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.

Switzerland). alkaloids and amines). and stems or roots were cut into pieces (10 mm in length). quinones. 2004). stem. Zheng and Wang 2001). and tannins). four in the Polygonaceae. Tan and Zou 2001. LC-MS. Folin-Ciocalteu reagent and HPLC-grade organic reagents were from BDH (Dorset. Medicinal plants contain a wide variety of free radical scavenging molecules. The main objective of this study was to explore the endophytic fungi from medicinal plants. 2007). nitrogen compounds (e. Traditional Chinese medicinal plants have been used for pharmaceutical and dietary therapies for several millennia (Cai et al. Authentic standards. as a novel antioxidant source. a total of 1. antidiabetic agents.g. UK). 1999.5. no comparative investigation has been carried out for their endophytes. 1999. flowers. or root) from the medicinal plants were first washed in running water. Despite numerous studies on antioxidant activities and phenolic contents in plants. Louis. and other endogenous metabolites (Cai et al. All the chemicals and reagents used in this study were of the analytical grade. Chan. or fruits were cut into segments (5 x5 mm). terpenoids. phenolic acids. K~ihk6nen et al. Natural compounds isolated from these medicinal plants are a rich source of novel drugs with multiple biological activities including antioxidant properties. 2002). lignans.. The relationships between the total antioxidant capacities and total phenolic contents in the 29 host plants were concurrently investigated for comparison with their endophytes. CHEMICALS AND REAGENTS The chemicals 2. such as phenolic compounds (e. The collected plants represent 29 species from six families. antibiotics. and GC-MS). with both studies showing highly significant positive linear correlations between the total antioxidant capacities and phenolic contents. The plant species and their collection sites are listed in Table 1. S. seven in the Asclepiadaceae. 2004). and Trolox (6-hydroxy-2. and sodium carbonate were purchased from Sigma/Aldrich (St. Re et al. Only the 292 morphologically distinct fungal isolates were used for investigation of the total antioxidant capacities. The medicinal plant species were authenticated by Mr. 2004. insecticidal products.2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS). All the plant samples were surface-sterilized by dip- . Wiyakrutta et al. antiviral compounds. flavonoids.7. 2004. stilbenes.: ENDOPHYTIC FUNGI 15 roids.g. There have been many studies on the antioxidant activities of various plants of medicinal use (e. a local plant expert at the University of Hong Kong. and their relationships. 2004.2007] HUANG ET AL. and other chemicals and reagents used in this study were obtained from Sigma/Aldrich. MO). which can in vitro produce bioactive compounds with potent antioxidant activity. 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. anticancer agents. however. ISOLATION OF ENDOPHYTIC FUNGI A total of 20 samples of plant parts (leaf. xanthones. 1999. potassium persulfate. IC~hk6nen et al. 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. including 13 species in the Apocynaceae. Cai et al. Antibiotics. and medicinal plants have been recognized as a repository of endophytes with novel metabolites of pharmaceutical importance (Strobel et al. All the flesh samples were taken to the laboratory and treated within 24 hours. terpenoids. vitamins. three in the Asteraceae. In the present study. and one each from the Lamiaceae and the Solanaceae. fruit. Tapiero et al. tetralones. and similar findings were reported for 133 Indian medicinal plants (Surveswaran et al. and others (Tan and Zou 2001). lignin. Surface sterilization and isolation of endophytic fungi followed a modified method as described by Schulz et al. The leaves. Zheng and Wang 2001). flower.g. T. immunosuppressive compounds as well as antioxidants have been reported from endophytic metabolites (Strobel et al.160 endophytic fungal isolates were obtained from different tissues of 29 traditional Chinese medicinal plant species..8-tetramethylchromate2-carboxylic acid) from Fluka Chemie AG (Buchs.. (1993). (2004) reported that phenolic compounds were the dominant antioxidant components in 112 traditional Chinese medicinal plants associated with combating cancer. phenolic contents. coumarins.

~ ~ ~ ~ ' ~ "e. ~ +1 § ~ § +1 § ~ o § § +1 z M Z +1 § § +1 § +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 ~. ~ ~ ~"~ ~ ~ ~ ~ ~ ~ ~ .~ ~ ~ ~ . ~ ~ ~ ~ ~ .~ ~: ~ ~ ~ ' ~ ~ ~ "~ ~ .a.~ < < ~ 9 . . ~ .~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ . ._~ ._I . . .2~*~-~ ~ ~. ~.16 ECONOMIC BOTANY [ V O L .~ Z LL z vo N Z Z ~ . 61 o z r~ z Z < z o o o o o o o +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 o o o +1 +1 +1 +1 5 M < Z .

