Bioassay-Guided Investigation of the Tanzanian Plant Pyrenacantha

Biosciences, Council for Scientific and Industrial Research, Pretoria, South Africa. § Chemistry Department, University of Dar es Salaam, DUCE, P.O. ...
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Bioassay-Guided Investigation of the Tanzanian Plant Pyrenacantha kaurabassana for Potential Anti-HIV-Active Compounds Justin J. Omolo,†,‡,§ Vinesh Maharaj,*,‡ Dashnie Naidoo,‡ Thomas Klimkait,⊥ Hamisi M. Malebo,∥ Samwel Mtullu,∇ Herbert V. M. Lyaruu,# and Charles B. de Koning*,† †

Molecular Sciences Institute, School of Chemistry, University of the Witwatersrand, P.O. Wits 2050, Johannesburg, South Africa Biosciences, Council for Scientific and Industrial Research, Pretoria, South Africa § Chemistry Department, University of Dar es Salaam, DUCE, P.O. Box 2329, Dar es Salaam, Tanzania ⊥ InPheno AG, Vesalgasse 1, CH-4051, Basel, Switzerland, and Department of Biomedicine, University of Basel, Switzerland ∥ Department of Traditional Medicine Research, National Institute for Medical Research, P.O. Box 9653, Dar es Salaam, Tanzania ∇ Tanga Aids Working Group, P.O. Box 1374, Tanga, Tanzania # Department of Botany, College of Natural and Applied Sciences, University of Dar es Salaam, P.O. Box 35060, Dar es Salaam, Tanzania ‡

S Supporting Information *

ABSTRACT: Two new anti-HIV xanthones, 6,7,11-trihydroxy-10methoxy-9-(7-methoxy-3-methyl-1-oxoisochroman-5-yl)-2-methyl12-oxo-12H-benzo[b]xanthene-4-carboxylic acid (1) and 6,7dihydroxy-10,11-dimethoxy-9-(7-methoxy-3-methyl-1-oxoisochroman-5-yl)-2-methyl-12-oxo-12H-benzo[b]xanthene-4-carboxylic acid (2), and a new hexadecahydrochrysen-3-ol (3) were isolated from the tubers of Pyrenacantha kaurabassana. Compounds 1 and 2 showed moderate anti-HIV activity when tested in the deCIPhR assay on HIV virus type NL4-3, with IC50 values of 21 and 2 μg/mL, respectively.

T

he role of plant natural products is well-documented.1 As a result, the use of plants to treat numerous diseases by traditional healers is still common practice, particularly in many African and Asian countries. In Tanzania for example, a survey estimated that 21% of the people in Dar es Salaam will first consult a traditional healer.1 It has been suggested that in the Kagera region of Tanzania, about 80% of HIV-infected patients receive medication from a traditional healer.2−4 In Tanga, a northeastern region on the Tanzanian coast, German doctors working in a district hospital noted that medication prescribed by traditional healers did prolong the life of HIV-infected patients.5 The physicians informed the health authority in the Tanga region about the existence of an herbal remedy that showed promising potency in fighting opportunistic infections or by slowing HIV replication in patients with confirmed HIV/ AIDS infection. On the basis of these findings, a working group comprising traditional and modern health practitioners was formed to provide a new strategy for health care by using the promising herbal drug for HIV/AIDS patients. The team thus organized themselves into a nongovernmental organization with the name “The Tanga AIDS Working Group” (TAWG), which was registered in 1994 in Tanzania for the purpose of providing health care services to people living with HIV and AIDS. Against this background, scientists became interested in determining the underlying chemical structures and pharmacophores of the compounds that are responsible for these © 2012 American Chemical Society and American Society of Pharmacognosy

prominently life-prolonging observations. The traditional medicines could be acting either by combating the opportunistic infections seen in immune-compromised HIV patients or alternatively by slowing the replication of the HIvirus in the patients.5 In this paper we report on research conducted on one of the four plants used by traditional healers. We used bioassay-guided fractionation methodologies in an attempt to isolate the active principles through a validated HIV entry inhibitor assay (InPheno, Basel). In particular, the structural elucidation of two new xanthones from the tubers of Pyrenacantha kaurabassana Baill is reported. Their anti-HIV activity as entry inhibitors is also reported.

