A Potential Antioxidant Resource: Endophytic Fungi from Medicinal Plants I

Wt3-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 Methods

COLLECTION 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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ping in 75% ethanol for 1 min, 2.5% sodium hypochlorite for 15 min, and again 75% ethanol for 1 min, followed by rinsing in sterile water three times. In each petri dish (9cm diameter), four processed segments or pieces were evenly spaced onto the surface of water agar (WA) medium supplemented with 200 U/ml penicillin G and 200 I~g/ml streptomycin sulfate for suppressing the growth of bacteria, followed by incubation at 28~ until the mycelium or colony originating from the sample appeared. Hyphal tips were transferred and cultured on potato carrot agar (PCA) plates, and the purified strains were preserved on potato dextrose agar (PDA) plates at 4~ in the Laboratory of Department of Zoology, the University of Hong Kong.
CULTIVATION OF ENDOPHYTIC FUNGI

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Fresh mycelia (grown on PDA plates at 28~ for 3-6 days) of morphologically different endophytes, approximately equal in quantity, were inoculated in 100 ml flasks, each containing 50ml of broth (sucrose, 30g; NaNOy 3g; K2HPO 4, 1 g; yeast extract, 1 g; KCI, 0.5 g; MgSO4.7H20, 0.5g; FeSO4, 0.01g; H20, 1,000ml), followed by incubation with a Shaking Incubator (Daihan Labtech Co., Ltd.) at 140rpm for 15 days at 28~ The culture broth of each endophytic fungus was centrifuged at 1,670g for 10 min and filtered using a Millipore filter with a 0.22-~m nylon membrane under vacuum at room temperature (-23~ to remove mycelium. The filtrate sample was stored at 4~ until use within 24 hours.
EXTRACTION OF HOST PLANT SAMPLES

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The plant samples were air-dried in a ventilated oven at 40~ to constant weight and ground to fine powder using a Kenwood MultiMill (Kenwood, Ltd., UK) and passed through a 24-mesh sieve. The sample (2g each) was extracted with 50ml of 80% methanol at room temperature for 24 hr in a water bath shaker (Shaking Bath 5B-16) (Techne, Ltd., UK). The extract was also filtered by a Millipore filter with a 0.22-~m nylon membrane under vacuum at room temperature, and stored at 4~ (Cai et al.

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TOTALANTIOXIDANT CAPACITYASSAY Total antioxidant capacity was assayed using the improved ABTS method (Cai et al. 2004; Re et al. 1999). The ABTS'+ radical cation was gen-

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erated by reacting 7mm ABTS and 2.45mm potassium persulfate after incubation at room temperature in darkness for 16 hours. The ABTS'* solution was diluted with 80% ethanol to an absorbance of 0.700 + 0.005 at 734nm. The tested sample was diluted with 80% ethanol so as to give 20-80% inhibition of the blank absorbance with 0.1 ml of sample. ABTS'* solution (3.9 ml; absorbance of 0.700 + 0.005) was added to 0.1 ml of the tested samples and mixed thoroughly. The reactive mixture was allowed to stand at room temperature for 6 min and the absorbance was immediately recorded at 734 nm. Different levels of trolox standard solution in 80% ethanol were prepared and assayed under the same conditions. The absorbance of the resulting oxidized solution was compared to that of the calibrated trolox standard. Results were expressed in terms of trolox equivalent antioxidant capacity (TEAC), i.e., gtmol trolox/100ml culture of endophytic fungus or mmol trolox/100g dry weight (DW) of plant. DETERMINATION OF TOTAL PHENOLIC CONTENT Total phenolic content (TPC) was estimated using the Folin-Ciocalteu colorimetric method described by Liu et al. (2002) and Cai et al. (2004) with minor modification. Briefy, the appropriate dilutions of the sample (0.2 ml) were oxidized with 0.5 N Folin-Ciocalteu reagent for 4 min at room temperature. Then the reaction was neutralized with saturated sodium carbonate (75g/1). The absorbance of the resulting blue color was measured at 760 nm with the spectrophotometer after incubation for 2 hr at room temperature in darkness. Quantification was done on the basis of the standard curve of gallic acid. Results were expressed as gallic acid equivalent (GAE), i.e., mg gallic acid/100 ml culture or g gallic acid/100 g DW. R P - H P L C ANALYSIS RP-HPLC analysis was performed using a Hewlett-Packard HPLC System (HP 1100 series, Waldbronn, Germany), consisting of a binary pump and a diode-array detector (DAD), and equipped with a Nucleosil 100-C18 column (5~m, 250 with a Nucleosil 5 C18 guard column (5 ~m, 4 4 mm) (Agilent Technologies, Loveland, CO). Phenolic compounds in the samples were analyzed using our previous method (Cai et al. 2004) with slight modifica-

