To address these questions, we performed *in vitro* studies with hepatic and intestinal tissues of rats, fed garlic supplements for two weeks. This approach allowed us to distinguish the influence, which garlic supplements could exert on first-pass Saqs metabolism in the gut and in the liver. Based on the knowledge of short-term influence of garlic phytochemicals and/or supplements on transporter-enzyme interplay (Berginc et al, 2009; Berginc et al, 2010a) and clinical data for Saq (Piscitelli et al, 2002), the mechanisms behind

According to the clinical experience with Saq, a warning regarding the possibility of interactions with garlic or garlic supplements has been extended also to other HIV-PIs. Therefore, our research also aimed to identify, whether the "*in vitro*" transport and metabolism and perhaps therapeutic efficacy of Dar, a novel, second-generation HIV-PI, could really be affected by garlic consumption in a similar fashion. Furthermore, we hypothesized that differences may exist between the influences of different garlic supplements on HIV-PI pharmacokinetics. Rats were thus fed two garlic supplements with very distinct composition; aged garlic extract – AGE, a preferentially hydrophilic product rich in water-soluble -glutammyl derivatives of organosulfur compounds, flavonoids and other substances (Berginc et al, 2010b) and garlic oil macerate – GOM, containing preferentially oil-soluble allicin degradation products (i.e. various sulfides, ajoene, vinyl dithiins and other) (Yoschida et al, 1999). We also analyzed plasma samples of fed rats to identify potential garlic metabolites/phytochemicals, which could modify gene expression

Salts for incubation salines, sunflower seed oil from Helianthus annuu, Williams medium E, L-glutamine, insulin, gentamicin and ampicillin, analytical standards for constituents of garlic supplements (rutin, tangeretin, bergamottine, adenosine, S-allyl cisteine and quercetin) were from Sigma Aldrich Chemie (Deisenhofen, Germany). Saq and ritonavir (Rito) were purchased at Sequoia Research Products. All chemicals used in this study were of the highest grade available. Kyolic® liquid aged garlic extract (AGE) was produced by Wakanuga of America CO., LTD (Mission Viejo, CA, USA), lot number 5H04A. The AGE used in this study was standardized to 1.27 g/l S-allylcysteine content, the compound normally used for the standardization of AGE. Commercially available garlics soft-gelatin capsules filled with garlic oil macerate (GOM) were purchased in a local pharmacy. Dar was extracted from Prezista® tablets (300mg) by ethanol extraction, evaporation and reconstitution in DMSO. The purity of extracted Dar was confirmed by HPLC analysis at 240 nm; the Dar peak surface represented 97% of the total chromatogram surface area.

(Kis et al, 2009) and consequently to the therapeutic efficacy of HIV-PIs.

pharmacokinetic interactions between Saq and garlic were elucidated.

as suggested by Piscitelli (Piscitelli et al, 2002).

**2. Methods and materials**

**2.1 Materials**

#### **2.2.1 Test system**

The experiment was conducted with adult, at the start 12 weak old, male Wistar rats. The study conformed to the Law for the Protection of Animals (Republic of Slovenia) and was registered at the Veterinary Administration of Republic of Slovenia No. 34401-41/2008/2.

Animals were treated according to the principles of Convention ETS 123 (The convention for the protection of animals used for experimental and other scientific purposes) and Directive 86/609/EEC (Council directive on the protection of animals used for experimental and other scientific purposes).

N – The number of fed rats; Mbefore - the mean rat weight before supplementation; Mafter – the mean rat weight on day 15.

Table 1. The experimental design of chronic/long-term garlic supplementation to rats.

Animals were fed with pelletized feed Global Diet 2018 ad libidum and had free access to the drinking water through the experimental period. Animals were weighed before first application, on day 7 and at the end of the study. The first weighing was used for randomization, group formation and dose adjustments. From the first and last weighing of animals the mean body weight at the start and at the end of the study were calculated. The experiment was designed with 4 groups of animals. Two different garlic supplements (aged garlic extract – AGE or soft gelatin capsules with garlic oil macerates - GOM) were given orally to two groups daily by gavage for 14 days. The recommended human single oral doses of garlic supplements (2.5 mL of liquid AGE and 2 soft gelatin capsules of GOM, respectively) were adjusted according to the rats weight; AGE was diluted by saline and the content of soft gelatin capsules was diluted by sunflower seed oil (Table 1). Separately we also evaluated the impact of intragastric gavage of saline (sham-A group) and of sunflower seed oil (sham-B group), because they were used to dilute both garlic suspplements. The oil was chosen in compliance with OECD repeated dose methods where oil is recommended as vehicle for worse soluble items (OECD, 2008). Apart from intragastric gavage of different supplements/saline/sunflower seed oil, all groups had access to standard rat chow and water ad libidum. On day 15, rat small intestine, liver and plasma were obtained from rats after short fastening period before the sacrificing. After euthanasia and laparotomy, the intestine was rinsed with ice-cold 10 mM glucose Ringer solution and jejunum was used. Rat liver was preserved in ice-cold transportation medium, consisting of Williams medium E supplemented with L-glutamine (0.29

First-Pass Metabolism Changes After Long-Term Garlic Supplementation 361

contained 20 µM Saq or Dar. The samples of incubation medium were withdrawn after 30 and 60 min for Saq and after 45 and 90 min for Dar to avoid steady state. After the incubation, liver slices were weighted and homogenized. All the samples were immediately precipitated with methanol (1:3) containing haloperidol as internal standard (100 µg/L). The samples were afterwards prepared for the analysis in the same manner as described for permeability experiments. The LC/MS/MS analysis of the liver extract samples and of the withdrawn samples from the incubation medium at different time points was performed. The transport rate (the rate of metabolite concentration increase in the incubation medium) of two Saq and two Dar metabolites (Saq-M1, Saq-M3, Dar-M1, Dar-M4) was determined. In the liver extracts concentrations of Saq/Dar and their metabolites were measured. By comparing concentrations of the parent drugs and their metabolites in the intracellular cell compartment obtained by extraction and the metabolite transport rates with the corresponding reference values (i.e. concentrations of Saq/Dar and their metabolites or metabolite transport rates determined without garlic phytochemicals) we were able to determine the impact of garlic supplements on the activity of efflux/uptake transporters

Prior to the LC/MS/MS analysis, each 200L plasma sample was precipitated with 3 volumes of ice-cold methanol, briefly vortexed, left to stand on -20°C for two days and then centrifuged at 10 000 g for 10 min at 4°C. The supernatant was transferred to autosampler

LC/MS/MS analysis of the samples obtained in the *in vitro* transport/metabolism experiments was performed on a Varian 1200L triple-quadrupole LC–MS Varian (Palo Alto, CA, USA). The mass spectrometer was coupled to ProStar 210 binary pumps, a Varian 420 autosampler and Varian 510 column oven. For chromatographic separation, a Phenomenex Kinetex 50x2.0 mm C-18 column with 2.6 m particles was used for Dar, and a Phenomenex Gemini 150x2.0 mm C-18 column with 3 m particles for Saq. The injection volume was 10 L and the column temperature was 50 C. Mobile phase consisted of water and acetonitrile containing 0.1% of formic acid. The elution for Dar and its metabolites was performed using a linear gradient from 20% to 65% of organic phase in 4 minutes with a flow rate of 0.4 mL/min, whereas that of Saq was performed using a linear gradient from 20% to 36% organic phase over 12 minutes with a flow rate of 0.5 mL/min. For the purpose of this study, two Dar (Dar-M1 – Dar carbamate hydrolysis (tr 3.3 min), Dar-M4 – Dar hydroxylation (tr 5.2 min)) 12 and two Saq (Saq-M1 – Saq oxidation at (1,1 dimethylethyl)amino group (tr 8.1 min), Saq-M3 – Saq oxidation at benzyl group (tr 5.9 min))

Rat plasma samples were analyzed by a triple-quadrupole LC/MS/MS Agilent 6460 (Agilent Technologies, Waldbronn, Germany) equipped with a Jet-Stream ESI interface coupled to an Agilent 1290 Infinity UPLC. 1L of acetonitrile-precipitated rat plasma sample was injected on

**2.2.5 LC/MS/MS sample analysis from studies with rat jejunum and liver slices**

into bile or back into plasma) and CYP3A4.

vials for subsequent LC/MS/MS analysis.

(Fitzsimmons&Collins, 1996) metabolites were monitored.

**2.2.6 LC/MS/MS analyis of plasma samples**

**2.2.4 Plasma preparation**

mg/mL), insulin (0.13 I.U/mL), gentamicin (50 µg/mL) and ampicilin (100 µg/mL). Plasma samples of rats fed with AGE, GOM and of reference animals were withdrawn and stored at -70C until LC/MS/MS analysis.

#### **2.2.2 Saq and Dar permeability through rat jejunum**

The intestinal tissue was cut into 3 cm long segments, excluding visible Peyer's patches. Intestinal segments were opened along the mesenteric border, stretched onto inserts with exposed tissue area of 1 cm2 and placed between two compartments of EasyMount side-byside diffusion chambers (Physiologic Instruments, San Diego, USA). 2.5 mL of bathing solution (Ringer buffer, pH 7.4) on each side of intestine was maintained at 37°C and continuously oxygenated and circulated by bubbling with carbogen (95% O2, 5% CO2). 625 mM glucose and 625 mM mannitol were always added to the serosal (S) and mucosal (M) sides, respectively, to give 10 mM final concentrations. After 25 min of pre-incubation, incubation salines were replaced by donor solutions containing HIV-PIs at the highest concentrations, which could be prepared in Ringer buffer; Saq (25 µM) or Dar (100 µM). If monitoring mucosal-to-serosal (M-S) permeability, donor solution also contained 10 mM mannitol, whereas if monitoring serosal-to-mucosal (S-M) permeability, 10 mM glucose was present in the donor solution. 250 µL of samples were withdrawn from the acceptor (M or S) side every 20 min up to 80 min and replaced by the fresh Ringer buffer containing all necessary ingredients at appropriate concentrations. The donor solutions were also replaced before each sampling time to avoid the loss of investigated compound (Saq, Dar) due to its intestinal metabolism. Immediately after sampling, the withdrawn samples were precipitated by methanol (1:3), which contained haloperidol (100 µg/L) as internal standard, vortexed and kept on ice for at least 15 min. Afterwards the proteins were removed by centrifugation for 10 minutes at 15 000 *g* and 4C. The supernatant was analyzed by LC/MS/MS for the parent drugs (Saq or Dar) and their metabolites.

During the permeability experiments, the diffusion chambers were equipped with two pairs of Ag/AgCl electrodes for measuring transepithelial potential difference and short circuit current with a multi channel voltage-current clamp (model VCC MC6, Physiologic Instruments) enabling the evaluation of tissue integrity and viability during the experiments. Only tissues that retained their integrity and viability (described in details in Berginc et al (Berginc et al, 2010b)) were used for the analysis.

During *in vitro* permeability experiments short-term and long-term effects were monitored. The expression short-term used throughout the text refers to the instant influence the addition of AGE supplement in 1 v/v % to the mucosal side of intestine from AGE or sham-A group exerted (i.e. allosteric modifications). On the other hand, the expression long-term effects refer to the transcriptional/translation changes resulting in different protein expression due to 2 week supplementation of rats.