FeSO4. 2. 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.670g for 10 min and filtered using a Millipore filter with a 0. 0.e~ =999 k. followed by incubation with a Shaking Incubator (Daihan Labtech Co. Ltd.5g.% 2004). The filtrate sample was stored at 4~ until use within 24 hours. NaNOy 3g. { . 1 g.000ml). were inoculated in 100 ml flasks. 0.) at 140rpm for 15 days at 28~ The culture broth of each endophytic fungus was centrifuged at 1. UK).. The extract was also filtered by a Millipore filter with a 0. yeast extract..= e% < = E 0 +l+l+l+l +1 t'-.5% sodium hypochlorite for 15 min. followed by incubation at 28~ until the mycelium or colony originating from the sample appeared. and the purified strains were preserved on potato dextrose agar (PDA) plates at 4~ in the Laboratory of Department of Zoology.. 0.~ 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. CULTIVATION OF ENDOPHYTIC FUNGI e-. H20. and again 75% ethanol for 1 min. MgSO4.22-~m nylon membrane under vacuum at room temperature (-23~ to remove mycelium. 1999). Ltd. Ltd. ping in 75% ethanol for 1 min. and stored at 4~ (Cai et al.5 g..7H20. 2004. K2HPO 4.~ Fresh mycelia (grown on PDA plates at 28~ for 3-6 days) of morphologically different endophytes. UK) and passed through a 24-mesh sieve.01g.22-~m nylon membrane under vacuum at room temperature. KCI... 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. 30g. Hyphal tips were transferred and cultured on potato carrot agar (PCA) plates. approximately equal in quantity. the University of Hong Kong. 0 ~ od +1 t~ "'"0 +1+1+1+1 O P'-- ~_~ r 4 { E2• ~ .. followed by rinsing in sterile water three times.: ENDOPHYTIC FUNGI 17 . The ABTS'+ radical cation was gen- o ~ F~ .2007] HUANG ET AL. In each petri dish (9cm diameter). 1. TOTALANTIOXIDANT CAPACITYASSAY Total antioxidant capacity was assayed using the improved ABTS method (Cai et al. 1 g. Re et al. each containing 50ml of broth (sucrose.-< -d . EXTRACTION OF HOST PLANT SAMPLES 0 .

A gradient elution used was 0-5 rain. The flow rate was 0. 65 min. flavanols. 55% B. Headspace conditions were as follows: powder samples were kept in incubator vials in an oven at 110~ with shaking for 20 min. a SILl 0Avp auto-sampler. 100% B. Detection was monitored at different wavelengths for various phenolic compounds: 280nm for hydroxybenzoic acids.5% formic acid.700 + 0. 5% B. 15-40 min. The ESI voltage was 4.1% formic acid. The absorbance of the resulting oxidized solution was compared to that of the calibrated trolox standard. 2. LC conditions were as follows: solvent A. Japan). flavanones.5 kV in negative ion mode. Germany).005) was added to 0. and equipped with a Nucleosil 100-C18 column (5~m. a central controller. 40-60 min.25mm. A nebulizing gas of 1. DETERMINATION OF TOTAL PHENOLIC CONTENT Total phenolic content (TPC) was estimated using the Folin-Ciocalteu colorimetric method described by Liu et al. injection volume was 5-10 ~l. 95-96 min. and tannins. The tested sample was diluted with 80% ethanol so as to give 20-80% inhibition of the blank absorbance with 0. and solvent B. isoflavones. a photodiode-array detector. 2004) with slight modifica- tion.9 ml. terpenoids. Flow rate was 0. 50-55% B.5 N Folin-Ciocalteu reagent for 4 min at room temperature. 4 • 4 mm) (Agilent Technologies. consisting of a binary pump and a diode-array detector (DAD).1 ~m film thickness) was used with helium as a carrier gas at a fow rate of 2. 60-65 min. 65-90 min. 320 nm for hydroxycinnamic acids and flavones. 30% B.. gtmol trolox/100ml culture of endophytic fungus or mmol trolox/100g dry weight (DW) of plant.2 ml) were oxidized with 0.45mm potassium persulfate after incubation at room temperature in darkness for 16 hours.1 ml of sample. o with 0. 55-100% B. Seto. mg gallic acid/100 ml culture or g gallic acid/100 g DW. 5% B. Injection volume was 1 gtl. Then the reaction was neutralized with saturated sodium carbonate (75g/1). 0. A DB-5MS column (60mx0. 60 min. 5% B.46 ml/min.700 + 0. The reactive mixture was allowed to stand at room temperature for 6 min and the absorbance was immediately recorded at 734 nm. 90-95 rain. Loveland. 61 erated by reacting 7mm ABTS and 2. and a single quadrupole MS detector with electrospray ionization (ESI) interface (Shimadzu. Different levels of trolox standard solution in 80% ethanol were prepared and assayed under the same conditions. 5-15 min.5 min . The analytical column was VP-ODS C18 column (250 x 2. 5 gtm) (Narmura Chemical Co. Waldbronn.1 ml of the tested samples and mixed thoroughly. 96-100 min. Results were expressed as gallic acid equivalent (GAE). MeOH with 0. The ABTS'* solution was diluted with 80% ethanol to an absorbance of 0.18 ECONOM IC BOTANY [VOL.1% formic acid. absorbance of 0. CO). 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.H P L C ANALYSIS RP-HPLC analysis was performed using a Hewlett-Packard HPLC System (HP 1100 series. 100-5% B. the appropriate dilutions of the sample (0.005 at 734nm. The LC eluant was introduced directly into the ESI interface without flow splitting. LC-ESI-MS The LC-MS-2010A system used in this study consisted of a LC-10ADvp binary pump. 250• with a Nucleosil 5 C18 guard column (5 ~m..e.5 kV in positive ion mode and 3. and solution B. 30-40% B. Phenolic compounds in the samples were analyzed using our previous method (Cai et al. Japan). 40 min. The GC oven temperature was kept at 40~ for 0. Briefy. 40-50% B. Quantification was done on the basis of the standard curve of gallic acid. 40% B.e. 50% B. R P . i. ABTS'* solution (3. The following gradient elution program was used in the improved HPLC method (solution A. 100% B. The scan range of ESI-MS was m/z 160-800. GC-MS A GCMS-QP2010 system (Shimadzu.8 ml/min and injection volume was 20 ~1. (2004) with minor modification. and detection was at 280nm. 100% methanol): 0 min.2 ml/min. (2002) and Cai et al. Results were expressed in terms of trolox equivalent antioxidant capacity (TEAC).5 l/min and a drying gas of 10 l/min were applied for ionization using nitrogen in both cases.0 mm. 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. and 90-98 min.. 5-30% B. 370 nm for flavonols and chalcones. i. 15 min.

