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RESEARCH ARTICLE 39
*1
Department of Chemistry and Industrial Chemistry, College of Applied and Industrial Sciences, University of Bahri, Sudan.
2
Department of Chemistry, Faculty of Sciences, Sudan University of Sciences and Technology, Sudan.
3
Department of Botany, College of Sciences, Cairo University, Egypt.
4
College of Pharmacy, Khartoum University, Sudan
(Received July 09, 2013; Accepted September 20, 2013)
Powdered air-dried leaves (1.0 Kg) of Withania somnifera Preliminary Phytochemical screening of the Withania
were ground in a Warring Blender to a fine powder and somnifera
extracted with chloroform to remove chlorophyll, resinous and Crude Methanolic Extracts
waxy material. The ground material was extracted with 85% The methanolic extracts were tested for the presence of active
aqueous methanol and the slurry was allowed to stand at room constituents such as flavonoids, steroids, terpenoids, alkaloids,
temperature for 24 hours with occasional stirring. The solvent- saponins, and tannins. The following standard procedures were
containing extract was then decanted and filtered by vacuum used:
filtration. The extraction of the ground leaves were further
repeated twice with the same solvent and twice with 50% Flavonoids
aqueous methanol [13].The four filtrates from each extraction i. To about (3 ml) of the methanolic extract few drops of
was combined and the excess solvent was evaporated under methanolic aluminum chloride were added. Formation of a yellowish
reduced pressure at 40C using a rotary evaporator to give colour indicates the presence of flavonoids [15].
crude extracts, (53g ) of a solid mass and then kept into a glass
ii. To about 3ml of the methanolic few drops of aqueous potassium
container to use.
hydroxide solution were added. A dark yellow colour is taken as a
positive test for flavonoid compounds.
Micro-organisms collection and maintenance
The microorganisms used in the study: Bacillus subtilis, iii. Shinoda Test: Small pieces of Magnesium ribbon followed by
Escherichia coli, Neisseria gonorrhoeae, Pseudomonas few drops of concentrated hydrochloric acid were added to a small
aeruginosa and Staphylococcus aureus. were obtained from amount of methanolic extract of the plant material. Formation of a
stock culture in the Department of Botany, University of crimson red colour indicates the presence of flavones in glycosidic
Cairo, Cairo City, Egypt. The organisms were stored on agar linkage [16].
slant in McCartney bottles and kept in the refrigerator, prior to
subculture. Steroids and Terpenoids
(40 ml) aliquot of the methanolic extract was evaporated to
Preparation of standard test organisms dryness on a water bath and the cooled residue was stirred with
One ml aliquots of 24 hours broth cultures of test organisms petroleum ether to remove most of the colouring matter. The
(Bacillus subtilis, Staphylococcus aureus, Escherichia coli, residue was extracted with (20 ml) chloroform. The chloroform
Pseudomonas aeruginosa and Neisseria gonorrhoeae) were solution was dehydrated over anhydrous sodium sulphate.
aseptically distributed onto nutrient agar slopes and incubated
(5 ml) portion of the solution was mixed with (0.5 ml) acetic
at 37 C for 24 hours. The bacterial growth was harvested and
anhydride, followed by two drops of concentrated sulphuric acid.
washed off with sterile normal saline, to produce a suspension
A green colour was observed indicating the presence of steroids
containing about 108- 109 colony forming units per ml. The
and no pink-violet colour was observed indicating the absence of
suspension was stored in the refrigerator at 4 C till used. Serial
terpenoids [17].
dilution of stock suspension, were made in sterile normal
saline, and 0.02 ml volumes of the appropriate dilution were Alkaloids
transferred onto the surface of dried nutrient agar and the drop About (30 ml) aliquot of the methanolic extract was evaporated to
was left to dry, and then incubated at 37 C for 24 hours [14]. dryness on a water bath. (5 ml) of 2N hydrochloric acid were
added and the solution was heated with stirring in a water bath for
Testing for antibacterial activity 10 minutes. The mixture was cooled and filtered. (2 ml) of the
To determine the activity of methanolic extract and pure filtrate, were added to few drops of Mayer reagent (Potassium
component of Withania somnifera against the five standard mercuric iodide solution). A precipitate was observed indicating
organisms. The cup-plate agar was adopted with some minor the presence of alkaloids[18].