#### **2.2.3 Saq and Dar metabolism and transport in the rat liver slices**

Rat liver slices (250 µm thick) for the experiments were prepared and pre-incubated for 30 min in the incubation medium (Williams medium E supplemented with L-glutamine (0.29 mg/mL), insulin (0.13 I.U/mL), gentamicin (50 µg/mL) and ampicilin (100 µg/mL)) at 37C. Afterwards, each liver slice was incubated in 1.5 mL of the same medium, which also contained 20 µM Saq or Dar. The samples of incubation medium were withdrawn after 30 and 60 min for Saq and after 45 and 90 min for Dar to avoid steady state. After the incubation, liver slices were weighted and homogenized. All the samples were immediately precipitated with methanol (1:3) containing haloperidol as internal standard (100 µg/L). The samples were afterwards prepared for the analysis in the same manner as described for permeability experiments. The LC/MS/MS analysis of the liver extract samples and of the withdrawn samples from the incubation medium at different time points was performed. The transport rate (the rate of metabolite concentration increase in the incubation medium) of two Saq and two Dar metabolites (Saq-M1, Saq-M3, Dar-M1, Dar-M4) was determined. In the liver extracts concentrations of Saq/Dar and their metabolites were measured. By comparing concentrations of the parent drugs and their metabolites in the intracellular cell compartment obtained by extraction and the metabolite transport rates with the corresponding reference values (i.e. concentrations of Saq/Dar and their metabolites or metabolite transport rates determined without garlic phytochemicals) we were able to determine the impact of garlic supplements on the activity of efflux/uptake transporters into bile or back into plasma) and CYP3A4.

#### **2.2.4 Plasma preparation**

360 Phytochemicals – Bioactivities and Impact on Health

mg/mL), insulin (0.13 I.U/mL), gentamicin (50 µg/mL) and ampicilin (100 µg/mL). Plasma samples of rats fed with AGE, GOM and of reference animals were withdrawn

The intestinal tissue was cut into 3 cm long segments, excluding visible Peyer's patches. Intestinal segments were opened along the mesenteric border, stretched onto inserts with exposed tissue area of 1 cm2 and placed between two compartments of EasyMount side-byside diffusion chambers (Physiologic Instruments, San Diego, USA). 2.5 mL of bathing solution (Ringer buffer, pH 7.4) on each side of intestine was maintained at 37°C and continuously oxygenated and circulated by bubbling with carbogen (95% O2, 5% CO2). 625 mM glucose and 625 mM mannitol were always added to the serosal (S) and mucosal (M) sides, respectively, to give 10 mM final concentrations. After 25 min of pre-incubation, incubation salines were replaced by donor solutions containing HIV-PIs at the highest concentrations, which could be prepared in Ringer buffer; Saq (25 µM) or Dar (100 µM). If monitoring mucosal-to-serosal (M-S) permeability, donor solution also contained 10 mM mannitol, whereas if monitoring serosal-to-mucosal (S-M) permeability, 10 mM glucose was present in the donor solution. 250 µL of samples were withdrawn from the acceptor (M or S) side every 20 min up to 80 min and replaced by the fresh Ringer buffer containing all necessary ingredients at appropriate concentrations. The donor solutions were also replaced before each sampling time to avoid the loss of investigated compound (Saq, Dar) due to its intestinal metabolism. Immediately after sampling, the withdrawn samples were precipitated by methanol (1:3), which contained haloperidol (100 µg/L) as internal standard, vortexed and kept on ice for at least 15 min. Afterwards the proteins were removed by centrifugation for 10 minutes at 15 000 *g* and 4C. The supernatant was analyzed by

During the permeability experiments, the diffusion chambers were equipped with two pairs of Ag/AgCl electrodes for measuring transepithelial potential difference and short circuit current with a multi channel voltage-current clamp (model VCC MC6, Physiologic Instruments) enabling the evaluation of tissue integrity and viability during the experiments. Only tissues that retained their integrity and viability (described in details in

During *in vitro* permeability experiments short-term and long-term effects were monitored. The expression short-term used throughout the text refers to the instant influence the addition of AGE supplement in 1 v/v % to the mucosal side of intestine from AGE or sham-A group exerted (i.e. allosteric modifications). On the other hand, the expression long-term effects refer to the transcriptional/translation changes resulting in different protein

Rat liver slices (250 µm thick) for the experiments were prepared and pre-incubated for 30 min in the incubation medium (Williams medium E supplemented with L-glutamine (0.29 mg/mL), insulin (0.13 I.U/mL), gentamicin (50 µg/mL) and ampicilin (100 µg/mL)) at 37C. Afterwards, each liver slice was incubated in 1.5 mL of the same medium, which also

and stored at -70C until LC/MS/MS analysis.

**2.2.2 Saq and Dar permeability through rat jejunum**

LC/MS/MS for the parent drugs (Saq or Dar) and their metabolites.

Berginc et al (Berginc et al, 2010b)) were used for the analysis.

**2.2.3 Saq and Dar metabolism and transport in the rat liver slices**

expression due to 2 week supplementation of rats.

Prior to the LC/MS/MS analysis, each 200L plasma sample was precipitated with 3 volumes of ice-cold methanol, briefly vortexed, left to stand on -20°C for two days and then centrifuged at 10 000 g for 10 min at 4°C. The supernatant was transferred to autosampler vials for subsequent LC/MS/MS analysis.

#### **2.2.5 LC/MS/MS sample analysis from studies with rat jejunum and liver slices**

LC/MS/MS analysis of the samples obtained in the *in vitro* transport/metabolism experiments was performed on a Varian 1200L triple-quadrupole LC–MS Varian (Palo Alto, CA, USA). The mass spectrometer was coupled to ProStar 210 binary pumps, a Varian 420 autosampler and Varian 510 column oven. For chromatographic separation, a Phenomenex Kinetex 50x2.0 mm C-18 column with 2.6 m particles was used for Dar, and a Phenomenex Gemini 150x2.0 mm C-18 column with 3 m particles for Saq. The injection volume was 10 L and the column temperature was 50 C. Mobile phase consisted of water and acetonitrile containing 0.1% of formic acid. The elution for Dar and its metabolites was performed using a linear gradient from 20% to 65% of organic phase in 4 minutes with a flow rate of 0.4 mL/min, whereas that of Saq was performed using a linear gradient from 20% to 36% organic phase over 12 minutes with a flow rate of 0.5 mL/min. For the purpose of this study, two Dar (Dar-M1 – Dar carbamate hydrolysis (tr 3.3 min), Dar-M4 – Dar hydroxylation (tr 5.2 min)) 12 and two Saq (Saq-M1 – Saq oxidation at (1,1 dimethylethyl)amino group (tr 8.1 min), Saq-M3 – Saq oxidation at benzyl group (tr 5.9 min)) (Fitzsimmons&Collins, 1996) metabolites were monitored.

#### **2.2.6 LC/MS/MS analyis of plasma samples**

Rat plasma samples were analyzed by a triple-quadrupole LC/MS/MS Agilent 6460 (Agilent Technologies, Waldbronn, Germany) equipped with a Jet-Stream ESI interface coupled to an Agilent 1290 Infinity UPLC. 1L of acetonitrile-precipitated rat plasma sample was injected on

First-Pass Metabolism Changes After Long-Term Garlic Supplementation 363

limit above which drugs could be classified as highly permeable compounds according to our intra-laboratory experiments and FDA suggested highly permeable standards (Yu et al, 2002). Tested HIV-PIs were also profoundly effluxed from enterocytes into the lumen (high Rex values, Table 2A and 2B – 1st phase designated "No Garlic"), indicating that at clinical Dar doses or the highest Saq doses, efflux transporters (Pgp, Mrp-2) were not saturated. This is in accordance with the results obtained by Holmstock (Holmstock et al, 2010). Namely, their Caco-2 permeability experiments and *in situ* intestinal perfusions of Pgp knock-down mice indicated a profound participation of Pgp in Dars secretion from absorptive cells even at the highest tested - clinical 100 µM dose. Transporter saturation was also not achieved with 25 μM Saq, as evident from significantly higher Rex values (higher than 3, Table 2B) compared to the value 1, which usually indicates complete transporter inhibition. Since Pgp/Mrp-2 were not inhibited *in vitro*, the *in vivo* impact of efflux transporters on limiting drug absorption could also be significant, owing to the higher expression of transporters in human small intestine than in the rats (Cao et al, 2006). Therefore, gut represents an

> No garlic AGE addition M-S (\*10-7) S-M (\*10-6) Rex M-S (\*10-7) S-M (\*10-6) Rex

> No garlic AGE addition M-S (\*10-7) S-M (\*10-6) Rex M-S (\*10-7) S-M (\*10-6) Rex

important biological barrier in achieving therapeutic plasma HIV-PI levels.

**Sham-B group** 9.5 ± 2.1 17.9 ± 1.8\* 19 **GOM group** 8.1 ± 2.1 5.8 ± 1.7\*a 7.2

**Sham-B group** 6.9 ± 0.8 2.3 ± 0.2\* 2.9 **GOM group** 6.5 ± 2.7 4.9 ± 0.6\*a 7.5

addition of AGE are also given (AGE addition) in the table.

**Sham-A group** 7.7 ± 0.7 20.4 ± 1.0\* 26 10.4 ± 4.4 20.6 ± 1.6\* 20 **AGE group** 8.6 ± 0.5 13.4 ± 1.0\*a 16 9.2 ± 1.1 11.8 ± 3.1\* 13

**Sham-A group** 6.7 ± 0.3 2.6 ± 0.7\* 3.9 5.9 ± 0.4 3.7 ± 0.3\*b 6.3 **AGE group** 7.0 ± 2.0 5.6 ± 1.0\*a 4 5.9 ± 0.5 7.4 ± 0.4\*b 13

HIV-PIs permeability was determined in two phases; first the permeability was measured in Ringer buffer pH 7.4 (20 min sampling intervals for 80 min – No garlic) and afterwards AGE was added to the mucosal side of rat ileum to give 1 % final AGE concentration and the experiment continued for

\* - S-M Papp HIV-PI values are significantly higher than the corresponding M-S Papp values a – S-M Papp values of treated rats (AGE or GOM group) significantly higher/lower than reference S-M

b – S-M Papp values determined after the addition of AGE significantly changed compared to S-M Papp

Table 2. Saq and Dar intestinal permeability through rat jejunum determined in rats fed different garlic supplements (AGE or GOM – AGE and GOM group), saline (sham-A group) or sunflower seed oil (sham-B group). The permeability values of Saq and Dar after the

**Table 2A Darunavir** - Papp GIT (100 µM) [cm/s]

**Table 2B Saquinavir** - Papp GIT (25 µM) [cm/s]

additional 80 min (AGE addition).

values in the first phase

Papp values (sham-A and sham-B group)

a Phenomenex Synergi Polar-RP 100x3mm 2.5m column. The mobile phase A consisted of water with 0.1% formic acid and mobile phase B consisted of 98% acetonitrile with 2% water. The gradient elution was linear from 1% B to 90% B over 10 minutes and the flow rate was 0.4 mL/min. After the column, the eluent was mixed with 0.2mL/min 100% acetonitrile via a zero-dead volume mixing tee in order to facilitate the evaporation and ionization process in the ESI Jet-Stream interface. The multiple reaction monitoring mode was used for the identification and relative quantification of AGE and GOM constituents in rat plasma (Table 4).

#### **2.2.7 Data analysis**

The apparent permeability coefficient (*P*app) of HIV-PIs was calculated according to Eq. (1)

$$P\_{app} = \frac{dc}{dt} \frac{V}{c\_0 A} \tag{1}$$

where *dc*/*dt* represents changes in HIV-PI concentration in the acceptor compartment per unit time under steady state conditions, *V* is the volume of the acceptor compartment, *A* is the exposed surface area (1 cm2) and *c0* is the initial, donor concentration of HIV-PIs. Afterwards the ratios (Rex) were determined according to the Eq. (2) by dividing S-M Papp ( *S M Papp* ) values with the corresponding M-S Papp ( *<sup>M</sup><sup>S</sup> Papp* ) values.

$$R\_{ex} = \frac{P\_{app}^{\quad S-M}}{P\_{app}^{\quad M-S}} \tag{2}$$

Results in Tables and Figures are presented as means ± SD of at least 3 measurements. Data were evaluated statistically using SPSS 16.0 for Windows. Where appropriate, F-test for testing the equality of variances and, afterwards, 2-tailed Student t-test ( = 0.05), were used. Otherwise, one way ANOVA, followed by Bonferroni post-hoc test were applied.

