Abstract
Ethnopharmacological relevance
A substantial proportion of the population in Papua New Guinea (PNG) lives with human immunodeficiency virus (HIV). Treatment requires lifelong use of antiretroviral therapy (ART). The majority of people in PNG use traditional medicines (TM) derived from plants for all types of health promotions. Consequently, there is a concern that herb-drug interactions may impact the efficacy of ART. Herb-drug, or drug-drug, interactions occur at the level of metabolism through two major mechanisms: enzyme induction or enzyme inhibition. In this study, extracts of commonly-used medicinal plants from PNG were screened for herb-drug interactions related to cytochrome P450s (CYPs).
Materials and Methods
Sixty nine methanol extracts of TM plants were screened for their ability to induce CYPs by human aryl hydrocarbon receptor- (hAhR-) and human pregnane X receptor- (hPXR-) dependent mechanisms, utilizing a commercially available cell-based luciferase reporter system. Inhibition of three major CYPs, CYP1A2, CYP3A4, and CYP2D6, was determined using human liver microsomes and enzyme-selective model substrates.
Results
Almost one third of the TM plant extracts induced the hAhR-dependent expression of CYP1A2, the hPXR-dependent expression of CYP3A4, or both. Almost two thirds inhibited CYP1A2, CYP3A4, or CYP2D6, or combinations thereof. Many plant extracts exhibited both induction and inhibition properties.
Conclusions
We demonstrated that the potent and selective ability of extracts from PNG medicinal plants to affect drug metabolizing enzymes through induction and/or inhibition is a common phenomenon. Use of traditional medicines concomitantly with ART could dramatically alter the concentrations of antiretroviral drugs in the body; and their efficacy. PNG healthcare providers should counsel HIV patients because of this consequence.
Keywords: Traditional medicine Asia & Oceania, herb-drug interactions, cytochrome P450s, Antiretroviral Therapy, Papua New Guinea
1. Introduction
Papua New Guinea (PNG) is in the midst of an HIV epidemic. According to the UNAIDS 2013 Report on the Global AIDS Epidemic, an estimated 25,000 people in PNG are living with HIV (approximately 1% of the population) (UNAIDS, 2013). Standard treatment of HIV patients requires a lifelong regimen of antiretroviral therapy (ART). PNG is culturally diverse (Whitehead, 1994) and traditional medicines play a vital role in the well-being of the people (National Department of Health, 2007). Many people, especially in regions where access to Western medicine is limited, rely on medicinal plants to relieve an array of ailments (Rai, 2007). Medicinal herb-drug interactions have been studied in areas that rely heavily of traditional medicines around the world (Muller and Kanfer, 2011; Lau et al., 2013). However, little is known about herb-drug interactions that may arise from PNG medicinal plants in combination with ART.
Drug-drug interactions involving cytochrome P450s (CYPs) occur through two major mechanisms: enhanced enzyme expression, termed induction, and enzyme inhibition. With the exception CYP2D6, the CYPs responsible for drug metabolism in the liver are inducible. CYP induction and the resultant increased drug metabolism leads to a decrease in bioavailable drug and a decrease in drug efficacy. Inhibition of CYPs, on the other hand, decreases drug metabolism and results in an increase in bioavailability. Elevated levels of drugs may enhance their therapeutic benefits, but can result in toxicity.
Particularly worrisome is the possibility that traditional medicine usage may unknowingly render normal ART ineffective. We surveyed 69 methanol extracts of commonly-used medicinal plants from 7 PNG provinces for CYP induction and inhibition. Induction profiles were determined for CYP1A2 and CYP3A4, enzymes whose expression levels are elevated by two distinct pathways. The inhibition profiles were determined for CYP1A2, CYP3A4, and CYP2D6, which are estimated to be responsible for biotransformation of approximately two thirds of prescription drugs (Wrighton and Stevens, 1992). Almost one third of the medicinal plant extracts tested significantly induced CYP expression, while close to two thirds exhibited potent CYP inhibition.
2. Materials and Methods
2.1. Medicinal plant collection
As previously reported (Waruruai et al., 2011; Jorim et al., 2012), University of Papua New Guinea (UPNG) fourth year pharmacy students collected medicinal plants from their home communities. These were in the Eastern Highlands, Western Highlands, Southern Highlands, Enga, Western, Manus and Northern Bougainville (autonomous) provinces. These surveys served to fulfill requirements for a Bachelor of Pharmacy degree from UPNG. In accordance with the requirements, plant information, including local names and medicinal uses, was documented. Voucher samples were prepared for plant identification and the information was compiled into a formal report. Plants were identified by UPNG or National herbaria staff where vouchers are stored. The reported data are stored in the UPNG Traditional Medicines Database. A compilation of this information is presented in Appendix 1.
