Isolation, structural elucidation of flavonoid constituents from ...

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the isolation of six flavonoids, kaempferol-3-O-α-l-rhamnopyranosyl (1!!!→6! ... pyrotechnica; Asclepiadaceace; brine shrimp; antitumor activity; kaempferol;.
Pharmaceutical Biology, 2009; 47(6): 539–552

RESEARCH ARTICLE

Isolation, structural elucidation of flavonoid constituents from Leptadenia pyrotechnica and evaluation of their toxicity and antitumor activity Amal M. Youssef Moustafa 1, Ahmed I. Khodair1, and Mahmoud A. Saleh2 Chemistry Department, Faculty of Science, Suez Canal University, Ismailia, Egypt, and 2Environmental Chemistry and Toxicology Laboratory, Texas Southern University, Houston, Texas, USA

1

Abstract An investigation of Leptadenia pyrotechnica (Forsk.) Decne (Asclepiadaceace) chemical constituents led to the isolation of six flavonoids, kaempferol-3-O-α-l-rhamnopyranosyl (1999→699)-O-β-d-glucopyranoside (E-I.1), kaempferol-3-O-β-d-rhamnopyranosyl (1999→699)-O-β-d-glucopyranoside (E-I.2), texasin-7-O-β-dglucopyranoside E-II.2, kaempferol-3-O-β-d-glucopyranoside (E-III.1), kaempferol (E-IV.1) and kaempferide3-O-α-l-rhamnopyranosyl (1999→699)-O-β-d-glucopyranoside (E-I.1a). The isolation of these compounds was carried out using Sephadex LH-20 low pressure liquid chromatography (LPLC), preparative paper chromatography (PC), and high performance liquid chromatography (HPLC). The chemical structures of the isolated compounds were established by mass spectrometry (FAB- and EI- techniques), nuclear magnetic resonance NMR (1H-, 13C- and COSY) spectral data and ultraviolet (UV) spectroscopic techniques. The acute toxicity of total alcoholic and total flavonoid extracts were examined by brine shrimp. The LC50 values were 11.89 and 84.14 ppm for the total alcoholic and total flavonoid extracts, respectively. The mortality rates of the isolated flavonoid fractions of E-I, E-I.1, E-I.2 represent the higher percentages of mortality compared with the rest of the flavonoid fractions. The plant exhibited activity as an antitumor agent in the initial potato disc screen. Keywords:  Leptadenia pyrotechnica; Asclepiadaceace; brine shrimp; antitumor activity; kaempferol; texasin-7-O-β-d-glucopyranoside

Introduction Plants belonging to the Asclepiadaceae are frequently used in traditional medicine and have been reported to be rich in steroidal glycosides, cardenolides, alkaloids, flavonoids, triterpenes and polyoxypregnane derivatives (Bazzaz et al., 2003; Paulo & Houghton, 2003; Atta & Mouneir, 2005; Cioffi et al., 2006; Khanna & Kannabiran, 2007). The plants of this family are known to contain cytotoxic and tumoricidal C/D-cis-polyoxypregnane esters and glycosides. Leptadenia pyrotechnica (Forsk.) Decne (Asclepiadaceae) is a plant wild growing in the Sharm El-Sheikh region, southern Sinai, Egypt. The leaves and bark of the plant are used in folk medicine to prepare

antispasmodic, anti-inflammatory, antihistaminic, antibacterial diuretic, urolith expulsion, expectorant, gout, and rheumatism remedies (Cioffi et al., 2006; Panwara & Tarafdarb, 2006; Abd El-Ghani & Amer, 2003; Aquino et al., 1996). A previous study on the aerial parts of the plant led to the isolation of some phenolic compounds, flavonoids, quercetin-3-O-galactoside, alkaloids, pregnane glycosides, amino acids, sterols, -sitosterol, triterpenoids, taraxerol, fernenol, and leptadenol, fatty acids, and fatty alcohols (Cioffi et  al., 2006; Noor et  al., 1993; Abd EL-Ghani & Amer, 2003; El-Hassan et  al., 2003; Panwara & Tarafdarb, 2006; Moustafa et al., 2007). The flavonoids of 11 Egyptian species of the tribe Asclepiadeae were found to produce flavonol glycosides,

All images in this article are available to view in colour at: www.informapharmascience.com/phb Address for Correspondence:  Amal M. Youssef Moustafa, Chemistry Department, Faculty of Science, Suez Canal University, BP 41522, Ismailia, Egypt. Tel.: 002066-3727487; E-mail: [email protected] (Received 12 February 2008; revised 23 February 2008; accepted 29 February 2008) ISSN 1388-0209 print/ISSN 1744-5116 online © 2009 Informa UK Ltd DOI: 10.1080/13880200902875065

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540   A. M. Youssef Moustafa et al. flavonol sulfates, and disulfates (Heneidak et al., 2006). Previous studies on flavonoids of different Leptadenia species revealed the presence of quercetin ­3-O-galactoside, quercetin 3-O-glucoside, rutin, cosmosiin, luteolin 7-Oglucopyranoside, kaempferol 3,7-diglucoside, astragalin, isoquercitrin, apigenin, kaempferol, luteolin, chrysoeriol, Peonoside, quercetin, hyprin and diosmetin (Gibbs, 1974; Eisenreichová et al., 2004). A method, utilizing brine shrimp (Artemia salina LEACH) (Krishnaraju et  al., 2006; Poli et  al., 2006), is proposed as a simple bioassay for determining LC50 values in g/mL of extracts. Crown gall is a neoplastic disease of plants induced by specific strains of the Gram-negative bacterium named Agrobacterium tumefaciens (Lippincott et  al., 1977). This bacterium contains large Ti (tumor-­inducing) plasmids which carry genetic information (T-DNA) that transforms normal plant cells into autonomous tumor cells (Chilton et  al., 1980). Thus, certain tumorigenesis mechanisms, in both plants and animals, have in common the intracellular incorporation of extraneous nucleic acids and it could be anticipated that some antitumor drugs might inhibit tumor initiation and growth in both plant and animal systems. The development of a simple antitumor pre-screen, using convenient and inexpensive plant tumor systems, could offer numerous advantages as alternatives to extensive animal testing in the search for new anticancer drugs (Ferrigni et al., 1982). Crown gall tumorigenesis on discs of potato tuber Solanum tuberosum L. Solanaceae was proposed as an ideal system for investigating the transformation process (Ferrigni & McLaughlin, 1984). The action of the antitumor compounds is neither via antibiosis nor through inhibition of bacterial attachment to the tumor-binding sites (Bryant et al., 1994). This paper deals with the isolation, structural elucidation of flavonoid constituents in L. pyrotechnica and also evaluation of their antitumor activity and toxicity using potato disc assay and brine shrimp.

