Anti-HIV-1 integrase activity of Mimusops elengi leaf extracts

Abstract

Context: Integrase (IN) is one of the three human immunodeficiency virus type 1 (HIV-1) enzymes that, together with a reverse transcriptase and protease, allow the virus to reproduce itself after infecting the host cells. Any new knowledge on inhibitors of this enzyme could provide essential clues for the development of anti-HIV drugs.

Objective: To evaluate the anti-HIV-1 IN activity of some Thai medicinal plant extracts, and to isolate the active compounds from the extract that possessed the strongest anti-HIV-1 IN activity.

Materials and methods: Ethanol extracts of eight Thai medicinal plants (10–100 µg/mL) were evaluated for their inhibitory effect against HIV-1 IN. An extract of Mimusops elengi L. (Sapotaceae) leaves that possessed the strongest anti-HIV-1 IN activity was fractionated to isolate the active compounds by an anti-HIV-1 IN assay-guided isolation process.

Results and discussion: The leaf extract from M. elengi had the strongest anti-HIV-1 IN activity with an IC₅₀ value of 62.1 µg/mL. A bioassay-guided isolation of the active compounds from M. elengi leaf extract resulted in the isolation of active compounds, identified as a mixture of gallocatechin and epigallocatechin. This mixture of gallocatechin and epigallocatechin showed satisfactory anti-HIV-1 IN activity with an IC₅₀ value of 35.0 µM. A flavanol glycoside, mearnsitrin was also isolated but was inactive at a concentration of 100 µM.

• Epigallocatechin
• gallocatechin
• HIV
• mearnsitrin

Introduction

Acquired immunodeficiency syndrome (AIDS) is a disease of the human immune system caused by the human immunodeficiency virus (HIV) that causes profound immune suppression. It is a serious life-threatening health problem, and one of the most quickly spreading diseases known to man. HIV is now a leading cause of death worldwide. HIV-1 is the cause of a worldwide epidemic and is most commonly referred to as HIV (Singh et al., 2005). This growing health epidemic has focused the energies of many research organizations to identify new HIV inhibitors and their molecular targets.

Integrase (IN) is one of the three HIV-1 enzymes that together with a reverse transcriptase and protease allows the virus to reproduce itself after infecting the host cells. HIV-1 IN is the enzyme responsible for inserting the HIV proviral DNA into the host DNA. This enzyme functions in a two-step manner, it initially removes a dinucleotide unit from the 3′ ends of the proviral DNA (termed “3′-processing”), then the 3′-processed strands are transferred from the cytoplasm to the host nucleus, where they are introduced into the host DNA (termed “strand-transfer” or “integration”) (Ovenden et al., 2004). Compounds that interfere with this enzymatic integration mechanism might therefore selectively make a significant contribution toward discovering novel therapeutic agents for treating HIV/AIDS.

Currently, only one HIV-1 IN inhibitor, raltegravir, is available commercially. Thus, searching for HIV-1 IN inhibitors from natural sources has seemed like a logical approach. Over the past decade, there has been substantial progress in research on natural products that possess anti-HIV-1 IN activity. Bioassay-guided isolations of crude herbal extracts have provided lead molecules for possible discovery of anti-HIV-1 IN drug candidates. It is reported that an ethanol extract of Smilax corbularia Kunth (Smilacaceae) exhibited a potent anti-HIV-1 IN activity (IC₅₀ 1.9 µg/mL) (Tewtrakul et al., 2006). Several flavonoids isolated from plants have also exhibited anti-HIV-1 IN activity, such as orobol from Eclipta prostrata (L) L. (Asteraceae) (Tewtrakul et al., 2007), luteolin from Coleus parvifolius Benth. (Labiatae) (Tewtrakul et al., 2003), gal²-¹glc-sinapoyl and gal²-¹glc-feruloyl from Thevetia peruviana (Pers.) K. Schum. (Apocynaceae) (Tewtrakul et al., 2002), with IC₅₀ values of 8.1, 11.0, 7.0 and 5.0 µM, respectively. Naturally occurring lignans also showed anti-HIV-1 IN activity, such as globoidnan isolated from Eucalyptus globoidea Blakely (Myrtaceae) possessed HIV-1 IN inhibitory activity with an IC₅₀ of 0.64 µM (Ovenden et al., 2004). In addition, MAP30 and GAP31, two proteins isolated from Momordica charantia L. (Cucurbitaceae) and Gelonium multiflorum A. Juss. (Euphorbiaceae), respectively, showed anti-HIV-1 IN activity (Lee-Huang et al., 1990, 1995).