2.2. Medicinal plant extraction
Dried, plant samples (~10 g) were extracted in 100 mL of 100% methanol (MeOH) overnight. Extracts were then evaporated to dryness and dissolved in dimethyl sulfoxide (DMSO) to yield a final concentration of approximately 10 mg/mL. Fractionation of Evodia hortensis was accomplished by mixing MeOH extract with three times its dry weight of Diaion ® HP20SS resin (Sorbent Technologies). The extract and resin mixture was evaporated to dryness, loaded into a column, and extracted using stepwise increases of isopropanol (IPA). The fractions were eluted as follows: 10% IPA/H2O (FW), 25% IPA/H2O (F1), 50% IPA/H2O (F2), 75% IPA/H2O (F3), and 100% MeOH (F4) (Bugni et al., 2008). These were evaporated to dryness and dissolved in DMSO to a final concentration of approximately 10 mg/mL.
2.3. CYP Induction Assays
Plant extracts were screened for the ability to induce CYP1A2 and CYP3A4. 1A2DRE (human XRECYP1A2-luciferase promoter) and DPX2 (human PXRCYP3A4-luciferase promoter) hepatoma cell lines (Puracyp, Inc.) were grown in T75 flasks to near-confluency (~75%). The cells were trypsin-released and aliquoted into 96-well plates. Cells were allowed to adhere for 24 h. Plant extracts were added to the wells and incubated for another 24 h. Cell viability and luciferase activity were then determined using commercially-available kits (Promega). Viability was expressed relative to control wells exposed to DMSO (vehicle). Luciferase activity was normalized to cell number as determined from the viability assay; average result for quadruplicate determinations presented. Induction was compared to that seen for prototype inducers, 1 μM β-naphthoflavone for 1A2DRE and 10 μM rifampicin for DPX2. β-naphthoflavone produced ~10-fold induction and rifampicin produced ~8-fold induction. Criteria for “positive” plant samples, based on current Food and Drug Administration (FDA) guidelines and previously published methods, was set at ~40% of the response seen for the prototype inducers, i.e., > 4.0-fold increase in luciferase activity for CYP1A2 and > 3.5-fold increase for CYP3A4 (FDA, 2012; Kuzbari et al., 2013).
2.4. CYP Inhibition Assays
Plant extracts were screened for inhibitory activity towards CYP1A2, CYP3A4, and CYP2D6 using enzyme-selective model substrates. Methoxyresorufin (MR) was used to assess CYP1A2 activity, 7-benzyloxyquinoline (7-BQ) was used to assess CYP3A4 activity and 7-methoxy-4-(aminomethyl)-coumarin (MAMC) was used to assess CYP2D6 activity. Plant extracts and human liver microsomes (Celsis) were added to 96-well plates (in duplicate, the average is reported). Substrate was added to the wells at half-saturating (Km) or saturating (Vmax) concentrations. NADPH (60 mM) was added to catalyze the enzymatic reactions and the formation of fluorescent product was measured using a Biotek – Synergy 2 Microplate Reader (Lamb et al., 2010). Extracts showing >50% inhibition at half-saturating and/or saturating substrate concentrations were deemed inhibitory. Whether the inhibition was suggestive of noncompetitive or competitive mechanism was noted. Noncompetitive inhibitors displayed similar inhibition at saturating and half-saturating substrate concentrations. Competitive inhibitors exhibited significantly greater inhibition at half-saturating substrate concentrations than at saturating substrate concentrations.
2.5. Characterization of E. hortensis extract sub-fractions
The methanol extract of E. hortensis inhibited all three CYPs tested. Therefore, the extract was fractionated as described above and fractions were re-tested for CYP inhibition. Fraction 4 inhibited all three CYPs. It was separated further using an Agilent Technologies 1200 series HPLC system. Prior to HPLC injection, Fraction 4 was evaporated to dryness and dissolved in DMSO. The sample was then injected onto a Phenomenex column (Luna 5 μm C18 (2) 100Å; 250 × 10.00 mm). Sub-fractions were collected every 30 seconds at a flow rate of 2.5 mL/min and solvent gradient of 40% to 100% ACN/H2O over 50 minutes. They were evaporated to dryness, dissolved in DMSO, and assayed for CYP inhibition.