tion. The following gradient elution program was used in the improved HPLC method (solution A, 2.5% formic acid, and solution B, 100% methanol): 0 min, 5% B; 15 min, 30% B; 40 min, 40% B; 60 min, 50% B; 65 min, 55% B; and 90-98 min, 100% B. Flow rate was 0.8 ml/min and injection volume was 20 ~1. Detection was monitored at different wavelengths for various phenolic compounds: 280nm for hydroxybenzoic acids, flavanones, flavanols, isoflavones, terpenoids, and tannins; 320 nm for hydroxycinnamic acids and flavones; 370 nm for flavonols and chalcones. LC-ESI-MS The LC-MS-2010A system used in this study consisted of a LC-10ADvp binary pump, a SILl 0Avp auto-sampler, a photodiode-array detector, a central controller, and a single quadrupole MS detector with electrospray ionization (ESI) interface (Shimadzu, Japan). The analytical column was VP-ODS C18 column (250 x 2.0 mm, 5 gtm) (Narmura Chemical Co., Seto, Japan). LC conditions were as follows: solvent A, 0.1% formic acid, and solvent B, MeOH with 0.1% formic acid. A gradient elution used was 0-5 rain, 5% B; 5-15 min, 5-30% B; 15-40 min, 30-40% B; 40-60 min, 40-50% B; 60-65 min, 50-55% B; 65-90 min, 55-100% B; 90-95 rain, 100% B; 95-96 min, 100-5% B; 96-100 min, 5% B. The flow rate was 0.2 ml/min, injection volume was 5-10 ~l, and detection was at 280nm. The LC eluant was introduced directly into the ESI interface without flow splitting. The scan range of ESI-MS was m/z 160-800. The ESI voltage was 4.5 kV in positive ion mode and 3.5 kV in negative ion mode. A nebulizing gas of 1.5 l/min and a drying gas of 10 l/min were applied for ionization using nitrogen in both cases. GC-MS A GCMS-QP2010 system (Shimadzu, Japan) equipped with an AOC-5000 auto-injector with a headspace MSH02-00B and with a common syringe (10~1) was employed for analysis of volatile and aliphatic compounds. Headspace conditions were as follows: powder samples were kept in incubator vials in an oven at 110~ with shaking for 20 min. Injection volume was 1 gtl. A DB-5MS column (60mx0.25mm, o with 0.1 ~m film thickness) was used with helium as a carrier gas at a fow rate of 2.46 ml/min. The GC oven temperature was kept at 40~ for 0.5 min

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and programmed to 150~ at a rate of l~ to 250~ at a rate of 10~ and kept constant at 250~ for 10 min. Liquid injection method was also used in this study. A CP-WAX 52 CB column (50 m x 0.20 mm, o with 0.2 I~m film thickness) was used with helium as a carrier gas at a flow rate of 1 ml/min and injection volume was 1 I~1. The GC oven temperature was kept at 40~ for 0.5 min and programmed to 150~ at a rate of 6~ to 250~ at a rate of 8~ and kept constant at 250~ for 8 rain. Splitless injections were done in both headspace and liquid injection methods. The MS was operated in electron ionization mode (70 eV) with a scan range of m/z 40-600. The interface and ion source temperature were 260~ and 200~ respectively. A library search was carried out using NIST and SZTERP libraries.
STATISTICALANALYSIS

morphologically distinct isolates from the 29 medicinal plants were selected for antioxidant activity screening and total phenolic content assay (Table 1 and Fig. 1). The characteristics of colony or hyphal and microscopic morphology were used to differentiate among these isolates. The most endophytic fungi (83 strains) were isolated from Tabernaemontana divaricata (L.) R. Br. ex Roem. & Schutt., while the least (23 strains) from Rauvolfia verticillata (Lour.) Baill.
TOTALANTIOXIDANTCAPACITYAND PHENOLIC CONTENT

All sample determinations were conducted in triplicate and the results were calculated as mean standard deviation (SD) in this study. Coefficients of determination (R2) were calculated using Microsoft Excel 2000. Differences between mean values were compared by least significant difference (LSD) calculated using the Statistical Analysis System (SAS Institute, Inc., Cary, NC).