Materials and methods Plant material Fresh aerial parts of L. pyrotechnica were collected in September 2000, during the flowering stage, from Wadi Khashab and Wadi Matzos, Sharm El-Sheikh to El-Tur road, southern Sinai, Egypt. The identity was established by Samia Heneidak, Department of Botany, Faculty of Science, Suez Canal University. A voucher specimen (number AMYM-1004) has been deposited in the herbarium of the Department of Botany, Faculty of Science, Suez Canal University, Ismailia, Egypt.

General methods Melting points were determined on Büchi 535 melting ‥ point apparatus (Buchi, Germany) and are uncorrected. UV absorption spectra were obtained on computerized system Lambda 2S UV/Vis spectrophotometer (PerkinElmer, USA) in the region 240-400 nm. FAB-MS was performed on Mat 95 double focusing instrument operated at unit resolution. The samples of interest were mixed on the probe tip with glycerol as matrix. Xenon gas was used to generate the primary ionization beam from an Ion-Tech gun operated at 6 KV. Ion source accelerating potential was adjusted at 5 KV. EI spectra (at 70 eV) were obtained using GC-MSQP 1000 EX Schimädzu and HP-Model, MS-5988 spectrometer. Ions are given in m/z (%). Electrospray ion mass spectrometry (ESI-MS) set was equipped with a Finnigan ESI II ion source. 1H, 13C and two-dimensional (1H-1H-COSY) NMR spectra were recorded in DMSO-d6 with a GE NMR, QE-300, FT-NMR system at 300 and 100 MHz. Chemical shifts are given relative to Tetramethylsilane (TMS) as an internal standard. The 1H-NMR spectra of aglycones were recorded with a Jeol JNM-EX 270 FT-NMR spectrometer at 270 MHz. Column chromatography (glass column;  80 cm × 2 cm) was carried out on silica gel 60 (70-230 mesh, Merck). After complete swelling and degassing of Sephadex LH 20-100 (particle size 25-100 m, ICN Biomedicals), it was packed into a LPLC using a Spectra/Chrom LC column (ID: 2. 5 cm, Volume: 4.91 mL/cm) or Sigma® Chemical column (ID: 0. 7 cm, length:  64 cm). The flow rate was 5 mL/min. Fractions of 30 mL each were collected using a fraction collector (Rikakikai, Tokyo). HPLC separation was performed with a Perkin-Elmer model series 400 solvent delivery system. The instrument was equipped with Pharmacia LKB.UVICORD SII, Pharmacia LKB.Rec.102. Phenomenex® LUNA 5 C18 column (250 × 4. 60 mm internal diameter), particle size; 5.00 ± 0.30 m was used. Solutions to be used by HPLC were passed through membrane filters (0.5 m pore size) prior to injection. Two solvent mixtures were employed from elution. Eluent A: MeOH, and eluent B: water. Separation was achieved at ambient temperature with a flow rate of 1 mL min−1. The gradient began with 25% eluent A and 75% eluent B, and was held at this concentration for the first time for 0.1 min. This was followed by a linear gradient to 50% eluent A over the next 10 min and then a sharp transition to 100% eluent A over the next 5 min. Data were collected at 254 nm. Peaks were identified with authentic standards by accordance to retention times. Two-dimensional paper chromatography (TDPC), the chromatogram was developed ascending in the long direction, using ­n-butanol-acetic acid-water (BAW) (4:1:5) as solvent, then developed ascending in the second direction with 15% acetic acid solvent. Different Thin Layer Chromatography

Isolation, structural elucidation of flavonoid constituents from Leptadenia pyrotechnica   541 (TLC) and Paper Chromatography (PC) adsorbents; Merck pre-coated silica gel 60 F254 on aluminum foil, glass (Sigma-Aldrich) plates; 250 and 500 m layer thickness, polyamide-6 TLC, Whatman No. 1 and/or Whatman  3 mm chromatographic sheets were applied for the detection, isolation and purification of the different flavonoid components. Detection was achieved by UV light (254 nm), anhydrous AlCl3 and/or iodine solution reagents. Extraction, isolation, and characterization of flavonoids The aerial parts of the plant (leaves, flowers and stems) were air-dried and ground together as a fine powder. The phytochemical screening was performed in accordance with Association of Official Agricultural Chemist (AOAC) (1990). About 2 kg of the dried powdered of L. pyrotechnica plant was defatted with petroleum ether (40-60°C), yielding 75 g upon evaporation. The marc was percolated with methanol until exhaustion. The methanol extract

was evaporated in vacuo, yielded 950 g residue, followed by extraction with dichloromethane then ethyl acetate to yield 8.4 and 15 g from these extracts, respectively. The solvent systems (S1) methanol-acetic acid-water (90:5:5), polyamide, (S2) BAW (4:1:5), PC and (S3) acetic acid-water (15:85), PC, showed the best resolution and revealed the presence of six flavonoids in the ethyl acetate extract; three major, Rf values, 0.77, 0.66, and 0.19 and three minor, Rf values, 0.89, 0.46, and 0.11 (S1). Three minor flavonoid spots in the dichloromethane extract were detected as yellowish spots, in addition to one bluish spot (Rf value, 0.59). Therefore, we focused on the isolation of the flavonoid constituents from the ethyl acetate extract. It was subjected to LPLC on Sephadex LH-20 as adsorbent which fractionated into four fractions E(I-IV) as shown in Figure 1A,1B. Fraction E-I was fractionated into two major fractions E-I.1and E-I.2. About 4 g of the total ethyl acetate extract was applied on LPLC using 95% methanol/water as eluant. The flavonoid constituents of fraction E-I.1 (0.39 g, Rf; 0.77, 0.66; S1 and 0.59; S2) were subjected to further isolation on LPLC, then applied on preparative TLC and

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Figure 1.  Thin-layer chromatography of column chromatography fractions. (A) Detection under ultra-violet light. (B) Detection after spraying with AlCl3 reagent.

