Leaf flavonoid glycosides as chemosystematic characters in Ocimum

Biochemical Systematics and Ecology 30 (2002) 327–342 www.elsevier.com/locate/biochemsyseco

Leaf flavonoid glycosides as chemosystematic characters in Ocimum Rene´e J. Grayer a,*, Geoffrey C. Kite a, Nigel C. Veitch a, Maria R. Eckert a, Petar D. Marin 1,a, Priyanganie Senanayake b, Alan J. Paton c a

Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3DS, UK b Department of Botany, University of Kelaniya, Kelaniya, Sri Lanka c Herbarium, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3DS, UK Received 12 February 2001; accepted 26 April 2001

Abstract Thirty-one accessions of nine species belonging to three subgenera of Ocimum (basil, family Lamiaceae) were surveyed for flavonoid glycosides. Substantial infraspecific differences in flavonoid profiles of the leaves were found only in O. americanum, where var. pilosum accumulated the flavone C-glycoside, vicenin-2, which only occurred in trace amounts in var. americanum and was not detected in cv. Sacred. The major flavonoids in var. americanum and cv. Sacred, and also in all other species investigated for subgenus Ocimum, were flavonol 3-O-glucosides and 3-O-rutinosides. Many species in subgenus Ocimum also produced the more unusual compound, quercetin 3-O-(6⬙-O-malonyl)glucoside, and small amounts of flavone O-glycosides. The level of flavonol glycosides produced was reduced significantly in glasshouse-grown plants, but levels of flavone glycosides were unaffected. A single species investigated from subgenus Nautochilus, O. lamiifolium, had a different flavonoid glycoside profile, although the major compound was also a flavonol O-glycoside. This was identified as quercetin 3-O-xylosyl(1⵮→2⬙)galactoside, using NMR spectroscopy. The species investigated from subgenus Gymnocimum, O. tenuiflorum (=O. sanctum), was characterised by the accumulation of flavone O-glycosides. These were isolated, and identified as the 7-O-glucuronides of luteolin and apigenin. Luteolin 5-O-glucoside was found in all nine species of Ocimum studied, and is considered to be a key character for the genus.  2002 Elsevier Science Ltd. All rights reserved. * Corresponding author. Tel: +44-181-332-5312; Fax: +44-181-332-5310. E-mail address: [email protected] (R.J. Grayer). 1 Present address: Faculty of Biology, Botanical Institute and Garden, University of Belgrade, Studentski trg 16, 11000 Belgrade, Yugoslavia. 0305-1978/02/$ - see front matter  2002 Elsevier Science Ltd. All rights reserved. PII: S 0 3 0 5 - 1 9 7 8 ( 0 1 ) 0 0 1 0 3 - X

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Keywords: Ocimum; Basil; Lamiaceae; Flavonoid glycosides; Luteolin 5-O-glucoside; Quercetin 3-O-(6⬙O-malonyl)glucoside; Quercetin 3-O-xylosyl(1⬙⬘→2⬙)galactoside; Chemosystematics; Infraspecific chemical variation.

1. Introduction The genus Ocimum L. (family Lamiaceae) comprises several important economic, culinary and medicinal herbs, the most well known species being O. basilicum L., sweet basil. Several authors, including Pushpangadan and Bradu (1995), recognise more than 150 species in the genus. However, most of these taxa are based mainly on characters of leaf and habit, which are very variable. For instance, the shape of the leaves in O. basilicum and its close relatives varies from small and liniform to large and round. The colours of the leaves vary from yellow-green to grey-green, red or almost black. The essential oil profiles of this group of plants are also very variable, such that plants belonging to the same species may have different scents, and on this basis may have been described as different species. However, Paton et al. (1999) proposed that only 65 species of Ocimum should be retained, based on an extensive taxonomic study of plant material collected in Africa, Asia, and South America, and that the remainder of the names should be considered as synonyms. Some of the species closely related to O. basilicum, but still recognised as separate species, are O. kilimandscharicum Baker ex Gu¨ rke, O. minimum L., O.×citriodorum Vis. and O. americanum L. All these species belong to subgenus Ocimum, section Ocimum. The circumscription of the genus Ocimum is also problematic. In a recent cladistic analysis of species of Ocimum and closely related genera based on morphological and pollen characters, Paton et al. (1999) found that Ocimum is only monophyletic if Erythrochlamys, Becium and Orthosiphon subgenus Nautilus are incorporated into it. Recognition of any of these taxa as separate genera would make Ocimum paraphyletic and thus the genus Ocimum was extended to include them. Two types of flavonoids are present in Ocimum. Lipophilic flavonoid aglycones (external flavonoids), often highly methylated, are found in glandular hairs on the surface of the leaves, stems and inflorescences. These have been the subject of two recent studies (Grayer et al., 1996a, 2001). The second type of flavonoids are polar flavonoid glycosides, which are stored in the vacuoles of aerial plant parts. The aim of the present study was to determine whether flavonoid glycosides can be used as characters at different levels of classification in the genus Ocimum (i.e. to distinguish species, and to characterise sections or subgenera). In order to compare chemical profiles of species, it is essential to establish whether any infraspecific chemical variation occurs. For instance, five different essential oil profiles were found in O. basilicum (Grayer et al., 1996b), which makes it difficult to use these compounds as systematic characters to distinguish this species from related ones. External flavonoid profiles are much less variable in this species (Grayer et al., 1996a), in contrast to those in O. americanum, which vary strongly (Grayer et al., 2001). The present study describes the distribution and infraspecific variation of vacuolar flavonoid glycosides

