Botanical Studies (2007) 48: 55-61.

3

These authors contributed to this work equally.

*

Corresponding author: E-mai: bkkuai@fudan.edu.cn; Tel:

+86-21-65642648; Fax: +86-21-65642648.

INTRODUCTION

Ammopiptanthus mongolicus (Maxim.) S. H. Cheng

[Leguminosae] is the relics of the Tertiary Period,

dis-

tinctively distributed in the northwestern desert area of

China, where is marked by seasonally extreme drought

and temperatures (over 40¢XC in the summer and under -30

¢XC in the winter), poor soil

quality

with high salinity, and

extraordinarily high ultraviolet-irradiation. The unique

for A. mongolicus is that it remains evergreen for all four

seasons in the desert area. It is catalogued in Chinese Tra-

ditional Medicine as an anti-inflammatory, anti-infectious

and pain-killing medicinal plant since it has long been

used to treat respiratory disorders (lung diseases, cough,

and infected throat), to kill stomachache, and to cure cold-

caused wounds and chronic rheumatism (Li et al., 2004).

We initially assumed that its efficacy in treating the dis-

eases could be somehow related to its harsh environment,

possibly through the accumulation of some kinds of sec-

ondary compounds in its aerial parts.

It has been understood that, sessile plants, particularly

those living in the extremely adverse environment, are

under constant attack of excess reactive oxygen species

(ROS), and have evolved efficient anti-oxidation defense

systems, including antioxidative enzymes or non-enzy-

matic antioxidants. The level of the antioxidants, which

Extraordinary accumulations of antioxidants in

Ammopiptanthus mongolicus (Leguminosae) and

Tetraena mongolica (Zygophyllaceae) distributed in

extremely stressful environments

Wei WANG

1,3

, Jingjing CHEN

1,3

, Jining LI

2

, Yunhai ZHANG

1

, Zhiyu SHAO

1

, and Benke KUAI

1,

*

1

State Key Laboratory of Genetic Engineering, Department of Biochemistry, School of Life Sciences, Fudan University,

Shanghai 200433, P. R. China

2

Western Ecological Environment and Biological Resources Developing United Research Center, Ningxia University,

Yinchuan, Ningxia 750021, P. R. China

(Received February 20, 2006; Accepted August 9, 2006)

ABSTRACT.

Ammopiptanthus mongolicus (Leguminosae) and Tetraena mongolica (Zygophyllaceae) are

distinctively distributed in the northwestern Gobi and desert areas of China. Their environments involve

seasonally extreme drought and temperatures, extraordinarily high UV radiation and poor soil

qualities

with

high salinity. Ammopiptanthus mongolicus remains evergreen for all four seasons and has been traditionally

used as anti-inflammatory, anti-infectious and pain-killing medicines. All the extracts prepared from the

two species exhibited significantly higher scavenging activities against the ¡PO

2

-

than a control. Five selected

extracts also showed wider spectra of antioxidative capacities. An activity-guided fractionation led to

identification of four major compounds. Resveratrol, a super strong antioxidant, accounted for as high as 0.05%

of the dried weight of A. mongolicus. Two isoflavones isolated are also reported to be antioxidative and anti-

inflammatory. Our results imply that plant species living in the extremely stressful environments may become

an abundant natural resource of strong antioxidants. Considering the fact that oxidation is involved in the

processes of infections, inflammation and other disorders, these results collectively suggest that efficacies of A.

mongolicus in treating infections, inflammatory disorders and in killing pains may be attributed, at least partly,

to its significantly larger non-enzymatic antioxidative capacities.

Keywords: 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical; Active-oxygen scavenging activity;

Ammopiptanthus mongolicus; Electron paramagnetic resonance (EPR); Lipid peroxidation; Resveratrol;

Superoxide anion radical (¡PO

2

-

); Tetraena mongolica.

Abbreviations: DMSO, Dimethyl Sulfoxide; DPPH, 2,2-Diphenyl-2-picrylhydrazyl hydrate; EPR, Electron

paramagnetic resonance; MDA, Malondialdehyde; ¡PO

2

-

, Superoxide anion radical; ROS, Reactive oxygen

species.