3. Results
3.1. Inducers of CYP1A2 & CYP3A4
In our study, increases in luciferase activity reflected induction at the CYP1A2 and CYP3A4 promoters. This activity was compared to prototype inducers, β-naphthoflavone (CYP1A2) and rifampicin (CYP3A4). When luciferase activity was greater than 40% that of the prototype inducers, induction by the plant extract was considered significant (FDA, 2012). The results are summarized in Table 1. Paspalum conjugatum and Sida rhombifolia extracts induced exceptionally high levels of CYP1A2 expression (> 10-fold increase) but did not induce CYP3A4. Fourteen extracts induced CYP3A4 but not CYP1A2. These were: Alstonia scholaris, Bidens pilosa (em028 & cw004), Coleus blumei, Cordyline terminalis, Curcuma longa, Manihot esculenta, Pometia pinnata, Psidium guajava (sk002, nn011, in045, em009, &cw019), and Wedelia biflora. Five extracts induced both CYP1A2 and CYP3A4: Calophyllum inophyllum, Codiaeum variegatum, Erythrina variegata, E. hortensis, and Sphaerostephanos unitus. C inophyllum was a potent inducer of CYP1A2 and CYP3A4 (31.7-fold and 15.6 fold increase, respectively).
Table 1.
CYP1A2 and CYP3A4 inducers and their inhibitory activity
| Genus and Species | Voucher Number | Plant Part | PNG Province | CYP1A2 Induction (-fold) | CYP3A4 Induction (-fold) | Inhibits CYP1A2 | Inhibits CYP3A4 | Inhibits CYP2D6 |
|---|---|---|---|---|---|---|---|---|
| P. conjugatum | cw070 | spikelets | Western Highlands | 11.4 | -- | ✓ | ✓ | ✓ |
| S. rhombifolia | nn010 | leaves | Manus | 15.5 | -- | ✓ | ||
| A. scholaris | in012 | leaves | North Bougainville | -- | 4.7 | ✓ | ||
| B. pilosa | em028 | leaves | Southern Highlands | -- | 5.7 | |||
| B. pilosa | cw004 | leaves | Western Highlands | -- | 5.5 | ✓ | ||
| C. blumei | sk043 | whole plant | Eastern Highlands | -- | 4.0 | ✓ | ||
| C. terminalis | cw030 | leaves from shoots | Western Highlands | -- | 3.8 | ✓ | ✓ | |
| C. longa | jp1103 | leaves | Enga | -- | 5.8 | |||
| M. esculenta | cw023 | leaves | Western Highlands | -- | 3.7 | ✓ | ||
| P. pinnata | ab057 | bark | Western | -- | 4.2 | ✓ | ✓ | ✓ |
| P. guajava | sk002 | leaves | Eastern Highlands | -- | 5.2 | ✓ | ✓ | ✓ |
| P. guajava | nn011 | leaves | Manus | -- | 4.6 | ✓ | ✓ | |
| P. guajava | in045 | leaves | North Bougainville | -- | 8.7 | ✓ | ✓ | |
| P. guajava | em009 | leaves | Southern Highlands | -- | 5.8 | ✓ | ✓ | ✓ |
| P. guajava | cw019 | leaves | Western Highlands | -- | 3.5 | ✓ | ✓ | |
| W. biflora | in021 | leaves | North Bougainville | -- | 3.8 | |||
| C. inophyllum | nn021 | leaves | Manus | 31.7 | 15.6 | ✓ | ✓ | ✓ |
| C. variegatum | nn016 | leaves | Manus | 5.8 | 14.2 | ✓ | ✓ | |
| E. variegata | nn056 | bark | Manus | 4.5 | 3.6 | ✓ | ✓ | ✓ |
| E. hortensis | cw049 | leaves & fruits | Western Highlands | 13.7 | 7.1 | ✓ | ✓ | ✓ |
| S. unitus | in061 | shoots | North Bougainville | 14.9 | 5.0 | ✓ | ✓ |
Medicinal plant induction of CYP1A2 and/or CYP3A4. (--) activity was < 4.0-fold (CYP1A2) & < 3.5-fold (CYP3A4); (✓) indicates inducers that also inhibited CYP metabolism.
3.2. Inhibitors of CYP1A2, CYP3A4 & CYP2D6
Commercial preparations of human liver microsomes, enriched for the CYP of interest, were used to determine inhibitory activity. The ability of medicinal plant extracts to inhibit metabolism of CYP-specific substrates was determined at half-saturating (Km) and saturating (Vmax) substrate concentrations. Results are summarized in Table 2. Inhibitory activity that was similar at both substrate concentrations was interpreted as noncompetitive (Segel, 1968). Inhibitory activity that was greater at lower substrate concentrations was suggestive of competitive inhibition (Segel, 1968). Some plant extracts were selectively inhibitory towards one type of CYP: Premna obtusifolia and S. rhombifolia inhibited only CYP1A2. Inhibition by both extracts was suggestive of a competitive mechanism. Plant extracts that inhibited only CYP3A4, 10 total, all exhibited activity suggestive of a competitive mechanism. These were: Angiopetris evecta, Carica papaya, Centella asiatica, Crinum asiaticum, C. terminalis (jp1144), Kalanchoe pinnata, Macaranga aleuritoides, M. esculenta, Passiflora foetida, and Premna serratofolia. With respect to CYP2D6, A. scholaris, along with Alstonia spectabilis, Citrus limon, and C. blumei, showed selective inhibition suggestive of a noncompetitive mechanism. B. pilosa and Solanum torvum, showed selective inhibition suggestive of a competitive mechanism.
