A Potential Antioxidant Resource: Endophytic Fungi from Medicinal Plants IWt3-YANG HUANG 2, YI-ZHONG CAI 3, JIE )(LING4, HAROLD AND M E I SUN 2'*

CORKE3,

2 Department of Zoology, The University of Hong Kong, Pokfulam Road, Hong Kong, PR China 3 Department of Botany, The University of Hong Kong, Pokfulam Road, Hong Kong, PR China 4 Republic Polytechnic, Woodlands Avenue 9, Singapore 738964 *Corresponding author; e-mail: meisun@hku.hk

A Potential Antioxidant Resource: Endophytic Fungi from Medicinal Plants. Medicinal plants and their endophytes are important resources for discovery of natural products. Several previous studies have found a positive correlation between total antioxidant capacity (TAC) and total phenolic content (TPC) of many medicinal plant extracts. However, no information is available on whether such a relationship also exists in their endophyhc fungal metabolites. We investigated the relationship between TAC and TPC for 292 morphologically distinct endophytic fungi isolated from 29 traditional Chinese medicinal plants. The antioxidant capacities of the endophytic fungal cultures were significantly correlated with their total phenolic contents, suggesting that phenolics were also the major antioxidant constituents of the endophytes. Some of the endophytes were found to produce metabolites possessing strong antioxidant activities. Several bioactive constituents from the fungal cultures and host plant extracts were identified. This investigation reveals that the metabolites produced by a wide diversity of endophytic fungi in culture can be a potential source of novel natural antioxidants. Key Words: Endophytic fungi, metabolites, medicinal plants, antloxidant activity, phenolic compounds, Chinese medicinal plants, traditional Chinese medicine, TCM.

There is increasing evidence indicating that reactive oxygen species (ROS, e.g., 02- and OH-) and free radical-meditated reactions can cause oxidative damage to biomolecules (e.g., lipids, proteins, and DNA), eventually contributing to, for example, aging, cancer, atherosclerosis, coronary heart ailment, diabetes, Alzheimer's disease, and other neurodegenerative disorders (Finkel and Holbrook 2000; Halliwell 1994). Antioxidants are thought to be highly effective in the management of ROS-mediated tissue impairments. Many antioxidant compounds possess antiinflammatory, antiatherosclerotic, antitumor, antimutagenic, anticarcinogenic, antibacterial, or antiviral activities to a greater or lesser extent

1Received 4 August 2006; accepted 9 November 2006.Economic Botany, 61(1), 2007, pp. 14-30.

(Cozma 2004; Halliwell 1994; Mitscher et al. 1996; Owen et al. 2000; Sala et al. 2002). Naturally derived antioxidants have received much attention in recent years (Hu and Kitts 2000; Schulz et al. 2002). Endophytes are fungi or bacteria residing inside healthy plant tissues without any discernible infectious symptoms (Wilson 1995). They could be a potential source of novel natural products for medicinal, agricultural, and industrial uses. Because they are relatively unstudied, much attention is now being paid to endophytic biodiversity, the chemistry and bioactivity of endophytic metabolites, and the relationships between endophytes and host plants (Schulz et al. 2002; Tan and Zou 2001). Endophytes provide a wide variety of structurally unique bioactive natural products, such as alkaloids, benzopyranones, chinones, flavonoids, phenolic acids, quinones, ste-

9 2007, by The New York Botanical Garden Press, Bronx, NY 10458-5126 U.S.A.

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HUANG ET AL.: ENDOPHYTIC FUNGI

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roids, terpenoids, tetralones, xanthones, and others (Tan and Zou 2001). Antibiotics, antiviral compounds, anticancer agents, insecticidal products, antidiabetic agents, immunosuppressive compounds as well as antioxidants have been reported from endophytic metabolites (Strobel et al. 2004), and medicinal plants have been recognized as a repository of endophytes with novel metabolites of pharmaceutical importance (Strobel et al. 2004; Tan and Zou 2001; Wiyakrutta et al. 2004). Medicinal plants contain a wide variety of free radical scavenging molecules, such as phenolic compounds (e.g., phenolic acids, flavonoids, quinones, coumarins, lignans, lignin, stilbenes, and tannins), nitrogen compounds (e.g., alkaloids and amines), vitamins, terpenoids, and other endogenous metabolites (Cai et al. 2004; IC~hk6nen et al. 1999; Zheng and Wang 2001). Traditional Chinese medicinal plants have been used for pharmaceutical and dietary therapies for several millennia (Cai et al. 2004; Tapiero et al. 2002). Natural compounds isolated from these medicinal plants are a rich source of novel drugs with multiple biological activities including antioxidant properties. There have been many studies on the antioxidant activities of various plants of medicinal use (e.g., K~ihk6nen et al. 1999; Re et al. 1999; Zheng and Wang 2001). Cai et al. (2004) reported that phenolic compounds were the dominant antioxidant components in 112 traditional Chinese medicinal plants associated with combating cancer, and similar findings were reported for 133 Indian medicinal plants (Surveswaran et al. 2007), with both studies showing highly significant positive linear correlations between the total antioxidant capacities and phenolic contents. Despite numerous studies on antioxidant activities and phenolic contents in plants, however, no comparative investigation has been carried out for their endophytes. In the present study, a total of 1,160 endophytic fungal isolates were obtained from different tissues of 29 traditional Chinese medicinal plant species. Only the 292 morphologically distinct fungal isolates were used for investigation of the total antioxidant capacities, phenolic contents, and their relationships. The relationships between the total antioxidant capacities and total phenolic contents in the 29 host plants were concurrently investigated for comparison with their endophytes. Preliminary identification and screening of bioactive constituents from the en-

dophytic metabolites and host plant extracts were also carried out using several chromatographic and spectroscopic techniques (HPLC, LC-MS, and GC-MS). The main objective of this study was to explore the endophytic fungi from medicinal plants, which can in vitro produce bioactive compounds with potent antioxidant activity, as a novel antioxidant source.

Materials and MethodsCOLLECTION OF PLANT MATERIALS The traditional Chinese medicinal plant samples used for isolation of endophytic fungi were all collected from healthy living plants grown in Hong Kong from March to September of 2005. The collected plants represent 29 species from six families, including 13 species in the Apocynaceae, seven in the Asclepiadaceae, four in the Polygonaceae, three in the Asteraceae, and one each from the Lamiaceae and the Solanaceae. The medicinal plant species were authenticated by Mr. S. T. Chan, a local plant expert at the University of Hong Kong. The plant species and their collection sites are listed in Table 1. All the flesh samples were taken to the laboratory and treated within 24 hours. CHEMICALS AND REAGENTS The chemicals 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), potassium persulfate, and sodium carbonate were purchased from Sigma/Aldrich (St. Louis, MO). Folin-Ciocalteu reagent and HPLC-grade organic reagents were from BDH (Dorset, UK), and Trolox (6-hydroxy-2,5,7,8-tetramethylchromate2-carboxylic acid) from Fluka Chemie AG (Buchs, Switzerland). Authentic standards, antibiotics, and other chemicals and reagents used in this study were obtained from Sigma/Aldrich. All the chemicals and reagents used in this study were of the analytical grade. ISOLATION OF ENDOPHYTIC FUNGI A total of 20 samples of plant parts (leaf, stem, flower, fruit, or root) from the medicinal plants were first washed in running water. The leaves, flowers, or fruits were cut into segments (5 x5 mm), and stems or roots were cut into pieces (10 mm in length). Surface sterilization and isolation of endophytic fungi followed a modified method as described by Schulz et al. (1993). All the plant samples were surface-sterilized by dip-

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