A Potential Antioxidant Resource: Endophytic Fungi from

Medicinal Plants I
Wt3-YANG HUANG 2, YI-ZHONG CAI 3, JIE )(LING4, HAROLD CORKE3,
AND M E I SUN 2'*

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. Sev-
eral previous studies have found a positive correlation between total antioxidant capacity
(TAC) and total phenolic content (TPC) of many medicinal plant extracts. However, no in-
formation is available on whether such a relationship also exists in their endophyhc fungal
metabolites. We investigated the relationship between TAC and TPC for 292 morphologi-
cally distinct endophytic fungi isolated from 29 traditional Chinese medicinal plants. The an-
tioxidant capacities of the endophytic fungal cultures were significantly correlated with their
total phenolic contents, suggesting that phenolics were also the major antioxidant con-
stituents of the endophytes. Some of the endophytes were found to produce metabolites
possessing strong antioxidant activities. Several bioactive constituents from the fungal cul-
tures 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 re-
active oxygen species (ROS, e.g., 02- and OH-)
and free radical-meditated reactions can cause ox-
idative damage to biomolecules (e.g., lipids, pro-
teins, 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 manage-
ment of ROS-mediated tissue impairments.
Many antioxidant compounds possess anti-
inflammatory, antiatherosclerotic, antitumor, an-
timutagenic, anticarcinogenic, antibacterial, or
antiviral activities to a greater or lesser extent
1Received 4 August 2006; accepted 9 November
2006.
(Cozma 2004; Halliwell 1994; Mitscher et al.
1996; Owen et al. 2000; Sala et al. 2002). Natu-
rally derived antioxidants have received much at-
tention in recent years (Hu and Kitts 2000;
Schulz et al. 2002).
Endophytes are fungi or bacteria residing in-
side 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 at-
tention is now being paid to endophytic biodi-
versity, the chemistry and bioactivity of endo-
phytic 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, chi-
nones, flavonoids, phenolic acids, quinones, ste-

Economic Botany, 61(1), 2007, pp. 14-30.

9 2007, by The New York Botanical Garden Press, Bronx, NY 10458-5126 U.S.A.
2007] HUANG ET AL.: ENDOPHYTIC FUNGI
roids, terpenoids, tetralones, xanthones, and oth-
ers (Tan and Zou 2001). Antibiotics, antiviral
compounds, anticancer agents, insecticidal prod-
ucts, antidiabetic agents, immunosuppressive
compounds as well as antioxidants have been re-
ported from endophytic metabolites (Strobel et
al. 2004), and medicinal plants have been recog-
nized as a repository of endophytes with novel
metabolites of pharmaceutical importance (Stro-
bel 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 en-
dogenous metabolites (Cai et al. 2004; IC~hk6nen
et al. 1999; Zheng and Wang 2001). Traditional
Chinese medicinal plants have been used for phar-
maceutical and dietary therapies for several mil-
lennia (Cai et al. 2004; Tapiero et al. 2002). Nat-
ural compounds isolated from these medicinal
plants are a rich source of novel drugs with multi-
ple biological activities including antioxidant
properties. There have been many studies on the
antioxidant activities of various plants of medici-
nal 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 dom-
inant antioxidant components in 112 traditional
Chinese medicinal plants associated with combat-
ing cancer, and similar findings were reported for
133 Indian medicinal plants (Surveswaran et al.
2007), with both studies showing highly signifi-
cant positive linear correlations between the total
antioxidant capacities and phenolic contents. De-
spite 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 endo-
phytic fungal isolates were obtained from differ-
ent tissues of 29 traditional Chinese medicinal
plant species. Only the 292 morphologically dis-
tinct fungal isolates were used for investigation of
the total antioxidant capacities, phenolic con-
tents, and their relationships. The relationships
between the total antioxidant capacities and total
phenolic contents in the 29 host plants were con-
currently investigated for comparison with their
endophytes. Preliminary identification and
screening of bioactive constituents from the en-
15
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 medici-
nal 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 sam-
ples 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 Apocy-
naceae, 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 Uni-
versity 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-ethylbenzothia-
zoline-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-tetramethylchromate-
2-carboxylic acid) from Fluka Chemie AG
(Buchs, Switzerland). Authentic standards, an-
tibiotics, 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 isola-
tion of endophytic fungi followed a modified
method as described by Schulz et al. (1993). All
the plant samples were surface-sterilized by dip-
16 ECONOMIC BOTANY [ V O L . 61

