PHYTOCHEMICAL AND BIOLOGICAL STUDY OF ?· C Cpd. 4+ Cpd. 5 Cpd. 6 Cpd. 7* Cpd. 12 1 - 127.67 (s ...…

Download PHYTOCHEMICAL AND BIOLOGICAL STUDY OF ?· C Cpd. 4+ Cpd. 5 Cpd. 6 Cpd. 7* Cpd. 12 1 - 127.67 (s ...…

Post on 20-Jun-2018




2 download

Embed Size (px)


  • Bull. Pharm. Sci., Assiut University, Vol. 35, Part 1, 2012, pp. 67-81.

    Received in 29/4/2012 & Accepted in 15/8/2012

    * Corresponding author : Yaser G. Gouda, E-mail:


    Daoud W. Bishay, Enaam Y. Backheet, Yaser G. Gouda* and Shaymaa M. Mostafa

    Department of Pharmacognosy, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt


    )6()7( ----)8(-- --)9()10()11(). 13(---) 12(

    . 89



    Fractionation and purification of the alcoholic extract of the leaves and stem -bark ofVangueria edulis cultivated in Egypt afforded thirteen compounds identified as: -sitosterolacetate (1), stigmasterol (2), palmitic acid (3), scopoletin (4), p-coumaric acid (5),protocatechuic acid (6), esculetin (7), ethyl 1-O-glucosyl-4-O-(E) caffeoyl quinate (8),kaempferol 3-O-rhamnoside, 7-O-rutinoside (9), -sitosterol (10), ceryl alcohol (11), vanillicacid (12) and -sitosterol-3-O--D-glucopyranoside (13). Identification of these compounds hasbeen established by physical, chemical and spectral data as well as comparison with authenticsamples. Compounds 8 and 9 are firstly reported from a natural source while the rest of thecompounds have been isolated for the first time from the genus Vangueria. The LD50,antibacterial, antifungal, anti-inflammatory, antipyretic and the effect on CNS were studied.


    Vangueria edulis (Vahl) Vahl Variety:bainesii Heirn (Rubiaceae) is a perennial shrubor a small tree not exceeds 6 meters in height,cultivated as an ornamental plant and known asSpanish tamarind1. According to the availableliterature, no report about the phytochemicalconstituents of this plant could be traced thatencouragethis study which includes theisolation and identification of the chemicalconstituents in addition to the biologicalactivities of the leaves and stem-bark of thisplant.


    ExperimentalGeneral experimental procedure: Meltingpoint (uncorrected)was recorded onElectrothermal 9100 Digital Instrument(England Ltd.). ESI-MS was recorded onMicromass Quattro microTM API massspectrometer (Germany). 1H-NMR (700, 600and 400 MHz) and 13C-NMR (175, 150 and100 MHz) spectra were measured on BrukerAVANCE AVIII (Oxford), JOEL/ECP (Japan)and Varian unity INOVA spectrometers(England) respectively using TMS as aninternal standard. UV spectra were determined

  • Daoud W. Bishay, et al.


    on Ultrospec 1000, UV/visible Spectrometer,Pharmacia Biotech, Cambridge, England.Agilent spectrometer (USA) for GC/MSanalysis. Column chromatography wasperformed on silica gel (70-230 mesh) and RP-18 (E-Merck, Germany), precoated silica gelG60F254 and RP-18 plates for TLC (E-Merck,Germany).

    Plant materialThe leaves and stem-bark of Vangueria

    edulis were collected at the flowering stage(May, 2008) from Al-Zohria garden, Cairo,Egypt and was kindly identified by Prof. Dr.Momen Mostafa Mahmoud, Professor ofTaxonomy, Faculty of Science, AssiutUniversity, Assiut, Egypt.