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RESULTS AND DISCUSSION

One of the four plants used by traditional healers in the Tanga region of Tanzania for the treatment of HIV is P. kaurabassana Baill (family Icacinaceae). This family consists of trees with long-stemmed woody vines. The family is widespread and can be found in India and the Philippines, while 30 species are found mainly in the tropical regions of Africa.6 Six species are endemic to East Africa, and four other species are found only in Received: April 5, 2012 Published: September 24, 2012 1712

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were observed at δH 3.64 and 3.82 (each 3H, s) as well as three phenolic signals at δH 9.70, 11.94, and 12.32 (each 1H, bs) that were D2O exchangeable. Two coupled methylene proton signals at δH 2.56 [2H (m)] were evident, and, on the basis of the HSQC spectrum, the proton at δH 4.65 [1H (m)] was attached to the oxymethine C-3′ (δC 75.9). This indicated the presence of a δ-lactone. Inspection of the 13C NMR and DEPT spectra showed 31 carbon signals: two methyl (δC 22.2, 20.7), two methoxy (δC 56.6, 55.3), one methylene (δC 32.9), one aliphatic methine (δC 75.9), five aromatic methine (δC 124.8, 121.5, 103.3, 100.5, 98.8), and 20 quaternary carbon signals (δC 191.1, 181.9, 171.8, 164.4, 163.1, 163.0, 162.6, 161.6, 159.4, 148.9, 139.0, 135.3, 132.9, 132.4, 119.4, 117.0, 113.5, 111.1, 108.3, 99.1). In the HMBC spectrum, there were correlations between the phenolic signal at δH 12.32 and the carbon signals at δC 161.6 (C-11) and 119.4 (C-10a), suggesting that this phenolic group is attached to C-11. HMBC correlations between H-8 at δH 6.51 [1H (s)] and the two carbon signals at δC 159.4 (C-7) and 108.3 (C-9) suggested that the phenolic group at δH 9.70 was attached to C-7. The HMBC correlation between the phenolic group (7-OH) and C-7 confirmed phenolic group attachment at C-7. HMBC correlations between H-8 and C-7 and C-10 (δC 163.1) suggested the attachment of a methoxy group at C-10. The correlations between H-6′ and C-8′ (δC 103.3), C-7′ (δC 164.4), C-5′ (δC 117.0), and C-4a′ (δC 132.4) suggested the presence of a methoxy group at C-7′. Correlations between H4′ and C-8a′ (δC 99.1), C-4a′, and C-3′ (δC 75.9) indicated the presence of a methyl group at C-3′ and the lactone carbonyl group at C-1′ (δC 171.8). The two coupled methylene protons (H-4′) showed correlations to C-3′, C-5′, C-4a′, and C-8a′, further confirming the presence of a δ-lactone fused to an aromatic ring. This was further supported by HMBC correlation of H-8′ to C-1′. The HMBC correlation of the Omethyl protons [δH 3.64 3H (s)] with the carbon at δC 164.4 confirmed the presence of the C-7′ methoxy group. The second methoxy group was similarly confined to C-10 based on the HMBC correlation of the O-methyl resonance [δH 3.82, 3H (s)] with C-10 at δC 163.1. The COSY spectrum showed correlations between H-8′, H6′, and H-8, between H-1 and H-3, and between H-4′, H-3′, and CH3-3′. The NOESY spectrum showed association of the aromatic methyl group and the deshielded aromatic singlets at δH 7.05 [1H (s)] and 7.62 [1H (s)], the latter showing HMBC correlations with the hydroxycarbonyl carbon at C-4. The HMBC spectrum also confirmed the location of the carbonyl carbon of the C-4 carboxylic acid (δC 135.3) and the C-2 methyl group (δC 113.5). From the NMR spectroscopic data the molecular formula of the compound was determined to be C31H24O11, corresponding to a theoretical exact mass of 572.5157. The LRMS data showed a molecular ion of 570.5. HRMS data recorded two weeks later showed a molecular ion [M + H]+ peak at m/z 571.1224, indicating that the hydroquinone had oxidized to a quinone. The formation of 4 (Figure 2) may be due to the wellknown oxidation of a hydroquinone to the quinone in the presence of air. The UV spectrum showed absorptions at 223, 283, and 434 nm, suggesting the presence of a xanthoid.13 The IR spectrum showed absorption signals for a hydrogen-bonded phenolic group (νmax 3509−3588 cm−1) and carbonyl groups (νmax 1645−1652 cm−1). The 1H and 13C NMR spectra of compound 2 were similar to those of compound 1. The 13C NMR spectrum of

