Food Chemistry 115 (2009) 650–656 Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem Chicoric acid found in basil (Ocimum basilicum L.) leaves Jungmin Lee a,*, Carolyn F. Scagel b a United States Department of Agriculture, Agricultural Research Service, PWA, Horticultural Crops Research Unit Worksite, 29603 U of I Lane, Parma, ID 83660, USA b United States Department of Agriculture, Agricultural Research Service, Horticultural Crops Research Unit, Corvallis, OR 97330, USA article info abstract Article history: This is the first report to identify the presence of chicoric acid (cichoric acid; also known as dicaffeoyltar- Received 28 August 2008 taric acid, which is a caffeic acid derivatized with tartaric acid) in basil leaves. Rosmarinic acid, chicoric acid Received in revised form 18 December 2008 and caftaric acid (in the order of most abundant to least; all derivatives of caffeic acid) were identified in Accepted 19 December 2008 fresh basil leaves. Rosmarinic acid was the main phenolic compound found in both leaves and stems. Chic- oric acid was not detected in sweet basil stems, although a small amount was present in Thai basil stems. Other cinnamic acid monomers, dimers and trimers were also found in minor quantities in both stems and Keywords: leaves. Basil polyphenolic contents were determined by blanched methanol extraction, followed by HPLC/ Phenolics DAD analysis. The characterization of the polyphenolics found in the basil extracts were performed by Cichoric acid Caffeic acid derivatives HPLC/DAD/ESI–MS/MS and co-chromatographed with purchased standard. The influence of inoculation Dicaffeoyltartaric acid with an arbuscular mycorrhizal fungus (AMF), Glomus intraradices, on plant phenolic composition was Lamiaceae studied on two basil cultivars,‘Genovese Italian’ and ‘Purple Petra’. Inoculation with AMF increased total Glomus intraradices anthocyanin concentration of ‘Purple Petra’ but did not alter polyphenolic content or profile of leaves and stems, of either cultivar, compared to non-inoculated plants. In the US diet, basil presents a more acces- sible source of chicoric acid than does Echinacea purpurea, in which it is the major phenolic compound. Published by Elsevier Ltd. 1. Introduction 2007), but complete phenolic profiles of basil have not been re- ported. Of the caffeic acid derivatives in basil, the present study Culinary herbs have been reported to possess antioxidant activ- is the first to identify the presence of chicoric and caftaric acids ities (Yanishlieva, Marinova, & Pokorny, 2006) suggesting that they in basil, respectively the second and third major phenolic acids might have potential human health benefits. Basil (family Lamia- present in basil leaves. ceae) is a popular herb in the US and Mediterranean diets. Basil’s Chicoric acid is the dominant phenolic reported in Echinacea pur- importance as a culinary herb, its historic usage, essential oil com- purea (Molgaard, Johnsen, Christensen, & Cornett, 2003; Perry, position and phenolics have been well reviewed by Makri and Burgess, & Glennie, 2001) and has been found in all parts (flower Kintzios (2008). Basil has shown antioxidant and antimicrobial heads, leaves, stems and root) of the E. purpurea plant ( Molgaard activities due to its phenolic and aromatic compounds (Gutierrez, et al., 2003). Echinacea (family Asteraceae) has been reported to have Barry-Ryan, & Bourke, 2008; Hussain, Anwar, Sherazi, & Przybylski, potential antioxidant, anti-inflammatory, antiviral and immuno- 2008; Javanmardi, Khalighi, Kashi, Bais, & Vivanco, 2002; stimulating properties, arising from the naturally occurring Yanishlieva et al., 2006). alkamides, caffeic acid derivatives, polysaccharides and glycopro- The main phenolics reported in basil are phenolic acids and fla- teins (Barnes, Anderson, Gibbons, & Philipson, 2005; Charvat, Lee, vonol-glycosides (Javanmardi et al., 2002; Jayasinghe, Gotoh, Aoki, Robinson, & Chamberlin, 2006; Dalby-Brown, Barsett, Landbo, & Wada, 2003; Kivilompolo & Hyotylainen, 2007; Kosar, Dorman, & Meyer, & Molgaard, 2005; Molgaard et al., 2003; Perry et al., 2001). Hiltunen, 2005; Nguyen & Niemeyer, 2008; Tada, Murakami, However, in a well-summarized and detailed review of Echinacea Omoto, Shimomura, & Ishimaru, 1996). The presence of caffeic acid species’ possible health benefits, its effectiveness was no better than derivatives (phenolic acid class) has been reported in basil that of a placebo (Barnes et al., 2005 and references therein). (Jayasinghe et al., 2003; Kivilompolo & Hyotylainen, 2007; Nguyen Chicoric acid itself has been reported to inhibit HIV integrase & Niemeyer, 2008; Tada et al., 1996; Toussaint, Smith, & Smith, (Charvat et al., 2006) and to exhibit antioxidant activities (Dalby-Brown et al., 2005). As dietary supplements, Echinacea her- bal extracts have been very popular (US annual sales estimated at $100–200 million during the years 2000–2006; Tilburt, Emanuel, & * Corresponding author. Tel.: +1 208 722 6701x282; fax: +1 208 722 8166. E-mail addresses: [email protected] (J. Lee), [email protected] Miller, 2008) but, for US consumers, basil represents a more readily (C.F. Scagel). available and inexpensive source of these compounds. 