Table 2.
CYP1A2, CYP3A4, & CYP2D6 Inhibitors
| Genus and Species | Voucher Number | Plant Part | PNG Province | CYP1A2 % Inhibition | CYP3A4 % Inhibition | CYP2D6 % Inhibition | |||
|---|---|---|---|---|---|---|---|---|---|
| MR (2.0 μM) | MR (0.4 μM) | 7-BQ (500 μM) | 7-BQ (100 μM) | MAMC (100 μM) | MAMC (40 μM) | ||||
| P. obtusifolia | nn031 | leaves | Manus | 53 | 68 | -- | -- | -- | -- |
| S. rhombifolia | nn010 | leaves | Manus | 54 | 75 | -- | -- | -- | -- |
| A. evecta | cw055 | leaves | Western Highlands | -- | -- | -- | 50 | -- | -- |
| C. papaya | em016 | leaves | Southern Highlands | -- | -- | 54 | 78 | -- | -- |
| C. asiatica | jp1165 | whole plant | Enga | -- | -- | -- | 56 | -- | -- |
| C. asiaticum | in059 | leaves | North Bougainville | -- | -- | -- | 65 | -- | -- |
| C. terminalis | jp1144 | leaves | Enga | -- | -- | -- | 76 | -- | -- |
| K. pinnata | nn019 | leaves | Manus | -- | -- | -- | 55 | -- | -- |
| M. aleuritoides | in002 | leaves & shoots | North Bougainville | -- | -- | -- | 50 | -- | -- |
| M. esculenta | cw023 | leaves | Western Highlands | -- | -- | -- | 65 | -- | -- |
| P. foetida | nn034 | twigs | Manus | -- | -- | -- | 51 | -- | -- |
| P. serratifolia | in005 | leaves | North Bougainville | -- | -- | -- | 63 | -- | -- |
| A. scholaris | in012 | leaves | North Bougainville | -- | -- | -- | -- | -- | 53 |
| A. spectabilis | ab005 | bark | Western | -- | -- | -- | -- | 72 | 81 |
| B. pilosa | cw004 | leaves | Western Highlands | -- | -- | -- | -- | -- | 51 |
| C. limon | em010 | fruits | Southern Highlands | -- | -- | -- | -- | 53 | 61 |
| C. blumei | sk043 | whole plant | Eastern Highlands | -- | -- | -- | -- | -- | 57 |
| S. torvum | in017 | leaves | North Bougainville | -- | -- | -- | -- | -- | 52 |
| Acalypha sp. | cw043 | leaves from shoots | Western Highlands | 85 | 71 | -- | 71 | -- | -- |
| I. pes-caprae | nn036 | leaves | Manus | 56 | 69 | 58 | 60 | -- | -- |
| P. guajava | nn011 | leaves | Manus | -- | 55 | -- | 62 | -- | -- |
| P. guajava | cw019 | leaves | Western Highlands | 58 | 66 | -- | 66 | -- | -- |
| S. unitus | in061 | shoots | North Bougainville | 60 | 65 | 53 | -- | -- | -- |
| S. malaccense | nn040 | bark | Manus | 56 | -- | -- | 60 | -- | -- |
| C. alata | nn050 | leaves | Manus | -- | 54 | -- | -- | 56 | 61 |
| C. variegatum | nn016 | leaves | Manus | -- | -- | 67 | 97 | 50 | 60 |
| C. terminalis | cw030 | leaves from shoots | Western Highlands | -- | -- | -- | 88 | 50 | 61 |
| P. guajava | in045 | leaves | North Bougainville | -- | -- | -- | 50 | 51 | 51 |
| C. inophyllum | nn021 | leaves | Manus | 74 | 86 | -- | 79 | 52 | 60 |
| C. alata | in014 | leaves | North Bougainville | 58 | 75 | -- | 57 | 54 | -- |
| C. equisetifolia | nn026 | bark | Manus | 66 | 65 | 54 | 61 | -- | 54 |
| E. variegata | nn056 | bark | Manus | 74 | 74 | 57 | 85 | 66 | 64 |
| E. hortensis | cw049 | leaves & fruits | Western Highlands | 52 | 69 | 93 | 100 | -- | 51 |
| P. conjugatum | cw070 | spikelets | Western Highlands | 77 | 87 | 60 | 88 | 57 | 53 |
| P. pinnata | ab057 | bark | Western | 82 | 81 | 72 | 99 | 67 | 64 |
| P. guajava | sk002 | leaves | Eastern Highlands | 52 | 57 | 53 | 85 | 59 | -- |
| P. guajava | em009 | leaves | Southern Highlands | 57 | 72 | 55 | 79 | 52 | 58 |
| T. catappa | nn022 | bark | Manus | 96 | 92 | 64 | 80 | 67 | 81 |
Medicinal plant extract inhibition of CYP metabolism using CYP-specific substrate: methoxyresorufin (MR), 7-benzoxyquinoloine (7-BQ), 7-methoxy-4-(aminomethyl)-coumarin (MAMC). (--) activity was < 50% inhibition.