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in a number of Ocimum species. All three subgenera of Ocimum are represented; subgenus Ocimum (sections Ocimum, Gratissima and Hiantia), subgenus Nautochilus, and subgenus Gymnocimum.

2. Materials and methods 2.1. Plant material Varieties and cultivars of Ocimum species, except for two specimens of O. tenuiflorum from Sri Lanka, were grown for several months under glasshouse conditions at the Royal Botanic Gardens, Kew, after germination of the seeds in early spring. The plant accessions are listed in Table 1. In summer the young plants were transferred outside to expose them to natural sunlight for at least two months, with the exception of one specimen each of O.×citriodorum (Kew 1996-917/BI 4405) O. gratissimum L. (Kew 1993-1040/B14406), and O. selloi Benth. (Kew 1987-2003/BI 4409) which were left in the glasshouse. These specimens were used to compare possible differences in flavonoid production between glasshouse-grown plants and those grown outside. Plants were harvested for chemical investigation at the end of the summer. Leaves were detached from the stems, frozen at ⫺20°C and later freezedried. One or two flowering stems of each accession were pressed for voucher specimens and deposited in the Herbarium, Royal Botanic Gardens, Kew. Two accessions of O. tenuiflorum L. (=O. sanctum L.) were collected in 1996 from the wild in Kurunegala, Sri Lanka, and were air-dried. Vouchers have been deposited in the Herbarium of the Department of Botany, University of Kelaniya, Sri Lanka (Kln 768 and Kln 782). Specimens used for the isolation of compounds are indicated in Table 1. 2.2. Extraction 200 mg of freeze- or air-dried material was roughly pulverised in a test tube with a glass rod and 5 ml of 80% aqueous MeOH were added. The test tubes were heated in a water bath at 90°C until the solution had boiled for two minutes. The tubes were then cooled, and the leaf material was left to extract in the 80% MeOH solution for 20–24 h at room temperature. Subsequently, the extracts were filtered and evaporated in vacuo at 40°C. For the isolation of flavonoid glycosides, larger amounts of plant material were extracted in a similar way in conical flasks. Typically 20 g of dried pulverised leaves were used, extracting with either 80% MeOH or 70% EtOH. 2.3. Two-dimensional paper chromatography (2-D PC) Extracts for analysis were redissolved in 1 ml 80% MeOH and applied as 8–10 spots of c. 2 µl in the corner of quarter sheets of Whatman No. 1 chromatography paper. The chromatograms were run in descending mode in BAW (n-BuOH, HOAc, H2O=4:1:5; v/v; upper layer) for the first dimension, and in 15% aqueous HOAc for

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Table 1 Accession numbers of Ocimum species used for flavonoid studies Species name

Accession number

Remarks

O. americanum var. americanum Kew 1996-913/BI 4396 O. americanum cv Sacred Kew 1998-2105/BI 6442 O. americanum var. pilosum Kew 1996-914/BI 6419 O. basilicum var. basilicum O. basilicum var. basilicum O. basilicum var. basilicum O. basilicum var. basilicum O. basilicum var. basilicum O. basilicum var. basilicum Intermediate between var. basilicum and var. purpurascens O. basilicum var. purpurascens O. basilicum var. purpurascens O. basilicum var. difforme O.×citriodorum O.×citriodorum O.×citriodorum O. gratissimum