#### **3. Results and discussion**

#### **3.1 The impact of long-term garlic supplementation on HIV-PIs intestinal permeability**

The intestinal transporter-enzyme interplay (i.e. PGP and CYP3A4 or MRP-2 and UGT) can be responsible for lowering the fraction of the absorbed drugs that are subjected to the metabolism or transport with these proteins (Zhang et al, 2008). The PGP/MRP-2 mediated drug efflux from absorptive cells into the GIT lumen prolongs the overall residence time of drugs in the enterocytes through the more intense entero-enteric circulation, subjecting drugs to a more extensive intracellular phase I or phase II metabolism (Wu&Benet, 1999). The impact of long-term garlic supplementation on the activity of intestinal transporters and thus on the fraction of HIV-PIs (Saq, Dar) absorbed were assessed by evaluation of *in vitro* parent drug intestinal permeability through the rat jejunum (Table 2A, 2B). 100 µM Dar donor concentrations were chosen to simulate intraluminal Dar concentrations after peroral administration of clinical dose 12. Since low water-solubility limits achieving high Saq concentrations (Aungst, 1999), the highest possible Saq donor concentrations (25 µM), which could be prepared in Ringer buffer pH 7.4, were applied.

Both HIV-PIs exerted low intestinal permeability regardless of whether rats were fed garlic supplements or not. Their M-S permeabilities were below 10×10-6 cm/s (Table 2A, 2B), the

a Phenomenex Synergi Polar-RP 100x3mm 2.5m column. The mobile phase A consisted of water with 0.1% formic acid and mobile phase B consisted of 98% acetonitrile with 2% water. The gradient elution was linear from 1% B to 90% B over 10 minutes and the flow rate was 0.4 mL/min. After the column, the eluent was mixed with 0.2mL/min 100% acetonitrile via a zero-dead volume mixing tee in order to facilitate the evaporation and ionization process in the ESI Jet-Stream interface. The multiple reaction monitoring mode was used for the identification and relative quantification of AGE and GOM constituents in rat plasma (Table 4).

The apparent permeability coefficient (*P*app) of HIV-PIs was calculated according to Eq. (1)

*app dc V <sup>P</sup>*

where *dc*/*dt* represents changes in HIV-PI concentration in the acceptor compartment per unit time under steady state conditions, *V* is the volume of the acceptor compartment, *A* is the exposed surface area (1 cm2) and *c0* is the initial, donor concentration of HIV-PIs. Afterwards the ratios (Rex) were determined according to the Eq. (2) by dividing S-M Papp

*P*

Results in Tables and Figures are presented as means ± SD of at least 3 measurements. Data were evaluated statistically using SPSS 16.0 for Windows. Where appropriate, F-test for testing the equality of variances and, afterwards, 2-tailed Student t-test ( = 0.05), were used. Otherwise, one way ANOVA, followed by Bonferroni post-hoc test were applied.

**3.1 The impact of long-term garlic supplementation on HIV-PIs intestinal permeability**  The intestinal transporter-enzyme interplay (i.e. PGP and CYP3A4 or MRP-2 and UGT) can be responsible for lowering the fraction of the absorbed drugs that are subjected to the metabolism or transport with these proteins (Zhang et al, 2008). The PGP/MRP-2 mediated drug efflux from absorptive cells into the GIT lumen prolongs the overall residence time of drugs in the enterocytes through the more intense entero-enteric circulation, subjecting drugs to a more extensive intracellular phase I or phase II metabolism (Wu&Benet, 1999). The impact of long-term garlic supplementation on the activity of intestinal transporters and thus on the fraction of HIV-PIs (Saq, Dar) absorbed were assessed by evaluation of *in vitro* parent drug intestinal permeability through the rat jejunum (Table 2A, 2B). 100 µM Dar donor concentrations were chosen to simulate intraluminal Dar concentrations after peroral administration of clinical dose 12. Since low water-solubility limits achieving high Saq concentrations (Aungst, 1999), the highest possible Saq donor concentrations (25 µM), which

Both HIV-PIs exerted low intestinal permeability regardless of whether rats were fed garlic supplements or not. Their M-S permeabilities were below 10×10-6 cm/s (Table 2A, 2B), the

*R*

( *S M Papp* ) values with the corresponding M-S Papp ( *<sup>M</sup><sup>S</sup> Papp* ) values.

could be prepared in Ringer buffer pH 7.4, were applied.

0

 *S M app ex M S app*

*dt c A* (1)

*P* (2)

**2.2.7 Data analysis**

**3. Results and discussion**

limit above which drugs could be classified as highly permeable compounds according to our intra-laboratory experiments and FDA suggested highly permeable standards (Yu et al, 2002). Tested HIV-PIs were also profoundly effluxed from enterocytes into the lumen (high Rex values, Table 2A and 2B – 1st phase designated "No Garlic"), indicating that at clinical Dar doses or the highest Saq doses, efflux transporters (Pgp, Mrp-2) were not saturated. This is in accordance with the results obtained by Holmstock (Holmstock et al, 2010). Namely, their Caco-2 permeability experiments and *in situ* intestinal perfusions of Pgp knock-down mice indicated a profound participation of Pgp in Dars secretion from absorptive cells even at the highest tested - clinical 100 µM dose. Transporter saturation was also not achieved with 25 μM Saq, as evident from significantly higher Rex values (higher than 3, Table 2B) compared to the value 1, which usually indicates complete transporter inhibition. Since Pgp/Mrp-2 were not inhibited *in vitro*, the *in vivo* impact of efflux transporters on limiting drug absorption could also be significant, owing to the higher expression of transporters in human small intestine than in the rats (Cao et al, 2006). Therefore, gut represents an important biological barrier in achieving therapeutic plasma HIV-PI levels.

HIV-PIs permeability was determined in two phases; first the permeability was measured in Ringer buffer pH 7.4 (20 min sampling intervals for 80 min – No garlic) and afterwards AGE was added to the mucosal side of rat ileum to give 1 % final AGE concentration and the experiment continued for additional 80 min (AGE addition).

\* - S-M Papp HIV-PI values are significantly higher than the corresponding M-S Papp values a – S-M Papp values of treated rats (AGE or GOM group) significantly higher/lower than reference S-M Papp values (sham-A and sham-B group)

b – S-M Papp values determined after the addition of AGE significantly changed compared to S-M Papp values in the first phase

Table 2. Saq and Dar intestinal permeability through rat jejunum determined in rats fed different garlic supplements (AGE or GOM – AGE and GOM group), saline (sham-A group) or sunflower seed oil (sham-B group). The permeability values of Saq and Dar after the addition of AGE are also given (AGE addition) in the table.

First-Pass Metabolism Changes After Long-Term Garlic Supplementation 365

expression levels in (un)treated rats were not performed, because increased mRNA levels for Pgp have been found to correlate well with increased protein levels and its functionality (MacLean et al, 2008; Englund et al, 2006). Although such correlation between mRNA and protein levels for Mrp-2 has not yet been established (MacLean et al, 2008), diallyl disulfide, garlic sulfide present in oily (i.e. GOM) preparations and in smaller amounts also in aqueous/hydrophilic garlic supplements (i.e. AGE) (Berginc et al, 2009), induced transcription of Mrp-2, leading to increased Mrp-2 protein levels in rat renal brush-border membrane vesicles. Owing to the established positive correlation between increased Pgp/Mrp-2 levels (elevated protein and mRNA levels) and their functionality, the long-term impact of garlic constituents on the Saq and Dar intestinal permeability was assessed

A 14-day AGE or GOM supplementation to rats significantly affected intestinal permeability of both antiretrovirals (Table 2A and 2B – AGE, GOM group); the efflux of Saq significantly increased and that of Dar significantly decreased compared to reference values (S-M Papp values of sham-A group – Table 2; see column "No garlic"). M-S permeabilities in both cases did not change (M-S Papp values sham-A or sham-B group). Sunflower seed oil, used to

The increase of Saqs S-M Papp values observed in rats fed with both garlic supplements as mentioned, implies that the expression of efflux transporters in the mucosal enterocytic membrane must have increased during long-term garlic supplementation due to the repeated exposure of enterocytes to garlic phytochemicals or perhaps their metabolites. In fact, our results partially confirm a hypothesis which has been proposed by Piscitelli (Piscitelli et al, 2002) to explain lower amounts of absorbed Saq in a clinical study. Our results are in accordance with the part of hypothesis, which claims that the transporter expression changes could be induced by garlic phytochemicals or their metabolites. Since rats were not fed HIV-PIs and garlic supplements simultaneously as performed in clinical study by Piscitelli, the second part of his hypothesis that the parent drug (Saq) or its metabolites could also be responsible for the observed effects, could not been corroborated. However, in the case of Dar, in spite of the suggested increased transporter expression, the efflux of this HIV-PI was significantly lower than that determined for the untreated rats (sham-A group). As we mentioned previously, Dar and Saq Pgp binding sites are not identical, which was confirmed in previous *in vitro* study on rat intestine using ritonavir (Rito) as potent Pgp and Mrp-2 inhibitor (Berginc et al, 2010c). While Rito significantly inhibited Dar efflux from enterocytes regardless of the applied Rito concentration, that was not true for Saq. Instead of inhibiting Pgp-mediated Saq efflux, low Rito concentrations stimulated active Saq excretion and the inhibition was achieved only at saturable Rito concentrations. This undoubtedly indicated that Dar and Rito share identical binding site on Pgp, whereas Saq and Rito do not; only after Rito-Pgp binding site(s) are completely occupied by Rito, Rito can displace Saq from its binding site on Pgp. Therefore, significant decrease of Dar efflux after 2-week rat supplementation with garlic products observed in this study was most probably due to the occupation of Dar-binding site on Pgp by garlic phytochemicals or their metabolites with longer half-life(s). This indicates that the compounds from garlic supplements remained bound to intestinal transporters for at least

dilute GOM doses, had no impact on the permeability of tested HIV-PIs.

18h, additionally confirming Piscitelli′ assumptions (Piscitelli et al, 2002).

directly by measuring their permeabilities.