Some plant extracts were inhibitory towards two of the three CYPs tested. Six inhibited CYP1A2 and CYP3A4: Acalypha sp., Ipomea pes-caprae, P. guajava (nn011 & cw019), S. unitus, and Syzygium malaccense. Acalypha sp., P. guajava (nn011 & cw019), S. unitus, and S. malaccense showed inhibition suggestive of a noncompetitive mechanism for CYP1A2, whereas I. pes-caprae inhibited CYP1A2 in a manner indicative of a competitive mechanism. I. pes-caprae and S. unitus inhibited CYP3A4, suggesting a noncompetitive mechanism. Conversely, Acalypha sp., P. guajava (nn011 & cw019) and S. malaccense appeared to inhibit CYP3A4 by a competitive mechanism. Cassia alata (nn050) inhibited CYP1A2 in a manner suggestive of a competitive mechanism, while the same extract inhibited CYP2D6 under a noncompetitive mechanism. C. terminalis (cw030) and C. variegatum inhibited CYP3A4 and CYP2D6 by competitive mechanisms. P. guajava (in045) inhibited CYP3A4 competitively, however, the extract inhibited CYP2D6 in a manner suggestive of a noncompetitive mechanism.
Inhibition of CYP1A2, CYP3A4, and CYP2D6 could result in a broad impairment of drug metabolism. Ten plant extracts displayed this broad activity. Casuarina equisetifolia, E. variegata, P. pinnata, P. guajava (sk002) and Terminalia catappa inhibited CYP1A2, suggestive of a noncompetitive mechanism; whereas, C. inophyllum, C. alata (in014), E. hortensis, P. conjugatum, and P. guajava (em009) appeared to inhibit in a competitive fashion. C. equisetifolia inhibited CYP3A4, suggesting a noncompetitive mechanism, while the other nine plant extracts appeared to inhibit competitively. Only T. catappa inhibited CYP2D6 in a manner suggestive of a competitive mechanism. The other extracts inhibited CYP2D6 in a noncompetitive fashion, except for C. alata and P. guajava (sk002), which unexpectedly displayed less inhibition at the lower substrate concentration. The mechanism behind this observation is unknown.
3.3. Induction and Inhibition Overlap
Several extracts showed induction and inhibition towards various CYPs (Table 1). For instance, S. rhombifolia selectively induced and inhibited CYP1A2 and M. esculenta induced and inhibited only CYP3A4. A. scholaris and C. blumei only induced CYP3A4 expression and selectively inhibited CYP2D6. P. guajava and C. terminalis induced CYP3A4 and inhibited CYP3A4 and CYP2D6. P. conjugatum induced CYP1A2 and inhibited all three CYPs tested. P. pinnata induced CYP3A4 and inhibited all three CYPs.
C. inophyllum, E. variegata, and E. hortensis displayed broad inductive and inhibitory activity by inducing both CYP1A2 and CYP3A4 and inhibiting CYP1A2, CYP3A4, and CYP2D6. S. unitus induced and inhibited CYP1A2 and CYP3A4. C. variegatum induced both CYPs, but was inhibitory towards CYP2D6 and CYP3A4.
3.4. Inactive Plant Extracts
Several plant extracts did not exhibit either enzyme induction or enzyme inhibition. These are listed in Table 3.
Table 3.