Southern Africa, namely, P. grandif lora Baill, P. kaurabassana Baill, P. kamassana, and P. scandens Planch.6,7 Apart from the use of this plant by the TAWG project in the treatment of HIV/AIDS, we were unable to find any scientific literature on the medicinal use of P. kaurabassana Baill in Tanzania. However, a literature search from Flora Zambeziaca revealed that the same species may serve as a medicinal plant in Southern Africa: In Malawi, Mozambique, and Zimbabwe, the local people use the heated root stock as a poultice. Among the Zulu community, the plant is used as a prophylactic against miscarriage and premature birth. The root is also used as a remedy for impotency and barrenness.8,9 A literature survey shows that no organic compounds have been isolated from this species. However, the isolation of only a handful of compounds has been reported from other species within the genus. For example, in Nigeria, the leaves of P. staudtii Hutch and Dalz that are used for dysmenorrea and threatened abortion have been subjected to chromatography to yield 3-carbomethoxypyridine. However, it is not clear if this compound is responsible for the reported biological activities.10−12 In an attempt to verify the claims of the use of the plant for treating people suffering from HIV in the Tanga Region of Tanzania, HIV bioassay-guided fractionation was done on tubers of P. kaurabassana, whereby the crude EtOAc fraction showed moderate anti-HIV activity in the HIV entry inhibitor assay by displaying full inhibition at 25 μg/mL (HIV-1 prototype virus NL4-3). Hence this fraction was further investigated extensively for pure novel natural products. The dried EtOAc extract of the tubers of P. kaurabassana was subjected to column chromatography using normal silica gel as the stationary phase and an acetone/CH2Cl2 solvent step gradient. After a series of purifications, two new xanthones, the benzo[b]xanthene-4-carboxylic acid 1 and a second related one bearing one more O-methyl substituent, 2 (Figure 1), both with

Figure 1. Structures of compounds 1−3.

a conformationally labile biaryl bond linked to an isochromanone nucleus, were isolated. In addition, the isolation of a new hydrochrysen-2-ol (3) was also accomplished. The 1H NMR spectrum of compound 1 showed five aromatic proton signals at δH 6.08 [1H (s)], 6.51 [1H (s)], 7.05 [1H (s)], 7.50 [1H (s)], and 7.62 [1H (s)]. Surprisingly, the meta coupling was not even observed in the 1H NMR at 600 MHz. In addition, two methyl signals at δH 2.50 [3H (s)] and 1.39 [3H (d, J = 6.0 Hz)] were evident, with one being an aromatic methyl substituent. Two aromatic methoxy signals 1713

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The molecular formula of 2, C32H26O11, corresponded to the exact mass of 586.1475. This was confirmed by a molecular ion at m/z 586.1461 as observed in the HRMS. The UV spectrum showed absorptions at 225, 262, 285, and 376 nm, again indicating the presence of a xanthoid. The IR spectrum showed absorption peaks for hydrogen-bonded hydroxy (νmax 3498 cm−1) and carbonyl groups (νmax 1790 cm−1). Therefore, 2 was identified as the 11-O-methyl derivative of compound 1. We could not determine the absolute configuration of C-3′ in compounds 1 and 2.

Figure 2. Quinone 4.

compound 2 showed the presence of an additional carbon resonance compared to 1. The 1H NMR spectrum displayed an additional O-methyl signal at δH 3.95 [3H (s)], which correlated to the carbon signals at δC 61.3 and 145.4 (C-11) in the HSQC and HMBC spectra, respectively. This indicated that the C-11 hydroxy group in compound 1 was methylated in compound 2.