& Arn. Wiyakrutta et al. STATISTICALANALYSIS morphologically distinct isolates from the 29 medicinal plants were selected for antioxidant activity screening and total phenolic content assay (Table 1 and Fig. respectively. 1A. 50-100. A library search was carried out using NIST and SZTERP libraries. 95.) Hook. Strain AcapF3 isolated from the flower of Artemisia capillaris Thunb. cuspidatum (56. > 200) was shown in Fig. AcS6 isolated from the stem of A. and Polygonum cuspidaturn Sieb. 2004).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. and Cestrum nocturnum L. Re et al. Distribution (the number of isolates) of endophytic fungi isolated from each medicinal plant with different ranges of total antioxidant capacity (TEAC value. 1999). (269. (53. Melodinus suaveolens (Hance) Champ.93 [~mol trolox/100 ml culture. ex Benth.2007] HUANG ET AL.66 mmol/100g DW).-Ham. 1998. also showed high colonization rates. CrF2 isolated from the flower of Catharanthus roseus (L.7 ~mol/100 ml).. had endophytic fungi colonizing in all of the tested samples (CR values were 100%).<20.5%. C. Differences between mean values were compared by least significant difference (LSD) calculated using the Statistical Analysis System (SAS Institute. Out of the total 1. This was consistent with previous reports (Saikkonen et al. E.76 btmol trolox/100ml culture. (217.) Lem. 92. ex Roem. Table 1 showed that antioxidant activity of the medicinal host plants ranged from 2.60 mmol trolox/100g DW.35 ~mol/100 ml). Strophanthus divaricatus (Lout. followed by TwL3 isolated from the leaf of T. Trachelospermum jasminoides (Lindl. Polygonurn capitatum Buch. The interface and ion source temperature were 260~ and 200~ respectively. 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.) R. and their CR values were 92. The TEAC values of the 292 endophytic fungi cultures exhibited a significant variation.160 preliminary fungal isolates. possessed the strongest antioxidant capacity with the value of 526. D. TOTALANTIOXIDANTCAPACITYAND PHENOLIC CONTENT All sample determinations were conducted in triplicate and the results were calculated as mean standard deviation (SD) in this study. The GC oven temperature was kept at 40~ for 0..5%.0%. followed by P. Hoya carnosa (L. Br.160 endophytic fungal strains were isolated from the 29 traditional Chinese medicinal plants. The most endophytic fungi (83 strains) were isolated from Tabernaemontana divaricata (L. f.60 mmol/100g DW).) G. Coefficients of determination (R2) were calculated using Microsoft Excel 2000. NC).20 mm. Don (224. Cary.. 292 The improved ABTS method has been widely used to assess total antioxidant capacities of crude extracts (Cai et al. curassavica (267..5%. Br. and 92. A CP-WAX 52 CB column (50 m x 0. & Arn. Strobel and Daisy 2003.7%).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. suaveolens (54.5%.94 mmol/100g DW)..8 . 100-200. 1). ~mol trolox/100ml culture: A.) R. 95. ranging from 2.: ENDOPHYTIC FUNGI 19 and programmed to 150~ at a rate of l~ to 250~ at a rate of 10~ and kept constant at 250~ for 10 min. Most fungal strains (190 of 292 isolates.93 Ftmol trolox/100 ml culture. The endophytic fungi with more than 100 p~mol trolox 100/ml are given in Table 2. The characteristics of colony or hyphal and microscopic morphology were used to differentiate among these isolates.8 gzmol/100ml). o with 0. 97.1%) showed moderate activities (20-50 p~mol trolox/100ml culture).. All the plant samples were found to harbor various endophytic fungi with different colonization rates (CR). wightianus (298. M. 65.0 I~mol/100ml). PrL7 isolated from the leaf of Plumeria rubra L.) Baill. ex D. while the least (23 strains) from Rauvolfia verticillata (Lour. The MS was operated in electron ionization mode (70 eV) with a scan range of m/z 40-600.87 to 526.. whereas Asclepias curassavica L.24 to 74. and Polygonum chineme L. Cerbera manghas L. 20-50. Toxocarpus wightianus Hook. & Schutt. 2004. with a mean value of 49. showed the lowest CR (36. Using this method. B. Liquid injection method was also used in this study. Results ISOLATIONOF ENDOPHYTIC FUNGI A total of 1. Inc. AlL4 isolated from the leaf of Artemisia indica Willd.22 mmol/100g DW). & Zucc. Palyganum capitatum showed the highest TEAC value (74.. Don.0%. Splitless injections were done in both headspace and liquid injection methods.