List of inactive plant extracts
| Genus and Species | Voucher Number | Plant Part | PNG Province |
|---|---|---|---|
| A. conyzoides | sk013 | leaves & flowers | Eastern Highlands |
| A. conyzoides | em021 | leaves | Southern Highlands |
| B. cernua | cw085 | leaves | Western Highlands |
| C. papuana | em036 | bark | Southern Highlands |
| Cyathea sp. | jp1160 | leaves | Enga |
| E. indica | nn012 | leaves | Manus |
| E. indica | in060 | whole plant | North Bougainville |
| E. hirta | nn020 | whole plant | Manus |
| F. copiosa | em023 | leaves | Southern Highlands |
| F. septica | in008 | bark | Manus |
| F. septica | nn006 | leaves & shoots | North Bougainville |
| F. wasa | cw032 | leaves | Western Highlands |
| H. rosa-sinensis | em053 | twigs | Southern Highlands |
| H. tiliaceus | in018 | shoots | North Bougainville |
| L. decumana | jp1157 | leaves | Enga |
| M. peltata | in041 | leaves & shoots | North Bougainville |
| M. micrantha | in016 | whole plant | North Bougainville |
| M. citrifolia | in007 | leaves & fruits | North Bougainville |
| Passiflora sp. | jp1111 | flowers & fruits | Enga |
| R. klossii | em055 | whole plant | Southern Highlands |
Induction activity was < 4.0-fold (CYP1A2) & < 3.5-fold (CYP3A4); inhibition activity was < 50% inhibition
3.5. Variable inhibition and induction results from the same plant
For some plants in the study, multiple samples had been collected from different locations. It was found that the induction and/or inhibition characteristics occasionally showed some degree of variation. Multiple reasons that extracts from different batches of plants might display inconsistent activities could include geographical region (Ozcan and Chalchat, 2005; Conforti et al., 2011), season of collection (dry or rainy) (Murakami et al., 2009), drying and extraction techniques (Komes et al., 2011), and the plant parts included in the extraction. A summary of the inductive and inhibitory properties of plants gathered from different regions with different plant parts listed is given in Table 4.
Table 4.
Activity of replicate plant extracts listed by plant part and collection site
| Genus and Species | Voucher Number | Plant Part | PNG Province | CYP1A2 Induction (- fold) |
CYP3A4 Induction (- fold) |
CYP1A2 % Inhibition |
CYP3A4 % Inhibition |
CYP2D6 % Inhibition |
|||
|---|---|---|---|---|---|---|---|---|---|---|---|
| MR (2.0 μM) | MR (0.4 μM) | 7-BQ (500 μM) | 7-BQ (100 μM) | MAMC (100 μM) | MAMC (40 μM) | ||||||
| A. conyzoides | sk013 | leaves & flowers | Eastern Highlands | -- | -- | -- | -- | -- | -- | -- | -- |
| A. conyzoides | em021 | leaves | Southern Highlands | -- | -- | -- | -- | -- | -- | -- | -- |
| A. evecta | em046 | vines | Southern Highlands | -- | -- | -- | -- | -- | -- | -- | -- |
| A. evecta | cw055 | leaves | Western Highlands | -- | -- | -- | -- | -- | 50 | -- | -- |
| B. pilosa | em028 | leaves | Southern Highlands | -- | 5.7 | -- | -- | -- | -- | -- | -- |
| B. pilosa | cw004 | leaves | Western Highlands | -- | 5.5 | -- | -- | -- | -- | -- | 51 |
| C. papaya | in032 | whole plant | North Bouganville | -- | -- | -- | -- | -- | -- | -- | -- |
| C. papaya | em016 | leaves | Southern Highlands | -- | -- | -- | -- | 54 | 78 | -- | -- |
| C. papaya | cw026 | flowers, leaves & white sap | Western Highlands | -- | -- | -- | -- | -- | -- | -- | -- |
| C. alata | nn050 | leaves | Manus | -- | -- | -- | 54 | -- | -- | 56 | 61 |
| C. alata | in014 | leaves | North Bouganville | -- | -- | 58 | 75 | -- | 57 | 54 | -- |
| C. asiatica | jp1165 | whole plant | Enga | -- | -- | -- | -- | -- | 56 | -- | -- |
| C. asiatica | nn014 | leaves | Manus | -- | -- | -- | -- | -- | -- | -- | -- |
| C. asiatica | cw069 | leaves | Western Highlands | -- | -- | -- | -- | -- | -- | -- | -- |
| C. variegatum | nn016 | leaves | Manus | 5.8 | 14.2 | -- | -- | 67 | 97 | 50 | 60 |
| C. variegatum | in031 | leaves | North Bouganville | -- | -- | -- | -- | -- | -- | -- | -- |