4000 Rt; 2.39

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Figure 2.  HPLC of Component E-I.1 and E-I.1a. (A) Analytical HPLC. (B) Preparative HPLC.

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542   A. M. Youssef Moustafa et al.

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Time (min) Figure 4.  Analytical HPLC of component E-II.2.

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Isolation, structural elucidation of flavonoid constituents from Leptadenia pyrotechnica   543 Response (AU)

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Time (min) Figure 5.  HPLC of component E-III.1. (A) Analytical HPLC. (B) Preparative HPLC.

subjected to further purification on Sephadex LH-20 column chromatography. The main compound E-I.1 yielded 0.32 g (Rf; 0.59; S2 and 0.66; S3). The purity of compound E-I.1 was checked by HPLC to give two peaks, as shown in Figure 2A and 2B, possessing retention times (tR) of 2.39 and 2.92 min. The flavonoid constituents of fraction E-I.2 (0.52 g, Rf; 0.77 and 0.66; S1) were subjected to further isolation on preparative paper chromatography (PPC). The main compound isolated E-I.2 (0.48 g, Rf; 0.63; S2, and 0.71; S3) was subjected to further purification on Sephadex LH-20 column chromatography and TDPC analysis. Moreover, the purity of compound E-I.2 was checked by HPLC to give one peak, as shown in Figure 3, possessing tR, 4.22 min. The flavonoid constituents of fraction E-II (0.91 g, Rf; 0.77, 0.66, 0.59 and 0.46; S1) were fractionated into two fractions E-II.1 (0.32 g, Rf; 0.77 and 0.66; S1) and E-II.2 (0.38 g, Rf; 0.66 and 0.59; S1) using Sephadex LH-20 LPLC and PPC. The main compound E-II.2 (Rf; 0.55; S2 and 0.63; S3) was isolated using Sephadex LH-20 LPLC and TDPC techniques to give 0.30 g. The purity was checked by HPLC to give a single peak, tR, 6.49 (Figure 4). The flavonoid constituents of fraction E-III (0.72g, Rf; 0.46 and 0.19; S1) were isolated to two fractions E-III.1 (0.50g, Rf; 0.46; S1) and E-III.2 (0.11 g, Rf; 0.46 and 0.19;

S1) using PPC and Sephadex LH-20 LPLC. The main compound E-III.1 (0.50 g, Rf; 0.46; S1, 0.83; S2 and 0.43; S3) was checked by HPLC to give a single peak, as shown in (Figure 5A and B) possessing tR, 8.93 min. The flavonoid constituents of fraction E-IV (0.61 g, 0.19 and 0.11; S1) were isolated on PPC. The main compound E-IV.1 (0.38 g, Rf; 0.79; S2 and 0.17; S3) was purified using Sephadex LH-20 LPLC and TDPC analysis. Partial and complete acid hydrolysis was carried out as described elsewhere (Mabry et al., 1970; Hyun et al., 2006). Kaempferol-3-O--l-rhamnopyranosyl (1999→699)-O. -d-glucopyranoside (E-I.1). Yellow amorphous powder, 0.32 g, melting point (m.p.); 220-222°C, Rf; 0.59; S2 and 0.66; S3, tR; 2.39. UV (max nm) MeOH: 255, 267sh, 303sh, 357, NaOMe/MeOH: 273, 329, 414, AlCl3/MeOH: 268, 299sh, 305sh, 359, 399, AlCl3/HCl/MeOH: 268, 298sh, 305sh, 359, 400, NaOAc/MeOH: 271, 322, 404, NaOAc/ H3BO3/MeOH: 257, 267sh, 290, 361. Negative ionization FAB-MS m/z 593 and 285. ESI-MS m/z: 594, 577, 286, 285, 257. 1H-NMR (300 MHz, d6-DMSO,  (ppm)): 7.90 (H 29, 69, 2H, d, J= 8.7 Hz), 6.80 (39, 59, 2H, d, J = 8.7 Hz), 5.91 (8, 1H, d, J = 2.1 Hz), 5.75 (6, 1H, d, J = 2.1 Hz), 5.12 (glc. H-199, 1H, d, J = 7.5 Hz), 4.40 (rha. H-1999, 1H, d, J = 1.5 Hz), 3.66-3.07 (rha.glc., 10H, m), 0.98 (rha. CH3, 3H, d, J = 6.3 Hz). 13C-NMR (75 MHz, d6-DMSO,  (ppm)):

