phytochemical and biological study of ?· c cpd. 4+ cpd. 5 cpd. 6 cpd. 7* cpd. 12 1 - 127.67 (s...

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  • 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: ghallab68@yahoo.com

    PHYTOCHEMICAL AND BIOLOGICAL STUDY OF VANGUERIAEDULIS CULTIVATED IN EGYPT

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

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

    :)1()2()3()4()5(

    )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.

    INTRODUCTION

    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.

    MATERIAL AND METHODS

    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

    mailto:ghallab68@yahoo.com

  • Daoud W. Bishay, et al.

    68

    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).

    CarbonNo.

    (ppm)and

    multiplicityCarbon No.

    (ppm)and

    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.

    70

    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 multiplicit

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