Table 2. NMR Spectroscopic Data for Compound 2 (600 MHz, CDCl3) position

Table 1. NMR Spectroscopic Data for Compound 1 (600 MHz, CDCl3) position 1 2 2-Me 3 4 4a 4CO2H 5a 6 6a 7 8 9 10 10a 11 11a 12 12a 1′ 3′

δC, type 124.8, 113.5, 22.2, 121.5, 135.2, 148.9, 181.9,

CH C CH3 CH C C C

163.0, 162.6, 111.1, 159.4, 100.5, 108.3, 163.1, 119.4, 161.6, 132.9, 191.1, 139.0, 171.8, 75.9,

C C C C CH C C C C C C C C CH

3′-Me

20.7, CH3

4′

32.9, CH2

δH (J in Hz)

HMBC

COSY

7.05, s

C-2, C-2Me, C-3

H-3

2.50, s 7.62, s

C-1, C-3 C-1, C-2, C-4CO2H

H-1

6.51, s

C-7, C-9

H-6′

1.39, d (6.0) 2.56, m

C-3′

H-3′Me, H4′ H-3′, H-4′,

C-3′, C-4a′, C-5′, C8′a

H-3′Me, H4′

4′a 5′ 6′

132.4, C 117.0, C 98.8, CH

6.08, s

C-4a, C-5′, C-7′, C-8′, C-9

H-8

7′ 8′a 8′

164.4, C 99.1, C 103.3, CH

7.50, s

H-6′

3.64, s 3.82, s

C-1′, C-4′a, C-7′, C8a′ C-7′ C-10

12.32, s 11.94, s 9.70, s

C-10a, C-11 C-5a, C-6, C-6a C-7, C-8

7′-OMe 10OMe 11-OH 6-OH 7-OH

55.3, CH3 56.6, CH3

1

125.1, CH

2 2-Me 3

116.3, C 19.7, CH3 126.1, CH

4 4a 4CO2H 5a 6 6a 7 8 9 10 10a 11 11a 12 12a 1′ 3′

133.7, C 153.2, C 180.5, C

3′-Me 4′

4.65, m

δC, type

4′a 5′ 6′ 7′ 8′ 8′a 7′-OMe 10OMe 11OMe 6-OH 7-OH

158.4, 160.8, 111.1, 162.1, 99.4, 107.3, 162.0, 122.1, 145.4, 131.4, 186.3, 138.1, 170.8, 75.0,

C C C C CH C C C C C C C C CH

15.3, CH3 31.9, CH2 130.8, 118.0 97.9, 162.8, 101.2, 98.1, 55.5, 54.3,

δH (J in Hz)

HMBC

COSY

7.19 (under CHCl3) 2.42, s 7.94, s

C-1, C-4 C-2, C-3, C-4a, C8CO2H

6.51, s

C-7, C-9

H-6′

1.38, d (6.0) 2.58, m

C-3′ C-3′, C-4a′, C-5′, C-8a′

H-3′Me, H-4′, H-3′, H-4′ H-3′Me, H-4′

6.10, s

C-5′, C-7′, C-9

H-8 H-6′

4.66, m

C CH C CH C CH3 CH3

7.45, s

C-1′

3.80, s 3.63, s

C-7′ C-10

61.3, CH3

3.95, s

C-11

9.71, s 13.09, s

C-5a, C-6, C-8 C-6a, C-7

The 1H NMR spectrum of compound 3 (Figure 1) showed the presence of two methine proton signals in the downfield region at δH 5.36 [1H (m)] and 3.51 [1H (m)]. Other signals were observed in the aliphatic upfield region, which include methylene {δH 2.28 [2H (m)]}, methine {δH 1.95 [1H (m)] and 1.61 [1H (s)]}, and nine methyl singlet signals (δH 1.61, 1.28, 1.23, 1.22, 1.14, 1.08, 0.97, 0.84, and 0.80). The 1H NMR, 13 C NMR, and DEPT spectra showed the presence of nine 1714