544   A. M. Youssef Moustafa et al. 133.00 (C-3), 98.00 (C-6), 93.00 (C-8), 104.00 (C-10), 120.62 (C-19), 130.59 (C-29), 115.26 (C-39), 115.26 (C-59), 130.59 (C-69), 102.72 (C-199), 74.28 (C-299), 76.62 (C-399), 69.85 (C-499), 75.63 (C-599), 66.93 (C-699), 101.08 (C-1999), 70.42 (C-2999), 70.42 (C-3999), 72.08 (C-4999), 68.45 (C-5999) and 17.88 (C-6999). EI-MS m/z (rel. int. %) for aglycone: 286 (100), 285 (33.56), 258 (5.57), 153(4.97), 152 (0.60), 121 (14.97), 93 (4.59). Kaempferol 49-methyl ether 3-O--d-rutinoside (kaempferide 3-O--d-rutinoside) (E-I.1a). tR; 2.92 min. 1 H-NMR (300 MHz, DMSO-d6,  (ppm)): 7.99 (29, 69, 2H, d, J = 9 Hz), 6.98 (39, 59, 2H, d, J = 9 Hz), 5.88 (8, 1H, d, J = 2.1 Hz), 5.72 (6, 1H, d, J = 2.1 Hz), 5.05 (glc. H-199, 1H, d, J = 7.5 Hz), 4.37 (rha. H-1999, 1H, d, J = 1.5 Hz), 3.66-3.07 (rha. glc., 10H, m), 0.98 (rha. -CH3, 3H, d, J = 6.3 Hz), 3.94 (-OCH3, 3H, s). Kaempferol-3-O--d-glucopyranosyl (1999→699)-O-d-glucopyranoside (E-I.2). Yellow amorphous powder, 0.48g, Rf; 0.63; S2, and 0.71; S3, tR; 4.22 min. UV (max nm) MeOH: 267, 300sh, 350, NaOMe: 277, 324, 403, AlCl3: 274, 304, 352, 398, AlCl3/HCl: 274, 304, 350, 398, NaOAc: 274, 312, 392, NaOAc/H3BO3: 266, 294sh, 352. NIFAB-MS m/z: 609, 285. 13C-NMR (75 MHz, DMSO-d6,  (ppm)): 158.26 (C-2), 132.00 (C-3), 176.00 (C-4), 160.93 (C-5), 98.00 (C-6), 164.00 (C-7), 92.86 (C-8), 158.26 (C-9), 106.90 (C-10), 121.52 (C-19), 129.31 (C-29), 114.90 (C-39), 160.93 (C-49), 114.23 (C-59), 129.31 (C-69), 100.61 (C-199), 72.91 (C-299), 77.15 (C-399), 72.23 (C-499), 77.15 (C-599), 67.03 (C-699), 106.90 (C-1999), 74.41 (C-2999), 77.15 (C-3999), 67.03 (C-4999), 77.15 (C-5999) and 60.60 (C-6999). EI-MS m/z (rel. int. %) for aglycone: 286 (100), 285 (24.6), 258 (11.6), 153 (7.4), 152 (1.2), 121 (25.6), 93 (11.7). 1H-NMR spectrum for aglycone (270 MHz, DMSO-d6,  (ppm)): 8.04 (2H, d, J = 8.5 Hz), *6.93 (2H, d, J = 8.5 Hz), 6.45 (1H, d, J = 2.1 Hz) and 6.2 (1H, d, J = 2.1 Hz), 12.45 due to H-bonded hydroxyl group. Texasin 7-O--d-glucopyranoside (E-II.2). 0.30 g, Rf; 0.55; S2 and 0.63; S3, tR; 6.49 min. UV Spectral Data max nm (MeOH): 258, 279sh, 326, NaOMe/MeOH: 258sh, 278sh, 289, 365, AlCl3/MeOH: 258, 278sh, 326, AlCl3/HCl/ MeOH: 258, 279sh, 325, NaOAc/MeOH: 258, 275sh, 332, NaOAc/H3BO3/MeOH: 258, 275sh, 326. ESI-MS spectra m/z: 448, 446, 428, 285, 284, 257, 254, 253, 226,153,132, 129,124. EI-MS m/z (rel. int. %) for aglycone: 286 (100), 285 (24.6). Kaempferol 3-O--d-glucopyranoside (E-III.1). Yellow amorphous powder, 0.49 g, m.p. 175-177°C, Rf; 0.83; S2, and 0.43; S3, tR; 8.93 min. UV (max nm) MeOH: 267, 347, NaOMe/MeOH: 273, 322sh, 397, AlCl3/MeOH: 272, 300sh, 349, 397, AlCl3/HCl/MeOH: 273, 300sh, 349, 385sh, 397, NaOAc/MeOH: 273, 310sh, 366, NaOAc/H3BO3/ MeOH: 267, 343. EI-MS m/z (rel. int. %) for aglycone: 286, 285. Kaempferol-3-O--d-glucopyranoside (E-III.2). 0.06 g, Rf; 0.46, S1, showed to be identical with flavonoid

E-III.1 which was previously isolated and considered as an additional amount of it. Kaempferol (3,5,7-trihydroxy-2-[4-hydroxyphenyl]4H-1-benzopyran-4-one). (EIV. 1). Yellow amorphous powder, 0.38 g, m.p. 279-281°C, Rf; 0.79; S2, and 0.17; S3. UV (max nm) MeOH: 253sh, 266, 294sh, 350, NaOMe: 275, 325, 406 (dec.), AlCl3/MeOH: 274, 303sh, 353, 399, AlCl3/HCl/MeOH: 274, 303sh, 352, 398, NaOAc/MeOH: 274, 303, 397, NaOAc/H3BO3/MeOH: 266, 290sh, 320sh, 353. EIMS m/z (rel. int. %): 286 (100), 285 (33.56), 258, (5.57) 153 (4.97), 152 (0.6), 121 (14.97), 93 (4.59). Brine shrimp lethality bioassay Cyotoxic effect of total flavonoids, some flavonoid fractions, methanol extract, defatted methanol extract, of L. pyrotechnica were evaluated by LC50 values of the brine shrimp lethality test (Krishnaraju et al., 2006; Poli et al., 2006; Ho et al., 2005; Pisutthanana et al., 2004). The eggs of brine shrimp were obtained from San Francisco Bay Brand, Newark, New Jersey. The tested samples were dissolved in methanol, and three graded doses, 10, 100 and 1000 g/mL, respectively, were used for 5 mL of seawater containing 10 brine shrimp nauplii in each group. The number of survivors was counted in each well after 6 h. Counting of the chronic LC50 was begun after 24 h from the start of the test. LC50 was determined by Probit analysis described by Meyer et al. (1982). The experiment was carried out in five replicates, and mean LC50 values were measured. Control discs were prepared using only methanol. Five replicates were prepared for each dose level. The negative control solution was simply the same saline solution used to prepare the stock test sample solution. Potassium dichromate was used as standard toxicant and dissolved in artificial seawater, to obtain concentrations of 1000, 100, and 10 ppm. Antitumor screening: potato disc assay The potato disc bioassays for the plant extracts were carried out as described elsewhere (Ferrigni et al., 1982; Ferrigni & McLaughlin, 1984; Anderson et al., 1988, 1992; Bryant et al., 1994). Tumors were initiated on potato discs (usually Pontiac red or red Russet variations). Fresh, disease-free potato tubers were obtained from local markets. A. tumefaciens strain B6 was maintained on solid slants under refrigeration. Subcultures were grown in 0.8% nutrient broth (Difco) supplemented with 0.5% sucrose and 0.1% yeast extract. Controls were made in the following way: 0.5 mL of DMSO was filtered through Millipore filters (0.22 m) into 1.5 mL of sterile distilled water and added to tubes containing 2 mL of the same A. tumefaciens strain B6. A standard solution of camptothecin was made as follows:  8 mg of camptothecin was dissolved in 2 mL of DMSO. This solution was filtered

Isolation, structural elucidation of flavonoid constituents from Leptadenia pyrotechnica   545 through 0.22 m Millipore filters into a sterile tube. 0.5 mL from this solution was added to 1.5 mL of sterile water and 2 mL of the same broth culture of A. tumefaciens strain B6. A blank solution was made in the following way: 0.5 mL of DMSO was added to 1.5 mL of sterile water. Using a sterile disposable pipette, 1 drop (0.05 mL) from these tubes was used to inoculate each potato disc, spreading it over the disc surface. The medium was solidified as required with 1.5% agar (Difco, Detroit, USA). The results were expressed as ± percentages versus the number of tumors on the control discs; inhibition was expressed as a negative percentage and stimulation was expressed as a positive percentage. Significant activity was indicated when two or more independent assays gave consistent negative values of approximately 20% or greater inhibition.