In this study, the anti-HIV-1 IN activity of ethanol extracts from eight Thai medicinal plants was investigated. An extract of Mimusops elengi leaves that possessed the strongest anti-HIV-1 IN activity was then fractionated to isolate the active compounds as detected by bioassay of fractions.

Materials and methods

Plant materials

The plant materials were collected from the Hat-Yai District, Songkhla Province, Thailand, in August 2010. The plants were identified by Associate Professor Pharkphoom Panichayupakaranant and deposited at the Herbarium of the Southern Center of Traditional Medicine, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Thailand, where herbarium specimens are kept. The pants used in this study were Brassica oleracea L. var. capitata L. (Cruciferae) leaves (specimen no. SKP 057 02 15 01), Coccinia grandis (L.) Voigt (Cucurbitaceae) leaves (specimen no. SKP 058 03 07 01), Diplazium esculentum (Retz.) Sw. (Athyriaceae) leaves (specimen no. SKP 020 04 05 01), Illicium verum Hook.f. (Illiciaceae) fruits (specimen no. SKP 090 09 22 01), Millingtonia hortensis L.f. (Bignoniaceae) leaves (specimen no. SKP 025 13 08 01), M. elengi L. (Sapotaceae) leaves (specimen no. SKP 171 13 05 01), Morinda coreia Ham. (Rubiaceae) leaves (specimen no. SKP 165 13 03 01) and Moringa oleifera Lam. (Moringaceae) leaves (specimen no. SKP 118 13 15 01). These plants were dried at 50 °C for 24 h in a hot air oven and then powdered using a grinder.

Enzyme and chemicals

The HIV-1 IN protein was kindly provided by Dr. Robert Craigie, National Institute of Health, Bethesda, Maryland, USA. This enzyme was expressed in Escherichia coli and purified according to the method described by Goldgur et al. (1999), and stored at −80 °C until used. Oligonucleotides of a long terminal repeat donor DNA (LTR-D) and the target substrate (TS) DNA were from QIAGEN Operon, Alameda, CA, and stored at −25 °C until used. The sequence of the biotinylated LTR donor DNA and its unlabeled complement were 5′-biotin-ACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGT-3′ (LTR-D1) and 3′-GAAAATC AGTCACACACCTTTTAGAGATCGTCA-5′ (LTR-D2), respectively, while those of the target substrate DNA (digoxigenin-labeled target DNA, TS-1) and its 3′-labeled complement were 5′-TGACCAAGGGCTAATTCACT-digoxigenin and digoxigenin-ACTG-GTTCCCGATTAAGTGA-5′ (TS-2), respectively.

Preparation of plant extracts

Dried plant powders were extracted twice with ethanol under reflux conditions for 1 h. The two extracts were combined and concentrated under reduced pressure to produce the ethanol extracts. The extracts were dissolved in 50% dimethyl sulfoxide (DMSO) to form stock solutions of 10 mg/mL before testing.

