J. Biol. Macromol., 20 (1), 17-22 (2020)

ArticleInhibition of HIV-1 Reverse Transcriptase Activity by the Extracts of Indian Plants
Keiko Ishizuka,1 Yuto Tsutsumi,1 Misato Baba,1 Radhika Biyani,2 Chen Wei Meng,3Manish Biyani,2,4 Masahiro Takagi,2 Jyotsna Jaiswal,5 Bechan Sharma,5 Kenji Kojima1,Teisuke Takita1, and Kiyoshi Yasukawa1,*1Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyoku, Kyoto 606-8502, Japan

2School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi,Ishikawa 923-1292, Japan3

School of Biological Science, Nanyang Technological University, Singapore

4

Department of Biotechnology, Biyani Girls College, Sector-3, Vidhyadhar Nagar, Jaipur 302039, India

5

Department of Biochemistry, University of Allahabad, Allahabad 211002, UP, India

Received November 19, 2019; Accepted December 24, 2019

As the currently available therapies for HIV-1 have high costs and are associated with theappearance of multi-drug resistant HIV-1 strains, the Indian traditional medicine system Ayurveda
may provide useful alternatives. In this study, we screened the inhibitory activities of various
extracts of five Indian plants for inhibitory effects on HIV-1 reverse transcriptase (RT). Water
extracts of the leaves of Argemone mexicana strongly inhibited the DNA polymerase activity of HIV1 RT, which indicated that they contained substances with inhibitor activity. Neither heat treatment
at 100ºC nor proteinase K treatment of the extracts abolished the inhibitory activity, suggesting thatthe inhibitory substance was an organic compound rather than a protein.
Key words: Argemone mexicana; HIV-1; inhibition; Parthenium hysterophorus; reverse transcriptase.

Introduction

and a 51-kDa p51 subunit. The former includes

Reverse transcriptase (RT) [EC 2.7.7.49] fromthe fingers, palm, thumb, and connection

human immunodeficiency virus type 1 (HIV-1)

subdomains and the RNase H domain, while thepossesses RNA- and DNA-dependent DNA

latter lacks the RNase H domain and has the otherpolymerase activities and RNase H activity. It is afour subdomains. The fingers, palm, and thumbheterodimer consisting of a 66-kDa p66 subunit

subdomains are involved in the DNA polymerase
activity, and the RNase H domain is in the RNase*H activity.

Correspondence author: Kiyoshi Yasukawa.

In the current HIV-1 therapy,

Phone: +81-75-753-6266
Fax: +81-75-753-6265

nucleoside/nonnucleoside RT inhibitors, proteaseE-mail: yasukawa@kais.kyoto-u.ac.jp

inhibitors, and integrase inhibitors are used,17

Inhibition of HIV-1 Reverse Transcriptase

which is highly expensive. In addition, the long-addition to chemosterilant and wound-healing

term use of such inhibitors has caused theactivities [6]. It is found in Ethiopia, Mexico,

emergence of multi-drug resistant HIV-1 strains,

United States, and many warm places in India. S.

which is a major issue. Therefore, thexanthocarpum is a species of herb. It is adevelopment of a novel and cheap HIV-1 RT

medicinal plant that is used mostly in India. Some

inhibitor is anticipated.

parts of the plant, especially the fruit, are

We previously screened the inhibitory

poisonous [7]. C. procera is a species ofactivities of 25 edible plants against the DNA

flowering plant. The milky sap contains a

polymerase activity of HIV-1 RT and found thatcomplex mix of chemicals, some of which arethe ethanol and water extracts of Brasenia

steroidal heart poisons [8]. T. peruviana is anschreberi (Junsai) exhibited strong inhibitory

evergreen shrub or small tree. The plant is used

activities [1, 2]. Purification and structural

widely in folk medicine. In India, it has become

determination of the constituent compounds areestablished as a household remedy for several

currently in progress.

diseases [9].

Ayurvedic medicine is an Indian traditional

In this study, we screened for HIV-1 RT

medicine system, which offers inexpensive,

inhibitory activity in these five Indian plants. The

natural, orally prescribed remedies of extracts ofresults showed that the water extracts of thethe leaves, flowers, and fruits of plants. However,leaves of A. mexicana strongly inhibited HIV-1

little is known about the application of Ayurvedic

RT activity.

medicine to HIV-1 therapy. In this study, weselected five Indian plants whose extracts wereMaterials and Methods

available in University of Allahabad as researchtargets: Parthenium hysterophorus (Gajar Ghas),

Materialsp(dT)15 was purchased fromArgemone mexicana (Satyanashi), Solanum

Fasmac (Tokyo, Japan). [methyl-3H]dTTP (1.52xanthocarpum (Kantakari), Calotropis proceraTBq/mmol) and poly(rA) were from GE(Safed aak), and Thevetia peruviana (PeeliHealthcare (Buckinghamshire, UK). Recombinant