Results and discussion Compound E-I.1 was identified as kaempferol-3-O--lrhamnopyranosyl (1999→699)-O--d-glucopyranoside. This evidence was supported by UV analysis with different diagnostic shift reagents. UV absorption spectrum of E-I.1 in methanol (MeOH) exhibited two absorption maxima at 357 nm (band-I, cinnamoyl system) and 255 nm (band-II, benzoyl system), which indicated that it was a ­flavonol-3-O-glycoside. The sodium methoxide (NaOMe)/MeOH solution of E-I.1 spectrum showed a bathochromic shift of 57 nm in band-I with increase in intensity and slow degeneration indicating the presence of 3, 49-dihydroxy flavone. Band-II appeared two peaks, designated IIa and IIb, with IIa being the peak at longer wavelength. Flavonols exhibited two absorption peaks, one maximum at 273 nm with a shoulder at 329 nm. This indicated the presence of 3,49-hydroxy groups. NaOMe/MeOH solution spectrum showed a shoulder peak at 329 nm which was an indicative to the presence of 7-hydroxy group. The addition of aluminum chloride (AlCl3) produced two peaks (Ib and Ia). The AlCl3/MeOH solution spectrum showed a bathochromic shift (42 nm) in band-I, indicated to the existence of 5-hydroxy group. AlCl3/HCl/MeOH solution spectrum exhibited slightly bathochromic shift (42 nm) in band-I relative to MeOH spectrum which confirmed the presence of 5-hydroxy group. This flavonoid followed Class II and group A based on the max AlCl3/MeOH value of 399 nm (Budavari, 2001; Eisenreichová et  al., 2004). According to the max AlCl3/HCl/MeOH value of band Ia (395 nm) and within group A, it followed the second category of flavones which had a maximum in the presence of AlCl3/HCl/MeOH at wavelengths greater than 395 nm. According to the shape, peak Ia at 400 nm reunites groups 14-19. In addition, band II in methanol (secondary band) was less than 271 nm. This was typical of

3-substituted flavone with a phloroglucinol type A-ring (group 14). The band II in methanol of 3-substituted flavones represented double peaks at 255 and 267 nm. These data were characterized to 3-substituted flavones. Sodium acetate (NaOAc)/MeOH spectrum showed a bathochromic shift (16 nm) in band-II which indicated the presence of a free 7-hydroxy group. Negative ionization FAB-MS spectra of compound E-I.1 exhibited a quasi molecular ion at m/z 593 and a fragment ion peak at 285 which were ascribed to [M-H]˙ and [M-H-146162]˙, suggesting the presence of two glycosyl moieties, hexose and deoxyhexose (glucose and rhamnose). The fragment ion peak of the aglycone at m/z 285 (24.6%) is in accordance with the molecular formula C15H9O6 of tetra-hydroxy substitution pattern. ESI-MS mass spectral data are in agreement with those reported for kaempferol (Eisenreichová et al., 2004; Tokuşoğlu et al., 2003; Hadizadeh et al., 2003). Hence, the fragmentation pathway undergoes the Retro-Diels Alder reaction giving rise to ring-A fragment at m/z 153 (59.32%) and 152 (10.53%). However, the hydrogen transfer ion at m/z 153 is much more intense than that of the normal fragment ion at m/z 152 which indicated to the presence of 5,7dihydroxy group. Furthermore, loss of CO directly from the molecular ion [M-H-146-162]˙-CO was also shown, leading to the phenylbenzofuran fragment ion at m/z 257 (11.6%), which further fragments giving rise to the benzoyl ion at m/z 121 (25.6%) and then m/z 93 (11.7%). 1 H-NMR spectrum showed the expected signals of a 1, 4-disubstituted B ring protons as two ortho-coupled resonances at  (ppm) 7.9 (2H, d, J = 8.7 Hz) and  6.80 (2H, d, J = 8.7 Hz) for H-29, H-69 and H-39, H-59, respectively. The presence of a 5,7-dihydroxy A-ring was deduced from the typical two meta-coupled resonances of H-8 and H-6 at  5.91 (1H, d, J = 2.1 Hz) and  5.72 (1H, d, J = 2.1 Hz), respectively (Makarevich, 1972; Bučková et al., 1988; Smolarz, 2002). The up-field shift of H-8 and H-6 resonances, relative to their normal positions ~6.4 and 6.2 ppm, respectively, was attributed to salt effect of an inorganic salt contamination from the plant extract (Kopp et al., 1996). The presence of a -glucopyranosyl moiety directly attached to the aglycone was detected from the relatively downfield -anomeric proton resonance at  5.12 (1H, d, J = 7.5 Hz). The terminal attachment of -rhamnopyranosyl moiety to C-699 on glucose was evidenced from the resonance of the anomeric proton H-1999 at  4.40 (1H, d, J = 1.5 Hz) and CH3-resonance at  0.98 (3H, d, J = 6.3 Hz). In addition, the value of  at 3.66-3.07(m, 10H) was assigned to the remaining rhamnosylglucosyl moiety, 10 protons. 1H-1H-chemical shift correlation (COSY) spectrum (Figure 6) showed the cross peaks which correlated H-29/69 and H-39/59, H-1″ and H-2″, H-1″9 and H-2″9, and H-59″ and -CH3 of compound E-I.1. Comparison of the 13C-NMR spectral data of compound E-I.1 with its aglycone, kaempferol, showed

546   A. M. Youssef Moustafa et al. 8

6

4

2

0

PPM

8 10

(H-2`/6`and H-3`/5`) (F-l.1a).