    Extraction and isolationThe air-dried powdered leaves (3 kg) and

    stem-bark (1 kg) were separately extracted with70% ethanoltill exhaustion. The combinedextract was evaporated till dryness to give 300g of leaf extract and 70 g of stem-bark extract.The obtained extracts were separately digestedin small amounts of distilled water andsuccessively extracted with n-hexane, CHCl3,EtOAc(for both leaf and stem-bark) and finallyn-butanol (for leaf)and dried over anhydroussodium sulphate to yield (100 g, leaf; 0.6 gstem-bark), (2.5 g leaf; 3 g stem-bark), (160 gleaf; 3.5 g stem-bark) and (7 g leaf) for n-hexane, CHCl3, EtOAc and n-butanol fractionsrespectively.

    1- The leaf extractThirty grams of the dried hexane fraction

    was chromatographed over silica gel column(900 g) and gradiently eluted with hexane-EtOAc. Fractions (250 ml each) were collectedand monitored using TLC [hexane:EtOAc(90:10) and (80:2) solvent systems]and 10%H2SO4 as spraying reagent; similar fractionswere pooledto give six fractions (F-I to F-VI).F-III (5.5g) from hexane-EtOAc (9:1) and F-IV(8 g) from hexane-EtOAc (85:15) wereseparately re-chromatographed on silica gel CCusing hexane-EtOAc gradient which affordedcompounds 1 (60 mg) and 2 (150 mg)respectively. F-VI (3 g) eluted with hexane-EtOAc (7:3) afforded compound 3 (25 mg)after repeated silica gel CC using hexane-acetone gradient.

    The dried chloroform fraction (2.5 g) waschromatographed over silica gel column (75 g)and eluted with CHCl3-MeOH gradiently.Similar fractions were combined to give threefractions (F-I to F-III). F-I (1 g) eluted frompure CHCl3 was re-chromatographed on silicagel CC using hexane-EtOAc gradient whichafforded compound 4 (60 mg).

    Thirty grams of the EtOAc fraction wastransferred to a Buchner [14 (id) 10 (l) cm]packed with silica gel; The elution startedinitially with pure CHCl3 followed by CHCl3-EtOAc gradiently, EtOAc and finally withEtOAc-MeOH. Similar fractions were pooledto give five fractions (F-I to F-V). F-II (8 g)eluted with CHCl3:EtOAc (70:30) waschromatographed on silica gel CC and elutedgradiently with hexane-EtOAc giving 3subfractions that by repeated silica gel CC,purification and crystallization affordedcompounds 5 (45 mg), 6 (20) and 7 (25 mg). F-III (12 g) eluted with CHCl3:EtOAc (30:70)was chromatographed on silica gel CC andeluted with CHCl3-MeOH gradient giving 2subfractions (F-IIIa and F-IIIb). F-IIIa waschromatographed over silica gel CC using n-hexane-EtOAc gradient, afforded compound 8(70 mg) while F-IIIb was chromatographedover RP-18 CC using 30% MeOH affordedcompound 9 (25 mg).

    2- The stem-bark extractParts of the chloroform (2.5 g) and EtOAc

    extracts (30 g) wereseparately subjected tosilica gel CC and gradiently eluted withhexane-EtOAc. Similar fractions werecombined together(four fractions each).Fraction II eluted with n-hexane-EtOAc (90:10)of CHCl3 extract after repeated silica gel CCyieldedcompound 10 (80 mg). EtOAc fractionII eluted with n-hexane-EtOAc (70:30)andfraction III eluted with n-hexane-EtOAc(50:50) after repeated silica gel CC yieldedcompounds 11 (30 mg), 12 (70 mg) and 13(120 mg).

    Compound (4): yellow crystals (MeOH). m.p.206-208C. 1H-NMR (CDCl3, 600 MHz): 6.20 (1H, d, 9.0 Hz, H-3), 7.55 (1H, d, 9.0 Hz,H-4), 6.84 (1H, s, H-5), 6.79 (1H, s, H-8), 3.88(3H, s, OCH3).

    13C-NMR (Table 1).

  • 69

    Table 1: 13C-NMR spectral data of compounds 4-7 and 12 in CD3OD (100 MHz).