Results
ISOLATIONOF ENDOPHYTIC FUNGI

A total of 1,160 endophytic fungal strains were isolated from the 29 traditional Chinese medicinal plants. All the plant samples were found to harbor various endophytic fungi with different colonization rates (CR). This was consistent with previous reports (Saikkonen et al. 1998; Strobel and Daisy 2003; Wiyakrutta et al. 2004). Melodinus suaveolens (Hance) Champ. ex Benth., Strophanthus divaricatus (Lout.) Hook. & Arn., and Polygonum cuspidaturn Sieb. & Zucc. had endophytic fungi colonizing in all of the tested samples (CR values were 100%). Cerbera manghas L., Trachelospermum jasminoides (Lindl.) Lem., Hoya carnosa (L. f.) R. Br., Toxocarpus wightianus Hook. & Arn., Polygonurn capitatum Buch.-Ham. ex D. Don, and Cestrum nocturnum L. also showed high colonization rates, and their CR values were 92.5%, 95.0%, 95.0%, 92.5%, 97.5%, and 92.5%, respectively, whereas Asclepias curassavica L. showed the lowest CR (36.7%). Out of the total 1,160 preliminary fungal isolates, 292

The improved ABTS method has been widely used to assess total antioxidant capacities of crude extracts (Cai et al. 2004; Re et al. 1999). Using this method, we measured the total antioxidant capacities of the methanolic extracts from the 29 medicinal plants as well as the cultures of the 292 endophytes isolated from these plants. Table 1 showed that antioxidant activity of the medicinal host plants ranged from 2.24 to 74.60 mmol trolox/100g DW. Palyganum capitatum showed the highest TEAC value (74.60 mmol/100g DW), followed by P. cuspidatum (56.22 mmol/100g DW), M. suaveolens (54.94 mmol/100g DW), and Polygonum chineme L. (53.66 mmol/100g DW). The TEAC values of the 292 endophytic fungi cultures exhibited a significant variation, ranging from 2.87 to 526.93 Ftmol trolox/100 ml culture, with a mean value of 49.76 btmol trolox/100ml culture. Most fungal strains (190 of 292 isolates, 65.1%) showed moderate activities (20-50 p~mol trolox/100ml culture). Distribution (the number of isolates) of endophytic fungi isolated from each medicinal plant with different ranges of total antioxidant capacity (TEAC value, ~mol trolox/100ml culture: A,<20; B, 20-50; C, 50-100; D, 100-200; E, > 200) was shown in Fig. 1A. The endophytic fungi with more than 100 p~mol trolox 100/ml are given in Table 2. Strain AcapF3 isolated from the flower of Artemisia capillaris Thunb. possessed the strongest antioxidant capacity with the value of 526.93 [~mol trolox/100 ml culture, followed by TwL3 isolated from the leaf of T. wightianus (298.35 ~mol/100 ml), PrL7 isolated from the leaf of Plumeria rubra L. (269.7 ~mol/100 ml), AcS6 isolated from the stem of A. curassavica (267.0 I~mol/100ml), CrF2 isolated from the flower of Catharanthus roseus (L.) G. Don (224.8 gzmol/100ml), AlL4 isolated from the leaf of Artemisia indica Willd. (217.8

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Fig. 1. Number of endophytic fungi isolated from each medicinal plant and screening of endophytic fungal metabolites for trolox (A) equivalent antioxidant capacity (TEAC) and (B) total phenolic content (TPC). Code names in the figure correspond with the code names of medicinal host plants in Table 1.

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TABLE 2. TOTALANTIOXIDANTCAPACITYAND PHENOLIC CONTENT OF METABOLITESFROM 22 SELECTED ENDOPHYTICFUNGI (>100 lilMOLTROLOX/100ML CULTURE). Codeof Endophyte AcaL5 AcS6 AcapF1 AcapF3 AcapF4 AiL 1 AlL3 AiL4 CmL3 CnoL3 CnoS 1 CnoS4 CrF2 CrL7 HcL2 MsL4 NoS3 PcuS7 ProS7 PrL2 PrL7 TwL3 Control LSD (p < 0.05)* HostPlant
Allamanda cathartica L. Asclepias curassavzca L. Artemism capillaris Thunb. Artemma capillaris Thunb. Artemisia capdlarzs Thunb. Artemisia indica Willd. Artemisia indica Will& Artemism indica Willd. Cerbera manghas L. Cestrum nocturnum L. Cestrum nocturnum L. Cestrum nocturnum L. Catharanthus roseus (L.) G. Don Catharanthus roseus (L.) G. Don Hoya carnosa (L. f.) R. Br. Melodinus suaveolens (Hance) Champ. ex Benth. Nerlum oleander L. Polygonum cuspldatum Sieb. & Zucc. Polygonum multzflorum Thunb. Plumeria rubra L. Plumeria rubra L. Toxocarpus wightianus Hook. & Am.

