroquinone 1.03 0.90 0.49
99 9.600 4-Ethylguaiacol 1.21 1.20 0.25
100 9.703 Orcinol 1.56 1.23 0.45
101 9.789 Solerone 7.27 - 0.12
102 9.876 Lactide 0.20 - -
103 10.121 4-Vinylguaiacol 0.68 0.75 0.58
104 10.249 4-Butoxy-1-butanol 0.23 - -
105 10.345 L-Glutamine - 0.59 -
106 10.484 4-Methylcatechol 0.87 - 0.56
107 10.558 Propylthiophene 0.19 - -
108 10.646 Syringol 5.56 6.75 0.83
109 10.678 2,3-Xylenol - 0.54 0.13
110 10.740 Eugenol 0.14 0.26 -
111 10.796 3,4-Dimethoxyphenol 032 - -
112 10.876 Dihydroeugenol 0.26 0.22 -
113 10.975 3,7,11,15-Tetramethyl-1-hexadecyn-3-ol 0.08 - -
114 11.052 4-Ethylcatechol 0.68 0.49 -
115 11.092 (E)-Dodec-2-enyl ethyl carbonate - 0.46 -
116 11.092 Unknown - - 0.50
117 11.233 2,3,5-Trimethylphenol 0.07 - -
118 11.285 4-Methoxythiophenol 0.14 - -
119 11.357 Vanillin 0.43 0.58 -
120 11.462 Isoeugenol 0.16 1.02 -
121 11.527 Sinenofuranol 0.18 - -
122 11.626 1-Decyne 0.27 0.72 -
123 11.965 Homovanillyl alcohol 2.56 2.78 0.08
124 12.027 Trans-Isoeugenol 0.91 - -
125 12.544 Allo-Inositol 4.86 0.15 -
126 13.017 5-tert-Butylpyrogallol 1.71 1.71 -
127 13.121 4-vinylsyringol 1.10 1.04 0.17
128 13.216 1,2-Anhydro-3,4,5,6-alloinositol 0.84 4.26 0.08
129 13.347 Dodecanoic acid 1.09 - -
130 13.557 4-vinyl-2,6-dimethoxyphenol 1.34 0.19 -
131 13.857 Amol 0.05 - -
132 13.935 Unknown 0.48 - -
133 14.106 3-Hydroxy-4-methoxycinnamic acid 0.40 - -
134 14.204 Senkyunolide 0.32 - -
135 14.544 3-sulfanylheptanal 0.21 - -
136 14.746 2,4-Dimethoxyphenol - 0.50 -
137 14.910 Methoxyeugenol 0.71 0.37 -
138 15.095 Syringaldehyde; 0.38 0.37 -
139 15195 (Z)-p-Methoxy-cinnamic acid - 1.32 -
140 15.390 3,4,5-Trimethoxyacetophenone 0.20 - -
141 15.599 Heptadecanoic acid 0.06 - -
142 15.713 Unknown 1.14 - -
143 15.980 Unknown 0.16 - -
144 16.225 Xanthoxylin 0.81 1.1 -
145 16.382 Tetradecanoic acid 0.66 - -
146 16.684 Cerulignol - 0.19 -
147 16.721 Homosyringic acid 1.80 2.40 -
148 16.806 Nonadecanol 0.05 - -
149 17.314 Unknown 0.50 - -
150 17.399 Unknown 0.19 - -
151 17.828 Cymol 0.27 - -
152 18.146 2,4-Dimethoxybenzyl alcohol 0.12 - -
153 18.284 Lidocaine 0.18 - -
154 18.337 Hexadecanoic acid 0.09 0.93 -
155 18.451 2-Imidazolidinethione - - 0.89
156 18.700 Unknown 1.57 - -
157 28.922 Plastoquinone 3 - 0.28 0.31
158 29.193 Tetrapentacontane - - 3.18
159 31.051 Hexadecanoic acid, hexadecyl ester - - 62.83
160 31.606 Unknown - - 2.31
Total - - 100 100 100

Fig. 1: GC-MS chromatogram of A. seyal stem dry distillate

Fig. 2: GC-MS chromatogram of A. seyal stem wood dry distillate

Fig. 3: GC-MS chromatogram of A. seyal stem bark dry distillate

Fig. 4: The common and major compounds in the dry distillates of A. seyal stem, stem wood and stem bark 1-59 = compounds number (table 3): 6,8,15,17,19,20,22,23,28,29,35,37,38,39,40,46,47,49,51,56,57,58,59,62,63,66,68,74,79,82,85,86,87,88,96,97,98,99,100,101, 103,108,110,119,120,122,123,125,126,127,128,130,137,138,144,145,146,147,148

DISCUSSION

The various phytochemical compounds found in the plants are known to have beneficial importance in industrial and medical sciences [20]. The preliminary phytochemical analysis of A. seyal stem, stem wood and stem bark (table 1) revealed the presence of tannins, terpenoids, cardiac glycoside and reducing sugar in all assessed plant materials. Flavonoids, alkaloids and steroids are detected in the stem and stem wood, whereas, they are absence in the stem bark. Saponnins are not detected in any of the three plant materials. The results showed that the stem and stem wood parts which are traditionally used by Sudanese women [1] have the same secondary metabolites, whereas, the stem bark which is slightly used showed different in secondary metabolites. The presence of flavonoids, alkaloids, tannins, steroid, terpeniods, cardiac glycosides and reducing sugars are compatible with the secondary metabolites of Fabaceae family [21]. These findings are reported for the first time about the secondary metabolites of A. seyal stem, wood and bark.