modification. (2 ml) Of the standard bacteria stock suspension
were mixed with 200 ml of sterile molten nutrient agar which Saponins
was maintained at 45 C. (20 ml) Aliquots of incubated agar 20 ml of water was added to 0.25 g of the extract in 100 ml beaker
were distributed into sterile Petri dishes. The agar was left to and boiled, filtered and then the filtrates used for the tests:
settle and each plate was cut and agar discs were removed. Froth Test: 5 ml of the filtrate was diluted with 20 ml of water and
Alternated cups were filled with 0.1 ml sample of each of the shaken vigorously. A stable froth (foam) up on standing indicates
extract (pure component) and allowed to diffuse at room the presence of saponins [19].
temperature for two hours. The Petri dishes were then
incubated in the up right position at 37 C for 18 hours. After Tannins
incubation, the diameter of the resultant growth inhibition To about (25 ml) of the methanolic extract was evaporated to
zones were measured, and averaged. dryness on a water bath and the residue was extracted with n-
hexane and filtered. The hexane- insoluble portion was stirred with
(10 ml) of hot saline solution (0.9% w/v of sodium chloride and
freshly prepared distilled water).
J OURNAL OF FOREST PRODUCTS & INDUSTRIES, 2013, 2(5), 39-45 ISSN:23254513(PRINT) ISSN 2325 - 453X (ONLINE )
41
The mixture was cooled and filtered and the volume adjusted totreated with few drops of ferric chloride solution, a blue colour was
(10 ml) with more saline solution. (5 ml) of this solution wasformed indicating the presence of tannins [20].
Isolation of flavonoids with water as an eluted solvent to give two major sub fractions,
The dry extract was loaded on a polyamide 6S column then each of them was separately fractionated on a Sephadex
chromatography (80 x 3 cm). The column was eluted with LH-20 to yield pure compound W3 (27 mg) and compound W4
water, and then water -Methanol mixtures of decreasing (21 mg) .
polarity and 7 fractions were collected. The major fractions
obtained were combined into three fractions after III. RESULTS AND DISCUSSION
chromatographic analysis. Fraction A (5 g) was fractionated by
The phytochemical screening of Withania somnifera
PC, using Whatman No 3 sheets and (n-butanol: acetic acid:
constituents in methanolic extracts revealed the presence of
water) upper layer as solvent system followed by Sephadex LH-
alkaloids, Steroids, saponins and phenolic compounds
20 column eluted with methanol (100%) and methanol/ water
(flavonoids, and tannins). The weight and percentage yield of
(9: 1), to afford two pure compounds W 1 (29 mg) and
pure compounds extracted from Withania somnifera are shown
compound W2 (36mg). Fraction B (3 g) was subjected to
in Table 1.
column chromatography on cellulose and n-BuOH saturated
Table 1
Weight and percentage yield of some compounds extracted from W. somnifera leaves
components weight Percentage yield
Compound W1 (mg) 29 0.58
Compound W1 (mg) 36 0.72
Compound W1 (mg) 27 o.77
The Rf values and colour reactions of pure compounds are shown in Table 2.