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).20 ECONOMIC BOTANY [VOL. Code names in the figure correspond with the code names of medicinal host plants in Table 1. 1. 61 Fig. .

255 22. Cestrum nocturnum L. Polygonum cuspldatum Sieb.00 mg/100 ml).133 26.12 _+ 4.83.00-5.00 mg/100 ml) mostly possess weaker antioxi- .58 _+0.37. Plumeria rubra L.00_+0.368 224.321 127.067 115.05)* HostPlant Allamanda cathartica L.22_+ 2.44 + 1.74_+0.71 _+0. Nerlum oleander L.88_+0.67-+ 0.892 109.109 18. 1.280 13.76_+ 0.036 526.43 _+0.00_+ 1.27mg/100ml (all more than 20 mg/100 ml). Cestrum nocturnum L.045 164. and their T P C values were 49. ranging from 0. Melodinus suaveolens (Hance) Champ. Artemism capillaris Thunb.37_+0.35 _+0.06 + 0.28_+0. Asclepias curassavzca L.40 _+0.956 4. Artemisia capdlarzs Thunb.963 267. and MsL4 isolated from the leaf of M.40_+ 0.541 19.777 117.718 102.93_+ 7. 24.69 g of gallic acid per 100 g DW. B. The endophytic fungi with higher phenolic content are given in Table 2. CrF2.) R.) G.035 6.915 204.22.41 _+2.27 _+0.095 0.942 177.01 _+0.: ENDOPHYTIC FUNGI 21 TABLE 2. respectively.557 125.17 -+0. C.40_+ 0. AcS6. >10. PrL7. and 21.90_+0. RELATIONSHIP BETWEEN TOTAL ANTIOXIDANT CAPACITY AND PHENOLIC CONTENT Figure 1 also shows that the endophytic fungi containing a lower phenolic content (TPC< 1.28. the control broth without endophytic fungi showed nearly no activity (1.28 to 8. mg/100ml: A. 26.35. 32.28 TPC (naggalhc aud/100m1culture) 7. Don Catharanthus roseus (L. Don Hoya carnosa (L.83 + 0.00-10.28 Ixmol/lOOml).081 139. 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. The distribution of endophytic fungi isolated from each medicinal plant with different ranges of total phenolic con- tent (TPC value. Br.418 127.69 _+0. 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) 124.76 _+2.119 100.33_+0. In contrast. <1.2007] HUANG ET AL.3%) contained medium levels of phenolics (1. Artemma capillaris Thunb.02 mg/100 ml) (Table 2).30_+0.081 269. Cerbera manghas L. Polygonum multzflorum Thunb.210 9. and AiL4) with higher antioxidant capacity also contained more phenolic compounds.) G. Seven endophytic isolates (AcapF3.00.95 _+0. least significant &fference.33-+0.797 150.633 * LSD (p< 0.190 49. & Am.232 24. MsL4.00_+ 3.022 9.925 298. It was found that the medicinal plants contained 0. D.21 + 2.20 _+3.22mg gallic acid per 100 ml culture.969 217. Catharanthus roseus (L. & Zucc.79 _+2.70_+ 3.25 _+ 2. f. suaveolens (204.4 Ixmol/lOOml). 5. 22.80_+2.00. Ixmol/lO0 ml).43 mg gallic acid/100 ml culture. TOTALANTIOXIDANTCAPACITYAND PHENOLIC CONTENT OF METABOLITESFROM 22 SELECTED ENDOPHYTICFUNGI (>100 lilMOLTROLOX/100ML CULTURE).12 to 49.005. We estimated the total phenolic content of the 292 selected endophytic fungal cultures and their medicinal host plants using the classical FolinCiocalteu colorimetric method.129 21. Total phenolic contents of the 292 endophytic fungal cultures showed a huge variation.128 6.195 6.0) was also shown in Fig.05). Toxocarpus wightianus Hook.053 11.20 _+0. Artemisia indica Will& Artemism indica Willd.604 1.0.26.617 13.60 _+1. Two hundred and seventeen of the endophytic fungal isolates (74. ex Benth. 1. TwL3.100 27.26_+ 0.was used for significantdifference comparisonamong means of various endophytic fungal cultures.02 0.166 12. with a mean value of 3.910 183. The T P C value of control broth without endophytic fungi was close to zero (0. Cestrum nocturnum L. Plumeria rubra L.562 152. Artemisia indica Willd.058 10.35 _+3.099 8.441 18.30_+ 1. 27.082 32.