The short-term impact (i.e. instant/allosteric changes of transporter/enzyme activities) induced by AGE phytochemicals on intestinal rats ABC transporters (Pgp, Mrp-2), involved in HIV-PI (Saq, Dar) absorption, have been investigated in our previous studies, where no feeding protocols were applied (Berginc et al, 2009; Berginc et al, 2010c). In the presented study these effects were re-investigated with the intestine of AGE fed rats to elucidate the (ir)reversibility of garlic phytochemicals or phytochemical metabolites binding to the efflux transporters. We reported previously that activities of Pgp and Mrp-2 significantly changed owing to allosteric modulations exerted in the presence of AGE components (Berginc et al, 2009; Berginc et al, 2010c). The impact of Pgp on the absorption of HIV-PIs was found to exceed that of Mrp-2, which has also been corroborated by Usansky (Usansky et al, 2008) in their *in vivo* study with Sprague-Dawley rats and Saq. In the case of Dar, mixed reports can be found regarding MRP-2/Mrp-2 importance in Dar absorption (Berginc et al, 2010c; Kakuda&Kiser, 2006). Based on this and the Mrp-2 expression pattern along GIT tract in rats (Mrp-2 expression decreases in more distal parts of rats small intestine) (Berginc et al, 2010c; Usansky et al, 2008), the participation of Mrp-2 in HIV-PIs intestinal absorption is minor and the observed short-term effects in our studies were predominately caused by modified Pgps activity. The noted short-term activity changes of the studied ABC transporters in our previous studies were explained by multiple binding sites in these transporters (four in Pgp and two in MRP-2) (Martin et al, 2000), which allow simultaneous binding of more substrates leading to allosteric modifications. Allosteric modifications induced by AGE phytochemicals in these studies were observed either as competitive inhibition between compounds (phytochemicals and drug) for an identical binding site (i.e. Dar – unchanged efflux) or as positive-cooperative effect and increased efflux (i.e. Saq), when the preferences of all involved substances for binding sites are distinct (Berginc et al, 2009; Berginc et al, 2010c). Similar allosteric modification of intestinal transporters involved in Saq and Dar efflux were also observed using small intestine of AGE fed rats in this study; Saq efflux significantly increased, whereas Dar efflux remained unchanged (slight but statistically insignificant decrease - Table 2A and 2B – phase "AGE addition"). The content of softgelatin capsules containing GOM could not be tested for their short-term influence on efflux transporters due to its poor miscibility with the incubation buffer, which would prevent adequate diffusion of oily phytochemicals from emulsion to the mucosal surface. The shortterm modifications of efflux transporter activities were equally pronounced in rats, fed AGE, and in the control rats (sham-A group). Namely the average ratios between S-M Papp values after and before AGE addition were ca 1.0 for Dar and 1.5 for Saq, regardless which rats were used (treated or untreated – Table 2; sham-A and AGE group). This means that in the time-frame from the last intragastric gavage of garlic supplement and animal euthanization (18 h), garlic phytochemicals must have completely dissociated from intestinal efflux transporters to determine identical magnitude of permeability changes. Intestinal transporters in fed rats therefore could remain occupied with garlic phytochemicals after intragastric gavage but for at most 18 h per day. This could be sufficient to change transporter expression in the long run. However, transcriptional/translational effects and consequently elevated/decreased transporter expression could also be induced by binding of the absorbed xenobiotics on the transporters gene promoters or to the corresponding transcriptional factors. Both scenarios could thus contribute to potential long-term expression changes. In the presented study mRNA or protein levels to quantify Pgp/Mrp-2

The short-term impact (i.e. instant/allosteric changes of transporter/enzyme activities) induced by AGE phytochemicals on intestinal rats ABC transporters (Pgp, Mrp-2), involved in HIV-PI (Saq, Dar) absorption, have been investigated in our previous studies, where no feeding protocols were applied (Berginc et al, 2009; Berginc et al, 2010c). In the presented study these effects were re-investigated with the intestine of AGE fed rats to elucidate the (ir)reversibility of garlic phytochemicals or phytochemical metabolites binding to the efflux transporters. We reported previously that activities of Pgp and Mrp-2 significantly changed owing to allosteric modulations exerted in the presence of AGE components (Berginc et al, 2009; Berginc et al, 2010c). The impact of Pgp on the absorption of HIV-PIs was found to exceed that of Mrp-2, which has also been corroborated by Usansky (Usansky et al, 2008) in their *in vivo* study with Sprague-Dawley rats and Saq. In the case of Dar, mixed reports can be found regarding MRP-2/Mrp-2 importance in Dar absorption (Berginc et al, 2010c; Kakuda&Kiser, 2006). Based on this and the Mrp-2 expression pattern along GIT tract in rats (Mrp-2 expression decreases in more distal parts of rats small intestine) (Berginc et al, 2010c; Usansky et al, 2008), the participation of Mrp-2 in HIV-PIs intestinal absorption is minor and the observed short-term effects in our studies were predominately caused by modified Pgps activity. The noted short-term activity changes of the studied ABC transporters in our previous studies were explained by multiple binding sites in these transporters (four in Pgp and two in MRP-2) (Martin et al, 2000), which allow simultaneous binding of more substrates leading to allosteric modifications. Allosteric modifications induced by AGE phytochemicals in these studies were observed either as competitive inhibition between compounds (phytochemicals and drug) for an identical binding site (i.e. Dar – unchanged efflux) or as positive-cooperative effect and increased efflux (i.e. Saq), when the preferences of all involved substances for binding sites are distinct (Berginc et al, 2009; Berginc et al, 2010c). Similar allosteric modification of intestinal transporters involved in Saq and Dar efflux were also observed using small intestine of AGE fed rats in this study; Saq efflux significantly increased, whereas Dar efflux remained unchanged (slight but statistically insignificant decrease - Table 2A and 2B – phase "AGE addition"). The content of softgelatin capsules containing GOM could not be tested for their short-term influence on efflux transporters due to its poor miscibility with the incubation buffer, which would prevent adequate diffusion of oily phytochemicals from emulsion to the mucosal surface. The shortterm modifications of efflux transporter activities were equally pronounced in rats, fed AGE, and in the control rats (sham-A group). Namely the average ratios between S-M Papp values after and before AGE addition were ca 1.0 for Dar and 1.5 for Saq, regardless which rats were used (treated or untreated – Table 2; sham-A and AGE group). This means that in the time-frame from the last intragastric gavage of garlic supplement and animal euthanization (18 h), garlic phytochemicals must have completely dissociated from intestinal efflux transporters to determine identical magnitude of permeability changes. Intestinal transporters in fed rats therefore could remain occupied with garlic phytochemicals after intragastric gavage but for at most 18 h per day. This could be sufficient to change transporter expression in the long run. However, transcriptional/translational effects and consequently elevated/decreased transporter expression could also be induced by binding of the absorbed xenobiotics on the transporters gene promoters or to the corresponding transcriptional factors. Both scenarios could thus contribute to potential long-term expression changes. In the presented study mRNA or protein levels to quantify Pgp/Mrp-2 expression levels in (un)treated rats were not performed, because increased mRNA levels for Pgp have been found to correlate well with increased protein levels and its functionality (MacLean et al, 2008; Englund et al, 2006). Although such correlation between mRNA and protein levels for Mrp-2 has not yet been established (MacLean et al, 2008), diallyl disulfide, garlic sulfide present in oily (i.e. GOM) preparations and in smaller amounts also in aqueous/hydrophilic garlic supplements (i.e. AGE) (Berginc et al, 2009), induced transcription of Mrp-2, leading to increased Mrp-2 protein levels in rat renal brush-border membrane vesicles. Owing to the established positive correlation between increased Pgp/Mrp-2 levels (elevated protein and mRNA levels) and their functionality, the long-term impact of garlic constituents on the Saq and Dar intestinal permeability was assessed directly by measuring their permeabilities.

A 14-day AGE or GOM supplementation to rats significantly affected intestinal permeability of both antiretrovirals (Table 2A and 2B – AGE, GOM group); the efflux of Saq significantly increased and that of Dar significantly decreased compared to reference values (S-M Papp values of sham-A group – Table 2; see column "No garlic"). M-S permeabilities in both cases did not change (M-S Papp values sham-A or sham-B group). Sunflower seed oil, used to dilute GOM doses, had no impact on the permeability of tested HIV-PIs.

The increase of Saqs S-M Papp values observed in rats fed with both garlic supplements as mentioned, implies that the expression of efflux transporters in the mucosal enterocytic membrane must have increased during long-term garlic supplementation due to the repeated exposure of enterocytes to garlic phytochemicals or perhaps their metabolites. In fact, our results partially confirm a hypothesis which has been proposed by Piscitelli (Piscitelli et al, 2002) to explain lower amounts of absorbed Saq in a clinical study. Our results are in accordance with the part of hypothesis, which claims that the transporter expression changes could be induced by garlic phytochemicals or their metabolites. Since rats were not fed HIV-PIs and garlic supplements simultaneously as performed in clinical study by Piscitelli, the second part of his hypothesis that the parent drug (Saq) or its metabolites could also be responsible for the observed effects, could not been corroborated. However, in the case of Dar, in spite of the suggested increased transporter expression, the efflux of this HIV-PI was significantly lower than that determined for the untreated rats (sham-A group). As we mentioned previously, Dar and Saq Pgp binding sites are not identical, which was confirmed in previous *in vitro* study on rat intestine using ritonavir (Rito) as potent Pgp and Mrp-2 inhibitor (Berginc et al, 2010c). While Rito significantly inhibited Dar efflux from enterocytes regardless of the applied Rito concentration, that was not true for Saq. Instead of inhibiting Pgp-mediated Saq efflux, low Rito concentrations stimulated active Saq excretion and the inhibition was achieved only at saturable Rito concentrations. This undoubtedly indicated that Dar and Rito share identical binding site on Pgp, whereas Saq and Rito do not; only after Rito-Pgp binding site(s) are completely occupied by Rito, Rito can displace Saq from its binding site on Pgp. Therefore, significant decrease of Dar efflux after 2-week rat supplementation with garlic products observed in this study was most probably due to the occupation of Dar-binding site on Pgp by garlic phytochemicals or their metabolites with longer half-life(s). This indicates that the compounds from garlic supplements remained bound to intestinal transporters for at least 18h, additionally confirming Piscitelli′ assumptions (Piscitelli et al, 2002).

First-Pass Metabolism Changes After Long-Term Garlic Supplementation 367

fed high fat diet would also increase, explaining the effects observed in this study on the

**Table 3A EXTRACT (mol/mg) TRANSPORT RATE**

**Sham-A group** 2.9 ± 0.3 4.6 ± 1.0 2.3 ± 0.1 3.8 ± 0.4 3.7 ± 0.3 **AGE group** 8.2 ± 0.2\* 3.9 ± 0.7 2.4 ± 0.4 3.4 ± 0.3 3.2 ± 0.2 **Sham-B group** 8.1 ± 0.4\* 4.9 ± 0.2 4.7 ± 0.7\* 5.6 ± 0.6\* 6.4 ± 0.6\* **GOM group** 10.3 ± 0.4\*a 7.8 ± 0.7\* 4.3 ± 0.9\* 4.8 ± 0.1\* 6.0 ± 0.3\*

**Table 3B EXTRACT (mol/mg) TRANSPORT RATE**

**Sham-A group** 7.8 ± 0.6 2.5 ± 0.2 3.4 ± 0.2 3.6 ± 0.8 2.1 ± 0.2 **AGE group** 10.3 ± 1.0\* 1.7 ± 0.0\* 1.7 ± 0.6\* 2.7 ± 0.5 1.8 ± 0.3 **Sham-B group** 8.5 ± 0.5\* 2.8 ± 0.3 6.0 ± 0.4\* 6.1 ± 1.4\* 5.9 ± 0.1\* **GOM group** 10.2 ± 1.1\*a 3.2 ± 0.2\* 5.0 ± 0.7\* 4.7 ± 0.5 4.5 ± 0.3\* \* - the amounts of HIV-PIs and of the corresponding metabolites in the rat liver extracts or metabolite transport rates are significantly higher/lower than the corresponding reference values (sham-A and B

a – significantly higher HIV-PI amounts in the extract compared to the amounts of HIV-PIs, determined

Table 3. Saq (Table 3A) and Dar (Table 3B) hepatic *in vitro* pharmacokinetics evaluated in rat liver slices obtained from rats fed with different garlic supplements (AGE or GOM groups), saline (sham-A group) or sunflower seed oil (sham-B group). The amounts of HIV-PIs and of the corresponding metabolites in the rat liver extracts and the metabolite transport rates

Higher intrahepatic retention of HIV-PIs after prolonged garlic consumption could thus be explained by two factors. First, hepatocytes were obviously exposed to lower concentration of xenobiotics or their metabolites, responsible for the induction of Pgp compared to enterocytes. Namely after peroral absorption of garlic phytochemicals, lower amounts of garlic phytochemicals and their corresponding metabolites could reach hepatocytes due to incomplete/low xenobiotic absorption through the intestinal mucosa, binding to enterocytes mucosal membranes, instability in GIT lumen and intraluminal metabolism, which altogether contributed to less profound effect on the expression of efflux transporters in hepatocytes than in the jejunum (Ioannides, 2003; Lawson&Wang, 2005b). Besides efflux transporters, Saq and Dar also utilize OATP1A2 (OATP-A), 1B1 (OATP-C) and 1B3 (OATP-8) solute uptake transporters to distribute into the human liver (Hartkoorn et al, 2010). The sinusoidal rat hepatocyte membrane embodies numerous Oatp isoforms with Oatp1, Oatp2 and Oatp4 being the most important ones (Treiber et al, 2004; Meier et al, 1997b). Because AGE has been shown to affect Oatp activity (Berginc et al, 2010b), increased intrahepatic HIV-PI concentrations observed in this study could also correspond to the increased Oatp expression in the liver, which by its influence on the HIV-PIs pharmacokinetics could have

in the extracts of rat liver extracts from control sham-A group.

exceeded the effects of efflux transporters.

from the rat liver slices into the incubation medium were determined.

group).