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5
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z

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2007] HUANG ET AL.: ENDOPHYTIC FUNGI 17

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
-d three times. In each petri dish (9cm diameter),
four processed segments or pieces were evenly
spaced onto the surface of water agar (WA)
...=
e% medium supplemented with 200 U/ml penicillin
G and 200 I~g/ml streptomycin sulfate for sup-
< pressing the growth of bacteria, followed by incu-
bation at 28~ until the mycelium or colony
originating from the sample appeared. Hyphal
= E tips were transferred and cultured on potato car-
0 rot agar (PCA) plates, and the purified strains
+l+l+l+l +1 were preserved on potato dextrose agar (PDA)
t'-.
plates at 4~ in the Laboratory of Department of
Zoology, the University of Hong Kong.
e-,
CULTIVATION OF ENDOPHYTIC FUNGI
0 ~
od Fresh mycelia (grown on PDA plates at 28~
for 3-6 days) of morphologically different endo-
"'"0
+1+1+1+1 +1 phytes, approximately equal in quantity, were in-
O P'--

t~ ~_~ oculated 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
r Labtech Co., Ltd.) at 140rpm for 15 days at
4 28~ The culture broth of each endophytic fun-
{ gus was centrifuged at 1,670g for 10 min and fil-
tered using a Millipore filter with a 0.22-~m
nylon membrane under vacuum at room temper-
E2•
..e~~ ature (-23~ to remove mycelium. The filtrate
k.~ sample was stored at 4~ until use within 24
=999 hours.
EXTRACTION OF HOST PLANT SAMPLES
The plant samples were air-dried in a venti-
lated oven at 40~ to constant weight and
0 ground to fine powder using a Kenwood Multi-
Mill (Kenwood, Ltd., UK) and passed through a
24-mesh sieve. The sample (2g each) was ex-
tracted 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.
2004).
{
TOTALANTIOXIDANT CAPACITYASSAY
..% Total antioxidant capacity was assayed using
F~ the improved ABTS method (Cai et al. 2004; Re
o ~ et al. 1999). The ABTS'+ radical cation was gen-
18 ECONOM IC BOTANY
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 ab-
sorbance 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 thor-
oughly. The reactive mixture was allowed to stand
at room temperature for 6 min and the ab-
sorbance 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 re-
sulting oxidized solution was compared to that of
the calibrated trolox standard. Results were ex-
pressed in terms of trolox equivalent antioxidant
capacity (TEAC), i.e., gtmol trolox/100ml cul-
ture 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 ap-
propriate 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 spec-
trophotometer 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 equiva-
lent (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 Tech-
nologies, Loveland, CO). Phenolic compounds in
the samples were analyzed using our previous
method (Cai et al. 2004) with slight modifica-
[VOL. 61
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. De-
tection 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 SIL-
l 0Avp auto-sampler, a photodiode-array detector,
a central controller, and a single quadrupole MS
detector with electrospray ionization (ESI) inter-
face (Shimadzu, Japan). The analytical column
was VP-ODS C18 column (250 x 2.0 mm, 5 gtm)
(Narmura Chemical Co., Seto, Japan). LC condi-
tions 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 inter-
face 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 nega-
tive ion mode. A nebulizing gas of 1.5 l/min and
a drying gas of 10 l/min were applied for ioniza-
tion 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
2007] HUANG ET AL.: ENDOPHYTIC FUNGI
and programmed to 150~ at a rate of l~
to 250~ at a rate of 10~ and kept con-
stant 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 vol-
ume 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 oper-
ated 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~ re-
spectively. A library search was carried out using
NIST and SZTERP libraries.
STATISTICALANALYSIS
All sample determinations were conducted in
triplicate and the results were calculated as mean
standard deviation (SD) in this study. Coeffi-
cients of determination (R2) were calculated using
Microsoft Excel 2000. Differences between mean
values were compared by least significant differ-
ence (LSD) calculated using the Statistical Analy-
sis 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 medici-
nal 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). Melod-
inus suaveolens (Hance) Champ. ex Benth., Stro-
phanthus divaricatus (Lout.) Hook. & Arn., and
Polygonum cuspidaturn Sieb. & Zucc. had endo-
phytic fungi colonizing in all of the tested sam-
ples (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 val-
ues were 92.5%, 95.0%, 95.0%, 92.5%, 97.5%,
and 92.5%, respectively, whereas Asclepias curas-
savica L. showed the lowest CR (36.7%). Out of
the total 1,160 preliminary fungal isolates, 292
19
morphologically distinct isolates from the 29 me-
dicinal plants were selected for antioxidant activ-
ity 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
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 sig-
nificant 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 mod-
erate activities (20-50 p~mol trolox/100ml cul-
ture). Distribution (the number of isolates) of en-
dophytic fungi isolated from each medicinal
plant with different ranges of total antioxidant
capacity (TEAC value, ~mol trolox/100ml cul-
ture: 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. pos-
sessed the strongest antioxidant capacity with the
value of 526.93 [~mol trolox/100 ml culture, fol-
lowed by TwL3 isolated from the leaf of T. wight-
ianus (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
20 ECONOMIC BOTANY [VOL. 61