NY NY NY NY NY NY NY

B B B B B B B

17/BI 2746 122/BI 2747 130W/BI 2748 145-BI 2749 147-97/BI 2750 SW/BI 2751 130P/BI 2752

O. gratissimum O. gratissimum O. kilimandscharicum

Kew 1993-1040/BI 4406 Kew 1993-1040/BI 5783 Kew 1996-916/BI 6420

O. lamiifolium

Kew 1997-6521/BI 6200

O. minimum

Kew 1995-3474/BI 4394

O. O. O. O. O.

minimum minimum selloi selloi tenuiflorum (=O. sanctum)

Kew Kew Kew Kew Kew

O. O. O. O.

tenuiflorum tenuiflorum tenuiflorum tenuiflorum

Kew 1997-5919/BI 5592 Kew 1997-5922/BI 6447 Kln 768/BI 9005 Kln 782/BI 9006

NY B 74/BI 2753 NY B 76/BI 2754 NY B 203/BI 2756 Kew 1996-917/BI 4392 Kew 1996-917/B 4405 Kew 1998-2104/BI 6446 Kew 1997-1387/BI 5583

1998-2097/BI 1998-2096/BI 1987-2003/BI 1987-2003/BI 1995-3473/BI

6443 6444 4409 6421 5789

Grown outside Grown outside Grown outside; also used for isolation work Grown outside Grown outside Grown outside Grown outside Grown outside Grown outside Grown outside Grown outside Grown outside Grown outside Grown outside Glasshouse-grown Grown outside Grown outside; also used for isolation work Glasshouse-grown Grown outside Grown outside; also used for isolation work Grown outside; also used for isolation work Grown outside; also used for isolation work Grown outside Grown outside Glasshouse-grown Grown outside Grown outside; also used for isolation work Grown outside Grown outside Collected from the wild; air-dried Collected from the wild; air-dried

the second. After drying, the chromatograms were viewed under UV light at 360 nm, and again after fuming with NH3 vapour. Colours of spots were recorded and Rf-values calculated. 2.4. Preparative paper chromatography (PPC) Prior to HPLC analysis, crude extracts were partially purified by PPC to remove rosmarinic acid and caffeic acid from the flavonoid fraction. Each extract was applied

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as a narrow band at the top of a quarter sheet of Whatman No. 3 chromatography paper, and the papers were run in descending mode in BAW. After drying the chromatograms and viewing them in UV light, the bottom quarter of each paper (containing rosmarinic and caffeic acids, which have high Rf-values in BAW and fluoresce blue under UV light) was removed. The remainder of the paper containing the flavonoid glycosides was cut into 1 cm2 pieces, which were eluted in 80% MeOH. After 24 h the eluate was filtered, evaporated and redissolved in 1 ml 80% MeOH, for HPLC analysis. PPC was also used for the purification and isolation of flavonoid glycosides. The crude extracts were applied onto whole sheets of Whatman No. 3 paper and developed in BAW. The bands containing flavonoids were cut out, eluted and subjected to a second stage of PPC using 15% HOAc. The purity of the flavonoids in the fractions was monitored by HPLC. 2.5. Analytical and semi-preparative HPLC with diode array detection (HPLCDAD) The HPLC system consisted of a Waters LC 600 pump and 996 photodiode array detector. Merck LiChrospher 100RP-18 (5 µm) columns were used; 4.0 mm (i.d.)×250 mm for analytical work and 10.0 mm (i.d.)×250 mm for semi-preparative isolations. Gradient profiles based on two solvents, denoted A and B, were employed. For analytical work A was 2% aqueous HOAc and B was MeOH–HOAc–H2O, 18:1:1, and for semi-preparative work A was H2O and B was MeOH. Initial conditions were 75% A, 25% B, with a linear gradient reaching B=100% at t=20 min. This was followed by isocratic elution (B=100%) to t=24 min, after which the programme returned to the initial solvent composition. Column temperature was maintained at 30°C and flow rates of 1.0 ml min⫺1 and 4.5 ml min⫺1 were used for analytical and semi-preparative HPLC, respectively. For analytical HPLC, 20 µl injections were made by autosampler. Retention times and UV spectra of flavonoids were compared with those of standards. 2.6. HPLC with atmospheric pressure chemical ionisation mass spectrometry (APCI LC-MS) For APCI LC-MS, chromatography was performed in a similar manner to analytical HPLC except that the concentration of acetic acid in the mobile phases was 1%. Mass spectra were recorded using a quadrupole ion trap mass spectrometer (Finnigan LCQ) with the sample being ionised by an APCI source using a vaporiser temperature of 550°C, sheath and auxillary nitrogen flow pressures of 80 and 20 psi, respectively, a capillary temperature of 150°C and a needle current of 5 µA. The mass spectrometer was controlled by Xcalibur 1.1 software (Finnigan) which was programmed to record the first order mass spectra and then the collision induced dissociation (CID) spectra of three or four of the most intense ions in each first order spectrum by means of the dynamic exclusion facility. CID spectra were obtained by prior isolation of the parent ion in the trap (isolation width 5 amu) and then applying a collision energy of 45% (without wideband activation). Dynamic exclusion allowed