Saq (\*10-11) Saq-M1 (\*10-13) Saq-M3 (\*10-12) Saq-M1 (\*10-9) Saq-M3 (\*10-8)

Dar (\*10-11) Dar-M1 (\*10-11) Dar-M4 (\*10-12) Dar-M1 (\*10-8) Dar-M4 (\*10-8)

**(µmol/min\*mg)**

**(µmol/min\*mg)**

hepatic HIV-PI pharmacokinetics in sunflower seed oil fed animals (sham-B group).

#### **3.2 The impact of long-term garlic supplementation on HIV-PIs hepatic pharmacokinetics**

Various high-throughput screening studies using human hepatic microsomes (Foster et al, 2001) and recombinant CYP3A4 enzymes (Pal&Mitra, 2006) indicated that lipophilic garlic supplements, AGE and individual garlic phytochemicals could significantly inhibit CYP3A4 activity (short-term effect) and consequently impair HIV-PI (Saq, Dar) metabolism. The short-term effects of AGE on the hepatic HIV-PI distribution and metabolism in rat liver slices have also been investigated in our previous studies, where we have shown that AGE allosterically activated Pgp transport (i.e. it decreased intracellular concentrations of Pgp substrate - Dar) in the same manner as in the intestine but inhibited hepatic Mrp-2 transport. We determined that Mrp-2 inhibition observed in those studies was caused by metabolism of garlic flavonoids to the corresponding glucuronide metabolites, which are good MRP-2/Mrp-2 substrates. These glucuronide metabolites bind to Mrp-2 and competitively displace other Mrp-2 substrates (i.e. Saq) from these transporters, causing an increase of intrahepatic Saq concentrations. The inhibition of CYP3A4 HIV-PI metabolism by AGE was also confirmed in that study (Berginc et al, 2010d).

However after 14 days of AGE supplementation, the concentrations of both tested HIV-PIs in rat liver slices significantly increased (Table 3). Similar results were obtained also with rats fed with GOM, but the impact of GOM supplementation on the hepatic pharmacokinetics was obscured by the high influence of reference treatment - sunflower oil alone (sham-B group). Namely, if one compares intracellular concentrations of parent drugs and of the corresponding metabolites in the extracts from the liver slices of rats fed only sunflower oil (Table 3, sham-B group) with the corresponding values obtained from liver slices of rats fed saline (Table 3: sham-A group), it is obvious that sunflower oil itself exhibited pronounced effects on HIV-PI hepatic distribution and metabolism. The effect of GOM was thus more difficult to evaluate than that of AGE (Table 3; sham-A and AGE group). The daily quantities of the sunflower seed oil used to dilute GOM for sufficiently exact dosing and in the appropriate reference group was ca. 1 mL. According to the guidelines, this amount added to the rat's dietary fat intake does not influence the animal's condition regarding the clinical examination, hematological, biochemical and histological parameters (OECD, 2008). However, the increased daily fat intake was obviously sufficient to induce transcriptional/translational changes, which finally led to changes in HIV-PIs hepatic distribution and metabolism (significantly higher intracellular HIV-PI concentrations in rat liver slices and significantly higher formation and excretion rate of metabolites) compared to the reference values (Sham A group). Sunflower seed oil represents a good source of monounsaturated fatty acids (i.e. oleic acid), which have been known to reversibly increase the transcription of enzymes (among them also CYP) in liver and gut (Niot et al, 1997; Chen et al, 2001). The intake of higher fat quantities also interferes with endogenous lipid synthesis in metabolism, leading to changes in the plasma lipids and bile acid composition. To avoid cytotoxicity of the increased amounts of bile acids formed; these acids need to be excreted into the bile. Therefore, the increased production of bile acids had to be accompanied with the increased transporter expression to ensure cell survival. The excretion of bile acids into gallbladder involves the uptake of these endogenous compounds from plasma into hepatocytes with the aid of uptake transporters (i.e. NTCP, OATPs) and their excretion into the gallbladder by sinusoidal efflux proteins (Pgp, Mrp-2, S-Pgp, MDR3) (Meier et al, 1997a). Therefore the expression of these transporters in organisms

Various high-throughput screening studies using human hepatic microsomes (Foster et al, 2001) and recombinant CYP3A4 enzymes (Pal&Mitra, 2006) indicated that lipophilic garlic supplements, AGE and individual garlic phytochemicals could significantly inhibit CYP3A4 activity (short-term effect) and consequently impair HIV-PI (Saq, Dar) metabolism. The short-term effects of AGE on the hepatic HIV-PI distribution and metabolism in rat liver slices have also been investigated in our previous studies, where we have shown that AGE allosterically activated Pgp transport (i.e. it decreased intracellular concentrations of Pgp substrate - Dar) in the same manner as in the intestine but inhibited hepatic Mrp-2 transport. We determined that Mrp-2 inhibition observed in those studies was caused by metabolism of garlic flavonoids to the corresponding glucuronide metabolites, which are good MRP-2/Mrp-2 substrates. These glucuronide metabolites bind to Mrp-2 and competitively displace other Mrp-2 substrates (i.e. Saq) from these transporters, causing an increase of intrahepatic Saq concentrations. The inhibition of CYP3A4 HIV-PI metabolism by AGE was

However after 14 days of AGE supplementation, the concentrations of both tested HIV-PIs in rat liver slices significantly increased (Table 3). Similar results were obtained also with rats fed with GOM, but the impact of GOM supplementation on the hepatic pharmacokinetics was obscured by the high influence of reference treatment - sunflower oil alone (sham-B group). Namely, if one compares intracellular concentrations of parent drugs and of the corresponding metabolites in the extracts from the liver slices of rats fed only sunflower oil (Table 3, sham-B group) with the corresponding values obtained from liver slices of rats fed saline (Table 3: sham-A group), it is obvious that sunflower oil itself exhibited pronounced effects on HIV-PI hepatic distribution and metabolism. The effect of GOM was thus more difficult to evaluate than that of AGE (Table 3; sham-A and AGE group). The daily quantities of the sunflower seed oil used to dilute GOM for sufficiently exact dosing and in the appropriate reference group was ca. 1 mL. According to the guidelines, this amount added to the rat's dietary fat intake does not influence the animal's condition regarding the clinical examination, hematological, biochemical and histological parameters (OECD, 2008). However, the increased daily fat intake was obviously sufficient to induce transcriptional/translational changes, which finally led to changes in HIV-PIs hepatic distribution and metabolism (significantly higher intracellular HIV-PI concentrations in rat liver slices and significantly higher formation and excretion rate of metabolites) compared to the reference values (Sham A group). Sunflower seed oil represents a good source of monounsaturated fatty acids (i.e. oleic acid), which have been known to reversibly increase the transcription of enzymes (among them also CYP) in liver and gut (Niot et al, 1997; Chen et al, 2001). The intake of higher fat quantities also interferes with endogenous lipid synthesis in metabolism, leading to changes in the plasma lipids and bile acid composition. To avoid cytotoxicity of the increased amounts of bile acids formed; these acids need to be excreted into the bile. Therefore, the increased production of bile acids had to be accompanied with the increased transporter expression to ensure cell survival. The excretion of bile acids into gallbladder involves the uptake of these endogenous compounds from plasma into hepatocytes with the aid of uptake transporters (i.e. NTCP, OATPs) and their excretion into the gallbladder by sinusoidal efflux proteins (Pgp, Mrp-2, S-Pgp, MDR3) (Meier et al, 1997a). Therefore the expression of these transporters in organisms

**3.2 The impact of long-term garlic supplementation on HIV-PIs hepatic**

also confirmed in that study (Berginc et al, 2010d).

**pharmacokinetics**

fed high fat diet would also increase, explaining the effects observed in this study on the hepatic HIV-PI pharmacokinetics in sunflower seed oil fed animals (sham-B group).

\* - the amounts of HIV-PIs and of the corresponding metabolites in the rat liver extracts or metabolite transport rates are significantly higher/lower than the corresponding reference values (sham-A and B group).

a – significantly higher HIV-PI amounts in the extract compared to the amounts of HIV-PIs, determined in the extracts of rat liver extracts from control sham-A group.

Table 3. Saq (Table 3A) and Dar (Table 3B) hepatic *in vitro* pharmacokinetics evaluated in rat liver slices obtained from rats fed with different garlic supplements (AGE or GOM groups), saline (sham-A group) or sunflower seed oil (sham-B group). The amounts of HIV-PIs and of the corresponding metabolites in the rat liver extracts and the metabolite transport rates from the rat liver slices into the incubation medium were determined.

Higher intrahepatic retention of HIV-PIs after prolonged garlic consumption could thus be explained by two factors. First, hepatocytes were obviously exposed to lower concentration of xenobiotics or their metabolites, responsible for the induction of Pgp compared to enterocytes. Namely after peroral absorption of garlic phytochemicals, lower amounts of garlic phytochemicals and their corresponding metabolites could reach hepatocytes due to incomplete/low xenobiotic absorption through the intestinal mucosa, binding to enterocytes mucosal membranes, instability in GIT lumen and intraluminal metabolism, which altogether contributed to less profound effect on the expression of efflux transporters in hepatocytes than in the jejunum (Ioannides, 2003; Lawson&Wang, 2005b). Besides efflux transporters, Saq and Dar also utilize OATP1A2 (OATP-A), 1B1 (OATP-C) and 1B3 (OATP-8) solute uptake transporters to distribute into the human liver (Hartkoorn et al, 2010). The sinusoidal rat hepatocyte membrane embodies numerous Oatp isoforms with Oatp1, Oatp2 and Oatp4 being the most important ones (Treiber et al, 2004; Meier et al, 1997b). Because AGE has been shown to affect Oatp activity (Berginc et al, 2010b), increased intrahepatic HIV-PI concentrations observed in this study could also correspond to the increased Oatp expression in the liver, which by its influence on the HIV-PIs pharmacokinetics could have exceeded the effects of efflux transporters.

First-Pass Metabolism Changes After Long-Term Garlic Supplementation 369

in the case of Pgp transporter. This resulted in altered Dar but not Saq metabolism. However this hypothesis will be difficult to confirm until more structural and functional information

The plasma levels of AGE and GOM constituents in plasma samples taken 24 hours after the last dosing were very low, resulting in signals near the limit of detection on a top-of-the line LC/MS/MS system. This fact primarily confirms that we were really observing the longterm influences of AGE and GOM exerted through altered enzyme and transporter expression in our experiments with the tissue samples rather than short-term allosteric or competitive effects of garlic constituents remaining in the isolated animal tissues. Nevertheless, significantly higher levels of garlic constituents were obtained in the plasma from AGE group compared to the plasma of GOM group (Table 4). The levels of S-allyl-Lcysteine, tangeretin and quercetin were 2, 5.4 and 8 times higher, respectively, in the plasma of AGE group compared to the GOM group. The relatively long period between the last dose and the plasma sampling allowed the majority of garlic-related metabolites to be excreted, so the only metabolite that was confidently quantified, was bergamottine glucuronide and it was present in the AGE group. The much higher concentration of garlic constituents and metabolites in the plasma of AGE group is in accordance with the results from the rat liver slices experiment, where it was shown that the influence of AGE supplementation on the Saq and Dar liver disposition was beyond that of GOM's (Table 3). It could be argued that the majority of GOM constituents and their metabolites were already eliminated especially through the lungs (they are volatile) before we performed experiments with the liver slice and before the plasma samples were taken, since the rats were fasting for 18 hours before the sacrifice. On the other hand, the long term changes of hepatic protein expression caused by GOM supplementation should have remained relatively unchanged after 2 weeks in spite of short period of food and GOM deprivation. Therefore, the conclusion of AGE's greater influence on HIV-PI's hepatic disposition remains unchanged.