    C Cpd. 4+ Cpd. 5 Cpd. 6 Cpd. 7* Cpd. 121 - 127.67 (s) 123.39 (s) - 121.74 (s)2 161.49 (s) 131.07 (d) 115.74 (d) 161.28 (s) 114.39 (d)3 113.24 (d) 116.79 (d) 145.89 (s) 111.89 (d) 147.21 (s)4 143.35 (d) 160.97 (s) 151.31 (s) 144.91(d) 151.18 (s)5 107.55 (d) 116.79 (d) 117.71 (d) 112.73 (d) 112.37 (d)6 150.18 (s) 131.07 (d) 123.88 (d) 143.36 (s) 123.83 (d)7 144.08 (s) 146.64 (d) 170.61 (s) 150.93 (s) 168.69 (s)8 103.13 (d) 115.71 (d) - 103.07 (d) -9 149.76 (s) 167.00 (s) - 148.96 (s) -

    10 111.44 (s) - - 111.16 (s) -OMe 56.38 (q) - - - 54.97(q)

    *Data in DMSO-d6 + Data in CDCl3 (150 MHz)

    Compound (5): yellow crystals (MeOH). m.p.131-133C. 1H-NMR (CD3OD, 400 MHz): 7.37 (2H, d, 8.4 Hz, H-2, 6), 6.73 (2H, d, 8.4Hz, H-3, 5), 7.52 (1H, d, 16.0 Hz, H-7), 6.21(1H, d, 16.0 Hz, H-8). 13C-NMR (Table 1).

    Compound (6): yellow crystals (MeOH). m.p.178-180C. 1H-NMR (CD3OD, 400 MHz): 7.43 (1H, d, 2.0 Hz, H-2), 6.80 (1H, d, 8.0 Hz,H-5), 7.40 (1H, dd, 2.0, 8.0 Hz, H-6). 13C-NMR(Table 1).

    Compound (7): yellow crystals (acetone). m.p.269-271C. 1H-NMR (DMSO-d6, 400 MHz): 6.16 (1H, d, 9.4 Hz, H-3), 7.86 (1H, d, 9.4 Hz,H-4), 6.97 (1H, s, H-5), 6.73 (1H, s, H-8). 13C-NMR (Table 1).

    Compound (8): yellow amorphous powder.ESI-MS m/z (% rel. int.) 544 [M]+ (45), 530(97), 516 (100), 501 (28). 1H-NMR (DMSO-d6,700 MHz): Quinic acid: 1.92 (1H, dd, 3.5,13.5 Hz, H-2ax), 2.10 (2H, m, H-2eq and H-6eq),3.57 (1H, m, H-3), 5.02 (1H, m, H-4), 3.88 (1H,m, H-5), 1.76 (1H, dd, 9.5, 12.5 Hz, H-6ax);Caffeoyl moiety: 7.02 (1H, d, 2.0 Hz, H-2'),6.76 (1H, d, 8.8 Hz, H-5'), 6.97 (1H, dd, 2.0,8.8 Hz, H-6'), 7.37 (1H, d, 15.9 Hz, H-7'), 6.01(1H, d, 15.9 Hz, H-8'); Glucose moiety: 4.08(1H, d, 7.6 Hz, H-1''), 3.42-4.39 (6H, m, H-2''-H-6''); Ethyl ester: 1.15 (3H, t, 7.0 Hz, CH3-1'''), 4.00 (2H, q, CH2-2''').

    13C-NMR (Table 2).