2.9041) and their host plants (y=8. 2. dant activity (TEAC values< 20 p~mol/100ml).00mg/100ml). R2 = 0.3211x 2.1524x + 18.9150).0mg/100ml) also have high total antioxidant activity (most of TEAC values>100 I~mol/100 ml).00-5. compared to the concentration of cultivated fungal strains.0333. These correlations suggest that the phenolic compounds are most likely responsible for the antioxidant activities of the fungal cultures and the host plants. R2=0. although fungal colonization itself . As shown in Fig. this does not imply that the phenolic compounds of the host plants are actually produced by their fungal endophytes in situ. 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. while those isolates with high total phenolic content (TPC> 10. 61 Fig.41. The small number of fungal cells in planta. is unlikely to make a major contribution to the quantity of phenolics of the host plant.22 ECONOMIC BOTANY [VOL. and the endophytic isolates with moderate antioxidant activities (TEAC values within 20-50 p~mol/100ml) normally contain medium levels of phenolics (1. 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. 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. However.

MW 154).60 mmol/100g DW).: ENDOPHYTIC FUNGI 23 may induce the host plant to produce a different phenolic profile. vegetables. Thus identification of these endophytic fungal isolates was not performed in this study. Most of the isolated endophytic fungi showed no reproductive structures or no distinctive features when cultured on common mycological medium. Wiyakrutta et al. A major volatile composition. Maynard.160 endophytic fungal isolates. 4B). 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. 2004. In this study. The RP-HPLC methods have also been well developed for most categories of phenolic compounds in plants (Cai et al. The improved ABTS method was not only a rapid and reliable test of total antioxidant capacity in vitro. divaricata. Furthermore. Zheng and Wang 2001). In addition to traditional morphological identification methods. Gamboa and Bayman 2001. Different phenolic compounds normally possess specific chromatographic behavior and UVvis spectral characteristics (Cai et al. 2004. fruits. and vascular plants (Arnold et al. the endophytic fungi did exhibit characteristic culture colony or hyphal and microscopic morphology that could be differentiated amongst the isolates. ferns. 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. 2003. 2005). especially in 77. Shah et al. with morphologically distinct fungal isolates ranging from 23 in R. phenolic terpenoids. 5). Representative bioactive compounds identified in this study are given in Table 3. flavonoids. indica) Discussion The ubiquity of endophytes in the plant kingdom has been well established as they have been found in all species investigated. 2005). Most of the selected medicinal host plants and endophytic fungal metabolites were also analyzed using LC-ESIMS and GC-MS. 2000. 1999. Sakakibara et al. Sakakibara et al. Santos-Buelga and Williamson 2003. and Gilbert 2001. Our results were consistent with previous reports that phenolic compounds are major antioxidant constituents in medicinal herbs. In this study. 2004. Chlorogenic acid (5-O-caffeoylquinic acid) was a major phenolic compound from medicinal plants widely distributed in the families of Apocynaceae and Asclepiadaceae. 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. For example. 2000. Shan et al. it is rather difficult to characterize every compound and elucidate its structure. 4). Shan et al. Because of the diversity and complexity of the natural mixtures of phenolic compounds in the large number of samples. Tan and Zou 2001). 