BIOCHEMISTRY

56

Botanical Studies, Vol. 48, 2007

may account for the efficacies of medicinal plants, varies

widely among species (Reinert et al., 1982; Bennett et al.,

1984). On the other hand, more and more studies demon-

strate that ROS are involved in the pathogenesis during

infections and various degenerative disorders, such as car-

diovascular disease and neuro-degenerative diseases (Har-

man, 1994; Cox and Cohen, 1996; Ames, 1998;

Finkel and

Holbrook, 2000). Oxidation is also involved in the early

stages of carcinogenesis (Vuillaume, 1987). Based on

these understandings and findings, a working hypothesis

was further postulated that the efficacy of A. mongolicus

in the traditional treatments of the various diseases might

be attributed to its extraordinary antioxidative capacity

derived from extraordinarily accumulated antioxidants

under the extremely stressful environment. To justify this

hypothesis, we set out to analyze the antioxidative capaci-

ties of various extracts from its aerial parts, with a hope to

identify distinctive antioxidants.

To further verify the assumption that an extraordi-

nary non-enzymatic antioxidative capacity might be a

characteristics of plants living in extremely stressfully

environments, extracts from a super xerophyte, Tetraena

mongolica Maxim. (Zygophyllaceae) were also analyzed

in parallel. Tetraena mongolica, predominantly distributed

in Gobi-like dry area, is an otherwise similar bushy plant

species. Due to its limited distribution, it has been listed

as an endangered species by the State. To make the analy-

ses experimentally justifiable, Daphniphyllum paxianum

Rosenth., a species naturally distributed in the milder

environment of southern part of China, was analyzed as a

negative reference.

In this study, all the extracts from A. mongolicus as

well as from T. mongolica exhibited significantly higher

antioxidative capacities against superoxide anion radical

than those from D. paxianum. Five selected extracts also

showed different magnitude of counteracting capacities

against DPPH and lipid peroxyl radicals. Three major

antioxidants were identified, and resveratrol, in particular,

accounted for as high as 0.05% of the dried weight of A.

mongolicus.

MATERIALS AND METHODS

Plant materials

Ammopiptanthus mongolicus and T. mongolica, were

collected from the northern Gobi and desert areas of the

Ningxia Hui and Inner Mongolia Autonomous Regions,

and D. Rosanth from the milder environment of northern

Guangdong province. They were identified by Dr.

Yulong Ding (Department of Botany, Nanjing Forestry

University).

Preparation of extracts and isolation of pure

compounds

Powdered aerial parts of A. mongolicus, T. mongolica

and D. paxianum were exhaustively extracted four

times with ethanol, and the resultant extracts (A

0

, T

0

,

and D

0

) were then partitioned within ethyl acetate and

H

2

O. The ethyl acetate-soluble portions were repeatedly

chromatographed over silica gel and Sephadex LH-20

column, and samples A

1

, A

3

, A

4

, A

5

, A

6

, T

1

, T

3

, T

4

and D

1

were subsequently obtained. The H

2

O-soluble

portion was further partitioned within n -Butanol and

H

2

O. The n -Butanol-soluble portion was repeatedly

chromatographed over silica gel and Sephadex LH-20

column, and samples A

2

, A

7

, T

2

, T

5

and D

2

were then

obtained. Finally, 5, 7, 4¡¦-trihydroxyisoflavone, maackiain

and 7, 3¡¦-dihydroxy-4¡¦-methoxyisoflavone were isolated

from A

4

, and resveratrol from A

5

(with structure formula).

The whole procedure was outlined in Figure 1.

Sup plements. 5, 7, 4¡¦-trih ydroxyisoflavon e: C

15

H

10

O

5

,

1

H

NMR (CD

3

OD, 400 MHz) £_ : 12.0 (

1

H, s, 5-OH) 8.13 (1H, s,

H-2), 7.72 (2H, d, J= 8.5 Hz, H-2¡¦, 6¡¦), 7.29 (2H, d, J= 8.5 Hz,

H-3¡¦, 5¡¦), 6.76 (1H, d, J= 2.1 Hz,, H-8), 6.68 (1H, d, J= 2.1 Hz,,

H-6);