    Table 2: 13C-NMR spectral data of compound8 (DMSO-d6, 175 MHz).



    multiplicityCarbon No.


    multiplicityQuinic acid

    C-1 72.5 (s) C-7' 145.5 (d)C-2 35.5 (t) C-8' 114.3 (d)C-3 69.5 (d) C-9' 165.9 (s)

    C-4 71.5 (d) Glucosyl moiety

    C-5 67.2 (d) C-1'' 103.8 (d)C-6 37.5 (t) C-2'' 73.5 (d)C-7 173.5 (s) C-3'' 77.0 (d)

    Caffeoyl moiety C-4'' 70.0 (d)C-1' 125.8 (s) C-5'' 76.4 (d)C-2' 114.9 (d) C-6'' 63.6 (t)C-3' 148.9 (s) Ethyl esterC-4' 146.0 (s) C-1''' 14.3 (q)C-5' 116.2 (d) C-2''' 60.7 (t)C-6' 121.7 (d)

    Compound (9): yellow amorphous powder.1H-NMR (CD3OD, 400 MHz): 8.12 (2H, d,8.8 Hz, H-2', 6'), 6.90 (2H, d, 8.8 Hz, H-3', 5'),6.78 (1H, d, 2.0 Hz, H-8), 6.48 (1H, d, 2.0 Hz,H-6), 5.10 (1H, d, 7.6 Hz, glu-1), 4.52 (1H, d,1.6 Hz, rham`.-1), 4.50 (1H, d, 1.6 Hz, rham-1),3.2-3.9 (m, sugar protons), 1.26 (3H, d, 6.0 Hz,rham' 6-CH3), 1.18 (3H, d, 6.4 Hz, rham 6-CH3). UV/Vis. spectral data (Table 3).

    13C-NMR (Table 4).

  • Daoud W. Bishay, et al.


    Table 3: UV/Vis. spectral data of compound 9 in methanol and with different ionizing and complexingreagents.

    max and max nmMeOH NaOMe NaOAc NaOAc/H3BO3 AlCl3 AlCl3/HClBand

    max max max max max max II 268 270 +2 268 - 266 - 274 +6 273 +5I 343 384 +41 345 +2 343 - 393 +50 392 +49

    Table 4: 13C-NMR spectral data of compound 9 (CD3OD, 100 MHz).

    C- atom (ppm) and ultiplicity Carbon No. (ppm) and multiplicity2 158.01 (s) rham -4 73.52b (d)3 135.80 (s) rham -5 71.38 (d)4 179.76 (s) rham -6 17.95c (q)5 162.72 (s) glu-1 100.65 (d)6 99.81 (d) glu -2 74.85 (d)7 163.64 (s) glu -3 78.01 (d)8 95.71 (d) glu -4 71.26 (d)9 159.84 (s) glu -5 77.91 (d)

    10 104.83 (s) glu -6 67.36 (t)1' 122.39 (s) rham '-1 101.14 (d)

    2' and 6' 132.54 (d) rham '-2 72.05 (d)3'and 5' 116.23 (d) rham '-3 72.92a (d)

    4' 161.68 (s) rham '-4 73.78b (d)rham-1 101.75 (d) rham '-5 69.68 (d)rham -2 71.61 (d) rham '-6 18.10c (q)rham -3 72.25a (d)

    Compound (12): light yellow crystals(MeOH). m.p. 210-213C. 1H-NMR (CD3OD,400 MHz): 7.56 (1H, dd, 2.0, 8.0 Hz, H-6),7.55 (1H, br s, H-2), 6.83 (1H, d, 8.0 Hz, H-5),3.80 (3H, s, OMe). 13C-NMR (Table 1).

    Preparation for the fatty acidsTen g of the n-hexane fractionof the leaves

    were saponified by refluxing with 50 ml of 0.5N ethanolic KOH for 3 hrs on a boiling waterbath. The alcohol was distilled off and theaqueous liquid was extracted with ether tillexhaustion. The alkaline aqueous solution thatremained after removal of the unsaponifiablematter was acidified with sulphuric acid andthe liberated fatty acids were extracted withether (3x50 ml). The combined ether extractswere washed with distilled water to remove anyacidity and dried over anhydrous sodiumsulphate. The solvent was distilled off underreduced pressure to give a viscous residue of

    the free fatty acids, which have yellowishbrown colour. A part of the residue wassubjected to esterification with methanol asfollow:

    One gram of the fatty acids was dissolvedin 150 ml of 10% H2SO4 in MeOH

    2 and thenrefluxed for 5 hrs. The solvent was distilled offand the residue was taken in 10 ml distilledwater, the aqueous solution was renderedalkaline with dilute ammonium hydroxide,where an oily layer was separated and extractedwith ether till exhaustion. The ethereal extractswere combined and distilled to give ayellowish-brown residue. A part of this residuewas analyzed by GC.