2005). all the 29 tested medicinal plants were colonized by various endophytic fungal strains. including algae. Interestingly. Sakakibara et al. The preliminary results showed that major types of bioactive compounds in both the medicinal host plants and endophytic fungal metabolites were phenolic acids. mosses. A. 2003. Shah et al. was detected in both AiL4 (an endophytic fungal culture) and its host plant (A. divaricata which had the highest level of chlorogenic acid and strong antioxidant activity (TEAC value was 26. Santos-Buelga and Williamson 2003. However. PRELIMINARY IDENTIFICATION AND COMPARISON OF BIOACTIVE CONSTITUENTS both contained a high level of chlorogenic acid (Fig. indica) (Fig. Arnold. modern molecular biology techniques are required for characterization and classification of these isolates. 2004. Therefore. volatile constituents. verticillata to 83 in 77. This is a common problem concerning the identification of endophytes (Arnold et al.2007] HUANG ET AL. 2004. and aliphatic compounds. AiL1 (an endophytic fungal culture) and its host plant (A. and spices (Cai et al. 2004). Gamboa and Bayman 2001). High levels of chlorogenic acid could be detected in most of the plant samples in these two families (Fig. 2003. quinones. but also an advantageous assay applicable to both hydrophilic and lipophilic antioxidants or systems (Cai et al. indica also contained high levels of caffeoylquinic acid derivatives (di-O-caffeoylquinic acids) (Fig. tentatively named artemisin (C12H10. 2005. 3). Re et al. tannins. 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 . certain compounds were found in both the endophytic fungal culture and the corresponding host plant extract (Table 3).

N. pictum. using PR-HPLC. capitatum. divar~cata A. CrF2*. divaricata A.14. PrL7*. mdzca. cathartica*. 2) provides support for the hypothesis that phenolic compounds are also responsible for the total antioxidant capacity of the endophytic fungal cultures. cuspidatum P. indlca. methyl ester 9. C. P. methyl ester LC-MSObserved AdductIons(m/z)or GC-MSData [M-HI (353). rubra* A. anticancer. The correlation between TEAC and T P C values (Fig. The categories of bioactive compounds identified in the host plant extracts were similar to those of 112 traditional Chinese medicinal plants (Cai et al. oleander.12-Octadecadienoic acid. P. and endophytic fungal metabolites represent an abundant source of bioactive and chemically novel compounds (Strobel et a[. 2004). A. CrS5*. C21H3602 ALL4* CrF2*. 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. [M + H] + (449). Endophytes are a poorly investigated group of microorganisms. peruviana * Compounds were found m both endophytic fungi and their host plants. P. phenolic acids. C. S.17-Eicosatrienoic acid. CI9H3402 MW: 320. PrL3* AcaL5* Endophyte~ AiLI* McdmmalHostPlants A. 61 TABLE 3. [M + Na] + (539) [M-HI (609). [M +Na] + (471) [M -H]-(518). C. cuspzdatum*. oleander. The results showed that phenolic compounds (e. C. cathartica*. sinensis.g. [M + Na] + (633) [M-HI. SdL2* ASS3. P. divaricata* A.(447). roseus*. multiflorum A. T. P. G. 1~ rubra*. H. CI7H3202 MW: 294. CI7H3402 MW: 268. Z divaricata P. and phenolic terpenoids) and certain volatile or aliphatic constituents were major substances in the endophyte cultures and host plant extracts. flavonoids. nocturnum. AND GC-MS. cuspzdatum P. P. nocturnum. S. PcuS7 AcaL5*. N. indica*. tannin constituents. (2004) reported that the metabolites produced by endophytic fungi from Thai medicinal plants exhibited antimicrobial. carnosa. multiflorum P. Endo- . chinense. cuspidatum. P. [M +H] + (355). [M + Na] § (541) [M -H]-(283) [M -H]-(269). but this association has never been investi- gated in endophytic fungi. PcuL7*. T.CrS5. [M + H]+ (271) MW: 154. roseus*. REPRESENTATIVEBIOACTIVE COMPOUNDS IN MOST OF THE TESTED ENDOPHYTIC FUNGAL METABOLITESAND MEDICINALHOST PLANTSIDENTIFIEDBY RP-HPLC. indica* C. LC-MS. nocturnum. methyl ester 11. methyl ester 9-Hexadecenoic acid. and antimalarial activities. cuspidatum *. cuspidatum. Ci2Hl0 MW: 270. 2004). PcuL7*. [M + Na]+ (377) [M-H]-(515). and some were identified by LC-ESI-MS and GC-MS. Phenolics have long been associated with antioxidant capacity in plants. cuspidatum.. P. CrL6*.24 ECONOMIC BOTANY [VOL. Wiyakrutta et al. hydroxyanthraquinones.