Kaner). P. hysterophorus is an annual weed

HIV-1 RT was expressed in Escherichia coli andutilized as an ethnomedicine for the treatment ofpurified from the cells as described previouslydifferent infectious and chronic diseases. In[10]. The RT concentration was determined usingaddition, extracts of P. hysterophorus have been

Protein Assay CBB Solution (Nacalai Tesque,found to be useful for the treatment of anemia,

Kyoto, Japan) with bovine serum albuminfever, wound healing, heart troubles, and sores. In(Nacalai Tesque) as standard.

contrast, the plant has the ability to kill otherPreparation of extracts of Indian plants

plants growing in its vicinity, and causes allergic

Freeze-dried leaves, flowers, and fruits werediseases in humans and animals [3, 4]. It is foundcrushed into a powdered form by using a blender.

in almost all parts of the world. A. mexicana is a

The powders were then extracted with water,

species of poppy. Despite being poisonous, allmethanol, ethyl acetate, or hexane at roomparts of this plant have been used in traditionaltemperature (20-25ºC) or at higher temperatures,

medicine to treat chronic diseases of the skin,equivalent to the boiling point of the respective

gastrointestinal tract, and respiratory tract. In

solvents, by using a Soxhlet apparatus. The

addition, it exhibits allelopathic, larvicidal,

solution of the extracts thus prepared werenematicidal [5], and antimicrobial potential, in

evaporated to dryness at room temperature.

18TheK. Ishizuka et al.

dried extracts were dissolved in the required

the glass filterGF/C 2.5 cm (Whatman,volume of the corresponding solvent.

Middlesex, UK). The amounts of [3H]dTTP

Measurement of HIV-1 RT activity using

incorporated was counted, and the reaction rate3[ H]-dTTP HIV-1 RT was incubated withwas determined.

extracts: 80 l of the HIV-1 RT solution (55 nM

Measurement of HIV-1 RT activity using

in 20 mM potassium phosphate buffer (pH 7.2), 2fluorescent dye PicoGreenmM dithiothreitol (DTT), 10% v/v glycerol

incubated with extracts as described above. Then,(buffer A)) and 32 l of extract were mixed andthe activity was measured with EnzChek Reverse

incubated at 37ºC for 5 min. Then, the activity

Transcriptase Assay Kit (Thermo Fisher

was measured as described previously [11].

Scientific, Waltham, MA) as described previously

Briefly, the reaction mixture (52 l) was carried[11]. Briefly, PicoGreen solution was prepared byout in 25 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.0200-fold diluting PicoGreen dsDNAmM DTT, 5.0 mM MgCl2, 25 µM poly(rA)-quantification reagent with 1×TE buffer (10 mMp(dT)15 (this concentration is expressed as that ofTris-HCl buffer (pH 7.5), 1 mM EDTA).

3

HIV-1 RT wasp(dT)15), 0.2 mM [ H]dTTP, 5 nM HIV-1 RT, andPoly(rA)-p(dT)16 solution was prepared by mixing

8% v/v extract at 37ºC. An aliquot (20 µl) waspoly(rA) (5 l) and p(dT)16 (5 l) and incubatingtaken from the reaction mixture at afor 1 h at room temperature followed by thepredetermined time and immediately spotted onto

dilution with 2 ml of polymerization buffer (60(A)(B)) Using [3H]-dTTP

Plant name1234567891011121314151617181920

Parthenium
hysterophorus

Argemonemexicana

Solanum
xanthocarpum
Calotropis
procera
Thevetia
peruviana

Part ofplant

Solvent

Water
Ethyl acetate
Leaf
HexaneEthyl acetate
Ethyl acetate
Flower Methanol
Ethyl acetate
Methanol
Ethyl acetate
Leaf
Methanol
Water
Ethyl acetate
Fruit
Ethyl acetate
Leaf
HexaneEthyl acetate
Fruit
Ethyl acetate
WaterWater
FlowerEthyl acetate
Fruit

Water

Temperature atextraction
RR
H
H
R
HH
R
H
H
H
H
R
H
H
R
R
H
H

Using fluorescent dye PicoGreen

1

1223344556677889910101111

1212

1313

14

14

15

15

1616

1717

18

18

19

1920

H

200

20

4060

80

Relative activity (%)

100

020

4060

80

100

Relative activity (%)

Fig. 1. Inhibition of HIV-1 RT activity by 20 extracts.
(A) Origin and preparation method of the extracts. R and H indicates room temperature and temperaturesequivalent to the boiling point of the respective solvents, respectively. (B) HIV-1 RT activity in thepresence of extracts. The results of the assay using [3H]-dTTP and that using fluorescent dye PicoGreenare shown. The reaction was carried out at 37°C with HIV-1 RT at 5 nM and extracts at 8% v/v. The
reaction without extracts was also carried out as a control. Relative activity was defined as the ratio ofthe reaction rate with extracts to that without it. One of the representative data of the average of triplicate
determinations with SD value is shown.