Source leaf stem flower flower flower leaf leaf leaf leaf leaf stem stem flower leaf leaf leaf stem stem stem leaf leaf leaf

TEAC(lamol trolox/100mlculture) 124.00_+ 1.963 267.00_+ 3.321 127.41 _+2.036 526.93_+ 7.942 177.12 _+ 4.119 100.20 _+0.718 102.21 + 2.969 217.76 _+2.081 139.44 + 1.418 127.40_+ 0.557 125.40 _+0.562 152.60 _+1.368 224.80_+2.910 183.30_+0.777 117.90_+0.915 204.40_+ 0.797 150.79 _+2.045 164.20 _+3.067 115.30_+ 1.892 109.25 _+ 2.081 269.70_+ 3.925 298.35 _+3.604
1.28

TPC (naggalhc aud/100m1culture) 7.67-+ 0.133 26.83 + 0.280 13.71 _+0.190 49.22_+ 2.441 18.43 _+0.195 6.88_+0.099 8.69 _+0.129 21.27 _+0.166 12.00_+0.210 9.06 + 0.022 9.58 _+0.058 10.17 -+0.082 32.37_+0.541 19.33-+0.128 6.74_+0.100 27.35 _+0.617 13.95 _+0.109 18.01 _+0.035 6.33_+0.053 11.76_+ 0.255 22.26_+ 0.232 24.28_+0.095 0.02 0.956

4.633

* LSD (p< 0.05), least significant &fference,was used for significantdifference comparisonamong means of various endophytic fungal cultures.

Ixmol/lO0 ml), and MsL4 isolated from the leaf of M. suaveolens (204.4 Ixmol/lOOml). In contrast, the control broth without endophytic fungi showed nearly no activity (1.28 Ixmol/lOOml). We estimated the total phenolic content of the 292 selected endophytic fungal cultures and their medicinal host plants using the classical FolinCiocalteu colorimetric method. It was found that the medicinal plants contained 0.28 to 8.69 g of gallic acid per 100 g DW. Total phenolic contents of the 292 endophytic fungal cultures showed a huge variation, ranging from 0.12 to 49.22mg gallic acid per 100 ml culture, with a mean value of 3.43 mg gallic acid/100 ml culture. Two hundred and seventeen of the endophytic fungal isolates (74.3%) contained medium levels of phenolics (1.00-5.00 mg/100 ml). The distribution of endophytic fungi isolated from each medicinal plant with different ranges of total phenolic con-

tent (TPC value, mg/100ml: A, <1.00; B, 1.005.00; C, 5.00-10.0; D, >10.0) was also shown in Fig. 1. The endophytic fungi with higher phenolic content are given in Table 2. Seven endophytic isolates (AcapF3, CrF2, MsL4, AcS6, TwL3, PrL7, and AiL4) with higher antioxidant capacity also contained more phenolic compounds, and their T P C values were 49.22, 32.37, 27.35, 26.83, 24.28, 22.26, and 21.27mg/100ml (all more than 20 mg/100 ml), respectively. The T P C value of control broth without endophytic fungi was close to zero (0.02 mg/100 ml) (Table 2). RELATIONSHIP BETWEEN TOTAL ANTIOXIDANT CAPACITY AND PHENOLIC CONTENT Figure 1 also shows that the endophytic fungi containing a lower phenolic content (TPC< 1.00 mg/100 ml) mostly possess weaker antioxi-

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Fig. 2. Relationshipsbetween total antioxidant capacities and phenolic contents of (A) metabolites of 292 endophytic fungal cultures selected from the 29 host plants and of (B) methanolicextracts from the 29 Chinese medicinal host plants.

dant activity (TEAC values< 20 p~mol/100ml), and the endophytic isolates with moderate antioxidant activities (TEAC values within 20-50 p~mol/100ml) normally contain medium levels of phenolics (1.00-5.00mg/100ml), while those isolates with high total phenolic content (TPC> 10.0mg/100ml) also have high total antioxidant activity (most of TEAC values>100 I~mol/100 ml). As shown in Fig. 2, a correlative relationship was present between the total antioxidant capacity and total phenolic content in the 292 endophytic fungal isolates as well as in their host plants. A highly positive linear correlation between total antioxidant capacity (y) and total phe-

nolic content (x) was established for both the endophytic fungal isolates (y=9.1524x + 18.41, R2=0.9041) and their host plants (y=8.3211x 2.0333, R2 = 0.9150). These correlations suggest that the phenolic compounds are most likely responsible for the antioxidant activities of the fungal cultures and the host plants. However, this does not imply that the phenolic compounds of the host plants are actually produced by their fungal endophytes in situ. The small number of fungal cells in planta, compared to the concentration of cultivated fungal strains, is unlikely to make a major contribution to the quantity of phenolics of the host plant, although fungal colonization itself