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.
2007] HUANG ET AL.: ENDOPHYTIC FUNGI 21

TABLE 2. TOTALANTIOXIDANTCAPACITYAND PHENOLIC CONTENT OF METABOLITESFROM 22 SELECTED

ENDOPHYTICFUNGI (>100 lilMOLTROLOX/100ML CULTURE).

Codeof TEAC(lamol TPC (naggalhc

Endophyte HostPlant Source trolox/100mlculture) aud/100m1culture)
AcaL5 Allamanda cathartica L. leaf 124.00_+ 1.963 7.67-+ 0.133
AcS6 Asclepias curassavzca L. stem 267.00_+ 3.321 26.83 + 0.280
AcapF1 Artemism capillaris Thunb. flower 127.41 _+2.036 13.71 _+0.190
AcapF3 Artemma capillaris Thunb. flower 526.93_+ 7.942 49.22_+ 2.441
AcapF4 Artemisia capdlarzs Thunb. flower 177.12 _+4.119 18.43 _+0.195
AiL 1 Artemisia indica Willd. leaf 100.20 _+0.718 6.88_+0.099
AlL3 Artemisia indica Will& leaf 102.21 + 2.969 8.69 _+0.129
AiL4 Artemism indica Willd. leaf 217.76 _+2.081 21.27 _+0.166
CmL3 Cerbera manghas L. leaf 139.44 + 1.418 12.00_+0.210
CnoL3 Cestrum nocturnum L. leaf 127.40_+ 0.557 9.06 + 0.022
CnoS 1 Cestrum nocturnum L. stem 125.40 _+0.562 9.58 _+0.058
CnoS4 Cestrum nocturnum L. stem 152.60 _+1.368 10.17 -+0.082
CrF2 Catharanthus roseus (L.) G. Don flower 224.80_+2.910 32.37_+0.541
CrL7 Catharanthus roseus (L.) G. Don leaf 183.30_+0.777 19.33-+0.128
HcL2 Hoya carnosa (L. f.) R. Br. leaf 117.90_+0.915 6.74_+0.100
MsL4 Melodinus suaveolens (Hance) leaf 204.40_+ 0.797 27.35 _+0.617
Champ. ex Benth.
NoS3 Nerlum oleander L. stem 150.79 _+2.045 13.95 _+0.109
PcuS7 Polygonum cuspldatum stem 164.20 _+3.067 18.01 _+0.035
Sieb. & Zucc.
ProS7 Polygonum multzflorum Thunb. stem 115.30_+ 1.892 6.33_+0.053
PrL2 Plumeria rubra L. leaf 109.25 _+2.081 11.76_+ 0.255
PrL7 Plumeria rubra L. leaf 269.70_+ 3.925 22.26_+ 0.232
TwL3 Toxocarpus wightianus leaf 298.35 _+3.604 24.28_+0.095
Hook. & Am.
Control 1.28 0.02
LSD (p < 0.05)* 4.633 0.956
* LSD (p< 0.05), least significant &fference,was used for significantdifference comparisonamong means of various endo-
phytic fungal cultures.

