J. Biol. Macromol., 14(1), 59-65 (2014)
ArticleInhibition of HIV-1 Reverse Transcriptase Activity
by Brasenia schreberi (Junsai) Components
Tetsuro Hisayoshi,1 Mayu Shinomura,1 Atsushi Konishi,1 Junji Tanaka,2 Hiroshi Shimoda,2Keishi Hata,3 Saori Takahashi,3 and Kiyoshi Yasukawa1,*
1

Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University,
Sakyo-ku, Kyoto 606-8502, Japan
2

Oryza Oil & Fat Chemical, 1 Numata Kitagatacho, Ichinomiya, Aichi 493-8001, Japan3

Akita Research Institute of Food and Brewing, 4-26 Sanuki, Arayamachi, Akita 010-1623, Japan

Received April 03, 2014; Accepted April 29, 2014
We screened for inhibitory activities of 25 wild vegetables and fruits for human immunodeficiency
virus type 1 (HIV-1) reverse transcriptase (RT). Among them, ethanol- and water-extracts ofBrasenia schreberi (Junsai) and water-extract of Petasites japonicus (Fuki) strongly inhibited theHIV-1 RT activity to incorporate dTTP into poly(rA)-p(dT)15. We tested HIV-1 RT inhibitory
activities of 15 polyphenols, isolated from Brasenia schreberi. Among them, gossypetin and hypolaetin
7-O-glucoside inhibited the activity.
Key words: Brasenia schreberi, HIV-1; inhibition, reverse transcriptase.

Introduction

inhibitor is anticipated.

Reverse transcriptase (RT) [EC 2.7.7.49] is the

Variousenzymeinhibitors

have

been

enzyme responsible for replication of retrovirus. It

identified in natural product. In the case withpossesses RNA- and DNA-dependent DNA

HIV-1 RT, 5,6,7-trihydroxyflavone (baicalein) [3]polymerase activities and RNase H activity.

and catechins containing a galloyl moiety such as

Human immunodeficiency virus type 1 (HIV-1) RT(-)-epicatechin 3-gallate (ECG) [4] and (-)-

is a heterodimer consisting of a 66-kDa p66

epigallocatechin 3-gallate (EGCG) [5] were

subunit and a 51-kDa p51 subunit [1, 2]. In thereported to inhibit the RNA-dependent DNA

HIV-1 RT therapy, nucleoside RT inhibitors

polymerase (reverse transcriptase) activity of(NRTIs)inhibitors

HIV-1 RT. However, it was pointed out that their(NNRTIs) are used. However, the emergence of RT

inhibitory activities were non-specific, due to theirinhibitor-resistant HIV-1 variants is a major issue.

binding abilities to various proteins [6]. Indeed, it

Therefore, the development of a novel HIV-1 RT

was recently reported that methyl gallate, purified*

andnon-nucleoside

RT

Correspondence author: Kiyoshi Yasukawa.

Phone: +81-75-753-6266
Fax: +81-75-753-6265
E-mail: yasukawa@kais.kyoto-u.ac.jp

from Pholiota adipose(Mushroom), completely

inhibited HIV-1 RT activity [7].

60HIV-1 reverse transcriptase inhibitor from Junsai

We previously prepared recombinant RTs

activityforincorporation

of

dTTP

into

from HIV-1 group M, spread all over the world,poly(rA)-p(dT)15 was measured as describedand HIV-1 group O, restricted to Cameroon andpreviously [8, 9]. In the experiment with extracts

neighboring countries in West Central Africa, andof foodstuffs, pre-incubation (20 l) was initiatedcompared their activities and stabilities [8, 9]. In

by mixing 18 l of the HIV-1 RT solution (220 nM

the present study, we screened for HIV-1 RT

in 20 mM potassium phosphate buffer (pH 7.2), 2

inhibitory activity in various foodstuffs using themM dithiothreitol (DTT), 10% v/v glycerol (buffer

HIV-1 group M RT and found the strong inhibitoryA)) and 2 l of ethanol- or water-extract ofactivity in ethanol- and water-extracts of Brasenia

foodstuffs. In the experiment with compounds,

schreberi (Junsai).

pre-incubation (20 l) was initiated by mixing 18
l of the HIV-1 RT solution (220 nM in buffer A)
and 2 l of the compound solution (0−110 M in

Materials and Methods

dimethyl sulfoxide (DMSO)). After the preMaterials

p(dT)15 was purchased from Life

incubation at room temperature for 3 min, theJapan

Japan).

reaction was carried out in 25 mM Tris-HCl buffer[methyl- H]dTTP (1.52 TBq/mmol) and poly(rA)(pH 8.3), 50 mM KCl, 2.0 mM DTT, 5.0 mM

were from GE Healthcare (Buckinghamshire, UK).