13

C NMR (CD

3

OD, 100 MHz) £_: 153.5 (d, C-2), 122.5

(s, C-3), 181.4 (s, C-4), 158.8 (s, C-5), 94.7 (d, C-6), 166.1 (s,

C-7) 100.3 (d, C-8), 159.4 (s, C-9), 106.0 (s, C-10), 124.1 (s,

C-1¡¦), 131.2 (d, C-2¡¦, 6¡¦), 116.4 (d, C-3¡¦, 5¡¦), 163.8 (s, C-4¡¦);

maackiain: C

16

H

12

O

5

,

1

H NMR (CD

3

OD, 400 MHz) £_ : 7.24 (1H,

d, J= 8.4 Hz, H-1), 6.47 (1H, dd, J= 8.4, 2.3 Hz, H-2), 6.29 (1H,

d, J= 2.3 Hz, H-4), 4.18 (1H, d, J= 11 Hz, H-6£\), 3.53 (1H, t, J=

11, 4.8 Hz, H-6£]), 3.43 (1H, m, H-6a), 6.77 (1H, s, H-7), 6.35

(1H, s, H-10), 5.85 (1H, d, J= 11 Hz, H-OCH

2

O-), 5.84 (1H,

d, J= 11 Hz, H-OCH

2

O-), 5.42 (1H, d, J= 6.9 Hz, H-11a);

13

C

NMR (CD

3

OD, 100 MHz): 133.6 (d, C-1), 111.2 (d, C-2), 160.6

(s, C-3), 104.6 (d, C-4), 158.5 (s, C-4a), 67.9 (t, C-6), 42.1 (d,

C-6a), 120.3 (s, C-6b), 106.4 (d, C-7), 143.6 (s, C-8), 149.9 (s,

C-9), 94.7 (d, C-10), 156.1 (s, C-10a), 80.5 (d, C-11a), 113.4 (s,

C-11b), 103.0 (t, C-OCH

2

O-); EIMS m/z 284 (100) 162 (20),

267 (15), 175 (13); 7, 3¡¦-dihyd roxy-4¡¦-methoxyisoflavone:

C

16

H

12

O

5

,

1

H NMR (C

5

D

5

N, 400 MHz) £_: 8.52 (1H, d, J= 8.5

Hz, H-5), 8.26 (1H, s, H-2), 7.88 (1H, d, J= 2.3Hz, H-2¡¦), 7.41

(1H, dd, J= 8.5, 2.3 Hz, H-6¡¦), 7.30 (1H, dd, J= 8.5 Hz, H-6),

7.18 (1H, d, J= 2.3 Hz, H-8 ), 7.13 (1H, d, J= 8.5 Hz, H-5¡¦ ),

3.85 (3H, s, H-OCH

3

);

13

C NMR (C

5

D

5

N, 100 MHz) £_ : 175.8

(s, C-4), 164.2 (s, C-7), 159.6 (s, C-9), 152.9 (d, C-2), 148.8 (s,

C-3¡¦), 148.2 (s, C-4¡¦), 128.4 (d, C-6¡¦), 126.5 (s, C-3), 125.1 (s,

C-1¡¦), 120.6 (d, C-5), 118.2 (s, C-10), 118.0 (d, C-6), 116.0 (d,

C-5¡¦), 112.5 (d, C-2¡¦), 103.2 (d, C-8), 56.1 (q, C-OCH

3

).

WANG et al. ¡X

Accumulations of antioxidants in

Ammopiptanthus

mongolicus

and

Tetraena

mongolica

57

Determination of scavenging activities against

superoxide anion radical with EPR

EPR assay was carried out as described by Arudi

with some modifications (1981). One hundred £gl sample

dissolved in DMSO at different concentrations was added

into a test tube containing 890 £gl of DMSO. Ten £gl of 50

mmol/L NaOH were then added. Forty £gl of the mixture

were added into a long aqueous quartz tube, which was

allowed to incubate at room temperature for 30 minutes

before it was transferred into liquid nitrogen immediately.

