Phenolics of selected species of Persicaria and Polygonum (Polygonaceae) in Egypt

2.1 General

UV spectra were recorded on Shimadzu, model 2401. EIMS spectra were carried out on Finnigan-Mat SSQ 7000 spectrometer. ESIMS spectra on Micromass Quattro-LC triple quadruple mass spectrometer equipped with a Z-Spray electro-spray ion source while NMR measurements were carried out using Jeol EX-500 spectroscopy; 500 MHz (¹H NMR) and 125 MHz (¹³C NMR) and Joel JNM-EX 270 spectroscopy; 270 MHz (¹H NMR) and 67.5 MHz (¹³C NMR). Sugars of O-glycosides were identified by enzymatic hydrolysis (β-glucosidase and β-galactosidase) or acid hydrolysis followed by co-chromatography with reference standards. C-glycoside flavonoids were determined using ferric chloride degradation (Mabry et al., 1970). Authentic samples were obtained from the department of phytochemistry and plant systematics, NRC.

2.2 Plant material

Four selected plant species were collected from different localities representing Egyptian taxa of the family Polygonaceae viz Persicaria salicifolia (Brouss. ex Willd.) Assenov, P. senegalensis (Meisn.) Soják, Polygonum bellardii All. and P. equisetiforme Sm. The investigated taxa were identified according to Boulos (1999), and voucher specimens were deposited in the herbarium of the National Research Centre (CAIRC). Details of the collected samples are given below, with an asterisk (*) indicating herbarium samples: P. salicifolia (Brouss. ex Willd.) Assenov, Ismailia Canal and Nile-Delta region 15 Nov. 2008, leg. S.R. Hussein and U.I.A. EL-Magly, s.n.623. P. senegalensis (Meisn.) Soják El-Kanater, Nile-Delta region 2 Oct. 2007, leg. S.R. Hussein and U.I.A. EL-Magly, s.n.532. P. bellardii^∗ All. Shakshouk near lake Qarun along a canal 24 Mar. 1981, leg. L. Boulos s.n.1725 and P. equisetiforme Sm. El-Arish & Rafah Desert Road. 25 Apr. 2007 leg. S.R. Hussein and U.I.A. EL-Magly, s.n.513.

2.3 Extraction and isolation

The aerial parts of P. senegalensis and P. equisetiforme were dried in the shade and ground. Each powder was extracted twice with 70% methanol. The extract was filtered and concentrated then partitioned with solvents of increasing polarity (petroleum ether, diethyl ether, chloroform, acetone, ethyl acetate, methanol and water). Isolation of flavonoids was carried out using column chromatography (MN-polyamide 6S) and paper chromatography (Whatman No. 1 and 3MM) using butanol–acetic acid–water 4:1:5 (BAW upper phase), water and 15% AcOH (water–acetic acid 17–3), (Harborne, 1967; Mabry et al., 1970). The compounds were further purified on a Sephadex LH-20 column with standard solvent systems (Markham, 1982). Trace compounds were identified by co-chromatography with authentic samples. Details of the isolated compound are outlined below.

3.1 Botanical investigations

3.2 Chemical investigations

Seventeen compounds (14 flavonoids and 3 phenolic acids) were isolated from the investigated taxa and identified using chemical and physical investigations. Ten compounds were isolated from P. senegalensis and identified as: Apigenin (5,7,4′-trihydroxyflavone) (1), 3,6-dimethoxy-kaempferol (2), 3,7,4′-trimethoxy-kaempferol (3), calycopterin (5,4′-dihydroxy-3,6,7,8-tetramethoxy-flavone) (4), quercetin (3,5,7,3',4'-pentahydroxy-flavone) (5), quercetin-3-O-glucopyranoside (6), quercetin-3-methoxy-3′-O-glucopyranoside (7), 5-hydroxy-7-methoxy-isoflavone (8), gentisic acid-5-O-(6′-O-galloyl)-glucopyranoside (9) and gentisic acid-5-O-(2′-O-glucopyranosyl)-rhamnoside (10).

Eight compounds were isolated from P. equisetiforme and identified as: quercetin-3-O-glucopyranoside (6), Apigenin-6-C-arabinopyranosyl-8-C-glucopyranoside (11), apigenin-6,8-di-C-glucopyranosyl (12), quercetin-3,7-di-O-glucopyranoside (13), rhamnetin-3-O-rhamnopyranoside (14), myricetin (3,5,7,3',4',5'-pentahydroxy-flavone) (15), myricetin-3-O-glucopyranoside (16) and gallic acid (17).