Tripterygium wilfordii. and the endophytic fungal cultures possessed antiproliferative activity on human peripheral blood mononuclear cells (Kumar et al.ion at m/z 353. HPLC chromatograms (280 nm) of methanohc extracts from the selected medicinal plant species in the family Apocynaceae and Asclepiadaceae. The dominant peak (retention time. (2005) reported that a phenolic compound (graphislactone A) with strong free radical-scavenging and antioxidant activity was isolated and identified from . phytic fungi were also found in a Chinese medicinal plant. [M + H] § ion at m/z 355. 2004).3 min) in HPLC profiles of all selected plants was identified as chlorogenic acid ([M-H]. .2007] HUANG ET AL. and [M + Na] + ion at m/z 377) by ESI-MS and by comparison with the related authenuc standard. 3.: ENDOPHYTIC FUNGI 25 Fig.20. Song et al.

Nerium oleander L. capillaris.. 5. Also. 210. and 77. no endophytic fungal culture with strong antioxidant activity was found from the host plant R capitatum. and TwL3) with strong antioxidant activities were isolated from those medicinal hosts showing a high antioxidant capacity. PmS7. indica). 3. both with a high antioxidant capacity. di-caffeoylquinicacid isomers (MW 516). which also showed strong antioxidant activity using the DPPH method by Jung et al. ALL1. 4. In the present study. I. wightianus (Table 1 and Table 2). This could . which showed the highest antioxidant capacity of all the plants studied. NoS3.26 ECONOMIC BOTANY [VOL. ALL3. (2004) showed that plants with medicinal values provide the best opportunities to isolate novel endophytic microorganisms as well as ones making novel bioactive products. AcapF3. Strobel et al. most of the endophytic fungal cultures showed antioxidant capacity to some extent. 8. chlorogenicacid (5-O-caffeoylquinicacid) (MW 354). PcuS7. and AiL4 from the leaves ofA. unknown phenolics (MW 212. the endophytic fungi (AcapF1 and AcapF4 from the flowers of A. The strongest antioxidant fungal strain. R cuspidatum. apigenin 7-O-glucuronide (MW 446). HPLC chromatograms (280 nm) and ESI-MS data of phenolics from (A) an endophytic fungus (ALL1)and (B) its host plant (A.. and 7. 2. 6. and 240). an endophytic fungus isolated from the root of Trachelospermum jasminoides (Apocynaceae). the culture of Cephalosporium sp. In this study. (2004). capillaris. indica) possessing strong antioxidant activities were isolated from two Artemisia medicinal plants. was found in A. 61 Fig. These findings show that the metabolites of endophytic fungal cultures can be a potential source of natural antioxidants. such as M. and those possessing high phenolic contents exhibited very strong antioxidant activities. we found that several endophytic fungi (MsL4. R multiflorum. In contrast. suaveolens. and 4.

