J Nutr Sci Vitaminol, 59, 336–342, 2013 Altitudinal Variation of Antioxidant Components and Capability in Indocalamus latifolius (Keng) McClure Leaf Qinxue NI1, Zhiqiang WANG1, Guangzhi XU1, Qianxin GAO1, Dongdong YANG1, Fumiki MORIMATSU2 and Youzuo ZHANG1,* 1 The Key Laboratory for Quality Improvement of Agricultural Products of Zhejiang Province, Zhejiang Agriculture and Forestry University, Lin’an 311300, China 2 R & D Center, Nippon Meat Packers Inc., Tsukuba, Ibaraki 300–2646, Japan (Received February 7, 2013) Summary Indocalamus latifolius (Keng) McClure leaf is a popular food material in East Asia due to its antioxidant and anticorrosive activities. To utilize it more effectively, we inves- tigated the discrepancy of antioxidant activities and active compound content in Indocala- mus latifolius leaf along with the altitude change. Total flavonoids, phenolics, titerpenoids and eight characteristic active constituents, i.e, orientin, isoorientin, vitexin, homovitexin, p-coumaric acid, chlorogenic acid, caffeic acid, and ferulic acid, were determined by UV- spectrophotometer and synchronous RP-HPLC, respectively. Antioxidant activity was mea- sured using DPPH and FRAP methods. Our data showed that the content of TP and TF, DPPH radical scavenging ability and ferric reduction power of Indocalamus latifolius leaf changed as altitude altered, with the trends of decreasing gradually when lower than 700 m and then increasing to 1,000 m. Chlorogenic acid and orientin were the main characteristic compounds in Indocalamus latifolius leaf and were also affected by altitude. Our result indi- cated that higher altitude with an adverse environment is conducive to secondary metabo- lite accumulation for Indocalamus latifolius. It would provide a theoretical basis to regulate the leaf collection conditions in the industrial use of Indocalamus latifolius leaf. Key Words Indocalamus latifolius (Keng) McClure, altitude, secondary metabolites, bam- boo characteristic compounds, antioxidant activities Bamboo is an evergreen and perennial plant of the latifolius leaves are mainly attributable to its abundant Gramineae family, which has a tropical and subtropical secondary metabolites, such as flavonoids, phenolic distribution. It is one of the most primitive and diverse components and triterpenoid (5). Among them, orien- taxa in the world and widely used as an important com- tin, isoorientin, vitexin, homovitexin, chlorogenic acid, modity in Southeast Asia and pacific islands, for exam- caffeic acid, p-coumaric acid and ferulic acid are eight ple, as building material, handicraft articles, food mate- characteristic compounds in bamboo leaves and were rial and traditional medicine. In particular, bamboo confirmed with antioxidant, anti-cancer, anti-inflam- leaves have been used in traditional Chinese medicine mation and radioprotective activities (6–10) (Fig. 1). for over 1,000 y for the treatment of fever, inflamma- Secondary metabolites are the major ingredients tion and detoxification. To date, bamboo leaf extracts that have been associated with the biological activity have also been used in food additives and pharmaceu- of plants. Their synthesis and accumulation pathways tical products, mainly to lessen or cure stomachache, in plants are the results of long-term natural selection diarrhea, vomiting, restlessness and excessive thirst (1, under certain conditions, which are closely related to 2). the ecological environment. The composition and con- Indocalamus latifolius (Keng) McClure is a kind of tents of secondary metabolites in the same plant show bamboo grass with a large leaf and dwarf stalk, which a significant difference in different ecological environ- is widely distributed in the south of China. Because of ments (11, 12). Altitude is an important environmen- its advantages of large biomass, easy collection and tal factor that affects the accumulation of secondary various biological activities, the leaves have been used metabolites due to the change of light, temperature, as the wrap of rice tamale, a popular traditional Chi- moisture and other ecological factors. The valid impact nese food, for several thousand years. It can make rice of altitude on the content of secondary metabolites has be more fragrant, less prone to rancidness and healthful been confirmed by several studies (13, 14). by its aroma components and antibacterial, antioxidant Yet, till now, the changes in secondary metabolite compounds (3, 4). Our previous study revealed that the composition and content and the physiological activities biological activities and functions of the Indocalamus of Indocalamus latifolius leaf along the altitudinal gradi- ent have rarely been reported. In order to improve the * To whom correspondence should be