19Inhibition of HIV-1 Reverse Transcriptase

mM Tris-HCl buffer (pH 8.1), 60 mM KCl, 8 mM

Relative activity (%)

MgCl2, 13 mM DTT, 100 M dTTP). Thereaction was started by adding the pre-incubatedsolution of HIV-1 RT and extract (24 l) to thepoly(rA)-p(dT)16 solution (96 l). An aliquot (25l) was taken from the reaction mixture at 2.5,5.0, and 7.5 min, to which 2 l of 200 mM EDTA
was immediately added and incubated on ice for30 min. Blank solution was prepared by mixing

100806040
200050poly(rA)-p(dT)16 solution (20 l), 200 mM EDTA(2 l), and HIV-1 RT solution (5 l). To each100 150 200 250 400[Extract] (µg/mL)

Fig. 2. Inhibition of HIV-1 RT by waterextracts of the leaf of P. hysterophorus(circle) and A. mexicana (triangle), andwater extracts of B. schreberi (square).
The results of the assay using [3H]-dTTP
are shown. Relative activity was definedas the ratio of the reaction rate withextracts to that without it. One of therepresentative data of the average of
triplicate determinations with SD value isshown.

solution (27 l), PicoGreen solution (173 l) wasadded. The tubes were wrapped with aluminum
foil and left at room temperature for 10 min. The
fluorescence at 523 nm was measured withEnSight (PerkinElmer, Waltham, MA) with theexcitation wavelength of 502 nm.
Results and Discussion

Relative activity (%)

Screening of HIV-1 RT inhibitory activity
We tested the inhibitory activities of 20
extracts on HIV-1 RT by using [3H]-dTTP or the
fluorescent dye PicoGreen (Fig. 1). The relative
activity was defined as the ratio of the reaction
rate with 8% v/v extract to that without it. TheExtracts

water extracts of the leaves of P. hysterophorus

100806040200None

Thermal treatment
Proteinase K(No. 1) and A. mexicana (No. 11) inhibited HIV-1
RT activity by more than 90%, whereas the ethyl

A. mexicana

B. schreberi－ ＋ － － ＋ －－ － ＋ － － ＋Fig. 3. Effects of thermal treatment andproteinase treatment on the inhibitory
activity. The results of the assay using[3H]-dTTP is shown. Relative activity wasdefined as the ratio of the reaction ratewith extracts to that without it. The
concentrations of the extracts of A.
mexicana and B. schreberi were 100 and400 g/ml, respectively, in the thermal
treatment at 100°C for 5 min, 160 and 400
g/ml, respectively, in the proteinase K
treatment (50 g/ml in 20 mM Tris-HCl
buffer (pH 7.5) at 37°C for 60 min), and64 and 160 g/ml, respectively, in the RT
reaction. One of the representative data of
the average of duplicate determinations areshown.

acetate, hexane, and methanol extracts inhibitedHIV-1 RT activity by 17%-58%. The difference ininhibitory activity between water extracts andother extracts was probably due to the different
constituents that were extracted by water andother solvents. The extracts of the leaves, fruits,and flowers of S. xanthocarpum, C. procera, andT. peruviana inhibited HIV-1 RT activity by 10%80%.
The water extract of the leaves of P.
hysterophorus (No. 1) and water extract of theleaves of A. mexicana (No. 11) showed thestrongest inhibitory activity. Thus, these two20

K. Ishizuka et al.

extracts were subjected to further analysis.

epigallocatechin 3-gallate (EGCG) [15] werereported to inhibit the DNA polymerase activity

HIV-1 RT inhibitory activity of water extracts ofof HIV-1 RT. However, India has numerous plant

the leaves of P. hysterophorus and A. mexicana

resources, and our results have suggested that

We examined the effect of increasing

there may be as yet unidentified Indian plants thatconcentrations of water extracts of the leaves of P.

possess strong HIV-1 RT inhibitory activity. In

hysterophorus and A. mexicana on HIV-1 RT

combination with Ayurvedic techniques, theseactivity (Fig. 2). B. schreberi (Junsai) was also

plants may be developed as a novel, inexpensive

examined as a comparator. The reaction rates

pharmaceutical agent for HIV-1 therapy.

decreased as the concentrations of the extracts
increased. The relative activity was 20% at 160

Referencesg/ml for P. hysterophorus, 0% at 240 g/ml for A.

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Calotropis procera [Internet], Available
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21

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Funding
This study was supported in part by Grants-in-Aidfor Scientific Research (no. 18J14339 (MisatoBaba), no. 18K19839 (Manish Biyani and K.Y.),and no. 18KK0285 (K.K., T.T., and K.Y.)) fromJapan Society for the Promotion of Science andKyoto

University Amgen

Scholar

Program(C.W.M. and K.Y.),
Communicated by Yasukawa Kiyosh22