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the CID spectra of co-eluting compounds to be recorded automatically by the instrument without prior knowledge of their molecular masses. 2.7. NMR spectroscopy NMR spectra were acquired using a Varian 500 MHz instrument. Samples were dried carefully, dissolved in DMSO-d6, and referenced to the residual solvent resonance at dH 2.50 ppm or dC 39.50 ppm as appropriate. All experiments were carried out at 37°C. 2.8. Identification of the flavonoids After isolation, the main flavonoid glycosides (1–8) from the leaves of Ocimum species were examined by UV spectroscopy with shift reagents (Mabry et al., 1970), acid hydrolysis and chromatographic analysis of the aglycones and sugars (Harborne, 1998), and APCI LC-MS (Grayer et al., 2000). On the basis of these procedures and by comparison with authentic standards, 1 (from O. americanum var. pilosum BI 6419) was identified as apigenin 6,8-di-C-glucoside (=vicenin-2), 6 (from O. kilimandscharicum BI 6420) as quercetin 3-O-rutinoside (=rutin), and 10 (from O. tenuiflorum BI 5789) as apigenin 7-O-glucuronide. Compounds 2, 3, 4, 7 and 8 were purified further using semipreparative HPLC and analysed by NMR spectroscopy. This enabled 2 (isolated from O. lamiifolium BI 6200) to be identified as quercetin 3-O-xylosyl(1⵮→2⬙)galactoside; 3 (from O. kilimandscharicum BI 6420) as luteolin 5-O-glucoside (=galuteolin); 4 (from O. tenuiflorum BI 5789) as luteolin 7-O-glucuronide; 7 (from O. minimum BI 4394) as quercetin 3-O-glucoside (=isoquercitrin); and 8 (also from O. minimum BI 4394) as quercetin 3-O-(6⬙-O-malonyl)glucoside. A further six compounds (5, 9, 11–14) were tentatively identified using data from HPLC-DAD and APCI LC-MS (Grayer et al., 2000) as summarised in Table 2. Quercetin 3-O-xylosyl(1⵮→2⬙)galactoside (2) has been identified previously in a range of species, including Armoracia rusticama (Larsen et al., 1982), Leuconotis eugenifolius (Abe and Yamauchi, 1994) and Saxifraga spp. (Miller, 1980; Chevalley et al., 1999) but only 13C NMR assignments have been presented (solvent not stated) (Larsen et al., 1982). A complete set of 1H NMR assignments for 2 was obtained in the present study using standard procedures. Quercetin 3-O-β-D-xylopyranosyl(1⵮→2⬙)-β-D-galactopyranoside. 1H NMR (500 MHz, DMSO-d6, 37°C): d12.68 (1H, br s, 5-OH), 7.72 (1H, dd, J=8.5, 2.4 Hz, H6⬘), 7.50 (1H, d, J=2.4 Hz, H-2⬘), 6.79 (1H, d, J=8.5 Hz, H-5⬘), 6.31 (1H, br s, H8), 6.11 (1H, br s, H-6), 5.68 (1H, d, J=7.6 Hz, H-1⬙), 4.56 (1H, d, J=7.3 Hz, H1⵮), 3.76 (1H, dd, J=9.2, 7.6 Hz, H-2⬙), 3.71 (1H, dd, J=11.3, 4.9 Hz, H-5⵮), 3.69 (1H, br d, J=3.7 Hz, H-4⬙), 3.61 (1H, dd, J=9.5, 3.4 Hz, H-3⬙), 3.43 (1H, dd, J=10.4, 5.8 Hz, H-6⬙), 3.35 (1H, m, H-3⵮), 3.29 (1H, m, H-6⬙), 3.28 (1H, m, H-4⵮), 3.15 (1H, m, H-2⵮), 3.08 (1H, m, H-5⬙), 3.05 (1H, dd, J=11.3, 10.1 Hz, H-5⵮). 13C NMR (125 MHz, DMSO-d6, 37°C) (assignment of non-quaternary C atoms by HSQC): d122.0 (C-6⬘), 115.3 (C-2⬘), 114.9 (C-5⬘), 104.3 (C-1⵮), 98.6 (C-6), 98.1 (C-1⬙), 93.4