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methyls, one oxymethine, nine methylene, five methine, and six quaternary carbon signals. The HSQC and HMBC spectroscopic data showed two downfield methine proton signals at δH 5.36 and 3.51, which were assigned to C-12 (δC 121.7), and C-3 (δC 71.8), respectively. Methyl proton signals at δH 0.80, 0.84, 0.97, 1.08, 1.23, 1.22, 1.61, 1.14, and 1.28 were assigned to methyl signals attached to C-24 (δ 19.3), C-23 (δ 18.7), C-26 (δ 11.9), C-28 (δ 29.1), C-25 (δ 19.0), C-22 (δ 19.3), C-27 (δ 11.8), and C-29 and C-30 (δ 26.1 and 28.2), respectively. The structure of compound 3 was also compared with the reported spectroscopic data of the triterpenoid 5 and other related compounds belonging to the lupine derivatives (Figure 3).14 Three exceptions were noted; for compound 5 a side-chain hydroxymethylene, a carboxylic acid group, and one more double bond were present.

Therefore, compound 3 was identified as a new hexadecahydrochrysen-3-ol. Anti-HIV Assay. Xanthones 1 and 2 showed moderate antiHIV activity when tested in the deCIPhR assay15 (Table 4). Table 4. HIV Screening Results of Xanthones 1 and 2 in the deCIPhR Assaya sample xanthone 1 xanthone 2 enfuvirtide (positive control)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

37.2, 31.6, 71.8, 37.2, 56.7, 31.9, 33.9, 39.7, 50.1, 37.1, 29.1, 121.7, 140.7, 42.3, 33.9, 36.1, 56.0, 45.8, 42.3, 31.9, 31.9, 19.3, 19.3, 18.7, 19.0, 11.9, 11.8, 21.0, 28.2, 26.1,

CH2 CH2 CH C CH CH2 CH2 C CH C CH2 CH C C CH2 CH2 C CH CH C CH2 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3

δH

C-3

3.51, 1H (m)

C-4

5.36, 1H (m)

C-13

1.95, 1H (m)

C-13, C-28

1.22, 0.84, 0.80, 1.23, 0.97, 1.61, 1.08, 1.28, 1.14,

3H 3H 3H 3H 3H 3H 3H 3H 3H

(brs) (s) (s) (s) (s) (s) (brs) (s) (s)

>12.5 31 not done

15.5

TC90 (μg/ mL)

111 22 0.026

>12.5 154 not done

SI 7

The presence of an additional O-methyl group in compound 2 replacing the OH group in compound 1 improved both the activity and selectivity indices, as depicted in Table 4. However, these compounds showed relatively low selectivity indices (SI), as an SI value of >100 is essential for further development. In summary, three new compounds were isolated from P. kaurabassana. Assessment of xanthones 1 and 2 indicated that alone they are not effective as HIV-entry inhibitors.

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HMBC

2.28, 2H (m)

21 2 0.01

SI

IC90 (μg/ mL)

IC50 = 50% inhibitory concentration in anti-HIV assay; TC50 = 50% inhibitory concentration in cytotoxicity assay.

Table 3. NMR Spectroscopic Data for Compound 3 (600 MHz, CDCl3) δC, type

TC50 (μg/ mL)

a

Figure 3. Structure of 5.

position

IC50 (μg/ mL)

EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were determined on a Kofler micro-hot stage melting point apparatus and are uncorrected. Optical rotations [α]D were measured using a Jasco DIP-370 polarimeter at 16 °C in CH2Cl2. A quartz microcell with a tube length of 100 mm and a volume of 1.0 mL was used. The UV−vis spectra were measured on a Shimadzu UV-2101PC UV−vis scanning spectrophotometer. IR absorptions were measured on a Perkin-Elmer System 2000 FT-IR spectrophotometer using KBr pellets. The samples were dissolved in CH2Cl2 and analyzed on a NaCl window. The 1D (1H, 13C, and DEPT) and 2D (COSY, HMQC, HMBC) NMR spectra were acquired on a 600 MHz Varian VnmrJ 600 spectrometer and referenced to residual solvent signals. Mass spectra were obtained on MS/HPLC (Hewlett-Packard, 5973 mass selective detector and HP 6890 Series GC) and GS-MS (SQ Detector) mass spectrometer instruments. HRMS spectra were obtained on a Waters API Q-TOF Ultima. TLC experiments were run on a 0.25 mm thick layer of Merck silica gel 60 F254 coated aluminum foil and glass plates. TLC spots were detected under UV light (254 and 366 nm) and spraying with vanillinsulfuric acid. Normal column chromatography was conducted using different sizes of columns packed with Merck silica gel 60 (size 0.040−0.063 mm). PTLC was run on 0.5 mm thick layer Merck silica gel 60 HF254 20 × 20 cm glass plates. Plant Material. The tubers of P. kaurabassana Baill were collected by TAWG in collaboration with traditional healers from the Muheza district in Tanga (Tanzania) on March 22, 2009. A voucher specimen (voucher specimen number 4225 HOS) verified by Prof. H. V. M. Lyaruu, Department of Botany, University of Dar es Salaam (Tanzania), was deposited in the University Herbarium, Dar es Salaam, Tanzania. Extraction and Isolation. The air-dried powder of the tubers (1.980 kg) of P. kaurabassana was extracted sequentially with CH2Cl2, EtOAc, MeOH, and H2O. The EtOAc extract (600 mg) was subjected to CC using silica gel as the stationary phase and a solvent step gradient of CH2Cl2/acetone as the mobile phase (from 5% to 100% acetone: CH2Cl2). The 30% acetone/CH2Cl2 fraction was purified using 25% acetone in CH2Cl2 throughout to afford two yellow, crystalline solids, 1 (8 mg) and 2 (10 mg), as well as a white, crystalline compound, 3 (8 mg).

C-17 C-20

The NMR spectroscopic and HRMS data (m/z 428.4018) led to the assignment of a molecular formula of C30H52O2. 1715

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6,7,11-Trihydroxy-10-methoxy-9-(7-methoxy-3-methyl-1-oxoisochroman-5-yl)-2-methyl-12-oxo-12H-benzo[b]xanthene-4-carboxylic acid, 1: yellow, crystalline material (CHCl3); mp 290−293 °C; [α]16 −122 (c 0.5, CHCl3); UV (CHCl3) λmax (log ε) 223 (4.18), 238 (3.92), 434 (3.24) nm; IR (Nujol) νmax 3509−3588, 1645 cm−1; 1H (600 MHz, CDCl3) and 13C (150 MHz, CDCl3) data, Table 1; HRMS m/z 571.1224 (calcd for C31H24O11 [M+ + H] 571.1240). 6,7-Dihydroxy-10,11-dimethoxy-9-(7-methoxy-3-methyl-1-oxoisochroman-5-yl)-2-methyl-12-oxo-12H-benzo[b]xanthene-4-carboxylic acid, 2: yellow, crystalline material (CHCl3); mp 226−229 °C; [α]16 −49 (c 0.9, CHCl3); UV (CHCl3) λmax (log ε) 225 (3.98), 262 (3.52), 285 (3.52), 376 (3.29) nm; IR (Nujol) νmax 3498, 1790 cm−1; 1 H (600 MHz, CDCl3) and 13C (150 MHz, CDCl3) data, Table 2; HRMS m/z 586.1461 (calcd for C32H26O11 [M+] 586.1475). (3S,10S,17S,14R,8S)-18-(2,2-Dimethylbutyl)-8,10,14,17,23,24-hexamethyl-1,2,3,4,5,6,7,8,9,10,11,14,15,16,17,18-hexadecahydrochrysen-3-ol, 3: white, crystalline powder (CHCl3); mp 220−221 °C; [α]16 +2 (c 0.4, CHCl3); IR (Nujol) νmax 3408, 1638 cm−1; 1H (600 MHz, CDCl3) and 13C (150 MHz, CDCl3) data, Table 3; HRMS m/z 428.4019 (calcd for C30H52O2 [M+] 428.4018).