    Gas-liquid chromatography (GLC) of thefatty acid methyl esters

    GLC analysis of the fatty acid methylesters was performed using Agilentspectrometer. Gas chromatograph equipped

  • 71

    with flame ionization detector and fitted with3% OV-17 on Carbowax HP 80/100 (6` x 1/8``x 0.085``) SS column, programmed at 160Cfor 2 min. then increase by 15C/min. till300C and isothermal for 15 min. Injector anddetectortemperature were 250C and 320Crespectively.

    Authentic materialsAuthentic stigmasterol, -sitosterol

    acetate, palmitic acid, -sitosterol, cerylalcohol and -sitosterol glucoside fromPharmacognosy Dept., Faculty of Pharmacy,Assiut University.

    Biological studyThe biological studies were performed on

    the different fractions: n-hexane, chloroform,ethyl acetate, total ethanolic and aqueousextract of leaves and stem-bark in addition tothe n-butanol fraction of the leaves.

    1- Experimental animalsMale albino rats (100-120 g) and mice

    (20-25 g) were housed under standardizedenvironmental conditions in the pre-clinicalanimal house, Pharmacology Department,Faculty of Medicine, Assiut University and fedwith standard diet and allowed for free accessto water.

    2- Materials for biological studyNormal saline 0.9%, PEG-600 and

    Emmon's Sabouraud Dextrose Agar (El-NaserPharmaceutical and Chemical Co., Egypt)(ADWIC). Indomethacin (El-Nile Co., Cairo,Egypt). Yeast. Gentamicin and clotrimazole(Memphis Co., Egypt). Carbamazepine(Novartis Co., Switzerland). Carrageenin,Pentylene tetrazole, 2% Tween 80 andDimethylformamide (Sigma Chemical Co., St.Louis, USA). Bacterial strains:Bacillus cereus;Escherichia coli;Klebsiella pneumonia;Micrococcus luteus;Pseudomonas aeruginosa;Staphylococcus aureus and the fungus Candidaalbicans (Microbiology Department, Faculty ofMedicine, Assiut University)

    3- Preparation for toxicological studyCertain weights of the total ethanolic

    extracts of the leaves and stem-bark weresuspended in saline solution by the aid ofpolyethylene glycol-600 (PEG-600, 3% w/v)

    and the volume was completed to 100 ml withsaline solution.

    Determination of the LD50The LD50 of the total ethanolic extracts

    were determined according the reportedmethod3. The experiment was carried out intwo phases; the first phase involved an initialdose finding procedure, in which the animalswere divided into three groups (3 rats, each).Doses of 10, 100 and 1000 mg/kg wereadministered i.p., one dose for each group. Thetreated animals were monitored for 24 hrs formortality and general behavior. The secondphase, based on the result of the above step;four different doses were chosen andadministered i.p. to four groups (1 rat, each).The treated animals were again monitored for24 hrs. The LD50 was calculated as thegeometric mean of the lowest dose showingdeath and the highest dose showing nodeath3&4. The symptoms of the toxicity arecharacterized by irritability, writhing,hypothermia, loss of motor co-ordination,sedation and deep sleep followed by death.

    4- Preparation for antimicrobial study200 mg of the extracts were separately

    taken and reconstituted in a least amount ofdimethylformamide (DMF) and the volumewas completed to 2 ml with DMF (100 mg/ml).DMF served as a negative control.

    Antibacterial activityThe tested bacterial strains were

    inoculated into Muller Hinton broth mediumand incubated for 3-6 hrs at 35C in a shakerwater bath until the culture attained a turbidityof 0.5 McFarland units. The final inoculumswere adjusted to 5x105 cfu/ml. Antiba...


View more >