Assay for HIV-1 IN inhibitory activity

The integration reaction was evaluated using the method described by Tewtrakul et al. (2002). Briefly, a mixture (45 µL) composed of 12 µL of IN buffer [containing 150 mM 3-(N-morpholino) propane sulfonic acid, pH 7.2 (MOPS), 75 mM MnCl₂, 5 mM dithiothritol (DTT), 25% glycerol and 500 µg/mL bovine serum albumin], 1 µL of 5 pmol/µL digoxigenin-labeled target DNA and 32 µL of sterilized water were added into each well of a 96-well plate. Subsequently, 6 µL of a plant extract sample solution and 9 µL of a 1/5 dilution of the integrase enzyme was added to the plates and incubated at 37 °C for 80 min. After washing the wells three times with 270 mL PBS, 100 µL of 500 mU/mL alkaline phosphatase (AP) labeled anti-digoxigenin antibody were added and incubated at 37 °C for 1 h. The plates were washed again three times with 270 mL washing buffer containing 0.05% Tween 20 in PBS and then another three times with 270 mL PBS. Then, an AP buffer (150 µL) containing 100 mM Tris-HCl (pH 9.5), 100 mM NaCl, 5 mM MgCl₂ and 10 mM p-nitrophenyl phosphate was added to each well and incubated at 37 ^oC for 1 h. Finally, the absorbance in each well was measured with a microplate reader at a wavelength of 405 nm. A control was composed of a reaction mixture of 50% DMSO and integrase enzyme, while a blank was buffer-E containing 20 mM MOPS (pH 7.2), 400 mM potassium glutamate, 1 mM ethylenediaminetetra-acetate disodium salt (EDTA, 2Na) 0.1% Nonidet-P 40 (NP-40), 20% glycerol, 1 mM DTT and 4 M urea without the integrase enzyme. Suramin, an HIV-1 IN inhibitor, was used as a positive control. where OD = absorbance detected from each well.

Statistics

For statistical analysis, the results of the anti-HIV-1 IN activity were expressed as a mean ± S.D. from four determinations. The IC₅₀ values were calculated using the Microsoft Excel program. Dunnett’s test was used versus a control for calculation of the statistical significance.

Bioassay-guided isolation of M. elengi

The dried leaf powder of M. elengi (500 g) was extracted with ethanol (2.5 L × 4), under reflux conditions for 1 h, to obtain (after solvent evaporation) a greenish-brown extract (120.7 g). The crude ethanol extract was partitioned between water and ethyl acetate. The ethyl acetate fraction was pre-adsorbed in silica gel, then applied to the top of a silica gel column (13 cm in diameter and 6 cm in height), and the column was subsequently eluted with 500 mL of solvent with the aid of a vacuum pump. Mixtures of hexane and ethyl acetate were used for column elution, using a step-gradient elution starting from 100% hexane and moving toward 100% ethyl acetate, followed by mixtures of ethyl acetate and methanol, from 10% to 20% v/v methanol. Based on TLC chromatograms of each fraction, eight pooled fractions (fractions 1--8) were obtained. The fractions were then tested for anti-HIV-1 IN activity. The anti-HIV-1 IN active fraction (fraction 7) was further purified by silica gel column chromatography (1 g extract per 40 g silica gel) eluted with mixtures of chloroform and methanol (from 9.7:0.3 to 8:2, v/v, 50 mL fractions were collected) to give five pooled fractions (fractions I–V). The fractions were again tested for anti-HIV-1 IN activity. The anti-HIV-1 IN active fractions were fractions IV and V. The fraction V was further purified on a Sephadex® LH-20 column (1 × 20 cm) using methanol as eluent and 20 mL fractions were collected to afford a mixture of compounds 1 and 2 as a white amorphous powder (180 mg).

Fraction IV was also re-chromatographed on the same silica gel column to give four pooled fractions (A–D). The fraction C was further purified on a Sephadex® LH-20 column (1 × 35 cm) using methanol as eluent to afford compound 3 as yellow needles (69 mg).

Identification of compounds 1–3

Compounds 1, 2 and 3 were identified by ¹H NMR, ¹³C NMR and EI-MS and compared with data in the literature (Chung et al., 2004; Rösch et al., 2004).