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(5) Scheinman, D. Traditional Medicine in Tanga Today. Indigenous Knowledge: Local Pathways to Global Development; The World Bank, 2004. (6) Jesper, K. Am. J. Bot. 2001, 88, 2259−2274. (7) Potgieter, M. J.; Van Wyk, A. E. Bot. Bull. Acad. Sin. 1994, 35, 105−113. (8) Cunningham, A. B. The Resource Value of Indigenous Plants to Rural People in a Low Agricultural Area, Vol. 1. Ph.D. Thesis, University of Cape Town, 1985. (9) Mendes, E. J. Icacinaceae. Royal Botanic Gardens, Kew; Flora Zambeziaca, Vol. 2; 1963; Part 1. (10) Falodun, A.; Usifoh, C. O.; Nworgu, Z. A. M. Pak. J. Pharm. Sci. 2005, 18, 31−33. (11) Falodun, A.; Usifoh, C. O. Acta Pol. Pharm.-Drug Res. 2006, 63, 235−237. (12) Falodun, A.; Usifoh, C. O.; Nworgu, Z. A. M. Afr. J. Biotechnol. 2006, 5, 1271−1273. (13) (a) Ito, C.; Itoigawa, M.; Takakura, T.; Ruangrungsi, N.; Enjo, F.; Tokuda, H.; Nishino, H.; Furukawa, H. J. Nat. Prod. 2003, 66, 200−205. (b) Seo, E. -K.; Kim, N. -C.; Wani, M. C.; Wall, M. E.; Navarro, H. A.; Burgess, J. P.; Kawanishi, K.; Kardono, L. B. S.; Riswan, S.; Rose, W. C.; Fairchild, C. R.; Farnsworth, N. R.; Kinghorn, A. D. J. Nat. Prod. 2002, 65, 299−305. (14) (a) Lavaud, C.; Massiot, G.; Le Men-Olivier, L.; Viari, A.; Vigny, P.; Delaude, C. Phytochemistry 1992, 31, 3177−3181. (b) Hill, R. A.; Connolly, J. D. Nat. Prod. Rep. 2012, 29, 780−818. (15) The deCIPhR assay is a proprietary assay system of InPheno AG, Switzerland. Two HIV lab strains differing by their tropism of coreceptor utilization for viral entry, pNL4-3 for CXCR4-usage and pNL-AD87 for CCR5-usage, were used during the assessment. The system uses a fully replicative virus and hence is able to assess the activity of any antiretroviral substance acting at a specific step/target of HIV life cycle and on a wide variety of cellular factors. For references see: Vidal, V.; Potterat, O.; Louvel, S.; Hamy, F.; Mojarrab, M.; Sanglier, J. J.; Klimkait, T.; Hamburger, M. J. Nat. Prod. 2012, 75, 414−419. Klimkait, T.; Stauffer, F.; Lupo, E.; Sonderegger-Rubli, C. Arch. Virol. 1998, 11, 2109−2131.

ASSOCIATED CONTENT

S Supporting Information *

1

H and 13C NMR spectra of compounds 1−3 are free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: 27 11 7176749. Tel: +27 11 7176724. E-mail: [email protected]. Fax: 27 12 8414790. Tel: +27 12 8413295. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by SABINA (Southern African Biochemistry and Informatics for Natural Products Network), CSIR (Council for Scientific and Industrial Research), Biosciences in Pretoria, the National Research Foundation (NRF, GUN 2053652) and IRDP of the NRF (South Africa) for financial support provided by the Research Niche Areas Programme, Pretoria, and the University of the Witwatersrand (Science Faculty Research Council). The TAWG is acknowledged for providing plant materials. The National Institute for Medical Research (NIMR) and the Department of Botany, College of Natural and Applied Sciences at the University of Dar es Salaam (UDSM), are acknowledged for their scientific and technical support. Prof. W. A. L. van Otterlo and Dr. M. Stander (University of Stellenbosch) are thanked for providing the HRMS data. The associate editor Prof. D. Ferreira is thanked for providing valuable input in the preparation of the manuscript.

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REFERENCES

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