Ixmol/lO0 ml), and MsL4 isolated from the leaf
of M. suaveolens (204.4 Ixmol/lOOml). In con-
trast, 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 Folin-
Ciocalteu 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 hun-
dred and seventeen of the endophytic fungal iso-
lates (74.3%) contained medium levels of pheno-
lics (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, 1.00-
5.00; C, 5.00-10.0; D, >10.0) was also shown in
Fig. 1. The endophytic fungi with higher pheno-
lic 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-
22 ECONOMIC BOTANY [VOL. 61

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 antioxi-
dant activities (TEAC values within 20-50
p~mol/100ml) normally contain medium levels of
phenolics (1.00-5.00mg/100ml), while those iso-
lates with high total phenolic content
(TPC> 10.0mg/100ml) also have high total an-
tioxidant activity (most of TEAC values>100
I~mol/100 ml). As shown in Fig. 2, a correlative re-
lationship was present between the total antioxi-
dant capacity and total phenolic content in the
292 endophytic fungal isolates as well as in their
host plants. A highly positive linear correlation be-
tween total antioxidant capacity (y) and total phe-
nolic content (x) was established for both the en-
dophytic 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 re-
sponsible for the antioxidant activities of the fun-
gal cultures and the host plants. However, this
does not imply that the phenolic compounds of
the host plants are actually produced by their fun-
gal endophytes in situ. The small number of fun-
gal 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
2007] HUANG ET AL.: ENDOPHYTIC FUNGI
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
Different phenolic compounds normally pos-
sess specific chromatographic behavior and UV-
vis 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 mix-
tures of phenolic compounds in the large num-
ber of samples, it is rather difficult to character-
ize every compound and elucidate its structure.
Therefore, only a preliminary identification of
phenolic compounds and other bioactive com-
positions in the endophytic fungal metabolites
and their medicinal host plants was carried out
in this study. We analyzed major phenolic com-
pounds 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 re-
lated literature data (Cai et al. 2004; Sakakibara
et al. 2003; Tan and Zou 2001). Most of the se-
lected medicinal host plants and endophytic fun-
gal metabolites were also analyzed using LC-ESI-
MS and GC-MS.
The preliminary results showed that major
types of bioactive compounds in both the medic-
inal host plants and endophytic fungal metabo-
lites were phenolic acids, flavonoids, tannins,
quinones, phenolic terpenoids, volatile con-
stituents, and aliphatic compounds. Representa-
tive bioactive compounds identified in this study
are given in Table 3. Chlorogenic acid (5-O-caf-
feoylquinic acid) was a major phenolic com-
pound from medicinal plants widely distributed
in the families of Apocynaceae and Asclepi-
adaceae. 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 endo-
phytic fungal culture) and its host plant (A. indica)
23
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).
Discussion
The ubiquity of endophytes in the plant king-
dom 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; Gam-
boa 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. ver-
ticillata to 83 in 77. divaricata. Most of the iso-
lated endophytic fungi showed no reproductive
structures or no distinctive features when cul-
tured 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 cul-
ture colony or hyphal and microscopic morphol-
ogy that could be differentiated amongst the iso-
lates. In addition to traditional morphological
identification methods, modern molecular biol-
ogy 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 capac-
ity in vitro, but also an advantageous assay appli-
cable to both hydrophilic and lipophilic antioxi-
dants or systems (Cai et al. 2004; Re et al. 1999;
Shah et al. 2005). This method was also success-
fully used in the present study to screen antioxi-
dant capacity for the 292 morphologically differ-
ent fungi out of the total 1,160 endophytic
fungal isolates. The RP-HPLC methods have also
been well developed for most categories of phe-
nolic 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 metabo-
lites from some (bioactive) endophytic isolates
were preliminarily analyzed and screened by
24 ECONOMIC BOTANY [VOL. 61