Inhibition ratio was determined by comparing with

a control group. EPR spectra were obtained with a

Bruker ER 200D SRC spectrometers and ER 4111 VT

temperature controller using micro-sampling pipettes

under the following conditions: SF, 9.59 GH

Z

; SP, 20 mW;

MA, 5 G; MF,100 KH

Z

; GN, 2.5*10

4

; CF, 3360 G; SW,

300 G; TC, 200 mS; TI, 100 S; TE, 130 K. The inhibition

percentage was calculated as: inhibition %=100¡Ñ(ref-

extract)/ref, where ref is the reference signal (DMSO-

NaOH), extract is the test signal. Each value was the mean

of five measurements.

DPPH assay

A method similar to Cotelle¡¦s

was employed except

that methanol was used instead of ethanol (Cotelle et

al., 1996). Two ml of 0.2 mmol/L DPPH in methanol

were gently mixed with 2 ml solutions of the extracts at

different concentrations (diluted to final concentrations

of 500, 100, 50 and 10 £gg/ml). L-Ascorbic acid was

used as positive control. The inhibition percentage was

calculated as: inhibition %=100¡Ñ[1-(Ai - Aj)/Ac], where

Ac is the absorbance of DPPH without extracts, Aj is the

absorbance of extracts, and Ai is the absorbance of DPPH

with extracts.

Lipid peroxidation assay

Lipid peroxidation assay was carried out basically as

described by Buege and Aust with some modifications

(Buege and Aust, 1978). Sprague-Dawley mice (¡ñ or ¡ð,

220 ¡Ó 20 g) were purchased from the Centre of Experi-

mental Animals, The Second Military Medical University.

The liver tissue of SD mice was prepared as 10% tissue

homogenate. One volume of homogenate was mixed thor-

oughly with two volumes of the stock solution of 10% w/v

trichloroacetic acid, 0.375% w/v thiobarbituric acid and

0.25 mol/L hydrochloric acid.

The absorbance of the clear

supernatant was determined at 535 nm. The amount of

MDA produced by hydrolyzing TEP was used as standard

(Csallany et al., 1984). L-Ascorbic acid was used as posi-

tive control. The inhibition percentage was calculated as:

inhibition %=100¡Ñ(Abs

standard

-Abs

extract

)/Abs

standard

.

Statistical analysis

All values were expressed as mean ¡Ó SD. Student¡¦s

t-test was used to determine the significance of differences

in all analyses. p values of less than 0.05 were considered

to be statistically significant.

RESULTS

Preparations of extracts from A. mongolicus,

T.

mongolica and D. paxianum

Three hundred and fifty g A

1

, 190g T

1

and 200 g D

1

were obtained from 6400 g A

0

, 5600 g T

0

, and 5000 g D

0

respectively. Four extracts were further eluted from A

1

fraction by chromatography, A

3

(petroleum ether 100%,

30 g), A

4

(petroleum ether : ethyl acetate 6:4, v/v, 35 g),

A

5

(petroleum ether : ethyl acetate 1:1, v/v, 45 g) and A

6

(petroleum ether : ethyl acetate 2:8, v/v, 20 g) respectively;

and two extracts from T

1

, T

3

(petroleum ether : ethyl

acetate 8:2, v/v, 37 g) and T

4

(petroleum ether : ethyl

acetate 4:6, v/v, 40 g) respectively. From the H

2

O soluble

fractions, three n-BuOH soluble fractions were derived,

A

2

(190 g), T

2

(120 g)

and D

2

(150 g)

respectively. A

7

(chloroform : methanol 1:1, v/v, 35 g) and T

5

(chloroform

: methanol 1:1, v/v, 28 g) were finally eluted from A

2

and

T

2

respectively by chromatography (Figure 1).

Scavenging activities of the extracts against

superoxide anion radical

Upon the addition of a scavenger, a decrease in ¡PO

2

-

signal intensity is a reflection of its scavenging activity. In

this study, it was expressed as the IC

50

value (w/v) of the

scavenger, at which 50% ¡PO

2

-

generated was counteracted.