a famous anticancer compound isolated from the bark of Pacific yew. was also detected in both the extract ofAllarnanda cathartica L. A. 5. 7. 1997). was identified in both A. 10. methyl ester.2007] Ittt (xl00.17-eicosatrienoic acid. oL-caryophyllene. 4). eucalyptol.. such as antioxidant. o~-farnesene. annua) (Gao et al. . and Stierle 1993. D r~ L~ o 3 10 20 30 40 50 T6 60 70 80 90 % I00 i~In Int (x10. Strobel. This suggests that endophytic fungi might produce the same bioactive natural compounds as some of those found in their host plants. The 11.O00) ISO- i ~ (B) 07e- ~ o o 71 ~o ~i=7 . Volatile oils are important bioactive compositions in medicinal plant species of Artemisia (e.g.. But. chlorogenic acid (5O-caffeoylquinic acid). Li. lndica). indica. caryophyllene. There are previous reports that some endophytes isolated from plants producing certain bioactive compounds could make the same natural products as the hosts (e. Xiao et al. Strobel and Hess. Molecular weight and formula are noted for most peaks in the figure. Furthermore. 11. and the extract of its endophytic fungal isolate AcaL5 using GC-MS (Table 3). one kind of aliphatic compound with antioxidant and anticarcinogenic activities (Rameazni-Kharazi 2004). 1995.14.. 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. has also been obtained from its fungal endophyte (Taxornyces andreanae) in vitro (Stierle et al. Lee et al. 1993). a major volatile constituent in the genus Arternisia. capillaris. a major bioactive phenolic compound. A. - . indica and its endophytic fungus AlL1 isolated from the leaf using RP-HPLC and ESI-MS (Fig.O00.. l ~ . indica and AlL1 using GC-MS (Fig.000) HUANG ET AL. capitaturn. insect-. antimutagenic.9. immunomodulatory. A. occur because we might not have isolated all the endophytic fungi present in P. artemisin.g. 2005. dimethyl phlthalate. Tentative identification of major peaks: 1. Stierle. artemisin. was detected in both the host plant A. In the present study. taxol. or disease-resistance. and only six of the 48 fungal isolates from the host plant were screened for their antioxidant activity.: ENDOPHYTIC FUNGI 27 (A) o o 20. 8.~. . !~ 9 10 20 30 40 50 80 70 80 90 IOOmll~ Fig. such as enhancement of stress-. and antiviral activities (dos Santos et al. 2000). 2000. For example. 5). and produc- . Chlorogenic acid and its derivatives possess a wide range of bioactivities. GC chromatograms of volatile compounds from (A) an endophytic fungus (ALL4) and (B) its host plant (A. and Ooi 2005).

1993. However. These findings indicate that the metabolites produced by the endophytic fungi in culture were a potential source of natural . (1990) suggests a fungal basis for the ethnobotanical utilization of piripiri (medicinal species of Cyperus) from Amazonia. This is a specific rationale for the collection of medicinal plants for endophyte isolation and naturalproduct discovery. Recent molecular biological studies of Pestalotiopsis microspora. however. may have provided an explanation as to how some of the host plant genes may have been acquired. However. Without controlled experiments to test whether the host plants can produce the same compounds in the absence of their endophytes. but such a plant is difficult to find because every plant previously studied is host to one or more endophytes (Arnold et al. 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. an endophytic fungus of Taxus wallichiana. as we have known that both the seasons and places of collection are important factors affecting their medicinal values.28 ECONOMIC BOTANY [VOL. These ecological or seasonal variations also affect the potency of medicinal plants. The total antioxidant capacity of the endophytic fungal metabolites was significantly correlated with their total phenolic content. Limitations of the Study The relationship between fungal endophytes and their host plants is a complicated research topic. the number of fungal cells would be multiplied far beyond their normal concentration in planta. Concluding Statements A large number of morphologically distinct endophytic fungi were isolated from the 29 traditional Chinese medicinal plant species studied. an endophyte-free plant of the same species should be used to serve as control. 1998). 1991). On the other hand. However. Strobel et al. 2004). and replicated by the endophytes (Long et al. Gamboa and Bayman 2001. 2000. expressed. The traditional Chinese medicinal plants have a long ethnobotanical history. 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. Clearly. The study by Plowman et al. Strobel et al. it is unlikely that such compounds isolated from the host plants could be entirely produced by their endophytes in situ. were specifically recognized by the endophytic Xylarlaceae species as chemical signals during the establishment of fungus-plant interactions (Chapela et al. and Gilbert 2001. two monolignol glucosides produced by the host plant. 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. 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. it is also possible that the medicinal values of some of the plants may in fact come from their endophytes. 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. and are related to specific use or applications by indigenous peoples. Earlier findings also showed that coniferin and syringin. it is just the first step in the investigation of the relationship and interaction between the host plants and endophytes. Arnold. when the fungal endophytes were isolated and cultivated in vitro. We also isolated and screened several endophytic fungi which could produce the metabolites containing bioactive compounds with potent antioxidant capacity. Maynard. Spatial heterogeneity (Carroll 1995) or seasonal differences (Rodrigues 1994) also exist in terms of compositions and quantities of endophytic strains. and thus a measurable phenolic profile is possible. 2004). 61 tivity improvement (Tan and Zou 2001. Most of the endophytic fungal cultures exhibited antioxidant activities. the hypothesis that medicinal values of plants have a biological basis resulting from fungal infection needs further investigation. 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. Tan and Zou 2001). In contrast. Ideally. considering the small quantity of the endophytes within each host plant. 1992) and tissue specificity (Luginbuhl and Miiller 1980). our study has not used randomly chosen plants that are not used in traditional Chinese medicine as a control group for comparison. Some endophytes exhibit host preference (Petrini et al.

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