addressed. comprehensive utilization of bamboo leaf, the present E-mail: [email protected] study investigated the altitude variation of the total level 336 Altitudinal Variation of Antioxidant Composition in Bamboo Leaf 337 University. All samples from each altitude were investi- gated three times in order to decrease method-related measurement errors. The characteristics of the collection site where Indo- calamus latifolius grows need to be commented on. Mt. Longwang is located between Zhejiang and Anhui Prov- inces in the southeast of China (1,587.4 m elevation, (A) 30˚23′N, 119˚23′E), which could be characterized as a subtropical monsoon area. The average temperature R: from the foothills and hilltop is 15.1–8.9˚C. The tem- perature at lower altitudes would decrease by 0.48˚C per 100 m. Extreme maximal and minimal temperature registered in summer and winter of the year were 37˚C and 211.3–20.6˚C, respectively. The frost-free period through the year lasted 208 d with a precipitation (B) 1,647 mm/y and average annual sunshine of 1,550– 2,000 h around the whole mountain. The annual Flavonoids R1 R2 R3 Phenolic acids R4 R5 UV- irradiation on the region was stronger in spring- Orientin -H -Glu -OH Chlorogenic acid -OH -R summer than that in autumn-winter with an annual 2 Homoorientin -Glu -H -OH Caffeic acid -OH -OH UV-irradiation of about 150 MJ/m . Vitexin -H -Glu -H p-Coumaric acid -H -OH Extraction process. Bamboo leaves were washed, Isovitexin -Glu -H -H Ferulic acid -OCH3 -OH drained, and enzymes inactivated immediately (micro- wave treated at 640 W three times: 1 min each time), Fig. 1. Chemical structure of orientin, homoorientin, then dried in a vacuum drying oven (60˚C or 2 h). vitexin, isovitexin, chlorogenic acid, caffeic acid, p-cou- maric acid, and ferulic acid. (A) is the basic framework The dried bamboo leaves were then milled to a powder of 4 flavonoids; (B) is the basic framework of 4 phenolic of 40-mesh particle size. Bamboo leaf powder 1 g was acids. mixed with 150 mL ethanol-aqueous solution (70%, v/v), refluxed 3 times (2 h each time), filtered and evap- orated to a constant volume of 100 mL. The moisture content of each sample was measured with an infra- of phenolics (TP), flavonoids (TF) and triterpenoid (TT), red moisture analyzer (Ohaus, Pine Brook, NJ) before plus eight characteristic compounds in bamboo leaf, extraction, and all the results were calculated based on in addition to antioxidant activities. Simultaneously, dry materials. we also discussed the relationship between antioxidant Determination of total phenolic (TP), total flavonoid (TF) activity and secondary metabolites with the aim of pro- and total triterpenes (TT) content. The contents of total viding a theoretical basis for exploiting and managing phenolics (TP), total flavonoids (TF) and triterpenoids bamboo leaf resources more scientifically and efficiently. (TT) in Indocalamus latifolius leaf were investigated using spectrophotometer analyses methods as reported below MATERIALS AND METHODS (15). All measurements were reproduced in triplicate. Chemical reagents. Methanol (HPLC grade), aceto- The TP concentration of the samples was determined nitrile (HPLC grade), Folin-Ciocalteau reagent, vanillin, using the Folin-Ciocalteu colorimetric method. The diphenyl-2-picryl-hydrazyl (DPPH), tripyridyltriazine amount of TP was calculated as a p-hydroxybenzoic (TPTZ) and authentic standards of phenolic compounds acid equivalent from the standard curve (Y52.2582 (p-hydroxybenzoic acid, chlorogenic acid, caffeic acid, X10.037, r50.9989), and expressed as the percentage p-coumaric acid, ferulic acid) were purchased from of p-hydroxybenzoic acid in dry leaf weight (%, DW). Sigma-Aldrich Co. (St. Louis, MO). Authentic standards The aluminum nitrate-sodium nitrite colorimetric of flavonoids (rutin, orientin, isoorientin, vitexin, homo- method was used to determine the TF content of the sam- vitexin) were purchased from Extrasynthese Chemical ples. The amount of TF was calculated as a rutin equiv- S.A.S. (Lyon Nord, Genay Cedex, France). Other chemi- alent from the standard curve (Y50.014X10.0016, cals were of analytic grade. r50.9999), and expressed as the percentage of rutin in Plant material and collection site. Indocalamus lati- dry leaf weight (%, DW). folius leaves were collected from Mt. Longwang (An’Ji, The vanillin-glacial acetic acid colorimetric method Zhejiang Province, China) at different altitudes (100, was used to determine the TT concentration of samples. 300, 500, 700 and 1,000 m) on 17th May, 2011, on a The amount of TT was calculated as an ursolic acid equiv- sunny day at the temperature of 12–20˚C, and average alent from the standard curve (Y50.0048X20.0136, relative humidity of 54% . Three sample plots were ran- r50.9976),