Vicenin-2

Quercetin 3-Oxylosyl(1⬙⬘→2⬙)galactoside Luteolin 5-O-glucoside Luteolin 7-O-glucuronide Luteolin 7-O-glucoside Quercetin 3-rutinoside Quercetin 3-O-glucoside Quercetin 3-O-(6⬙-Omalonyl)glucoside Apigenin 5-O-glucoside Apigenin 7-O-glucuronide Apigenin 7-O-glucoside Kaempferol 3-O-rutinoside Kaempferol 3-O-glucoside Kaempferol 3-Omalonylglucoside

1

2

14.5 14.7 14.8 15.2 15.3 15.7

12.7 13.0 13.1 13.6 13.8 14.1

11.9

8.8

Rt (min)a

260, 268, 268, 265, 265, 265,

335 338 338 345 345 345

250, 345 256, 267sh, 350 256, 267sh, 350 256, 356 256, 356 256, 356

256, 356

270, 336

UV λmax (nm)

433 447 433 595 449 535

449 463 449 611 465 551

597

595

[M+H]+

491, 449

449

507, 465

465

577, 475, 457, 385, 355 465

[I+H]+

APCI-MS in positive mode (m/z)

271 271 271 287 287 287

287 287 287 303 303 303

303

[A+H]+

O. tenuiflorum

O. kilimandscharicum O. minimum O. minimum

O. kilimandscharicum O. tenuiflorum

O. americanum var. pilosum O. lamiifolium

Species from which isolated

a For composition of HPLC solvents, see Materials and Methods. [M+H]+, pseudomolecular ion in positive mode; [I+H]+, intermediate ions; [A+H]+, aglycone ion.

9 10 11 12 13 14

3 4 5 6 7 8

Identification

Compound

Table 2 Flavonoids identified in species of Ocimum, their HPLC retention times, UV absorption maxima and APCI mass spectraa R.J. Grayer et al. / Biochemical Systematics and Ecology 30 (2002) 327–342 333

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(C-8), 79.4 (C-2)⬙, 75.8 (C-2⵮) 75.6 (C-3⵮), 73.6 (C-5⬙), 73.3 (C-3⬙), 69.2 (C-4⵮), 67.5 (C-4⬙), 65.3 (C-5⵮), 59.7 (C-6⬙).

3. Results and discussion 3.1. Flavonoid glycosides identified in Ocimum species Seven flavonol O-glycosides (2, 6–8 and 12–14), six flavone O-glycosides (3–5 and 9–11) and one flavone C-glycoside (1) were identified in species of Ocimum (see Table 2 and Fig. 1). Many of these compounds have been reported from Ocimum species previously; 1, 3, 4 and 10 from O. tenuiiforum (as O. sanctum) (No¨ rr and Wagner, 1992; Nair and Gunasekaran, 1982), 1, 6 and 7 from O. basilicum (Skaltsa and Philianos 1986, 1990; Baritaux et al., 1991), and 1, 3, 5–7 and 11–13 from

Fig. 1. Flavonoid glycosides of Ocimum. All sugar residues occur in the β-D-pyranoside configuration. “X” indicates site of substitution unknown (14).