TABLE 3. REPRESENTATIVEBIOACTIVE COMPOUNDS IN MOST OF THE TESTED ENDOPHYTIC FUNGAL

METABOLITESAND MEDICINALHOST PLANTSIDENTIFIEDBY RP-HPLC, LC-MS, AND GC-MS.

CompoundCategories LC-MSObserved
and Representanvc AdductIons(m/z)or
Conmtuents GC-MSData Endophyte~ McdmmalHostPlants
Phenolic acids
Chlorogenic acid (5-O [M-HI (353), AiLI* A. indica*, G. pictum,
-caffeoylquinic acid) [M +H] + (355), H. carnosa, N. oleander,
[M + Na]+ (377) T. divar~cata
Di-O-caffeoylquinic acids [M-H]-(515), A. mdzca, N. oleander,
[M + Na] + (539) S. divaricata
Flavonoids
Quercetin 3-rutinoside [M-HI (609), A. indlca, C. nocturnum,
(rutin) [M + Na] + (633) Z divaricata
Quercetin 3-rhamnoside [M-HI- (447),
(quercitrin) [M + H] + (449), P. capitatum, P. chinense,
[M +Na] + (471) P. cuspidatum, P. multiflorum
Quinones
Anthraquione glycoside [M -H]-(518), P. cuspidatum
[M + Na] § (541)
Rehein [M -H]-(283) P. cuspzdatum
Emodin [M -H]-(269), P. cuspidatum, P. multiflorum
[M + H]+ (271)
Volatile compounds
Artemisin MW: 154, Ci2Hl0 ALL4* A. indica*
Aliphatic compounds
Hexadecanoic acid, MW: 270, CI7H3402 CrF2*, CrS5*, C. roseus*, C. nocturnum,
methyl ester PcuL7*, PrL7*, P. cuspidatum *, 1~ rubra*,
SdL2* S. divaricata*
9-Hexadecenoic acid, MW: 268, CI7H3202 ASS3,CrS5,
methyl ester PcuS7
9,12-Octadecadienoic MW: 294, CI9H3402 AcaL5*, CrF2*, A. cathartica*, C. roseus*,
acid, methyl ester CrL6*, PcuL7*, C. nocturnum, P. cuspzdatum*,
PrL3* P. rubra*
11,14,17-Eicosatrienoic MW: 320, C21H3602 AcaL5* A. cathartica*, A. sinensis,
acid, methyl ester 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, hydroxy-
anthraquinones, 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 be-
tween TEAC and T P C values (Fig. 2) provides
support for the hypothesis that phenolic com-
pounds are also responsible for the total antioxi-
dant capacity of the endophytic fungal cultures.
Endophytes are a poorly investigated group of
microorganisms, and endophytic fungal metabo-
lites 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-
2007] HUANG ET AL.: ENDOPHYTIC FUNGI 25