**3.3 Plasma levels of AGE and GOM constituents and their metabolites**

is available about the binding sites of CYP3A enzymes.

Analyte MRM [m/z]

N/D - the response was below the limit of detection

(polarity)

ret. time [min]

Rutin 611->303 (+) 4,81 34 N/D N/D Bergammotine 339->203 (+) 10,35 N/D N/D N/D S-allyl-L-cysteine 162->145 (+) 2,32 1174 593 152 Adenosine 268->136 (+) 2,62 3041 2079 515 Tangeretine 373->343 (+) 8,55 1227 229 306 Quercetine 301->151 (-) 6,37 412 51 116 Bergammotin-glucuronide 515->339 (+) 6,96 1088 N/D N/D

Table 4. The multiple reaction monitoring mode (MRM) transitions used for the LC/MS/MS identification and quantification of garlic constituents in rat plasma (first two columns) and the peak areas obtained from the plasma samples of rats fed with AGE, GOM and the reference group (the last three columns: Plasma-AGE, Plasma-GOM and Plasma-REF, respectively).

Plasma-AGE [Area cps]

Plasma-GOM [Area cps]

Plasma-REF [Area cps]

These results suggest that the observed hepatic physiological adaptations to supplementation of garlic products or sunflower seed oil were exerted by garlic phytochemicals, components of sunflower seed oil and/or the corresponding metabolites. Because AGE lacks oily compounds that constitute GOM and/or sunflower seed oil, the substances responsible for the changes in membrane transporter expression, were most probably different. The differences in garlic supplement composition and/or plasma levels of garlic phytochemicals notwithstanding, the same final effect regarding HIV-PIs and HIV-PI-metabolite hepatic distribution were observed. The composition dissimilarities of garlic supplements and consequently of the produced metabolites also affected HIV-PIs Cyp3A metabolism in the liver slices differently. According to our results (Table 3), less Saq and especially Dar metabolites were formed in AGE group compared to the control sham-A group. However, in the presence of GOM and sunflower seed oil, Cyp metabolism of both investigated drugs significantly increased (note the significant increase of Saq-M1 and Dar-M4 in Table 3). Our results are in accordance with experimental data obtained in studies with various oily garlic supplements and AGE (Fischer et al, 2007). Garlic oil constituents, diallyl sulfide, diallyl disulfide and diallyl trisulfide have been found to increase the transcription of several hepatic enzymes (Cyp1A1, 2B1 and 3A1, NAD(P)H quinine oxidoreductase 1, glutathione-S-transferase) in wild type Wistar-Kyoto rats (Fischer et al, 2007), similarly to our findings in GOM group (increased production of HIV-PI metabolites and consequently their faster transport from hepatocytes into the incubation media). However, another clinical study on volunteers taking garlic extract (GarliPure) twice daily for 3 weeks indicated no impact of garlic phytochemicals on CYP3A4 expression in intestine or liver, while at the same time PGP expression significantly increased (Hajdaa et al, 2010). GarliPure extract is labeled to contain -glutamylcysteines (12 mg), alliin (4.8 mg), sulfur (3.9 mg), thiosulfinates (3.8 mg) and to have an allicin releasing potential, meaning that after consumption, this product enables *in vivo* allicin synthesis from alliin in ca 3.6 mg quantities. AGE, which we used to feed rats in this study, also predominately containes glutamylcysteines with S-allyl-L-cysteine being the main representative. However, AGE does not contain allicin because during the aging process, the content of lipophilic allicin degradation products declines and the compounds responsible for distinct garlic odor and unwanted side effects in AGE are thus minimized. However, based on our analysis, small amounts of these lipophilic phytochemicals are still present in AGE (Berginc et al, 2009). A recent analysis by Lawson and Wang (Lawson&Wang, 2001a) indicated that only one of 24 marketed allicin-releasing brands enabled the release of the designated amount of allicin, whereas the release from other supplements reached only 10 to 15% of the declared values. Therefore, one can assume that the compositions of GarliPure and of AGE used in this study are very similar. Similar to observations in the study with GarliPure impact on Saq pharmacokinetics in humans (Hajdaa et al, 2010) we also determined no impact of long term AGE supplementation on Cyp3A Saq metabolism in rats, indicating that the expression of this enzyme remained constant in spite of AGE administration. However in the case of Dar, AGE significantly decreased its metabolism. Similarly to multiple binding sites in efflux transporters, the CYP3A4 kinetic studies by Shou (Shou et al, 1994) performed on CYP3A4 transfected HepG2 cells indicated the existence of two binding sites in this enzyme. It is thus possible, that Saq and Dar, although metabolized by the same enzyme (i.e. CYP3A4), do not share identical binding sites on CYP3A4, which would be similar to their interaction with Pgp transporters. Phytochemicals or metabolites present in or derived from AGE (but not GOM) could have occupied the Dar but not Saq binding site on CYP3A enzyme, similarly as

These results suggest that the observed hepatic physiological adaptations to supplementation of garlic products or sunflower seed oil were exerted by garlic phytochemicals, components of sunflower seed oil and/or the corresponding metabolites. Because AGE lacks oily compounds that constitute GOM and/or sunflower seed oil, the substances responsible for the changes in membrane transporter expression, were most probably different. The differences in garlic supplement composition and/or plasma levels of garlic phytochemicals notwithstanding, the same final effect regarding HIV-PIs and HIV-PI-metabolite hepatic distribution were observed. The composition dissimilarities of garlic supplements and consequently of the produced metabolites also affected HIV-PIs Cyp3A metabolism in the liver slices differently. According to our results (Table 3), less Saq and especially Dar metabolites were formed in AGE group compared to the control sham-A group. However, in the presence of GOM and sunflower seed oil, Cyp metabolism of both investigated drugs significantly increased (note the significant increase of Saq-M1 and Dar-M4 in Table 3). Our results are in accordance with experimental data obtained in studies with various oily garlic supplements and AGE (Fischer et al, 2007). Garlic oil constituents, diallyl sulfide, diallyl disulfide and diallyl trisulfide have been found to increase the transcription of several hepatic enzymes (Cyp1A1, 2B1 and 3A1, NAD(P)H quinine oxidoreductase 1, glutathione-S-transferase) in wild type Wistar-Kyoto rats (Fischer et al, 2007), similarly to our findings in GOM group (increased production of HIV-PI metabolites and consequently their faster transport from hepatocytes into the incubation media). However, another clinical study on volunteers taking garlic extract (GarliPure) twice daily for 3 weeks indicated no impact of garlic phytochemicals on CYP3A4 expression in intestine or liver, while at the same time PGP expression significantly increased (Hajdaa et al, 2010). GarliPure extract is labeled to contain -glutamylcysteines (12 mg), alliin (4.8 mg), sulfur (3.9 mg), thiosulfinates (3.8 mg) and to have an allicin releasing potential, meaning that after consumption, this product enables *in vivo* allicin synthesis from alliin in ca 3.6 mg quantities. AGE, which we used to feed rats in this study, also predominately containes glutamylcysteines with S-allyl-L-cysteine being the main representative. However, AGE does not contain allicin because during the aging process, the content of lipophilic allicin degradation products declines and the compounds responsible for distinct garlic odor and unwanted side effects in AGE are thus minimized. However, based on our analysis, small amounts of these lipophilic phytochemicals are still present in AGE (Berginc et al, 2009). A recent analysis by Lawson and Wang (Lawson&Wang, 2001a) indicated that only one of 24 marketed allicin-releasing brands enabled the release of the designated amount of allicin, whereas the release from other supplements reached only 10 to 15% of the declared values. Therefore, one can assume that the compositions of GarliPure and of AGE used in this study are very similar. Similar to observations in the study with GarliPure impact on Saq pharmacokinetics in humans (Hajdaa et al, 2010) we also determined no impact of long term AGE supplementation on Cyp3A Saq metabolism in rats, indicating that the expression of this enzyme remained constant in spite of AGE administration. However in the case of Dar, AGE significantly decreased its metabolism. Similarly to multiple binding sites in efflux transporters, the CYP3A4 kinetic studies by Shou (Shou et al, 1994) performed on CYP3A4 transfected HepG2 cells indicated the existence of two binding sites in this enzyme. It is thus possible, that Saq and Dar, although metabolized by the same enzyme (i.e. CYP3A4), do not share identical binding sites on CYP3A4, which would be similar to their interaction with Pgp transporters. Phytochemicals or metabolites present in or derived from AGE (but not GOM) could have occupied the Dar but not Saq binding site on CYP3A enzyme, similarly as in the case of Pgp transporter. This resulted in altered Dar but not Saq metabolism. However this hypothesis will be difficult to confirm until more structural and functional information is available about the binding sites of CYP3A enzymes.

#### **3.3 Plasma levels of AGE and GOM constituents and their metabolites**

The plasma levels of AGE and GOM constituents in plasma samples taken 24 hours after the last dosing were very low, resulting in signals near the limit of detection on a top-of-the line LC/MS/MS system. This fact primarily confirms that we were really observing the longterm influences of AGE and GOM exerted through altered enzyme and transporter expression in our experiments with the tissue samples rather than short-term allosteric or competitive effects of garlic constituents remaining in the isolated animal tissues. Nevertheless, significantly higher levels of garlic constituents were obtained in the plasma from AGE group compared to the plasma of GOM group (Table 4). The levels of S-allyl-Lcysteine, tangeretin and quercetin were 2, 5.4 and 8 times higher, respectively, in the plasma of AGE group compared to the GOM group. The relatively long period between the last dose and the plasma sampling allowed the majority of garlic-related metabolites to be excreted, so the only metabolite that was confidently quantified, was bergamottine glucuronide and it was present in the AGE group. The much higher concentration of garlic constituents and metabolites in the plasma of AGE group is in accordance with the results from the rat liver slices experiment, where it was shown that the influence of AGE supplementation on the Saq and Dar liver disposition was beyond that of GOM's (Table 3). It could be argued that the majority of GOM constituents and their metabolites were already eliminated especially through the lungs (they are volatile) before we performed experiments with the liver slice and before the plasma samples were taken, since the rats were fasting for 18 hours before the sacrifice. On the other hand, the long term changes of hepatic protein expression caused by GOM supplementation should have remained relatively unchanged after 2 weeks in spite of short period of food and GOM deprivation. Therefore, the conclusion of AGE's greater influence on HIV-PI's hepatic disposition remains unchanged.

N/D - the response was below the limit of detection

Table 4. The multiple reaction monitoring mode (MRM) transitions used for the LC/MS/MS identification and quantification of garlic constituents in rat plasma (first two columns) and the peak areas obtained from the plasma samples of rats fed with AGE, GOM and the reference group (the last three columns: Plasma-AGE, Plasma-GOM and Plasma-REF, respectively).

First-Pass Metabolism Changes After Long-Term Garlic Supplementation 371

Berginc, K., Trontelj, J., Kristl, A. (2010a) The influence of aged garlic extract on the uptake

Berginc, K., Žakelj, S., Kristl, A. (2010b). In vitro interactions between aged garlic extract and

Berginc, K., Trdan, T., Trontelj, J., Kristl, A. (2010c). HIV protease inhibitors: garlic

Berginc, K., Milisav, I., Kristl, A. (2010d). Garlic flavonoids and organosulfur compounds:

Sun, D. (2006). Why is it Challenging to Predict Intestinal Drug Absorption and Oral

Chen, H.W., Yang, J.J., Tsai, C.W., Wu, J.J., Sheen, L.Y., Ou, C.C., Lii, C.K. (2001).  Selective biotransformation of the human

Foster, B.C., Foster, M.S., Vandenhoek, S., Krantis, A., Budzinski, J.W., Arnason, J.T.,

Hartkoorn, R.C., Kwan, W.S., Shallcross, V., Chaikan, A., Liptrott, N., Egan, D., Sora, E.S.,

Pharmaceutical Sciences, Vol. 4, No. 2, pp. 176 – 184, ISSN 1482-1826. Hajdaa, J., Rentschc, K.M., Gublerd, C., Steinerte, H., Stiegera, B., Fattingerf, K. (2010). Garlic

No. 5 (December 2010), pp. 729 – 735, ISSN 0928-0987.