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may induce the host plant to produce a different phenolic profile. Our results were consistent with previous reports that phenolic compounds are major antioxidant constituents in medicinal herbs, vegetables, fruits, and spices (Cai et al. 2004; Shan et al. 2005; Zheng and Wang 2001).
PRELIMINARY IDENTIFICATION AND COMPARISON OF BIOACTIVE CONSTITUENTS

both contained a high level of chlorogenic acid (Fig. 4). A major volatile composition, tentatively named artemisin (C12H10, MW 154), was detected in both AiL4 (an endophytic fungal culture) and its host plant (A. indica) (Fig. 5). Furthermore, A. indica also contained high levels of caffeoylquinic acid derivatives (di-O-caffeoylquinic acids) (Fig. 4B).

Different phenolic compounds normally possess specific chromatographic behavior and UVvis spectral characteristics (Cai et al. 2004; Sakakibara et al. 2003; Santos-Buelga and Williamson 2003; Shah et al. 2005). Because of the diversity and complexity of the natural mixtures of phenolic compounds in the large number of samples, it is rather difficult to characterize every compound and elucidate its structure. Therefore, only a preliminary identification of phenolic compounds and other bioactive compositions in the endophytic fungal metabolites and their medicinal host plants was carried out in this study. We analyzed major phenolic compounds in selected endophytic fungi (TEAC > 50 txmol/100 ml) and all the medicinal host plants using RP-HPLC with DAD by comparison with over 100 authentic phenolic standards and related literature data (Cai et al. 2004; Sakakibara et al. 2003; Tan and Zou 2001). Most of the selected medicinal host plants and endophytic fungal metabolites were also analyzed using LC-ESIMS and GC-MS. The preliminary results showed that major types of bioactive compounds in both the medicinal host plants and endophytic fungal metabolites were phenolic acids, flavonoids, tannins, quinones, phenolic terpenoids, volatile constituents, and aliphatic compounds. Representative bioactive compounds identified in this study are given in Table 3. Chlorogenic acid (5-O-caffeoylquinic acid) was a major phenolic compound from medicinal plants widely distributed in the families of Apocynaceae and Asclepiadaceae. High levels of chlorogenic acid could be detected in most of the plant samples in these two families (Fig. 3), especially in 77. divaricata which had the highest level of chlorogenic acid and strong antioxidant activity (TEAC value was 26.60 mmol/100g DW). Interestingly, certain compounds were found in both the endophytic fungal culture and the corresponding host plant extract (Table 3). For example, AiL1 (an endophytic fungal culture) and its host plant (A. indica)

Discussion
The ubiquity of endophytes in the plant kingdom has been well established as they have been found in all species investigated, including algae, mosses, ferns, and vascular plants (Arnold et al. 2000; Arnold, Maynard, and Gilbert 2001; Gamboa and Bayman 2001). In this study, all the 29 tested medicinal plants were colonized by various endophytic fungal strains, with morphologically distinct fungal isolates ranging from 23 in R. verticillata to 83 in 77. divaricata. Most of the isolated endophytic fungi showed no reproductive structures or no distinctive features when cultured on common mycological medium. This is a common problem concerning the identification of endophytes (Arnold et al. 2000; Gamboa and Bayman 2001; Wiyakrutta et al. 2004). Thus identification of these endophytic fungal isolates was not performed in this study. However, the endophytic fungi did exhibit characteristic culture colony or hyphal and microscopic morphology that could be differentiated amongst the isolates. In addition to traditional morphological identification methods, modern molecular biology techniques are required for characterization and classification of these isolates. The improved ABTS method was not only a rapid and reliable test of total antioxidant capacity in vitro, but also an advantageous assay applicable to both hydrophilic and lipophilic antioxidants or systems (Cai et al. 2004; Re et al. 1999; Shah et al. 2005). This method was also successfully used in the present study to screen antioxidant capacity for the 292 morphologically different fungi out of the total 1,160 endophytic fungal isolates. The RP-HPLC methods have also been well developed for most categories of phenolic compounds in plants (Cai et al. 2004; Sakakibara et al. 2003; Santos-Buelga and Williamson 2003; Shan et al. 2005). In this study, the constituents in methanolic extracts of the 29 medicinal host plants and in the metabolites from some (bioactive) endophytic isolates were preliminarily analyzed and screened by