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O. gratissimum var. gratissimum (Grayer et al., 2000). However, this is the first time that quercetin 3-O-xylosyl(1⵮→2⬙)galactoside (2) and quercetin 3-O-(6⬙-Omalonyl)glucoside (8) have been isolated and identified from species of Ocimum. 3.2. Infraspecific variation of flavonoid glycosides in Ocimum species The flavonoid profiles of ten accessions of O. basilicum, five of O. tenuiflorum, three each of O. minimum and O. americanum, and two of O. gratissimum were examined for evidence of infraspecific variation using HPLC-DAD of purified extracts. The UV absorbance of the flavonoids at their lmax-values was recorded in order to estimate the relative proportions of each flavonoid glycoside in relation to the total amount of flavonoid glycosides in the plants. These results are presented in Table 3. All accessions of O. basilicum contained a mixture of quercetin 3-O-rutinoside (6) and quercetin 3-O-glucoside (7) as the major flavonoids. These compounds separated well by 2-D PC, but not by HPLC, so that it was difficult to quantify them. Thus in Table 3, the levels of 6 and 7 are combined, similarly with 12 and 13 (the 3-O-rutinoside and 3-O-glucoside of kaempferol, respectively). APCI LC-MS cannot be used to distinguish glucosides from their co-eluting rutinosides, because one of the MS-fragments of a rutinoside has the same m/z value as the protonated glucoside of the same aglycone (Grayer et al., 2000). The monoglycoside pairs 4 and 5 and 10 and 11 also co-eluted, but each component could be distinguished by APCI LCMS from the different m/z values of their [M+H]+ ions. APCI LC-MS showed that while most species contained the 7-O-glucosides of luteolin and apigenin (5 and 11, respectively), O. tenuiflorum accumulated the corresponding 7-O-glucuronides 4 and 10. Most accessions produced high levels of the malonylated quercetin 3-glucoside 8 and variable levels of kaempferol-3-O-rutinoside and 3-O-glucoside (12 and 13, respectively). Among the O. basilicum accessions, both quantitative and qualitative differences were observed with respect to the minor flavonoid glycosides, vicenin-2 (1), luteolin 5-O-glucoside (3), luteolin 7-O-glucoside (5), apigenin 7-O-glucoside (11) and kaempferol 3-O-malonylglucoside (14) (Table 3). The qualitative differences may not be significant, however, because these compounds were usually present in trace amounts only. In addition, some purified fractions still contained considerable amounts of caffeic acid derivatives, which made it more difficult to detect low amounts of co-eluting flavonoids. For example, apigenin 5-O-glucoside (which has the same retention time as rosmarinic acid) could only be detected by HPLC after several purification steps. However, it was easily detected by 2-D PC because of its blue fluorescence under UV light and characteristic Rf-values. The flavonoid glycoside profiles found among the ten accessions of O. basilicum do not add support to the classification of O. basilicum into three varieties, as the differences within the varieties were of the same order as those between them. The differences in flavonoid glycosides among the three accessions of O. americanum were more substantial, although they were mainly quantitative rather than qualitative, especially in relation to the concentration of vicenin-2 (1). This was the

basilicum basilicum basilicum basilicum basilicum basilicum basilicum basilicum basilicum basilicum

var. var. var. var. var. var. var. var. var. var.

basilicum BI 2746 basilicum BI 2747 basilicum BI 2748 basilicum BI 2749 basilicum BI 2750 basilicum BI 2751 purpurascens BI 2752 purpurascens BI 2753 purpurascens BI 2754 difforme BI 2756

b

a

± + ± ± +

+++ +++ +++ +++ +++

For identity of compounds 1–14, see Table 2. +++ ⬎30% of total flavonoid glycosides; ++ 15–30%; + 5–15%; ± ⬍5%.

5592 5789 6447 9005 9006

± ± ± ± ±

BI BI BI BI BI

O. O. O. O. O.

tenuiflorum tenuiflorum tenuiflorum tenuiflorum tenuiflorum

+ +

±

+ + +

+ +

+++

±

+

±

+ ±

± ±

±

5

±

+ +

+ ± ± ±

±

±

4

± ± ± ± ±

3

1b

O. gratissimum BI 5583 O. gratissimum BI 5783

O. minimum BI 4394 O. minimum BI 6443 O. minimum BI 6444

O. americanum var. americanum BI 4396 O. americanum Sacred BI 6442 O. americanum var. pilosum BI 6419

O. O. O. O. O. O. O. O. O. O.

Taxon and accession number

±

+

+++ +++

+++ +++ +++

+++ +++ ++

+++ +++ +++ +++ +++ +++ +++ +++ +++ +++

6/7

+++ +++ +++

+++ +++ ±

++ ++ ++ ++ ++ + ++ ++ + +

8

+

±

± ± ± ± ±

±

9

Table 3 Occurrence and relative proportions of flavonoid glycosides (1–14)a in different accessions of five Ocimum species