James, C.E., Gibbons, S., Bray, P.G.,

*and Pharmacokinetics*, Vol. 25, No. 3, pp. 307 – 313, ISSN 1347-4367.

*Journal of Nutrition*, Vol. 49, No.6, pp. 373 – 384, ISSN 1436-6207.

0142-2782.

Zhong, Y., Amidon, G.L., Yu, L.X.,

pp. 1675 – 1686, ISSN 0724-8741.

277, ISSN 0928-0987.

266, ISSN 0090-9556.

No. 5, pp. 1438 – 1443 ISSN 0022-3166.

of saquinavir and darunavir into HepG2 cells and rat liver slices. *Drug Metabolism*

drugs used for the treatment of cardiovascular and diabetic patients. *European*

supplements and first-pass intestinal metabolism impact on the therapeutic efficacy. *Biopharmaceutics & Drug disposition*, Vol. 31, No. 8-9, pp. 495 – 505, ISSN

impact on the hepatic pharmacokinetics of saquinavir and darunavir. *Drug Metabolism and Pharmacokinetics*, Vol. 25, No. 6, pp. 521 – 531, ISSN 1347-4367. Cao, X., Gibbs, S.T., Fang, L., Miller, H.A., Landowski, C.P., Shin, H.C., Lennernas, H.,

Bioavailability in Human Using Rat Model. *Pharmaceutical Research,* Vol. 23, No. 8,

and garlic oil independently regulate hepatic cytochrome P450 2B1 and the placental form of glutathione-S-transferase expression in rats. *Journal of Nutrition*, Vol. 131,

A., Artursson, P. (2006). Regional levels of drug transporters along human intestinal tract: Co-expression of ABC and SLC transporters and comparison with Caco-2 cells. *European Journal of Pharmaceutical Sciences*, Vol 29, No. 3-4, pp. 269 –

Lehman-McKeeman, L.D., Cherrington, N.J. (2007). Induction of drugmetabolyzing enzymes by garlic and allyl sulfide compounds via activation of constituitive androstane receptor and nuclear factor E2-related factor 2. *Drug Metabolism & Disposition*, Vol. 35, No. 6 (June 2007), pp. 995 – 1000, ISSN 0090-9556.

immunodeficiency virus protease inhibitor saquinavir by human small-intestinal cytochrome P4503A4. *Drug Metabolism and Dispossition*, Vol 25, No. 2, pp. 256 –

Gallicano, K.D., Choudri, S. An *in Vitro* Evaluation of Human Cytochrome P450 3A4 and P-glycoprotein Inhibition by Garlic. (2001). Journal of Pharmacy and

extract induces gastrointestinal P-glycoprotein, but exhibits no effect on intestinal and hepatic CYP3A4 in humans. *European Journal of Pharmaceutical Sciences*, Vol. 41,

Furthermore, based on the plasma profiles from rats exposed to different garlic supplements, it can be concluded that constituents and their metabolites, although structurally and chemically different, induced similar hepatic effects regarding the transporter expression but exerted significantly different effect on CYP3A metabolism of both compounds. The affinities of these metabolites or their parent phytochemicals from both supplements for CYP3A are evidently different, because AGE constituents/metabolites inhibited/did not affect Cyp3A HIV-PI metabolism, whereas HIV-PI Cyp3A metabolism increased after long-term GOM supplementation.

#### **4. Conclusion**

Although applied at the highest possible donor concentrations, both drugs (Saq, Dar) displayed very low permeability in the absorptive direction and were subjected to a profound efflux from enterocytes back into intestinal lumen, indicating that the intestinal wall represents an important biological barrier for attaining therapeutic HIV-PIs plasma levels.

A concomitant consumption of garlic supplements together with HIV-PIs places the infected patients at higher risk for therapy failure, because the components of garlic supplements and/or their metabolites readily bind to efflux transporters. This binding changes the activities of efflux transporters by allosteric modifications in short-term and leads to an increased expression in the long run. Although two garlic supplements with different compositions were used, ABC transporter activity in the intestine increased similarly in both cases. Long-term garlic supplementation also affected hepatic metabolism and distribution of both HIV-PIs. Contrary to the effects in the intestine, the up-regulation of efflux transporters in the liver was less important than the increased expression of uptake Oatps transporters, which resulted in the overall increased intrahepatic HIV-PI amounts that could be further subjected to Cyp3A metabolism. Although the effects of AGE and GOM were similar regarding the transporter expression, their effects on Cyp3A metabolism were significantly different. AGE inhibited Dar but not Saq Cyp3A metabolism, while in GOM treated rats the Cyp3A metabolism of both drugs significantly increased, highlighting important dissimilarities in plasma profiles of garlic phytochemicals and the corresponding metabolites, as shown in this study. Given the fact that garlic supplements are so widely used in the HIV infected population because of their protective cardiovascular and antiinfective effects, further research should be directed at the identification of high- and lowrisk supplement – drug combinations.

#### **5. References**

Furthermore, based on the plasma profiles from rats exposed to different garlic supplements, it can be concluded that constituents and their metabolites, although structurally and chemically different, induced similar hepatic effects regarding the transporter expression but exerted significantly different effect on CYP3A metabolism of both compounds. The affinities of these metabolites or their parent phytochemicals from both supplements for CYP3A are evidently different, because AGE constituents/metabolites inhibited/did not affect Cyp3A HIV-PI metabolism, whereas HIV-PI Cyp3A metabolism

Although applied at the highest possible donor concentrations, both drugs (Saq, Dar) displayed very low permeability in the absorptive direction and were subjected to a profound efflux from enterocytes back into intestinal lumen, indicating that the intestinal wall represents an important biological barrier for attaining therapeutic HIV-PIs plasma

A concomitant consumption of garlic supplements together with HIV-PIs places the infected patients at higher risk for therapy failure, because the components of garlic supplements and/or their metabolites readily bind to efflux transporters. This binding changes the activities of efflux transporters by allosteric modifications in short-term and leads to an increased expression in the long run. Although two garlic supplements with different compositions were used, ABC transporter activity in the intestine increased similarly in both cases. Long-term garlic supplementation also affected hepatic metabolism and distribution of both HIV-PIs. Contrary to the effects in the intestine, the up-regulation of efflux transporters in the liver was less important than the increased expression of uptake Oatps transporters, which resulted in the overall increased intrahepatic HIV-PI amounts that could be further subjected to Cyp3A metabolism. Although the effects of AGE and GOM were similar regarding the transporter expression, their effects on Cyp3A metabolism were significantly different. AGE inhibited Dar but not Saq Cyp3A metabolism, while in GOM treated rats the Cyp3A metabolism of both drugs significantly increased, highlighting important dissimilarities in plasma profiles of garlic phytochemicals and the corresponding metabolites, as shown in this study. Given the fact that garlic supplements are so widely used in the HIV infected population because of their protective cardiovascular and antiinfective effects, further research should be directed at the identification of high- and low-

Amagase, H. (2006) Clarifying the real bioactive constituents of garlic. *Journal of Nutrition*,

Aungst, B.J. (1999). P-glycoprotein, secretory transport, and other barriers to the oral

Berginc, K., Žakelj, S., Uršič, D., Kristl, A. (2009). Aged garlic extract stimulates P-

delivery of anti-HIV drugs. *Advanced Drug Delivery Reviews*, Vol. 39, No. 1-3

glycoprotein and Multidrug Resistance Associated Protein 2 mediated effluxes. *Biological&Pharmaceutical Buletin*, Vol.32, No. 4694, pp. 694 – 699, ISSN 0918-6158..

Vol. 136, No. 3, pp. 716S – 725S, ISSN 0022-3166.

(October 1999), pp. 105 – 116, ISSN 0169-409X.

increased after long-term GOM supplementation.

risk supplement – drug combinations.

**5. References**

**4. Conclusion**

levels.

First-Pass Metabolism Changes After Long-Term Garlic Supplementation 373

OECD guidelines for the testing of chemicals 407 - Repeated Dose 28-Day Oral Toxicity

Orenstein, J.M., Fox, C., Wahl, S.M. (1997). Macrophages as a Source of HIV During

Pal, D., Mitra, A.K. (2006). MDR- and CYP3A4-mediated drug–herbal interactions. *Life Sciences*, Vol. 78, No. 18 (March 1997), pp. 2131 – 2145, ISSN 0024-3205. Piscitelli, S.C., Burstein, A.H., Welden, N., Gallicano, K.D., Falloon, J. (2002). The effect of

Saksena, N.K., Wang, B., Zhou, L., Soedjono, M., Ho, Y.S., Conceicao, V. (2010). HIV

reservoir sites. *HIV/AIDS – Research and Pallative Care*, Vol. 2, pp. 103 – 122. Shou, M., Grogan, J., Mancewicz, J.A., Krausz, K.W., Gonzalez, F.J., Gelboin, H.V.,

CYP3A4: Evidence for the simultaneous binding of two substrates in a Cytochrome P450

Treiber, A., Schneiter, R., Delahaye, S., Clozel, M. (2004). Inhibition of Organic Anion

Hepatic Uptake Is the Major Determinant in the Pharmacokinetic Interaction between

*Therapeutics*, Vol. 308, No. 3 (March 2004), pp. 1121 – 1129, ISSN 0022-3565. Unger, R.H. (2003). Lipid overload and overflow: metabolic trauma and the metabolic

Usansky, H.H., Hu, P., Sinko, P.J. (2008). Differential roles of P-glycoprotein, Multidrug-

Vermeir, M., Lachau-Durand, S., Mannens, G., Cuyckens, F., van Hoof, B., Raoof, A. (2009).

*Metabolism and Disposition*, Vol. 37, No. 4, pp. 809 – 820, ISSN 0090-9556. Wu, C.Y., Benet, L.Z.(2005). Predicting drug disposition via application of BCS:

Yoshida, H., Katsuzaki, H., Ohta, R., Ishikawa, K., Fukuda, H., Fujino, T., Suzuki, A. (1999).

Yu, L.X., Amidon, G.L., Polli, J.E., Zhao, H., Mehta, M.U., Conner, D.P., Shah, V.P., Lesko,

*Diseases*, Vol. 34, No. 2, pp. 234 – 238, ISSN 1058-4838.

Korzekwa, K.R. (1994). Activation of

Transporting Polypeptide-Mediated

pp. 398 – 403, ISSN 1043-2760.

pp. 863 – 869, ISSN 0090-9556.

588 – 590, ISSN 0916-8451.

Vol. 22, No. 1, pp. 11 – 23, ISSN 0724-8741.

19, No. 7 (July 2002), pp. 921 925, ISSN 0724-8741.