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TABLE 3. REPRESENTATIVEBIOACTIVE COMPOUNDS IN MOST OF THE TESTED ENDOPHYTIC FUNGAL METABOLITESAND MEDICINALHOST PLANTSIDENTIFIEDBY RP-HPLC, LC-MS, AND GC-MS. CompoundCategories and Representanvc Conmtuents Phenolic acids Chlorogenic acid (5-O -caffeoylquinic acid) Di-O-caffeoylquinic acids Flavonoids Quercetin 3-rutinoside (rutin) Quercetin 3-rhamnoside (quercitrin) Quinones Anthraquione glycoside Rehein Emodin Volatile compounds Artemisin Aliphatic compounds Hexadecanoic acid, methyl ester 9-Hexadecenoic acid, methyl ester 9,12-Octadecadienoic acid, methyl ester 11,14,17-Eicosatrienoic acid, methyl ester LC-MSObserved AdductIons(m/z)or GC-MSData [M-HI (353), [M +H] + (355), [M + Na]+ (377) [M-H]-(515), [M + Na] + (539) [M-HI (609), [M + Na] + (633) [M-HI- (447), [M + H] + (449), [M +Na] + (471) [M -H]-(518), [M + Na] (541) [M -H]-(283) [M -H]-(269), [M + H]+ (271) MW: 154, Ci2Hl0 MW: 270, CI7H3402 MW: 268, CI7H3202 MW: 294, CI9H3402 MW: 320, C21H3602 ALL4* CrF2*, CrS5*, PcuL7*, PrL7*, SdL2* ASS3,CrS5, PcuS7 AcaL5*, CrF2*, CrL6*, PcuL7*, PrL3* AcaL5*

Endophyte~ AiLI*

McdmmalHostPlants
A. indica*, G. pictum, H. carnosa, N. oleander, T. divar~cata A. mdzca, N. oleander, S. divaricata

A. indlca, C. nocturnum, Z divaricata P. capitatum, P. chinense, P. cuspidatum, P. multiflorum P. cuspidatum P. cuspzdatum P. cuspidatum, P. multiflorum

A. indica* C. roseus*, C. nocturnum, P. cuspidatum *, 1~ rubra*, S. divaricata* A. cathartica*, C. roseus*, C. nocturnum, P. cuspzdatum*, P. rubra* A. cathartica*, A. sinensis, P. cuspidatum, T. peruviana

* Compounds were found m both endophytic fungi and their host plants.

using PR-HPLC, and some were identified by LC-ESI-MS and GC-MS. The results showed that phenolic compounds (e.g., phenolic acids, flavonoids, tannin constituents, hydroxyanthraquinones, and phenolic terpenoids) and certain volatile or aliphatic constituents were major substances in the endophyte cultures and host plant extracts. The categories of bioactive compounds identified in the host plant extracts were similar to those of 112 traditional Chinese medicinal plants (Cai et al. 2004). Phenolics have long been associated with antioxidant capacity in plants, but this association has never been investi-

gated in endophytic fungi. The correlation between TEAC and T P C values (Fig. 2) provides support for the hypothesis that phenolic compounds are also responsible for the total antioxidant capacity of the endophytic fungal cultures. Endophytes are a poorly investigated group of microorganisms, and endophytic fungal metabolites represent an abundant source of bioactive and chemically novel compounds (Strobel et a[. 2004). Wiyakrutta et al. (2004) reported that the metabolites produced by endophytic fungi from Thai medicinal plants exhibited antimicrobial, anticancer, and antimalarial activities. Endo-

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Fig. 3. HPLC chromatograms (280 nm) of methanohc extracts from the selected medicinal plant species in the family Apocynaceae and Asclepiadaceae. The dominant peak (retention time, - 20.3 min) in HPLC profiles of all selected plants was identified as chlorogenic acid ([M-H]- ion at m/z 353, [M + H] ion at m/z 355, and [M + Na] + ion at m/z 377) by ESI-MS and by comparison with the related authenuc standard.

phytic fungi were also found in a Chinese medicinal plant, Tripterygium wilfordii, and the endophytic fungal cultures possessed antiproliferative activity on human peripheral blood mononuclear

cells (Kumar et al. 2004). Song et al. (2005) reported that a phenolic compound (graphislactone A) with strong free radical-scavenging and antioxidant activity was isolated and identified from