++ +++ +++ +++ +++

10

±

±

± ±

± ± ± ± ±

±

11

±

±

+ + +

+ +

+ ± + ± + + + ± + +

12/13

±

±

±

14

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major flavonoid glycoside in var. pilosum BI 6419 and its level was twice as high as that of the quercetin 3-O-glycosides 6 and 7 when combined. In contrast, 1 was only a trace constituent of var. americanum and absent from cv. Sacred, whereas 6 and 7 were the major constituents of both. The quercetin malonyl glucoside 8 was only present in trace amounts in var. pilosum, whereas the levels of 8 were almost as high as those of 6 and 7 in the other two accessions of O. americanum. These three accessions of O. americanum also show substantial differences in their surface flavonoid profiles (Grayer et al., 2001). O. minimum, O. gratissimum and O. tenuiflorum showed only infraspecific variation with respect to minor flavonoid glycosides, while the profile of the major compounds was similar in all accessions belonging to the same species. Nair and Gunasekaran (1982) reported the same flavonoid glycosides in O. tenuiflorum (collected in India) as found in the present study, but in addition detected orientin and the 7methyl ether of vitexin. 3.3. Comparison of flavonoid glycoside profiles among different species and subgenera of Ocimum Extracts were prepared of one accession each of four additional species of Ocimum, viz. O.×citriodorum, O. kilimandscharicum, O. selloi and O. lamiifolium, and analysed by 2-D PC and HPLC-DAD. The results are presented in Table 4 together with typical flavonoid glycoside profiles of 1–3 accessions of the other five species. The species are arranged into three subgenera according to the classification of Paton et al. (1999). Subgenus Ocimum is further subdivided into several sections, three of which (Ocimum, Gratissima and Hiantia) are represented here. The major compounds in all seven species of subgenus Ocimum (apart from O. americanum var. pilosum) were flavonol 3-O-glycosides (quercetin 3-O-rutinoside, 6, and/or quercetin 3-O-glucoside, 7). These were accompanied by quercetin 3-O-(6⬙-O-malonyl) glucoside (8) in all species belonging to sections Ocimum and Hiantia. Vicenin-2, luteolin and apigenin 5-O- and 7-O-glucosides and kaempferol 3-O-glycosides were minor compounds in most species belonging to subgenus Ocimum. Although no malonylated quercetin glycosides were found in O. gratissimum (section Gratissima), trace amounts of a 7-O-malonylglucoside were found in this species during previous investigations (Grayer et al., 2000). Species in subgenus Ocimum clearly produce a similar spectrum of flavonoid types, although levels may vary (Table 4). The extent of this variation is often of the same order of magnitude both within and between species. This should be taken into account when using flavonoid glycosides as characters for the taxonomy of this subgenus. For example, most accessions of O. basilicum contained small amounts of the flavone 7-O-glycosides 5 and 11, whereas most accessions of the closely related O. minimum lacked them. However, these compounds were absent from the particular accession, O. basilicum BI 2747, which showed an almost identical flavonoid pattern to O. minimum BI 6443 (see Table 3). Moreover, the compounds were present in O. minimum BI 4394, which showed a similar profile to O. basilicum BI 2754. One constant difference did exist between all the accessions of the two species, namely the relative amount of the malonylated

b

a

For identity of compounds 1–14, see Table 2. +++ ⬎30% of total flavonoid glycosides; ++ 15–30%; + 5–15%; ± ⬍5%.

± ±

± +

++

subgenus Nautochilus (Bremek.) A.J. Paton O. lamiifolium BI 6200

subgenus Gymnocimum (Benth.) A.J. Paton O. tenuiflorum BI 5592 O. tenuiflorum BI 5789

±

subgenus Ocimum section Hiantia (Benth.) A.J. Paton O. selloi BI 6421 +++

+

+

± + + ± +

+

3

subgenus Ocimum section Gratissima (Benth.) A.J. Paton O. gratissimum BI 5583

2

±

± ± ± +++ ± ±

1b

subgenus Ocimum section Ocimum O. basilicum var. basilicum BI 2749 O. basilicum var. purpurascens BI 2754 O. basilicum var. difforme BI 2756 O. americanum var. americanum BI 4396 O. americanum var. pilosum BI 6419 O.×citriodorum BI 6446 O. kilimandscharicum BI 6420 O. minimum BI 4394 O. minimum BI 6444

Taxon and accession number

+++ +++

4

+

+

± ±

±

± ± ±

5

Table 4 Occurrence and relative proportions of flavonoid glycosides (1–14)a in nine different species of Ocimum