The scavenging activities of eight extracts from A .

mongolicus and six extracts from T. mongolica, as well

as three from D. paxianum, were determined accordingly,

Figu re 1. P reparations of extracts from A. mongolicus,T.

mongolica and D. paxianum. P owde red aeri al part s were

exhaustively extracted four times with ethanol, and the resultant

extracts (A

0

, T

0

, and D

0

) were then partitioned within ethyl

ac etate and H

2

O. The ethyl ac etat e-solubl e portions were

repeatedly chromat ographed over silic a gel and S epha de x

LH-20 column, and samples A

1

, A

3

, A

4

, A

5

, A

6

, T

1

, T

3

, T

4

and

D

1

were subsequently obtained. The H

2

O-soluble portion was

further partitioned within n-Butanol and H

2

O. The n-Butanol-

soluble portion was repeatedly chromatographed over silica gel

and Sephadex LH-20 column, and samples A

2

, A

7

, T

2

, T

5

and

D

2

were then obtained. A, T and D stand for A. mongolicus, T.

mongolica and D. paxianum respectively.

58

Botanical Studies, Vol. 48, 2007

and their IC

50

values were summarized in Table 1. All of

the extracts exhibited significantly scavenging activities

against the ¡PO

2

-

by EPR. Generally, the extracts from A.

mongolicus showed higher scavenging activities than the

extracts from T. mongolica, and, as expected, the extracts

from D. paxianum the lowest.

Interestingly, upon further partitioning and/or eluting of

the initial ethanol extract, a general tendency of enhanced

scavenging activity was observed (Table 1), particularly

as the ratio of ethyl acetate to petroleum ether in eluting

fluids was raised (Figure 3). A

5

showed the lowest IC

50

value among the all, implicating that more and/or stronger

¡PO

2

-

scavengers were concentrated in the extract.

With A

5

,

a scavenging activity against ¡PO

2

-

could still be detected at

a concentration as low as 0.006 g/ml. An EPR spectrum of

A

5

scavenging activities against ¡PO

2

-

was shown in Figure

2.

Scavenging activities of selected extracts

against DPPH radical

The inhibition percentages of five selected extracts

against stable DPPH radical were determined accordingly

and their inhibition percentages listed in Table 2. Different

Figu re 2. S cave nging activities of diluted A

5

a gains t ¡PO

2

-

detected by EPR. A series of dilutions (50%, 40%, 30%, 20%,

15% and 10% ) was made from i ts init ial concentrat ion of

0.06 g/ml, and their respective scavenging capacities (100%,

96.38%, 92.57%, 88.22%, 46.38%, 42.75% and 22.46%) against

superoxide anion radical (¡PO

2

-

) were determined accordingly,

with 100% counteracted by the undiluted initial A

5

(the top), and

0% by blank (the bottom).

Table 2. Inhibition percentages of selected extracts to DPPH radical.

Concentrations

(£gg/ml)

Extracts

A

1

A

2

T

1

T

2

A

5

L-Ascorbic acid

10

11.7¡Ó2.0

5.42¡Ó0.3

0

0

15.8¡Ó1.3

74.03¡Ó2.3

50

29.0¡Ó3.6

11.7¡Ó2.0

24.5¡Ó1.1

12.3¡Ó3.6

41.4¡Ó4.2

80.25¡Ó3.0

100

45.7¡Ó2.5

16.2¡Ó2.3

60.8¡Ó3.5

33.8¡Ó2.8

72.2¡Ó2.5

96.30¡Ó2.1

500

92.2¡Ó1.2

47.2¡Ó3.4

95.8¡Ó2.9

92.2¡Ó3.5

96.0¡Ó3.1

99.64¡Ó0.9

The scavenging effects were expressed as the percentage inhibition (means¡ÓS.D., n=4) compared to the blank (buffer instead of

extract). L-Ascorbic acid was used as a positive control.

Figu re 3. Compa riso n of IC

50

va lues of sc avengi ng ¡PO

2

-

activities of four extracts from A. mongolicus by EPR. The IC

50

values of A

3

, A

4

, A

5

and A

6

were 0.026, 0.029, 0.016 and 0.020

respectively. A stands for A. mongolicus.

Table 1. The IC

50

values of all the extracts against superoxide

anion radical with EPR.