Gallocatechin (1): white amorphous powder, UV (MeOH) λ_max (log ε) 221 (4.70), 269 (3.70), 321 (3.03) nm; IR (KBr) ν_max 3400, 1629, 1016 cm⁻¹; ¹H NMR (CD₃OD, 500 MHz) δ 2.50 (1H, dd, J = 7.78, 16.0 Hz, H-4α), 2.85 (1H, dd, J = 5.48, 16.92, H-4β), 3.96 (1H, m, H-3), 4.53 (1H, d, J = 7.32 Hz, H-2), 5.91 (1H, d, J = 2.29 Hz, H-8), 5.94 (1H, d, J = 2.29 Hz, H-6), 6.51 (1H, s, H-2′, 6′). ¹³C NMR (CD₃OD, 125 MHz) δ 28.05 (C-4), 68.73 (C-3), 82.83 (C-2), 95.91 (C-8), 96.31 (C-6), 100.76 (C-10), 107.24 (C-2′, 6′), 131.58 (C-1′), 134.00 (C-4′), 146.84 (C-3′, 5′), 156.81 (C-5), 157.63 (C-7), 157.78 (C-9). EIMS m.z (%): 138 (100), 306 (28), 281 (20), 264 (10), 168 (32), 126 (33), 112 (23), 98 (22), 72 (98).

Epigallocatechin (2): white amorphous powder, UV (MeOH) λ_max (log ε) 221 (4.70), 269 (3.70), 321 (3.03) nm; IR (KBr) ν_max 3400, 1629, 1016 cm⁻¹; ¹H NMR (CD₃OD, 500 MHz) δ 2.71 (1H, dd, J = 3.2, 16.69 Hz, H-4α), 2.82 (1H, dd, J = 4.12, 16.92 Hz, H-4β), 4.16 (1H, m, H-3), 4.75 (1H, s, H-2), 5.86 (1H, d, J = 2.29 Hz, H-8), 5.92 (1H, d, J = 2.28 Hz, H-6), 6.40 (1H, s, H-2′, 6′). ¹³C NMR (CD₃OD, 125 MHz) 29.10 (C-4), 67.48 (C-3), 79.87 (C-2), 95.55 (C-8), 96.43 (C-6), 100.13 (C-10), 107.03 (C-2′, 6′), 131.53 (C-1′), 133.60 (C-4′), 146.66 (C-3′, 5′), 157.28 (C-5), 157.55 (C-7), 157.93 (C-9). EIMS m.z (%): 138 (100), 306 (28), 281 (20), 264 (10), 168 (32), 126 (33), 112 (23), 98 (22), 72 (98).

Mearnsitrin or Myricetin 4′-methyl ether-3-O-rhamnoside (3): yellow needles, UV (MeOH) λ_max (log ε) 202 (4.99), 263 (4.32), 337 (4.11) nm; IR (KBr) ν_max 3417, 1634, 1022 cm⁻¹; ¹H NMR (CD₃OD, 500 MHz) δ 0.95 (3H, d, J = 5.5 Hz, Me-6″), 3.33 (1H, m, H-4″, 5″), 3.72 (1H, dd, J = 3.21, 8.69 Hz, H-3″), 3.87 (3H, s, OCH₃-4), 4.22 (1H, d, J = 1.83 Hz, H-2″), 5.37 (1H, d, J = 1.38 Hz, H-1″), 6.20 (1H, d, J = 2.0 Hz, H-6), 6.36 (1H, d, J = 2.0 Hz, H-8), 6.88 (1H, s, H-2′, 6′). ¹³C NMR (CD₃OD, 125 MHz) δ 17.71 (Me-6″), 60.93 (OMe-4), 71.90 (C-5″), 72.05 (C-2″), 72.13 (C-3″), 73.26 (C-4″), 94.79 (C-8), 99.93 (C-6), 103.71 (C-1″), 106.03 (C-10), 109.92 (C-2′, 6′), 127.02 (C-1′), 136.76 (C-3), 139.43 (C-4′), 151.92 (C-3′, 5′), 158.62 (C-9), 159.07 (C-2), 163.27 (C-5), 166.06 (C-7), 179.72 (C-4). EIMS m.z (%): 332 (100), 317 (85), 262 (25), 234 (7), 205 (7), 153 (15), 136 (10), 69 (25).