Compound 10 (Fig. 1) which was isolated from P. senegalensis and identified as gentisic acid-5-O-(2′-O-glucopyranosyl)-rhamnoside, is a new compound identified for the first time in nature. Six compounds 2, 3, 4, 7, 8 and 9 were isolated for the first time from the plant and the remaining three compounds were previously isolated from the same plant by Midiwo and Owuor (1992), Midiwo et al. (1992, 1994, 2002). Compounds 6, 11, 12, 13, 14, 15, 16 and 17 from P. equisetiforme were isolated for the first time from the plant. The distribution of these compounds was shown in Table 1.

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Figure 1

Structure of compound 10.

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Compound 10 was isolated from the H₂O fraction as white crystals after separation and purification on a Sephadex LH-20 column. Complete acid hydrolysis of 10 afforded rhamnose and glucose as sugar moiety, identified by co-chromatography with authentic samples. The ESI-MS of 10 showed the molecular ion peak as the base peak [M+H]⁺ at 463 m/z which is equal to the Mt 462 corresponding to the molecular formula C₁₉H₂₆O₁₃. ¹H NMR spectrum of 10 showed signals at δ 7.57, 6.87 and 6.52 assigned for H-6, H-4 and H-3, respectively, which is equivalent to gentisic acid protons (Sakushima et al., 1995). In addition, there is a broad signal at δ 5.41 for the rhamnose sugar and a doublet signal at δ 4.59 for the anomeric sugar protons with J = 8.6 Hz which is consistent for the glucose moiety. The ¹³C NMR spectrum confirmed that 10 is gentisic acid (Sakushima et al., 1995) with rhamnose and glucose sugar. The C-2′ of rhamnose was shifted downfield from 71 to 82.1 ppm and the upfield shift of C-1′ from 105 to 102.5 ppm, indicated that the interglycosidic linkage between the glucose and rhamnose is glucosyl (1″ → 2′) rhamnoside (Agrawal, 1989). From the above data, compound 10 was identified as gentisic acid-5-O-(2′-O-glucopyranosyl)-rhamnoside.

3.3 Chemosystematics

P. salicifolia and P. senegalensis are characterized by the presence of 3,7,4′-trimethoxy-kaempferol, 5-hydroxy-7-methoxy-isoflavone and gentisic acid glycosides. These compounds were not detected in the Polygonum species while quercetin 3,7-di-O-β-glucopyranoside and apigenin-6-C-α-arabinopyranosyl-8-C-β-glucopyranoside are present in P. salicifolia and P. equisetiforme. In addition, no methylated flavonoids were detected in P. bellardii and only one methylated flavonoid (rhamnetin-3-O-α-rhamnopyranoside) is detected in P. equisetiforme. P. equisetiforme is also characterized by the capability to form 3,4,5-trihydroxy nuclei in the form of myricetin and gallic acid.

The distribution of methylated flavonoids within the genus Persicaria in the present study agrees with the observations that methylated flavonols were detected in some species of sections Echinocaulon and Persicaria (Kuroyanagi and Fukushima, 1982; Kawasaki et al., 1986; Park, 1987). Furthermore; and according to Harborne (1977) and Kawasaki et al. (1986); methylation of flavonoids hydroxyl groups represents a more advanced step in the biosynthesis of flavonoids.

3.4 Numerical analysis

In order to determine the position of Persicaria compared to Polygonum, a dataset for numerical analysis was constructed. These data included 111 macro-, micromorphological and chemical characters. Each character used in the numerical analysis was treated as a binary character in data matrix using Minitab statistical software 13 (Minitab Inc., 1999).The dendogram formed based on the numerical analysis is illustrated in Fig. 2. At similarity level of 35.91%, the applied operational taxonomic units were separated into two main groups. Group (I) includes P. salicifolia and P. senegalensis while group (II) includes P. bellardii and P. equisetiforme. The taxa of group (I) separated at similarity level of 78.22% while the taxa of group (II) separated at similarity level of 66.76%. The grouping of Persicaria species and Polygonum species under investigation in two distinct groups is evidence for the treatment of Persicaria and Polygonum as two distinct entities.

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Figure 2

Dendrogram for the morphological, anatomical and chemical characters.

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