Concentrations (g/ml)

A

0

ethanol extract

0.088¡Ó0.015

A

1

ethyl acetate extracts

0.075¡Ó0.010

A

2

n-BuOH -eluted extracts

0.151¡Ó0.008

A

3

(petroleum ether 100%)

0.026¡Ó0.005

A

4

(petroleum ether : ethyl acetate 6:4) 0.029¡Ó0.005

A

5

(petroleum ether : ethyl acetate 1:1) 0.016¡Ó 0.009

A

6

(petroleum ether : ethyl acetate 2:8) 0.020¡Ó0.005

A

7

(chloroform : methanol 1:1)

0.024¡Ó 0.010

T

0

ethanol extract

0.110¡Ó 0.010

T

1

ethyl acetate extracts

0.093¡Ó0.009

T

2

n-BuOH-eluted extracts

0.085¡Ó 0.013

T

3

(petroleum ether : ethyl acetate 8:2) 0.035¡Ó0.011

T

4

(petroleum ether : ethyl acetate 4:6) 0.070¡Ó0.008

T

5

(chloroform : methanol (1:1)

0.020¡Ó0.005

D

0

ethanol extract

0.182¡Ó0.020

D

1

ethyl acetate extracts

0.210¡Ó0.015

D

2

n-BuOH-eluted extracts

0.156¡Ó0.011

WANG et al. ¡X

Accumulations of antioxidants in

Ammopiptanthus

mongolicus

and

Tetraena

mongolica

59

extracts showed varied inhibition percentages to DPPH

radical, with the A

1

and A

5

being relatively stronger.

A

5

showed stronger scavenging capacities particularly

at concentrations higher than 10 £gg/ml.

However,

its scavenging capacities were smaller than those of

L-ascorbic acid at comparable concentrations

.

Lipid peroxidation assay on selected extracts

Inhibitory effects of five selected extracts to the

production of MDA were determined using the mouse liver

tissue, with L-ascorbic acid as a positive control, and their

inhibition percentages at different concentrations were

shown in Figure 4. Although all the extracts examined

exhibited significantly antioxidative capacities at most

concentrations, their capacities were generally slightly

or significantly smaller than that of L-Ascorbic acid at

comparable concentrations. Only at concentrations lower

than 1 mg/ml, did A

5

and T

2

show significantly or slightly

higher anti-oxidative capacities than those of L-Ascorbic

acid, being 6.57% and 1.75% higher respectively at 0.1

mg/ml, and 11.53% and 9.84% at 0.01 mg/ml (Figure 4).

Identification of major pure compounds and

scavenging activity of resveratrol against ¡PO

2

-

Since A5 consistently exhibited the highest scavenging

activities or antioxidative capacities in the previous

assays, an attempt was made to obtain pure scavengers

or antioxidants from the extract. We isolated a major

compound from A

5

, which was identified as resveratrol

according to its IR absorption,

1

H NMR (Nuclear

Magnetic Resonance) spectrum and

13

C NMR signal

information. Purified resveratrol

from A

5

accounted for

0.05% of the dried weight of A. mongolicus. Other three

pure compounds (5, 7, 4¡¦-trihydroxyisoflavone, maackiain

and 7, 3¡¦-dihydroxy-4¡¦-methoxyisoflavone) were isolated

and identified from A

4

. In the scavenging activity assay

against ¡PO

2

-

, resveratrol exhibited a large magnitude of

antioxidative potency. At concentrations from 0.0375 to

0.0038 g/ml, over 90% of ¡PO

2

-

generated was counteracted,

compared to Vitamin E with which a two to three times

higher concentration (0.0521 to 0.0104 g/ml) was required

to achieve a similar inhibition ratio (Table 3). However,

when we tried to dilute them further, both of them

exhibited non-characteristic spectra of ¡PO

2

-

, which might

be due to the presence of excess hydroxyl groups in both

of the compounds. Further analysis is under way.

DISCUSSION

Over-generation of ROS is observed under diversified

stress conditions (Elstner et al., 1988; Foyer and Harbin-

son, 1994). ROS can break and destruct bio-molecules and

membranes. Some small molecules, such as phenolic and

flavonoid compounds, play a big part in scavenging ROS

(Foyer et al., 1994), and it has been observed that UV-B

radiation causes an increase in the synthesis of UV-absorb-

ing flavonoids (Gusman et al.,

2001

). However, to our best

knowledge, the correlation between the severity of envi-

ronmental conditions, with which plants grow naturally,

and their non-enzymatic scavenging activity against ROS

has not been meaningfully explored. In this study, we

found that various extracts from two distinctive species,

A. mongolicus and T. mongolica, stressed under season-

ally extreme environmental conditions and constant poor

soil

qualities

with high salinity in their lives, exhibited

significantly higher scavenging activities against the ¡PO

2

-

,

than those of the extracts from D. paxianum, a species

growing in the much milder environment with relatively

fertile soil in the northern mountain area of Guangdong

province, the southeast part of China. The results indicated

a preliminary positive correlation between the severity of

plant environmental conditions and their non-enzymatic

scavenging activities against the ¡PO

2

-

.