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 medic- cells (Kumar et al. 2004). Song et al. (2005) re-
inal plant, Tripterygium wilfordii, and the endo- ported that a phenolic compound (graphislactone
phytic fungal cultures possessed antiproliferative A) with strong free radical-scavenging and an-
activity on human peripheral blood mononuclear tioxidant activity was isolated and identified from
26 ECONOMIC BOTANY [VOL. 61

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 cul-
tures can be a potential source of natural antioxi-
dants.
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 an-
tioxidant capacity, such as M. suaveolens, Nerium
oleander L., R cuspidatum, R multiflorum, and 77.
wightianus (Table 1 and Table 2). Also, the endo-
phytic fungi (AcapF1 and AcapF4 from the flow-
ers of A. capillaris; ALL1, ALL3, and AiL4 from
the leaves ofA. indica) possessing strong antioxi-
dant 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 antioxi-
dant activity was found from the host plant R
capitatum, which showed the highest antioxidant
capacity of all the plants studied. This could
2007] HUANG ET AL.: ENDOPHYTIC FUNGI 27

Ittt (xl00.000)

(A)

o
o

20. D r~ L~ o

3 T6 %

10 20 30 40 50 60 70 80 90 I00 i~In

Int (x10,O00,O00)
ISO-

i ~ (B)

07e- ~ o o 71 ~o ~i=7

; - ~,., . l ~ , - . !~ 9
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~-far-
nesene.

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 endo-
phytes isolated from plants producing certain
bioactive compounds could make the same natu-
ral 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 (5-
O-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 composi-
tions 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 com-
pound with antioxidant and anticarcinogenic ac-
tivities (Rameazni-Kharazi 2004), was also de-
tected 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 bioac-
tive natural compounds as some of those found in
their host plants.
The colonization and propagation of endo-
phytes 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-
28 ECONOMIC BOTANY
tivity improvement (Tan and Zou 2001; Strobel
et al. 2004). Some endophytes exhibit host pref-
erence (Petrini et al. 1992) and tissue specificity
(Luginbuhl and Miiller 1980). Spatial hetero-
geneity (Carroll 1995) or seasonal differences
(Rodrigues 1994) also exist in terms of composi-
tions 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 Ama-
zonia. Earlier findings also showed that coniferin
and syringin, two monolignol glucosides pro-
duced by the host plant, were specifically recog-
nized 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 fun-
gal 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 en-
dophytes 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; Stro-
bel et al. 2004). Without controlled experiments
to test whether the host plants can produce the
same compounds in the absence of their endo-
phytes, however, we cannot conclude with cer-
tainty that the same compounds are in fact pro-
duced in vivo by the endophytes alone or by both
the endophytes and the host plants.
[VOL. 61
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 in-
vestigation of the relationship and interaction be-
tween the host plants and endophytes. It remains
unclear whether the hosts and endophytes ex-
change 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 recom-
bination of the endophyte with the host in evolu-
tionary 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 explana-
tion 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 ethnobo-
tanical history, and are related to specific use or
applications by indigenous peoples. This is a spe-
cific rationale for the collection of medicinal
plants for endophyte isolation and natural-
product 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 en-
dophytic fungi were isolated from the 29 tradi-
tional Chinese medicinal plant species studied.
Most of the endophytic fungal cultures exhibited
antioxidant activities. The total antioxidant ca-
pacity of the endophytic fungal metabolites was
significantly correlated with their total phenolic
content. We also isolated and screened several en-
dophytic fungi which could produce the metabo-
lites 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
2007] HUANG ET AL.: ENDOPHYTIC FUNGI
antioxidants and some novel bioactive com-
pounds. Unknown/novel compounds in the
tested endophytic fungal metabolites will be fur-
ther identified. Future investigations will be con-
ducted for those endophytic fungi with strong
antioxidant capacity to identify and elucidate the
bioactive constituents and their chemical struc-
tures, 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 as-
sistance in field collection and identification of
medicinal plants.
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