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Fig. 4. HPLC chromatograms (280 nm) and ESI-MS data of phenolics from (A) an endophytic fungus (ALL1)and (B) its host plant (A. indica). I, chlorogenicacid (5-O-caffeoylquinicacid) (MW 354); 2, 3, and 4, unknown phenolics (MW 212, 210, and 240); 5, 6, and 7, di-caffeoylquinicacid isomers (MW 516); 8, apigenin 7-O-glucuronide (MW 446).

the culture of Cephalosporium sp., an endophytic fungus isolated from the root of Trachelospermum jasminoides (Apocynaceae). In the present study, most of the endophytic fungal cultures showed antioxidant capacity to some extent, and those possessing high phenolic contents exhibited very strong antioxidant activities. These findings show that the metabolites of endophytic fungal cultures can be a potential source of natural antioxidants. Strobel et al. (2004) showed that plants with medicinal values provide the best opportunities to isolate novel endophytic microorganisms as well as ones making novel bioactive products. In this study, we found that several endophytic fungi (MsL4, NoS3, PcuS7, PmS7, and TwL3) with strong antioxidant activities were isolated

from those medicinal hosts showing a high antioxidant capacity, such as M. suaveolens, Nerium oleander L., R cuspidatum, R multiflorum, and 77. wightianus (Table 1 and Table 2). Also, the endophytic fungi (AcapF1 and AcapF4 from the flowers of A. capillaris; ALL1, ALL3, and AiL4 from the leaves ofA. indica) possessing strong antioxidant activities were isolated from two Artemisia medicinal plants, both with a high antioxidant capacity. The strongest antioxidant fungal strain, AcapF3, was found in A. capillaris, which also showed strong antioxidant activity using the DPPH method by Jung et al. (2004). In contrast, no endophytic fungal culture with strong antioxidant activity was found from the host plant R capitatum, which showed the highest antioxidant capacity of all the plants studied. This could

2007]
Ittt (xl00.000)

HUANG ET AL.: ENDOPHYTIC FUNGI

27

(A)
o
o

20.

r~

L~

3
10 20 30 40 50

T6
60 70 80 90

%
I00 i~In

Int (x10,O00,O00)
ISO-

i ~

(B)

07e-

71 ~o ~i=7

; - ~,., . l ~ , -

. !~

10

20

30

40

50

80

70

80

90

IOOmll~

Fig. 5. GC chromatograms of volatile compounds from (A) an endophytic fungus (ALL4) and (B) its host plant (A. lndica). Molecular weight and formula are noted for most peaks in the figure. Tentative identification of major peaks: 1, eucalyptol; 7, caryophyllene; 8, oL-caryophyllene;9, dimethyl phlthalate; 10, artemisin; 11, o~-farnesene.

occur because we might not have isolated all the endophytic fungi present in P. capitaturn, and only six of the 48 fungal isolates from the host plant were screened for their antioxidant activity. There are previous reports that some endophytes isolated from plants producing certain bioactive compounds could make the same natural products as the hosts (e.g., Stierle, Strobel, and Stierle 1993; Lee et al. 1995; Strobel and Hess, 1997). For example, taxol, a famous anticancer compound isolated from the bark of Pacific yew, has also been obtained from its fungal endophyte (Taxornyces andreanae) in vitro (Stierle et al. 1993). In the present study, chlorogenic acid (5O-caffeoylquinic acid), a major bioactive phenolic compound, was detected in both the host plant A. indica and its endophytic fungus AlL1 isolated from the leaf using RP-HPLC and ESI-MS (Fig. 4). Chlorogenic acid and its derivatives possess a wide range of bioactivities, such as antioxidant, antimutagenic, immunomodulatory, and antiviral activities (dos Santos et al. 2005; Li, But, and Ooi

2005). Furthermore, artemisin, a major volatile constituent in the genus Arternisia, was identified in both A. indica and AlL1 using GC-MS (Fig. 5). Volatile oils are important bioactive compositions in medicinal plant species of Artemisia (e.g., A. indica, A. capillaris, A. annua) (Gao et al. 2000; Xiao et al. 2000). The 11,14,17-eicosatrienoic acid, methyl ester, one kind of aliphatic compound with antioxidant and anticarcinogenic activities (Rameazni-Kharazi 2004), was also detected in both the extract ofAllarnanda cathartica L. and the extract of its endophytic fungal isolate AcaL5 using GC-MS (Table 3). This suggests that endophytic fungi might produce the same bioactive natural compounds as some of those found in their host plants. The colonization and propagation of endophytes may in some ways offer significant benefits to their host plants by producing a plethora of substances that provide protection or increase the fitness of the hosts, such as enhancement of stress-, insect-, or disease-resistance, and produc-