(H-2`/6`and H-3`/5`) (F-l.1).

6

(H-1```and H-2```)

(H-1``and H-2``)

4

(5```and -CH3)

2

0

PPM

10

Figure 6.  1H-1H-COSYT- experiment of component E-I.1.

an up-field shift of 2.6 ppm for C-3 signal which confirm the position of glycosylation at C-3 (Hamzah & Lajis, 1998; Komissarenko & Kovalev, 1988; Clause & Tyler, 1968; Bučková et  al., 1988). The (1″9→6″)-O-glycosidic linkage of the rhamnosyl on the glucoside moiety was evidenced from the fact that the C-6″ signal at 66.93 ppm was shifted downfield (6.33 ppm). This is related to the 60.60 ppm chemical shift of the corresponding carbon atom (C-6″9) of the terminal glucose (Bučková et  al., 1988). Moreover, the C-29″ signal of terminal rhamnose at 70.42 ppm was shifted up-field (1.18 ppm) of the corresponding carbon atom C-2″9 (71.60 ppm) of rhamnose directly attached with position-3 (Soliman et al., 2002). The configuration of the anomeric center of the rhamnopyranosyl moiety was determined to be  due to the

presence of anomeric carbon signal at 101.08 ppm in its 13C-NMR spectrum. Also, it is confirmed from small coupling constants (J = 1.5 Hz) for the anomeric proton signal of the rhamnosyl moiety in 1H-NMR spectrum (Mabry et al., 1970; Balbaa et al., 1976). The sugar moiety was identified as glucose and rhamnose after partial and complete hydrolysis of this compound and comparison with authentic references. Another analogue was observed from the 1H-NMR spectrum as a minor compound accompanying compound E-I.1. This compound E-I.1a which nearly has the same Rf value as compound E-I.1, was identified as kaempferol 49-methyl ether 3-O--d-rutinoside (kaempferide 3-O--d-rutinoside) on the basis of the comparison of its signals with the corresponding signals

Absorption

Isolation, structural elucidation of flavonoid constituents from Leptadenia pyrotechnica   547

1.000 0.95 0.90 0.85 0.80 0.75 0.70 0.65 0.60 0.55 0.50 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.000 199.5 220

MeOH MeOH + NaOMe

267.27 277.44

403.00

300.50 350.23 324.52

240

260

280

300

320

340

380

400

420

440

460

480 502.5

Absorption

Wavelength (nm)

1.000 0.95 0.90 0.85 0.80 0.75 0.70 0.65 0.60 0.55 0.50 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.000 199.5 220

MeOH MeOH + AICI3 MeOH + AICI3+ HCI

267.27 274.96 274.50 300.50

350.57 352.00

350.23 304.20 304.32

240

260

280

300 320 340 380 Wavelength (nm) Figure 7.  Continued.

393.04

400

398.00

420

440

460

480 502.5

548   A. M. Youssef Moustafa et al. 1.50 1.4

MeOH MeOH + NaOAc MeOH + NaOAc + H3BO3

1.3 1.2 1.1

266.98 274.96

Absorption

1.0 0.9

392.78

0.8 312.27

0.7 0.6

357.54 350.23

0.5 0.4

267.23

0.3 0.2 0.1 0.00 220.0 240

260

280

300

320

340

360

380

400

420

440

460

480 502.5

Wavelength (nm) Figure 7.  UV/Vis absorption spectra of compound E-I.2 in (A) MeOH and MeOH/NaOMe, (B) MeOH, MeOH/AlCl3 and MeOH/AlCl3/HCl and (C) MeOH, MeOH/NaOAc, and MeOH/NaOAc/H3BO3.

of compound E-I.1. The location of methoxy group at C-49 of the compound E-I.1a at  (ppm) 3.94 (3H, s) was suggested due to the downfield shift of the ortho douplet of H-39 and 59 relative to that of compound E-I.1. 1H-NMR spectral data showing the expected signals of the B ring protons as two ortho-coupled resonances at  7.99 (2H, d, J = 9 Hz) and  6.98 (2H, d, J = 9 Hz) for H-29/69 and H-39/59, respectively. The presence of 5,7dihydroxy A-ring was deduced from the typical two meta-coupled resonances of H-8 and H-6 at  5.88 (1H, d, J = 2.1 Hz) and  5.72 (1H, d, J = 2.1 Hz), respectively (Makarevich, 1972). The relative up-field shift of H-8 and H-6 resonances from the normal position was attributed to salt effect of an inorganic salt contamination from the plant extract (Kopp et al., 1996). The H-1″ of compound E-I.1a was overlapped with H-1″ of compound E-I.1. The presence of -glucopyranosyl moiety directly attached to the aglycone was detected from the relatively downfield -anomeric proton resonance at  5.05 (1H, d, J =7.5 Hz). The terminal attachment of a-rhamnopyranosyl moiety to C-6 on glucose was evidenced from the resonance of the anomeric proton H-1″9 at  4.37 (1H, d, J = 1.5 Hz) and CH3-protons at  0.98 (3H, d, J = 6.3 Hz). In addition the value of  3.66-3.07 (m, 10H) was assigned to rhamnosylglucosyl 10 protons. Moreover, 1H-1Hchemical shift correlation (COSY) spectrum (Figure 6) showed the cross peaks which correlated H-29/69 and H-39/59 resonances of compound E-I.1a. Hence the data

obtained from HPLC indicated that there are two components E-I.1 and E-I.2 have the retention time Rt ; 2.39 and 2.92 min, respectively, as shown in (Figure 2). Trials to separate each of them to get enough volume from compound E-I.1a by HPLC for carrying spectral analysis failed and the data obtained was confirmed by comparison of Rt values of authentic samples. Compound E-I.2 was identified as kaempferol-3-O-d-glucopyranosyl (19″→6″)-O--d-glucopyranoside. This evidence was supported by UV analysis with different diagnostic shift reagents. UV absorption spectrum in MeOH (Figure 7A-C), had two absorption maxima at 350 nm (band-I, cinnamoyl system) and 267 nm (band-II, benzoyl system), characteristic of flavonol skeleton. Addition of NaOMe solution produced a bathochromic shift of 52 nm in band-I with increase in intensity and slow degeneration pointing to substituted 3,49-positions. UV spectra with AlCl3, AlCl3/HCl and NaOAc/H3BO3 exhibited shifts that supported the presence of 7-hydroxy group and ruled out a dihydroxy system in either the A or B ring (Mabry et al., 1970; Nagy et al., 1984, 1986; Eisenreichová et al., 2004). The NIFAB-MS spectra (negative ion mode) of E-I.2, exhibited a quasi-molecular ion at m/z 609 and a fragment ion peak at m/z 285 which were ascribed to [M-H]˙ and [M-H-162-162]˙ suggesting the presence of two glycosyl moieties (two glucose). The fragment ion peak of the aglycone at m/z 285 is in accordance with the