| C. variegatum | in006 | leaves, stalk & sap | North Bouganville | -- | -- | -- | -- | -- | -- | -- | -- |
| C. terminalis | jp1144 | leaves | Enga | -- | -- | -- | -- | -- | 76 | -- | -- |
| C. terminalis | cw030 | leaves from shoots | Western Highlands | -- | 3.8 | -- | -- | -- | 88 | 50 | 61 |
| E. indica | nn012 | leaves | Manus | -- | -- | -- | -- | -- | -- | -- | -- |
| E. indica | in060 | whole plant | North Bouganville | -- | -- | -- | -- | -- | -- | -- | -- |
| F. septica | in008 | bark | Manus | -- | -- | -- | -- | -- | -- | -- | -- |
| F. septica | nn006 | leaves & shoots | North Bouganville | -- | -- | -- | -- | -- | -- | -- | -- |
| P. guajava | sk002 | leaves | Eastern Highlands | -- | 5.2 | 52 | 57 | 53 | 85 | 59 | -- |
| P. guajava | nn011 | leaves | Manus | -- | 4.6 | -- | 55 | -- | 62 | -- | -- |
| P. guajava | in045 | leaves | North Bouganville | -- | 8.7 | -- | -- | -- | 50 | 51 | 51 |
| P. guajava | em009 | leaves | Southern Highlands | -- | 5.8 | 57 | 72 | 55 | 79 | 52 | 58 |
| P. guajava | cw019 | leaves | Western Highlands | -- | 3.5 | 58 | 66 | -- | 66 | -- | -- |
| S. torvum | jp1126 | whole plant | Enga | -- | -- | -- | -- | -- | -- | -- | -- |
| S. torvum | in017 | leaves | North Bouganville | -- | -- | -- | -- | -- | -- | -- | 52 |
CYP Induction: (--) activity was < 4.0-fold (CYP1A2) & < 3.5-fold (CYP3A4); CYP Inhibition: (--) activity was < 50% inhibition; methoxyresorufin (MR), 7-benzoxyquinoloine (7-BQ), 7-methoxy-4-(aminomethyl)-coumarin (MAMC).
A. conyzoides, E. indica and F. septica were consistently non-active in both enzyme induction and enzyme inhibition assays, despite the different plant parts extracted. Among five leaf extracts of P. guajava, from different locations, all exhibited CYP3A4 induction and CYP3A4 inhibition; however, the inhibition of CYP1A2 and CYP2D6 varied (Table 4). Only one of three samples of C. variegatum showed significant enzyme-altering activity, as was the case for C. papaya. For the former, enzyme induction (CYP1A2 and CYP3A4) and inhibition (CYP3A4 and CYP2D6) was only seen in the sample from Manus, the two samples from North Bougainville were inactive. For C. papaya, one sample contained leaves only, one contained flowers, leaves and sap, and one contained whole plant. Only the sample composed of leaves inhibited CYP3A4.
3.6. Inhibitory characterization of E. hortensis
E. hortensis is a medicinal plant used for pain and inflammation in the Western Highlands Province of PNG. Screening results of E. hortensis revealed induction of CYP1A2 and CYP3A4 and inhibition of CYP1A2, CYP3A4, and CYP2D6 (Tables 1 & 2). Fractionation of the whole plant extract yielded high inhibitory activity towards all three CYPs in Fraction 4 (100% MeOH). Fraction 4 was further separated by HPLC. Interest was focused on the five major peaks that appeared in the 210 nm absorbance trace. HPLC fractions corresponding to this region of the chromatogram was assessed for CYP inhibition. An expansion of the 210nm chromatogram of the five major peaks and inhibition data are aligned in Figure 1. Error bars indicate SEM.
Figure 1.
Percent inhibition of CYP1A2, CYP3A4, and CYP2D6 following two-stage fractionation of an E. hortensis extract. Screening revealed inhibition of CYP1A2, CYP3A4, and CYP2D6. Fractionation of the methanol extract localized high inhibitory activity of all three CYPs in F4 (100% MeOH). F4 was separated by reversed-phase HPLC, collecting aliquots every 30 s over a 50 min run (H2O:ACN 40% to 100%). Expansion of five major peaks from 210nm chromatogram and inhibition data are aligned; the complete chromatogram is inset. Error bars indicate S.E.M.
Peak 1 (retention time (RT) - 18.78 min), Peak 2 (RT - 19.62 min), Peak 3 (RT - 20.20 min), and Peak 5 (RT - 22.95 min) showed preferential inhibition of CYP3A4. Peak 4 (RT - 21.38 min) preferentially inhibited CYP1A2. None of the fractions within this region inhibited CYP2D6.