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tivity improvement (Tan and Zou 2001; Strobel et al. 2004). Some endophytes exhibit host preference (Petrini et al. 1992) and tissue specificity (Luginbuhl and Miiller 1980). Spatial heterogeneity (Carroll 1995) or seasonal differences (Rodrigues 1994) also exist in terms of compositions and quantities of endophytic strains. These ecological or seasonal variations also affect the potency of medicinal plants, as we have known that both the seasons and places of collection are important factors affecting their medicinal values. The study by Plowman et al. (1990) suggests a fungal basis for the ethnobotanical utilization of piripiri (medicinal species of Cyperus) from Amazonia. Earlier findings also showed that coniferin and syringin, two monolignol glucosides produced by the host plant, were specifically recognized by the endophytic Xylarlaceae species as chemical signals during the establishment of fungus-plant interactions (Chapela et al. 1991). However, the hypothesis that medicinal values of plants have a biological basis resulting from fungal infection needs further investigation.

Limitations of the Study

The relationship between fungal endophytes and their host plants is a complicated research topic. There have been suggestions that many of the compounds that we typically think of as plant secondary metabolites may in fact be produced by fungal endophytes. However, it is unlikely that such compounds isolated from the host plants could be entirely produced by their endophytes in situ, considering the small quantity of the endophytes within each host plant. In contrast, when the fungal endophytes were isolated and cultivated in vitro, the number of fungal cells would be multiplied far beyond their normal concentration in planta, and thus a measurable phenolic profile is possible. Ideally, an endophyte-free plant of the same species should be used to serve as control, but such a plant is difficult to find because every plant previously studied is host to one or more endophytes (Arnold et al. 2000; Arnold, Maynard, and Gilbert 2001; Gamboa and Bayman 2001; Strobel et al. 2004). Without controlled experiments to test whether the host plants can produce the same compounds in the absence of their endophytes, however, we cannot conclude with certainty that the same compounds are in fact produced in vivo by the endophytes alone or by both the endophytes and the host plants.

Although our comparative analysis of the bioactive compositions of the host plants and the isolated endophytic fungi has led to the discovery of some unique natural products produced by the endophytic fungi, it is just the first step in the investigation of the relationship and interaction between the host plants and endophytes. It remains unclear whether the hosts and endophytes exchange metabolites or whether the endophytes can produce the same bioactive products through metabolic processes independently from the hosts. One mechanism proposed to explain why some endophytes produce the same bioactive compounds as their host plants is genetic recombination of the endophyte with the host in evolutionary time (Stierle et al. 1993; Tan and Zou 2001). Recent molecular biological studies of Pestalotiopsis microspora, an endophytic fungus of Taxus wallichiana, may have provided an explanation as to how some of the host plant genes may have been acquired, expressed, and replicated by the endophytes (Long et al. 1998). On the other hand, it is also possible that the medicinal values of some of the plants may in fact come from their endophytes. The traditional Chinese medicinal plants have a long ethnobotanical history, and are related to specific use or applications by indigenous peoples. This is a specific rationale for the collection of medicinal plants for endophyte isolation and naturalproduct discovery. However, our study has not used randomly chosen plants that are not used in traditional Chinese medicine as a control group for comparison. Clearly, further research is needed to ascertain whether traditional medicinal plants are more likely to harbor novel antioxidant fungi and their products than plants selected at random.

Concluding Statements
A large number of morphologically distinct endophytic fungi were isolated from the 29 traditional Chinese medicinal plant species studied. Most of the endophytic fungal cultures exhibited antioxidant activities. The total antioxidant capacity of the endophytic fungal metabolites was significantly correlated with their total phenolic content. We also isolated and screened several endophytic fungi which could produce the metabolites containing bioactive compounds with potent antioxidant capacity. These findings indicate that the metabolites produced by the endophytic fungi in culture were a potential source of natural

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antioxidants and some novel bioactive compounds. Unknown/novel compounds in the tested endophytic fungal metabolites will be further identified. Future investigations will be conducted for those endophytic fungi with strong antioxidant capacity to identify and elucidate the bioactive constituents and their chemical structures, and to determine the taxonomic identities of these endophytes.

Acknowledgments
This research was supported by grants from the University of Hong Kong (Seed Funding for Basic Research). We thank Mr. S. T. Chan for assistance in field collection and identification of medicinal plants.

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