The concentration-dependent scavenging activities of

A

5

dilutes implied the existence of strong active oxygen

scavengers in the extract, which lead to the isolation of a

strong antioxidant, resveratrol. It accounted for as high as

Figure 4. Inhibition percentages of five selected extracts to

the production of MDA at different concentrations. Each bar

represents mean ¡Ó S.D. (n=3). (*p<0.05, vs. Vc).

Table 3. Scavenging activities of resveratrol against ¡PO

2

-

.

Concentration (g/mL) Inhibition (%)

Resveratrol

0.0375

100

0.0300

97.86

0.0225

95.72

0.0113

93.58

0.0038

90.37

Vitamin E

0.0521

99.93

0.0208

97.74

0.0156

93.26

0.0104

90.26

60

Botanical Studies, Vol. 48, 2007

0.05% of the dried weight of A. mongolicus. The extraor-

dinarily high content is in a sharp comparison with that

(0.001-0.0005%) in the Vitis vinifera, which was reported

to be the most abundant natural source of resveratrol

(Langcake and Pryce, 1976). Resveratrol is a kind of non-

flavonoid polyphenol, and has been identified as a natural

antioxidant in red and white wine (Soleas et al., 1997;

German and Walzem, 2000). Plants synthesize resveratrol

in response to fungal infections and UV irradiation stress

(Langcake and Pryce, 1976; Jang et al., 1997), as well as

to nutrient limitation (Soleas et al., 1997). Taken together,

these results help to explain the toughness of A. mongo-

licu.

Resveratrol has been found to suppress NMBA-induced

rat esophageal tumorigenesis by targeting COXs and PGE

2

(Li et al., 2002). The two major isoflavone compounds

isolated have been reported to have strong antioxidative

and anti-inflammatory activities (Cao, 1997). Another

major compound, maackiain, has been known to be anti-

bacterial/fungal (Cachinero et al., 2002). In one of our

separate experiments, significant effects of these extracts

on the extensions of life span of D. melanogaster were ob-

served, with adequate concentrations varying from 1 mg

to 50 mg/100 g (data to be published elsewhere). All these

data support our working hypothesis about the efficacy of

A. mongolicus in the traditional treatment of respiratory

disorders, stomachache, cold-caused wounds and chronic

rheumatism.

DPPH and lipid peroxidation have been used as con-

venient tools for radical scavenging assays. Five selected

extracts with the lowest IC

50

against the ¡PO

2

-

also showed

antioxidative capacities against DPPH radical and lipid

peroxidation radical at different magnitudes of potency,

with the A

5

still being the strongest antioxidant. The

consistency of antioxidative capacities of these extracts

against different radicals indicates that these extracts have

wider spectra of scavenging activities.

In conclusion, the accumulation of antioxidants could

be stimulated to an extraordinarily high level in plant spe-

cies distributed in extremely stressful environments. And

the medicinal efficacy of A. mongolicus in the treatments

of respiratory disorders, stomachache, cold-caused wounds

and chronic rheumatism may be fundamentally related to

the antioxidative process. One implication of our results

is that some plant species living in the harsh environment

could become an abundant natural resource for the de-

velopment of medicines for anti-inflammatory and anti-

infectious, as well as for anti-degenerative disorders and

even anti-tumorigenesis.

It is also advisable to develop

new types of healthy food and / or food additives from the

edible parts of plants living in stressful environments or to

make food crops more nutritional and/or healthy by stress-

ing them before harvesting.

Acknowledgement. We thank Professor Shiming Chen

(Department of Chemistry, Fudan University) for his

assistance in electron paramagnetic resonance experiment.

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