+

+++

+++

+++ +++ +++ +++ ++ +++ +++ +++ +++

6/7

+

++ + + +++ ± +++ ± +++ +++

8

±

+

±

±

9

++ +++

10

±

± ±

± ± ±

11

±

±

+ ± +++ + +

± + +

12/13

±

±

14

338 R.J. Grayer et al. / Biochemical Systematics and Ecology 30 (2002) 327–342

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flavonol glycoside 8. This was always present at a higher level in O. minimum than in O. basilicum (see Table 3) and can be considered to be a distinguishing feature between these taxa. The flavonoid profile of the accession of O. kilimandscharicum differed from those of all other species in subgenus Ocimum because of its much higher proportion of kaempferol glycosides. The profiles of O. lamiifolium (subgenus Nautichilus) and O. tenuiflorum (subgenus Gymnocimum) were quite different from those found for species belonging to subgenus Ocimum. Although the main flavonoid in O. lamiifolium was also a flavonol 3-O-glycoside (quercetin 3-O-xylosyl(1⵮→2⬙)galactoside), both the sugars (xylose and galactose) and their interglycosidic linkage differed from those in subgenus Ocimum, where only flavonol 3-O-glucosides and 3-Orhamnosyl(1⵮→6⬙)glucosides (=rutinosides) were found. Furthermore, O. lamiifolium lacked the flavone C-glycoside 1, but produced larger amounts of flavone 5-O-glycosides than species belonging to subgenus Ocimum. All five accessions of O. tenuiflorum (subgenus Gymnocimum) accumulated flavone 7-O-glycosides rather than flavonol 3-O-glycosides, although low levels of the latter were detected in accessions BI 5592 and BI 6447. The flavone 7-O-glycosides were glycosylated with glucuronic acid, a sugar that was not detected in the other two subgenera. Thus, the flavonoid glycoside profile of O. tenuiflorum is different from those of the other Ocimum species investigated. Nevertheless, O. tenuiflorum does produce luteolin 5-O-glucoside, in common with all the other Ocimum species studied. This compound can therefore be considered to be a key character for the genus Ocimum, particularly because it is an uncommon constituent in the family Lamiaceae (Gil-Mun˜ os, 1993). It is of interest to note that this compound was not found in a pilot study of the closely related genus Plectranthus (Grayer, R.J., unpublished results). 3.4. Flavonoid glycoside profiles of plants grown under glass Certain steps of flavonoid biosynthesis require UV light and production of some flavonoids is increased following UV irradiation. For example, Markham et al. (1998) found that the levels of isoorientin increased dramatically in certain rice cultivars after exposure to UV-B irradiation, whereas isovitexin levels were hardly affected. Ocimum species are not hardy, and are usually grown under glass in Great Britain. However, UV light does not penetrate most types of glass, which could affect flavonoid production in plants. Prior to the flavonoid survey of Ocimum described here, the effect of growing plants under glass on flavonoid production was studied in three species, O.×citriodorum, O. gratissimum and O. selloi. In each case, one plant was grown in a glasshouse for two months after germination, whereas another plant from the same batch of seeds was grown outside in natural light for two months in the summer before the leaves were collected for extraction. Extracts of these plants were compared by means of 2-D PC and HPLC and the results are presented in Table 5. Production of flavone O- and C-glycosides was largely unaffected in plants grown in a glasshouse, whereas production of flavonol 3-O-glycosides was greatly reduced in all three species. For this reason, the flavonoid survey was carried out using plants

b

a

– –

± ±

Outside Glasshouse

O. selloi BI 6421 O. selloi BI 4409

+++ ±

+++ ±

+++ ±

6–8

– –

± ±

– –

11

For identity of compounds 1–14, see Table 2. Compounds 1, 3, 5 and 11 are flavone glycosides; 6–8 and 12–14 are flavonol glycosides. +++ high concentration in the plant; + medium concentration; ± trace amount.

– –

+ +

+ +

+ +

Outside Glasshouse

O. gratissimum BI 5783 O. gratissimum BI 4406

– –

+ +

± ±

Outside Glasshouse

O.×citriodorum BI 4392 O.×citriodorum BI 4405

5

3

1b

Taxon and accession number Growing conditions

Table 5 Differences in flavonoid profiles between plants grown outside and under glasshouse conditionsa

– –

± –

+ –

12–14

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that were grown outside. These results highlight the importance of considering light quality as a variable in any chemosystematic survey based on flavonoid distribution and concentration.

Acknowledgements A grant from the Royal Society to P.M. is gratefully acknowledged. Dr Eli Putievsky, Newe Ya’ar Research Centre, Haifa, Israel, is thanked for providing the seeds of most accessions, and Clive Foster for sowing the seeds and growing most of the plant material used for this study. We also thank Sarah Bryan, Janice Irwin, Louise Archer and Anna Price who contributed significantly to the present investigation during their university industrial placement years at the Royal Botanic Gardens, Kew. Access to NMR facilities was kindly provided by the Medical Research Council Biomedical NMR Centre, National Institute for Medical Research, Mill Hill, London.

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