Isolation, structural elucidation of flavonoid constituents from Leptadenia pyrotechnica   549 molecular formula C15H9O6 of tetra-hydroxy substitution pattern. The 13C-NMR spectral data of E-I.2 showed an upfield shift of 3.6 ppm for C-3 signal, and downfield shifts of 11.46 and 1.10 ppm for the C-2 and C-4 signals, respectively, in comparison with its aglycone (kaempferol). This pointed to the position of glycosylation at C-3 (Komissarenko & Kovalev, 1988; Clause & Tyler, 1968). (19″→6″)-O-glycosidic linkage was evidenced by the fact that the C-6″ signal at 67.03 ppm was shifted downfield (7.03 ppm), compared with the chemical shift value (60.00 ppm) of the corresponding carbon atom (C-6″9) of the terminal glucose (Clause, 1961). Moreover, the C-2″9 signal of terminal glucose at 74.41 ppm was shifted up-field (1.59 ppm) in compared to the corresponding carbon atom C-2″ (76.00 ppm) of glucose which directly attached to position-3 (Farnsworth, 1966). The sugar moiety was identified as two glucose after partial and complete hydrolysis of this compound and comparison with authentic references. EI-MS spectrum of the aglycone of compound E-I.2 showed a molecular ion (M+) at m/z 286 (100%) which corresponds to the molecular formula C15H10O6 of tetra-hydroxy substitution pattern. The mass spectral data are in agreement with those reported for kaempferol. Hence, the fragmentation pathway undergoes the Retro-Diels Alder reaction giving rise to ring-A fragment at m/z 153 and 152. The 1H-NMR spectrum showed the expected signals of 1, 4-disubstituted B ring protons as two ortho-coupled resonances at  (ppm) 8.04 (2H, d, J = 8.5 Hz) and  6.93 (2H, d, J = 8.5 Hz) for H-29, H-69 and H-39, H-59, respectively. The presence of a 5,7-dihydroxy A-ring was deduced from the typical two meta-coupled resonances of H-8 and H-6 at  6.45 (1H, d, J = 2.1 Hz) and  6.2 (1H, d, J= 2.1 Hz), respectively (Makarevich, 1972). The hydroxyl proton attached to C-3 appears as a singlet off set at  12.45 ppm due to Hbonded hydroxyl group. On the other hand, the other hydroxyl protons attached to C-7, C-49 and C-5 appeared at  9.35, 10.15, and 10.80 ppm, respectively, as singlet peaks which are exchangeable. The flavonoid constituents of fraction E-II (0.91 g) were fractionated into two fractions E-II.1 and E-II.2. Comparative TLC, PC and HPLC analysis of the major purified flavonoid compound E-II.1 (0.21 g, Rf; 0.63; S2) were shown to be identical with flavonoid compound E-I.2. The compound E-II.2 was identified as texasin ­7-O-glucoside. UV absorption spectrum in MeOH exhibited two characteristic absorption maxima of isoflavone at 326 nm (band-I, cinnamoyl system) and 258 nm (band-II, benzoyl system). Moreover, it gave the same absorbency in accordance with texasin 7-O-glucoside with different diagnostic shift reagents (Kaushal & Bhatia, 1982). The ESI-MS spectra exhibited a quasi molecular ion at m/z 448 and a fragment ion peak at 285 which were ascribed to [M+1]+ and [M-162]+ suggesting the presence of one glycosyl moiety (hexose). The fragment ion peak of the aglycone at m/z

285 is in accordance with the molecular formula C16H13O5 of dihydroxy and one methoxy substitution pattern. The mass spectral data are in agreement with those reported for texasin 7-O-hexoside (Kaushal & Bhatia, 1982). Hence, the fragmentation pathway underwent the Retro-Diels Alder reaction giving rise to ring-A fragment at m/z 153 and m/z 132, and there were further fragments giving rise to m/z 124. Furthermore, loss of H3CO- directly from the molecular ion [M-162]+ was also shown, leading to the fragment ion at 254, which lost CO giving m/z 226. In addition, loss of -CO directly from the molecular ion [M-162]+ and gave m/z 257 and m/2z 129. The complete hydrolysis of this compound revealed the presence of glucose as the sugar moiety and the aglycone was identified as texasin by comparison with authentic references. Fraction E-III was fractionated into two fractions E-III.1 and E-III.2. The compound E-III.1 was identified as kaempferol 3-O--d-glucopyranoside. UV absorption spectrum in MeOH exhibited two absorption maxima at 347 nm (band-I, cinnamoyl system) and 267 nm (band-II, benzoyl system), which indicated that it was a flavonol3-O-glycoside. Moreover, it gave the same absorbency in accordance with kaempferol 3-O--d-lucopyranoside with different diagnostic shift reagents (Eisenreichová et al., 2004; Skrzypczakova, 1967; Heneidak et al., 2006). The complete hydrolysis of this compound revealed the presence of glucose as the sugar moiety and the aglycone was identified as kaempferol by comparison with authentic references. The EI-MS spectra of aglycone E-III.1 exhibited a molecular ion at m/z 286 and a fragment ion peak at 285, which were ascribed to M+ and [M-H]+. These EI-MS data are in accordance with the presence of kaempferol as an aglycone (Nagy et al., 1984, 1986; Tokuşoğlu et al., 2003; Hadizadeh et al., 2003). Comparative TLC and PC of purified major flavonoid compound E-III.2 (kaempferol-3- O--dglucopyranoside), 0.06g, Rf; 0.46, S1, showed to be identical with flavonoid compound E-III.1 previously isolated and was considered as an additional amount of it. Compound E-IV.1 was identified as kaempferol (3,5,7trihydroxy-2-[4-hydroxyphenyl]-4H-1-benzopyran-4one). UV absorption spectrum in methanol exhibited two absorption maxima at 350 nm (band-I, cinnamoyl system) and 253sh and 266 nm (band-II, benzoyl system), which indicated that it was a flavonol. E-IV.1 gave the same absorbency in accordance with kaempferol with different diagnostic shift reagents (Eisenreichová et al., 2004; Budavari, 2001). The mass spectrum (EI-MS) showed a molecular ion (M+) at m/z 286 (100%) and a fragment ion (M+-1) at 285 which corresponds to the molecular C15H9O6 of tetrahydroxy substitution pattern. The mass spectral data are in agreement with those reported for kaempferol