MgCl2,

Recombinant HIV-1 group M RT was expressed inconcentration is expressed as that of p(dT)15), 0.2

Escherichia coli and purified from the cells as

mM [3H]dTTP, 2 M HIV-1 RT, and 1% v/v extract

described previously [8, 9]. The RT concentrationor 0−10 M compound at 37ºC. An aliquot (20 µl)

was determined using Protein Assay CBB Solution

was taken from the reaction mixture at a

(Nacalai Tesque, Kyoto, Japan) with bovine serum

predetermined time and immediately spotted onto

albumin (Nacalai Tesque) as standard.

the glass filter GF/C 2.5 cm (Whatman, Middlesex,

Technologies

Ltd.

(Tokyo,325µM

poly(rA)-p(dT)15(thisUK). The amounts of [3H]dTTP incorporated was
Preparationofextractsoffoodstuffs

counted in 2.5 ml of Ecoscint H (NationalFreeze-dried wild vegetables and fruits were

Diagnostics,

Atlanta,

GA)

with

a

liquidreduced to powder. In the preparation of ethanol-

scintillation counter LSC-5100 (Aloka, Mitaka,

extracts, the powder was extracted with ethanol

Japan), and the reaction rate was determined.

and evaporated to dryness. The evaporated sample
was dissolved in ethanol at a final concentration of

Results and Discussion

10-20 mg/ml. In the preparation of water-extracts,
the powder was added to water and autoclaved at

HIV-1 RT inhibitory activities of extracts of

105ºC for 30 min. After centrifugation, thefoodstuffs

supernatant were evaporated to dryness. The dry

We tested inhibitory activities of ethanol- andmatter was dissolved in water at a final

water-extracts of 25 wild vegetables or fruits forconcentration of 10-40 mg/ml.

HIV-1

Measurement of HIV-1 RT activity

RT

RT

to

incorporatedTTPpoly(rA)-p(dT)15 (Fig. 1). The relative activity

intoT. Hisayoshi, et al.

61

Wild vegetables and fruits

Ethanol extracts

Water extracts

Control
Cucumis melo (Muskmelon)
Hosta montana (Urui)
Citrullus lanatus (Watermelon)
Allium fistulosum (Negi)
Vigna unguiculata (Kurosasage)
Solanum lycopersicum (Tomato)
Bassia scoparia (Tonburi)
Colocasia esculenta (Eddoe)
Petasites japonicus (Fuki)
Chrysanthemum morifolium (Chrysanthemum)
Aralia cordata (Udo)
Asparagus officinalis (Asparagus)
Macrocybe gigantea (Nioushimeji)
Malus pumila (Apple)
Corchorus olitorius (Nalta jute)
Cucumis sativus (Cucumber)
Abelmoschus esculentus (Okura)
Denanthe stolonifera (Seri)
Pyrus pyrifolia (Pear)
Matteuccia struthiopteris (Kusasotetsu)
Glycine max (Soy)Spinacia oleracea (Spinach)Dioscorea batatas (Chinese yam)Zingiber mioga (Myoga)
Brasenia schreberi (Junsai)

0

20

40

60

80

Relative activity (%)

100 0

20

40

60

80

100

Relative activity (%)

Fig. 1. Inhibition of HIV-1 RT activity by extracts of 25 wild vegetables or fruits.
The reaction was carried out at 37°C with HIV-1 RT at 20 nM and ethanol- or water-extracts of wild
vegetables and fruits at 1% v/v. Control means the reaction with 1% ethanol instead of ethanol-extracts
in left panel and that with 1% water instead of water-extracts in right panel. The relative activity wasdefined as the ratio of the reaction rate with extracts to that of control (43.6 x 10-9 M s-1 in left panel and56.3 x 10-9 M s-1 in right panel).

was defined as the ratio of the reaction rate withschreberi and water-extract of Petasites japonicus

1% v/v extract to that without it. Among them,

inhibited HIV-1 RT activity by 32, 6, and 36%,ethanol- and water-extracts of Brasenia schreberi

respectively, compared with control (data not(Junsai) and water-extract of Petasites japonicus

shown), indicating that water-extract of Brasenia(Fuki) completely inhibited HIV-1 RT activity. In

schreberi had the strongest inhibitory activity.

all foodstuffs except for Macrocybe gigantea(Nioushimeji) and Dioscorea batatas (Chinese

HIV-1 RT inhibitory activities of compoundsyam), water-extract exhibited stronger inhibitory

isolated from Brasenia schreberi

activities than ethanol- extract.