Results and discussion

Among the ethanol extracts from the eight Thai medicinal plants investigated for anti-HIV-1 IN activity, the extract from M. elengi leaves exhibited the most potent inhibitory activity against HIV-1 IN with an IC₅₀ value of 62.1 µg/mL, followed by the leaf extract of M. hortensis (IC₅₀ = 77.7 µg/mL) (Table 1). The other plant extracts were not active at the concentration of 100 µg/mL. M. elengi is an evergreen tree belonging to the family Sapotaceae. Preparations from this plant have well-documented claims for possessing pharmacological activities including their action as an antioxidant, antibacterial, anticariogenic, antifungal, gastroprotective, hypotensive, antidiabetic, diuretic, antinociceptive and anticancer agent (Baliga et al., 2011). The leaves of this plant are well known for their analgesic and antipyretic activities (Gami et al., 2012; Sehgal et al., 2011). Although several biological activities of M. elengi leaf extracts have been reported, including free radical scavenging (Saha et al., 2008), antibacterial (Niyomkam et al., 2010) and antifungal (Satish et al., 2007) activities, this is the first report of its anti-HIV-1 IN activity. An ethanol extract of M. elengi leaves was therefore subjected to fractionation and isolation of the anti-HIV-1 IN compounds.

Table 1. Anti-HIV-1 IN activity of the selected plants.

Plant extracts/compound	IC50 (µg/mL) ± S.D.
Brassica oleracea	n.a.
Coccinia grandis	n.a.
Diplazium esculentum	n.a.
Illicium verum	n.a.
Millingtonia hortensis	77.7 ± 0.79
Mimusops elengi	62.1 ± 0.30
Morinda coreia	n.a.
Moringa oleifera	n.a.
Suramin (positive control)	3.4 ± 0.1

IC₅₀ values are expressed as mean ± S.D. (n = 4); n.a. = not active at 100 µg/mL.

On the basis of anti-HIV-1 assay-guided purification of the crude ethanol extract of M. elengi leaves, a mixture of gallocatechin and epigallocatechin was isolated as an anti-HIV-1 IN compounds, with an IC₅₀ value of 35.0 µM. An identification of the mixture of gallocatechin and epigallocatechin was performed by the ¹H NMR and ¹³C NMR data compared with data in the literature (Rösch et al., 2004) as well as an HPLC analysis of the mixture compared with the authentic gallocatechin and epigallocatechin. It has been reported that gallocatechin isolated from Nelumbo nucifera Gaertn. (Nelumbonaceae) rhizomes exhibited strong HIV-1 RT and IN inhibitory activities (Jiang et al., 2011). In addition, epigallocatechin gallate, a green tea catechin, has been shown to inhibit a broad spectrum of HIV-1 subtypes without harming human cells. Epigallocatechin gallate has been reported to inhibit HIV-1 replication by targeting several steps in the HIV-1 life cycle, such as interfering with the RT and protease activity, blocking gp120-CD4 interaction by binding to CD4 and inactivating virions (Nance et al., 2009). Four catechins with the galloyl moiety, including catechin gallate, epigallocatechin gallate, gallocatechin gallate and epicatechin gallate, were also found to inhibit HIV-1 IN effectively. These four catechins may reduce the activity of HIV-1 IN by disrupting its interaction with virus DNA (Jiang et al., 2010). In this study, we report that other catechins, gallocatechin and epigallocatechin also possess significant anti-HIV-1 IN activity. These natural antioxidant components with antiviral activities could be potentially important for anti-HIV-1 drug development and application to HIV-1 therapy. In addition, gallocatechin and epigallocatechin may be used as indicative markers for quality control of M. elengi leaf extract.

A flavonol rhamnoside, mearnsitrin, was also purified from the ethanol extract of M. elengi leaves. Mearnsitrin was first purified from the leaves of Acacia mearnsii (MacKenzie, 1967), and recognized as being a rare flavonol glycoside (Nair et al., 1999). This is the first report of mearnsitrin in M. elengi leaves.

Declaration of interest

The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.

Acknowledgements

The authors thank the Prince of Songkla University for support in the form of a research grant, and Dr. Robert Craigie, the National Institute of Health, Bethesda, Maryland, USA, for providing HIV-1 integrase enzyme. Also thanks to Dr. Brian Hodgson for assistance with the English.