550   A. M. Youssef Moustafa et al. (Eisenreichová et  al., 1985; Tokuşoğlu et  al., 2003; Hadizadeh et al., 2003). The flavonoid pattern of our studied L. pyrotechnica is more or less close to those of the related Leptadenia species and Asclepiadaceae. But, the compound E-II.2 (texasin 3-O-glucoside) was not isolated from this family before. Concerning quercetin 3-O-galactoside, it was isolated before from L. pyrotechnica (Gibbs, 1974) while not detected in our study. On the contrary, the other flavonoids isolated from this species in our study were detected for the first time. LC50 determinations The results obtained for all tested samples from the brine shrimp toxicity test were recorded and represented in

Table 1. The data of mortality rates point out that with a concentration 1,000 ppm the methanol extracts exhibited high mortality (100%). While the other extracts, defatted methanol and ethyl acetate (flavonoids), represented 98.91% and 90.59%, respectively. Moreover, with a concentration of 100 ppm, the extracts, methanol, defatted methanol, exhibited high mortality, and represented 88 and 82.35%, respectively. Ethyl acetate (flavonoids) represented a lower percentage, 52.24%. On the other hand, with a concentration of 10 ppm, ethyl acetate (flavonoids) represented the lowest percentage, 10.98%, but the other extracts were not detected. Also, the different fractions of flavonoids were detected using 1000 ppm concentration only, while 100 and 10 ppm were not detected. The mortality rates recorded in Table 1 point out that, with a concentration of 1000 ppm, fractions E-I

Table 1.  Mortality of brine shrimp at various concentration of the different extracts of Leptadenia pyrotechnica. Dosage log Accumulated Accumulated Ratio Dead: Plant extract Dose ppm dose Dead Alive Dead Alive Total Mortality % MeOH extract 1000 3 50 0 94 0 94/94 100 100 2 44 6 44 6 44/50 88 10 1 – – – – – – MeOH extract after 1000 3 49 1 81 1 81/82 98.78 defatting 100 2 32 18 32 19 32/51 62.74 10 1 – – – – – – Dichloromethane 1000 3 50 0 50 0 50/50 100 extract 100 2 – – – – – – 10 1 – – – – – – EtOAc extract 1000 3 42 8 70 8 70/78 89.74 (Flavonoids) 100 2 23 27 28 35 28/63 44.44 10 1 5 45 5 80 5/85 5.88 Flavonoid fraction 1000 3 – – – – – – E-I (11–13 ) 100 2 16 34 16 34 16/50 32 10 1 – – – – – – Flavonoid fraction 1000 3 – – – – – – E-II (14–17 ) 100 2 15 35 15 35 15/50 30 10 1 – – – – – – Flavonoid fraction 1000 3 – – – – – – E-III (18–19 ) 100 2 30 20 30 20 30/50 60 10 1 – – – – – – Flavonoid fraction 1000 3 – – – – – – E-I.1 ( 21–32 ) 100 2 13 37 13 37 13/50 26 10 1 – – – – – – Flavonoid fraction 1000 3 – – – – – – E-I.2 (33–38 ) 100 2 46 4 46 4 46/50 92 10 1 – – – – – – Flavonoid fraction 1000 3 – – – – – – E-II.1 ( 11–14 ) 100 2 37 13 37 13 37/50 74 10 1 – – – – – – Flavonoid fraction 1000 3 – – – – – – E-II.2 (15–16 ) 100 2 37 13 37 13 37/50 74 10 1 – – – – – – Flavonoid fraction 1000 3 – – – – – – E-IV (20–25) 100 2 33 17 33 17 33/50 66 10 1 – – – – – –

LD50 ppm –





101.74

















Isolation, structural elucidation of flavonoid constituents from Leptadenia pyrotechnica   551 and E-I.1 represented the highest percentages of mortality, 92% and 88%, while the other fractions, E-I.2, E-II, E-III, E-III.1, E-II.1, and E-IV, represented 76%, 74%, 74%, 66%, 64%, and 60%, respectively. The results of acute median lethal concentrations LC50 of some extracts were calculated using the Reed-Muench method. The results obtained (Table 1) revealed that the higher LC50 valuse were recorded in flavonoids and exhibited less toxicity compared with the other detected extracts, which represented 84.14 ppm. The estimated LC50 and its 95% confidence limits of flavonoids were 84.14 (45.85-154.41). It is probable that with a concentration of 100 ppm, the high percentages of mortality rates of fractions E-I, E-I.1 and E-I.2 (92%, 88%, and 76%, respectively) may be attributed to the long glycosidic moiety in the structure of these flavonoids. Results of potato disc assay The results obtained from the potato disc assay showed that the methanol and ethyl acetate extracts have the most activity as antitumor agent which represented -49.30 and -43.20%, respectively. The relative antitumor activity of L. pyrotechnica could be attributed mainly to its flavonoid constituents. On the other hand, with this procedure, crown gall tumors on potato discs could routinely be employed as a comparatively rapid, inexpensive, safe and statistically reliable pre-screen for antitumor activity. The methodology is simple and the assay can be performed in-house, with minimal technical training and equipment, to detect potentially useful compounds and active extracts.

Acknowledgements The authors are grateful to the Environmental Chemistry and Toxicology Laboratory, Texas Southern University, Houston, Texas, USA for the Fellowship that was provided to undertake this work.

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