Brasenia schreberi is an aquatic plant which

We compared the inhibitory activities of

lives in pure water. Gallic acid, quercetin, andethanol- and water-extracts of Brasenia schreberi

quercetin 7-O-glucoside were previously reported

and water-extract of Petasites japonicus. At 0.1%

to be isolated from leaves of Brasenia schreberi

v/v, ethanol- and water-extracts of Brasenia

produced in Canada [10]. Recently, 1562HIV-1 reverse transcriptase inhibitor from Junsai

ethyl gallate
caffeoyl glucose
kaempferol 3-O-(6” -galloyl) glucoside : R=H
quercetin 3-O-(6” -galloyl) glucoside : R=OH

coumaric acid: R=H
caffeic acid: R=OH
kaempferol 3-O-glucoside : R=H
quercetin 3-O-glucoside : R=OH

kaempferol : R=H
quercetin : R=OH

8

A

gossypetin

8Ahypolaetin 7-O-glucosidejunsainoside A

ferulic acidtilirosidecontrol
ethyl gallate
caffeoyl glucose
quercetin 3 -O-(6''-galloyl) -glucosidequercetin 3 -O-glucosidekaempherol 3-O-(6''-galloyl) -glucosidekaempherol 3-O-glucosidejunsainoside A
quercetin
tilirosidekaempherol
caffeic acidcoumaric acidferulic acidgossypetin
hypolaetin 7 -O-glucoside
0

20

40

60

80

100

Relative activity (%)
Fig. 2. Inhibition of HIV-1 RT activity by 15 compounds isolated from Brasenia schreberi
The reaction was carried out in 10% DMSO at 37°C with HIV-1 RT at 20 nM and compounds at 10
M. Control means the reaction without compounds. The relative activity was defined as the ratio of thereaction rate with compound to that without it (49.5 x 10-9 M s-1).

T. Hisayoshi, et al.

63

compounds were isolated from leaves of BraseniaM gossypetin were determined to be 40 ± 8, 31 ±schreberi produced in Akita, Japan (J. T., H. S.,

4, 24 ± 4, 10 ± 2, and 13 ± 2 M, respectively, andunpublished results) (Fig. 2). They include a new

the apparent kcat values (kcat,app) observed for 0, 1.0,

compound, named junsainoside A. We tested

2.5, 5.0, and 10 M gossypetin were determined toinhibitory activities of these 15 compounds forHIV-1 RT (Fig. 2). The relative activity wasdefined as the ratio of the reaction rate withcompound to that without it. Among them,acid inhibited HIV-1 RT activity by 52, 38, and19%, respectively, compared with control, whileother 12 compounds did not inhibit it. Hypolaetin
7-O-glucoside and gossypetin, unlike other 13
compounds, have the hydroxyl group at the 8
position of the A ring (Fig. 2), although it isunknown if it is responsible for the inhibition.
Ethanol- and water-extracts of Brasenia
schreberi inhibited the activity completely (Fig. 1),
while hypolaetin 7-O-glucoside or gossypetin did
not inhibit it completely (Fig. 2). It was reported
that gossypetin [11] and quercetin [12] inhibitedthe HIV-1 RT activity. The IC50 values of quercetin
was 60 M [12], while in this study, quercetin at
10 M did not exhibit the inhibitory activity (Fig.
2). As far as we know, there has been no report ofinhibition of HIV-1 RT by hypolaetin 7-Oglucoside. In regard of this, it was reported thathypolaetin, kaempferol, and quercetin inhibited theHIV-1 integrase activity [13].
Manner of inhibition of gossypetin in HIV-1
RT activity
We measured the reaction rates for theincorporation of dTTP into poly(rA)-p(dT)15, in thepresence and the absence of various concentrationsof gossypetin. All the plots showed saturated
profiles (Fig. 3A). The plot of [dTTP]/vo vs.
[dTTP] (Hanes-Woolf plot) in the presence and theabsence of gossypetin showed non-parallel lines
intersecting at the Y-axis, indicating that inhibition
was uncompetitive (Fig. 3B). The apparent Km
values (Km,app) observed for 0, 1.0, 2.5, 5.0, and 10

vo (nM s-1)

hypolaetin 7-O-glucoside, gossypetin, and ferulic

64HIV-1 reverse transcriptase inhibitor from Junsai

be 3.4 ± 0.2, 2.6 ± 0.1, 1.5 ± 0.1, 0.86 ± 0.05, and-1

Acknowledgments

0.68 ± 0.03 s , respectively. Based on this, theThis study was supported in part (K.Y.) by Grants-

reaction rate can be described by the following

in-Aid for Scientific Research (nos. 19580104 andequation:

21580110) from the Japan Society for the
Promotion of Science and the Towa foundation forfood research.
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Communicated by Tate Shinichi