Table 2
The Rf values and colour reactions of pure compounds
Comp. Rf values x 100 Colour reactions
Table 4
UV Spectrum data of pure compounds extracted from W. somnifera
Comp. MeOH NaOMe AlCl3 AlCl3/ NaOAc NaOAc/
No HCl H3BO3
W1 267, 300 275, 326 270, 303sh, 274, 303sh, 274, 303sh, 266, 297sh,
sh, 350 sh, 402 350, 400 355, 386 379 359
W2 257, 300sh 272, 327 273, 295 268, 300sh, 275, 310 260,300sh,
360 sh, 409 sh, 425 362, 400 sh, 380 375
W3 250, 296 240, 260 270, 10sh, 260, 350 260, 380, 255, 300sh,
sh, 350 sh, 380 340sh, 445 sh, 420 430 390
Table 5
The antibacterial activity of crude and pure compounds extracted from
Withania somnifera
Inhibition zone diameter (mm / mg sample)
Sample
B acillu Escherichia Neisseria Pseudomonas S taphylococcus
subtilis coli gonorrhoeae aeruginosa aureus
(G+) (G-) (G-) (G-) (G+)
Control: DMSO 0.0 0.0 0.0 0.0 0.0
Standard
Tetracycline
27 31 33 29 26
Antibacterial agent
Crude extract 9 13 9 13 13
W1 12 11 12 12 11
W2 9 9 8 9 9
W3 13 14 15 10 13
The methanolic extract of the leaves of W. somnifera was [22]. NaOMe exhibited a bathochromic shift of +52 nm
fractionated by polyamide column chromatography to give indicating a 4`- OH. NaOAc gave about +7 nm indicating the 7-
several fractions, which were further chromatogramphed on OH, also the shift of boric acid spectrum indicated a catechols
Sepadex LH 20 and cellulose to afford three compounds w1, w2 moiety [21]. 1H-NMR (500 MHz, DMSO-d6) figure (1) shows
and w3. The structures of these flavonoids were characterized (ppm): 3.0 3.5 (m, 3H) assigned for a methoxyl function;
on the basis of the IR show in table 1. The NaOMe, AlCl3, 4.2 5 (m, 6H) accounting for sugar protons; 6.04 (s, 1H)
AlCl3/ HCl, and fused NaOAc/ H3BO3 reagents were and 6.25 (d, 1H), accounting for C6- and C8- H's respectively.
separately added to the methanolic solution of the investigated The resonances at 6.84 and 7.98 ppm were assigned for the
material and UV measurements were then recorded in (table 4) aromatic H's of B- ring. The froth test [23] revealed the
to determine the position of hydroxyl groups. glycosidic nature of compound (W1), after completed acid
Compound W1 absorbs at max (MeOH) 267, 350 nm due to the hydrolysis and subsequent paper chromatography the sugar was
benzoyl and cinnamoyl chromophores of the flavonol moiety identify as galactose.
J OURNAL OF FOREST PRODUCTS & INDUSTRIES, 2013, 2(5), 39-45 ISSN:23254513(PRINT) ISSN 2325 - 453X (ONLINE )
43
OCH 3
HO O OH
O
O galactosyl
OH
OH
HO O
OH
O
galactosyl
OH O
The UV spectral data of compound W3 in MeOH characterized characterized by shift reagents of NaOAc and NaOAc/ H3BO3
a flavone with three hydroxyl groups. The addition of NaOMe respectively. The 1H-NMR spectrum of W3 (Figure: 3)
gave a bathochromic shift of 30 nm (table 4) indicated of 4`- showed: 3.2- 3.9 (m) assigned for sugar protons. Furthermore,
OH. Also 7-OH and catechol moiety in ring B were there was only one anomeric proton signal of sugar moiety
J OURNAL OF FOREST PRODUCTS & INDUSTRIES, 2013, 2(5), 39-45 ISSN:23254513(PRINT) ISSN 2325 - 453X (ONLINE )
44
attached to a flavonoid skeleton (doublet at 5.4) proving the The complete acid hydrolysis of compound W 3 and
presence of rhamnosyl moiety (with a methyl group at 1.1 subsequent UV studies of the resultant aglycone using the shift
ppm). The resonances at 6.15 and 6.7(d, 2H) were assigned reagent AlCl3 indicated a 3-hydroxyl and this is precisely the
for C6- and C8- H's also signals at 6.92 (s, 1H), 7.03 (s, 1H) cite of glycosylation. The aglycone of compound W3 seems to
and 7.41 (d, 1H) are typical for a flavone B-ring with 3`- and 4`- be trihydroxy flavone.
OH functions. The resonance at 8 ppm is characteristic of C5-
H.
OH
OH
HO O
O rhamnosyl
O
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