The diverse health benefit of the plants is usually known to be through the numerous phytochemicals extractions [22]. The dry distillation of A. seyal stem and stem wood is well known for its medicinal and cosmetic values among the Sudanese women [3]. The dry distillates of A. seyal stem, stem wood and stem bark were found to be oily pale brown to red in color, with specific aromatic odor and water soluble. The distillates yields (table 2) were; 15 %; 14.1%; 13.6% (v/w) and 83.87%; 48%; 53% (w/w) of the stem, stem wood and bark, respectively. Their specific gravity; refractive index; acid value; saponification value and ester value (table 2) were found to be (0.95g, 0.95, 0.95); (1.341, 1.352, 1.341); (23.6 ml/g, 24.6 ml/g, 21.7 ml/g); (50.9 ml/g, 58.9 ml/g, 37.8 ml/g) and (34.8 ml/g, 34.3 ml/g, 16.1 ml/g) in the stem, stem wood and bark respectively. The dry distillates of A. seyal stem and stem wood mainly used by Sudanese women [3] were found to be slightly different in their physiochemical properties, whereas, the physiochemical property of the slightly used distillate of stem bark is more different from that of stem and stem wood (table 2). These findings are reported for the first time about the percentage content and physiochemical properties of A. seyal dry distillates.

Regarding the GC-MS analysis, the difference between the chemical constituents of stem and stem wood distillates had been slightly, while, that of stem bark distillate was more difference from these two distillates (fig. 1, 2, 3, 4 and table 3). The identification of the constituents was done by the direct comparison of their retention times; peak areas; molecular weight; formula and fragmentation patterns according to the NIST library [19].

The analysis revealed the presence of many nitrogenous compounds which was compatible with the chemistry of the Fabaceae [20]. The dry distillate from the stem represented the presence of one hundred and twenty three constituents of which one hundred and seventeen of them have been identified, and the major constituents were found to be solerone (7.27%), furfural (7.15%), catechol (7.11%), syringol (5.56%), allo-inositol (4.86%), mequinol (4.81%), furfur alcohol (3.35%), 3-methyl-1,2-cyclopentanedione (3.24%), phenol (2.73%), homovanillyl alcohol (2.56%) and 3-cresol (2.11%). the stem wood dry distillate was composed of eighty compounds, all of them have been identified with major compounds of 1, 3-dimethyl-5-methoxypyrazol (10.61%), syringol (6.75%), furfur alcohol (5.24%), mequinol (4.49%), 1,2-anhydro-3,4,5,6-alloinositol (4.26%), 3-methyl-1,2-cyclopentanedione (3.42%), catechol (3.37%), 3-methoxycatechol (3.22%), homovanillyl alcohol (2.78%), homosyringic acid (2.40%), 3-cresol (2.18%), 3-methyl-2-cyclopentenone (2.44%) and 1,2-cyclopentanedione (2.03%). Sixty six compounds were detected in the dry distillate of the stem bark of which sixty-three have been identified and the main compounds were found to be hexadecanoic (62.83%), catechol (3.38%), tetrapentacontane (3.18%), phenol (2.72%), mequinol (2.30%) and 2-ethylfurane (2.22%). Some of the detected and identified compounds could be artefacts due to the process of dry distillation.

The main and common constituents found in the three distillates (fig. 4) were found to be proportional to their antioxidant and antimicrobial activity [1]. It is noteworthy to point out the relationship between these main constituents known of their antimicrobial and antioxidant properties and their content in the dry distillates. The different concentrations of these active constituents and their proportions in the three dry distillates explain clearly the potent antimicrobial activity of the stem [1] and its application in the fumigation traditions by Sudanese women. These findings about the chemical composition of A. seyal stem, stem wood and stem bark dry distillates are reported for the first time and adds to the current literature with regard to the presence of many nitrogenous compounds in the Fabaceae family [8] and abundance of essential oils, diterpenes, phytosterols and triterpenes in the Acacia genus.

CONCLUSION

These research conclude that the medicinal Sudanese Dokhan which is a fumigation process done by Sudanese women have good potential as sources of different bioactive compounds and antioxidants.

It is noteworthy to add that the chemical composition and content of detected components in the three dry distillates of the plant were proportional and comply with their uses in Sudanese traditional medicine. Sudanese women usually use stem wood for Dokhan, sometimes they use the stem and rarely the stem bark.

AUTHORS CONTRIBUTION

Design of the work was done by the first and second authors Dr. Ikram Mohamed Eltayeb and Dr. Itmad Awad Elhassan. Interpretation of data, writing and correction of the manuscript was done by the first author Dr. Ikram Mohamed Eltayeb. The experimental part of the work was done by Ms. Jihad Hasab Elrasoul and Ms. Eiman Salah Eldind.

CONFLICT OF INTERESTS

Declared none

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