0308-8146/$ - see front matter Published by Elsevier Ltd. doi:10.1016/j.foodchem.2008.12.075 J. Lee, C.F. Scagel / Food Chemistry 115 (2009) 650–656 651 Phenolic production in a plant can be affected by biotic and abi- with AMF by hand-mixing 52 g of the AMF inoculum into the otic factors (Hussain et al., 2008; Lee & Martin, 2009; Toussaint growing substrate in each pot before sowing seeds (AMF treat- et al., 2007; Yao, Zhu, & Zeng, 2007). Colonization by arbuscular ment). The same quantity of sterile (121 °C, 15 min) inoculum mycorrhizal fungi (AMF) is known to improve plant nutrient up- was mixed into the growing substrate for the remainder of the take and use (Clark & Zeto, 2000), and stress tolerance (Smith & plants before sowing seeds (control). Containers were placed in a Read, 1997). AMF colonization can also alter or enhance phenolic glass house and watered as needed. Supplemental light production within the host plant (Toussaint et al., 2007; Ganz, (720 lmol PAR mÀ2 sÀ1) was provided for 16 h dÀ1 by high-pres- Kailis, & Abbott, 2002). The relationship between AMF colonization sure multi-vapour lamps. Day/night temperatures were controlled and the phenolic compounds produced in plants is still not well at 24/15 °C during the experiment. After cotyledons had fully ex- understood (Toussaint, 2007; Yao et al., 2007). panded, plants were fertilized weekly with 50 ml of a liquid fertil- The objectives of this study were to better identify the phenolic izer (Peters Professional, Scotts Company, Maysville, OH, USA) compounds found in the aerial portions of the fresh basil plant, containing 380 mg N lÀ1, 150 mg P lÀ1, 380 mg P lÀ1,64mgSlÀ1, and to determine the impact of AMF on plant phenolics within two 100 mg Ca lÀ1,36mgMglÀ1, 4.6 mg Fe lÀ1,18mgCllÀ1, 0.55 mg cultivars. The polyphenolic contents of basil samples were deter- Mn lÀ1, 0.37 mg B lÀ1, 0.024 mg Zn lÀ1, 0.06 mg Cu lÀ1, and mined using a blanched methanol extraction procedure, followed 0.006 mg Mo lÀ1. Plants were pruned back, to three nodes, six by HPLC/DAD analysis. The characterization of the polyphenolics weeks after germination and flowers that began to develop nine found in the basil extracts was performed by HPLC/DAD/ESI–MS/MS. weeks after germination were removed from plants. 2.4. Plant harvest and colonization assessment 2. Materials and methods All plants were destructively harvested 16 weeks after germina- 2.1. Plant materials tion and separated into leaves, stems, and roots. Substrate was re- moved from roots by washing and samples of roots were taken for Common sweet basil and Thai basil (Ocimum basilicum, specific assessing AMF colonization. Samples of leaves and stems were ta- cultivar names unknown) were purchased from a local market in ken for phenolic analyses (described below). The samples of fresh Nampa, ID, USA. Edible portions (mainly leaves) were separated roots were cleared and stained, using a modified procedure of Phil- from stems, and both fractions were then stored frozen at À80 °C lips and Hayman (1970), in which lacto-phenol was replaced with prior to extraction. Purchased sweet basil will be referred to as lacto-glycerin, and assessed for AMF colonization. AMF coloniza- sweet basil, for clarification hereafter, to distinguish it from the tion was measured on 1 cm sections of root samples, using the other (sweet) basil samples of the AMF portion of this study. Biermann and Linderman (1980) method. Two basil cultivars (Genovese Italian and Purple Petra) were used to examine the effect of inoculation with AMF on plant phenolic 2.5. Sample preparation and extraction for polyphenolic analysis production. Echinacea whole plant herbal supplement extract was pur- Frozen samples were liquid nitrogen-powdered and extracted chased from a local shop (Nampa, ID, USA) and analyzed by HPLC. with acidified methanol (0.1% formic acid, v/v) as described in Lee The Echinacea herbal extract contained E. purpurea, according to and Finn (2007) with the following modification: An IKA M20 Uni- the manufacturer’s label. All chemicals for polyphenolic extraction versal mill (IKA works Inc., Wilmington, NC, USA) was used to grind and HPLC analysis were obtained from Sigma Chemical Co. (St. frozen samples. Frozen powder (5 g) with the first methanol addi- Louis, MO, USA) unless indicated otherwise. Solvents and chemi- tion was blanched in a boiling water bath for 5 min, then immedi- cals for this investigation were of analytical and high performance ately chilled in an ice bath for 10 min. This chilled mixture was liquid chromatography (HPLC) grade. Purified chicoric acid stan- centrifuged and the pellet was re-extracted, two additional times, dard was purchased from Indofine Chemical Company, Inc. with acidified methanol, as described in Lee and Finn (2007). Meth- (Hillsborough, NJ, USA). anol was evaporated with a RapidVap Vacuum Evaporation System (Labconco Corp., Kansas City, MO, USA) at 40 °C, and re-dissolved in 2.2.