4. Discussion
This investigation found that 21 out of 69 (~30%) of the PNG medicinal plant extracts tested induced CYP expression and 38 out of 69 (~55%) inhibited CYP-dependent metabolism. Many displayed both inhibition and induction, 18 out of 69 (~26%). Either outcome, alone or in combination, may impact the effective management of people on ART. Induction may have negative consequences by decreasing the overall concentrations of antiretroviral drugs in the body. Protease inhibitors are widely used in ART and are metabolized predominately by CYP3A4. Previous studies have shown that herbal remedies can reduce concentrations of protease inhibitors through CYP3A4 induction. St. John’s Wort is an herbal supplement used to treat depression, it has been reported to significantly reduce plasma level of indinavir upon coadministration (Piscitelli et al., 2000). Of the medicinal plants tested, 19 of 69 significantly induced CYP3A4. Ritonavir has demonstrated drug-drug interactions due to CYP1A2 induction (Yeh et al., 2006). Seven of the 69 medicinal plant extracts also induced CYP1A2. The inductive herbal preparations reported here have the potential to attenuate the efficacy of antiretroviral and other drugs.
Inhibition can have either positive or negative consequences. Inhibition of CYPs in the presence of ART may lead to decreases in metabolism prolonging therapeutic effects. Ritonavir, a potent inhibitor of CYP3A4, is given in conjunction with other protease inhibitors to enhance their pharmacokinetic profiles and beneficial effects (Hsu et al., 1998; King et al., 2004; Josephson, 2010). Recently, a potent CYP3A4 inhibitor, cobicistat, has been specifically developed for the same purpose (Shah et al., 2013). Conversely, excessive CYP inhibition can have negative consequences due to toxicity associated with the elevated concentrations of antiretroviral or other drugs in the body (Bressler, 2006).
The medicinal plant extracts in our study displayed a range of inhibitory activities. Some extracts were selective towards individual CYPs, like P. obtusifolia towards CYP1A2. Other extracts exhibited broad inhibitory activity towards multiple CYPs, as in the case of E. hortensis. Characterization of E. hortensis revealed that multiple compounds within the extract contribute to its broad activity. The attribution of inhibition to a given metabolite in an extract is difficult because of the demonstrable presence of multiple inhibitory compounds. Inhibitory mechanisms of the extracts evaluated were suggestive of both competitive and noncompetitive inhibition.
These crude extracts provide a representative sampling of medicinal plants likely consumed by an HIV patient in PNG. The ability of plant extracts to induce and/or inhibit CYPs was determined in vitro. It is not known whether the active plant constituents exert their activity in individuals consuming them. However, the data presented here suggest the “potential” of the identified medicinal plants to induce and/or inhibit ART metabolism in people living with HIV.
5. Conclusions
Medicinal plant usage in combination with ART is a likely scenario throughout much of PNG. As seen in this study, many PNG medicinal plants have the potential to induce and/or inhibit hepatic drug metabolizing enzymes, possibly resulting in unanticipated consequences on the management of HIV. For this reason, it is suggested that PNG healthcare providers counsel patients regarding concomitant medicinal plant usage, especially when a patient’s response to prescription therapy lies outside of that normally encountered. This knowledge will benefit PNG healthcare providers in managing the treatment regimens of their HIV patient population.
Supplementary Material
Acknowledgments
We would like to thank the UPNG students who participated in the collection of the medicinal plants and their local communities: Aron Bale and the villages of the East Awin area in the Kuinga Local Level Government (LLG), North Fly District, Western Province; Christy Wai and the people of the Anglimp Village in the Western Highlands Province; Enos Mais and the people of the Habare Village of Tari Pori in the Southern Highlands Province; Isaiah Norg and the people of the Buka region of North Bougainville; Joyce Pato and the villages of Nuignu, Amarail, Kendiyam, Tukusender, Yaimalha, and Comarepeka within the Laiagam District of the Enga Province; Noan Nembou and the people of Baluan in the Balopa LLG of the Manus Province; and Seva Korape and the people of the Unggai-Bena District of the Eastern Highlands Province. Funding for this project was provided by PNG National Aids Council Secretariat (JTA International Sponsor Award No. 10020310) and NIH Fogarty Int. Ctr. U01 (TW006671).
Footnotes
Competing interests
The authors have no competing interests to disclose.
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Contributor Information
Erica C. Larson, Email: erica.c.larson@utah.edu.
Laura B. Hathaway, Email: laura.hathaway@utah.edu.
John G. Lamb, Email: greg.lamb@hci.utah.edu.
Chris D. Pond, Email: chris.pond@hsc.utah.edu.
Prem P. Rai, Email: raipp@yahoo.com.
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Pius Piskaut, Email: piskautp@upng.ac.pg.
Louis R. Barrows, Email: lbarrows@pharm.utah.edu.
Michael R. Franklin, Email: michael.franklin@pharm.utah.edu.
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