Opportunistic Infections. *Science*, Vol.7, No. 5320 (June 1997), pp. 1857 – 1861, ISSN

garlic supplements on the pharmacokinetics of saquinavir. *Clinical Infectious*

reservoirs in vivo and new strategies for possible eradication of HIV from the

active site. *Biochemistry*, Vol. 33, No. 21 (May 1994), pp. 6450 – 6455, ISSN 0006-

Bosentan and Cyclosporin A in the Rat. *Journal of Pharmacology and Experimental*

syndrome. *Trends in Endocrinology and Metabolism*, Vol. 14, No. 9 (November 2003),

Resistance Associated Protein 2, and Cyp3A on Saquinavir oral absorption in Sprague-Dawley rats. *Drug Metabolism and Disposition*, Vol. 36, No. 5 (May 2008),

Absorption, metabolism, and excretion of darunavir, a new protease inhibitor, administered alone and with low-dose ritonavir in healthy subjects. *Drug*

Transporter/absorption/elimination interplay and development of a biopharmaceutics drug disposition classification system. *Pharmaceutical Research*,

An Organosulfur Compound Isolated from Oil-Macerated Garlic Extract, and Its Antimicrobial Effect. *Bioscience, Biotechnology and Biochemistry*, Vol. 63, No. 3, pp.

L.J., Chen, M.L., Lee, V.H.L., Hussain, A.S. (2002). Biopharmaceutics Classification System: The Scientific Basis for Biowaiver Extensions. *Pharmaceutical Research*, Vol.

Study in Rodents. 2008.

0036-8075.

2960.

Back, D.J., Khoo, S.H., Owen, A. (2010). HIV protease inhibitors are substrates for OATP1A2,

Hogg, R.S., Bangsberg, D.R., Lima, V.D., Alexander, C., Wood, S.E., Dong, W.W.Y.,

Emergence of drug resistance is associated with an increased risk of death among patients

Holmstock, N., Mols, R., Annaert, P., Augustujns, P. (2010). In situ intestinal perfusion in

Ioannides, C. (2003). Drug–phytochemical interactions. *Inflammopharmacology*, Vol. 11, No. 1,

Kakuda, T.N., Kiser, J.J. (2006). 7th international workshop on clinical pharmacology of HIV

Kis, O., Robillard, K., Chan, G.N.Y., Bendayan, R. (2009). The complexities of antiretroviral

Lawson, L.D., Wang, Z.J. (2001a). Low allicin release from garlic supplements: a major

Methyl Sulfide: Use in Measuring Allicin Bioavailability. *Journal of Agricultural and Food Chemistry*, Vol. 53, No. 6 (March 2006), pp. 1974 – 1983, ISSN 0021-8561. Löscher, W., Potschka, H. (2005). Drug resistance in brain diseases and the role of drug

MacLean, C., Moenning, U., Reichel, A., Fricker, G. (2008). Closing the gaps: A full scan of

Martin, C., Berridge, G., Higgins, C.F., Mistry, P., Charlton, P., Callagan, R. (2000).

*Pharmacology*, Vol. 58, No. 3 (September 2000), pp. 624-632, ISSN 0026-895x. Meier, P.J., Eckhardt, U., Schroeder, A., Hagenbuch, B., Stieger, B. (1997). Substrate

Niot, I., Poirier, H., Besnard, P.H. (1997). Regulation of gene expression by fatty acids:

(February-March 1997), pp. 129 – 133, ISSN 0300-9084.

*Chemistry*, Vol. 49, No. 5 (April 2001), pp. 2592 – 2599, ISSN 0021-8561. Lawson, L.D., Wang, Z.J. (2005b). Allicin and Allicin-Derived Garlic Compounds Increase

*Sciences*, Vol. 31, No. 1 (January 2009), pp. 22 – 35, ISSN 0165-6147.

No. 9 (September 2010), pp. 1407 – 1410, ISSN 0090-9556.

2010), pp. 112 – 120, ISSN 1744-6872.

ISSN 1549-1277.

pp. 7 – 42.

1533, ISSN 1472-4683.

Breath Acetone through Allyl

591 – 602, ISSN 1471-0048

9556.

0270-9139.

Montaner, J.S.G., Harrigan, P.R. (2006).

OATP1B1 and OATP1B3 and lopinavir plasma concentrations are influenced by SLCO1B1 polymorphisms. *Pharmacogenetics & Genomics*, Vol. 2, No. 2 (February

first starting HAART. *PloS Medicine*, Vol. 3, No. 9,(September 2006), pp. 1570 – 1578,

knockout mice demonstrates inhibition of intestinal P-glycoprotein by ritonavir causing increased darunavir absorption. *Drug Metabolism and Disposition,* Vol. 38,

therapy. Expert Opinion on Pharmacotherapy. Lisbon, Vol. 7, No. 11, pp. 1519 –

drug-drug interactions: role of ABC and SLC transporters. *Trends in Pharmacological*

problem due to the sensitivities of allinase activity. *Journal of Agricultural and Food*

efflux transporters*. Nature Reviews Neuroscience*, Vol. 6, No. 8 (August 2005), pp.

the intestinal expression P-glycoprotein, Breast Cancer Resistance Protein, and Multidrug Resistance Associated Protein 2 in male and female rats. *Drug Metabolism and Disposition*, Vol. 36, No. 7, (july 2008), pp. 1249 – 1254, ISSN 0090-

Communication between Multiple Drug Binding Sites on P-glycoprotein. *Molecular*

specificity of sinusoidal bile acid and organic anion uptake systems in rat and human liver. *Hepatology*, Vol. 26, No. 6 (December 1997), pp. 1667 – 1677, ISSN

special reference to fatty acid-binding protein (FABP). *Biochimie*, Vol. 97, No. 2-3

**16**

*Scotland*

**Anti-Trypanosomal Activity and Cytotoxicity**

John O. Igoli, Alexander I. Gray, Carol J. Clements and Hazar A. Mouad

Human African trypanosomiasis, also known as sleeping sickness, is a vector-borne parasitic disease. The parasites concerned are protozoa belonging to the *Trypanosoma* genus. They are transmitted to humans by tsetse fly (*Glossina* genus) which have acquired their infection from human beings or from animals harbouring the human pathogenic parasites. Rural populations living in regions where transmission occurs and which depend on agriculture, fishing, animal husbandry or hunting are the most exposed to the disease. Sleeping sickness threatens millions of people in 36 countries in sub-Saharan Africa. Many of the affected populations live in remote areas with limited access to adequate health services. Treatment depends on drugs that can cross the blood-brain barrier to reach the parasite. Such drugs are toxic and complicated to administer. Suramin, discovered in 1921, is one of the drugs used for the treatment. However, it provokes certain undesirable effects, in the urinary tract and allergic reactions. Other drugs in use are equally toxic and difficult to administer. Thus the need to develop new drugs is imperative. Natural products and medicinal plants (Antia et al, 2009; Sara et al, 2004; Bizimana et al., 2006; Okpekon et al., 2004) have continued to contribute to the search for new and potent anti-trypanosomal drugs with minimal side effects. Their continued relevance in drug discovery and development is due to their bioactivity, biodiversity and reported safety in their use. An analysis of new and approved drugs for the treatment of human diseases indicated that natural products have continued to play a highly significant role in the drug discovery and development process. Thus biologically active natural products from plants, their derivatives or analogues contributed up to 57% of top selling prescription drugs in the United States in 1997 (Newman et al., 1997). This dominant role is due to the leads for the development of drugs that natural products give. Thus expanding, not decreasing, the exploration of nature as a source of novel active agents that may serve as leads and scaffolds for elaboration into desperately needed efficacious drugs for a multitude of disease indications is advocated (Newman & Cragg, 2007). Ethnobotanical surveys have revealed the use of *Alcornea cordifolia*, *Euphorbia poisonii*, *Monodora myristica*, *Prosopis africana*, *Spondias mombim, Nauclea pobeguinii, Terminalia avicennioides, Cochlospermum planchonii, Nauclea latifolia* and *Withania somnifera* in the traditional medicine in parts of Nigeria (Igoli et. al., 2005). These plants are used as spices and medicinally for the treatment of diarrhoea,

**1. Introduction**

 **of Some Compounds and Extracts from**

*Natural Product Laboratories, SIPBS, University of Strathclyde, Glasgow*

**Nigerian Medicinal Plants**

Zhang, L., Zhang, Y.D., Strong, J.M., Reynolds, K.S., Huang, S.M. (2008). A regulatory viewpoint on transporter-based drug interactions. *Xenobiotica*, Vol. 38, No. 7-8, pp. 709 – 724, ISSN 0049-8254.

### **Anti-Trypanosomal Activity and Cytotoxicity of Some Compounds and Extracts from Nigerian Medicinal Plants**

John O. Igoli, Alexander I. Gray, Carol J. Clements and Hazar A. Mouad *Natural Product Laboratories, SIPBS, University of Strathclyde, Glasgow Scotland*

#### **1. Introduction**

374 Phytochemicals – Bioactivities and Impact on Health

Zhang, L., Zhang, Y.D., Strong, J.M., Reynolds, K.S., Huang, S.M. (2008). A regulatory

709 – 724, ISSN 0049-8254.

viewpoint on transporter-based drug interactions. *Xenobiotica*, Vol. 38, No. 7-8, pp.

Human African trypanosomiasis, also known as sleeping sickness, is a vector-borne parasitic disease. The parasites concerned are protozoa belonging to the *Trypanosoma* genus. They are transmitted to humans by tsetse fly (*Glossina* genus) which have acquired their infection from human beings or from animals harbouring the human pathogenic parasites. Rural populations living in regions where transmission occurs and which depend on agriculture, fishing, animal husbandry or hunting are the most exposed to the disease. Sleeping sickness threatens millions of people in 36 countries in sub-Saharan Africa. Many of the affected populations live in remote areas with limited access to adequate health services. Treatment depends on drugs that can cross the blood-brain barrier to reach the parasite. Such drugs are toxic and complicated to administer. Suramin, discovered in 1921, is one of the drugs used for the treatment. However, it provokes certain undesirable effects, in the urinary tract and allergic reactions. Other drugs in use are equally toxic and difficult to administer. Thus the need to develop new drugs is imperative. Natural products and medicinal plants (Antia et al, 2009; Sara et al, 2004; Bizimana et al., 2006; Okpekon et al., 2004) have continued to contribute to the search for new and potent anti-trypanosomal drugs with minimal side effects. Their continued relevance in drug discovery and development is due to their bioactivity, biodiversity and reported safety in their use. An analysis of new and approved drugs for the treatment of human diseases indicated that natural products have continued to play a highly significant role in the drug discovery and development process. Thus biologically active natural products from plants, their derivatives or analogues contributed up to 57% of top selling prescription drugs in the United States in 1997 (Newman et al., 1997). This dominant role is due to the leads for the development of drugs that natural products give. Thus expanding, not decreasing, the exploration of nature as a source of novel active agents that may serve as leads and scaffolds for elaboration into desperately needed efficacious drugs for a multitude of disease indications is advocated (Newman & Cragg, 2007). Ethnobotanical surveys have revealed the use of *Alcornea cordifolia*, *Euphorbia poisonii*, *Monodora myristica*, *Prosopis africana*, *Spondias mombim, Nauclea pobeguinii, Terminalia avicennioides, Cochlospermum planchonii, Nauclea latifolia* and *Withania somnifera* in the traditional medicine in parts of Nigeria (Igoli et. al., 2005). These plants are used as spices and medicinally for the treatment of diarrhoea,

Anti-Trypanosomal Activity and Cytotoxicity

for each test species and cell line.

**2.1.1 Anti-trypanosomal activity**

concentration after a 1:1 dilution of 20µg/ml / 20µM.

Table 1. Anti-trypanosomiasis assay plate layout.

the initial screen.

of Some Compounds and Extracts from Nigerian Medicinal Plants 377

Wells containing active compounds are easily identified as they remain blue in colour and have background levels of fluorescence. The test samples are initially screened at a single concentration and then MIC or IC 50 values are determined for the active compounds at n=2 or n=3. Positive and negative controls and a sterility checks are included in all assays. The incubation and treatment times, incubation conditions and seeding densities are optimised

Samples were tested against the bloodstream form of *Trypanosoma brucei brucei* (*T.b.brucei*) S427. The activity of the plant extracts and isolated compounds was determined using the well-established Alamar blue™ 96 well microplate assay. Samples were initially screened at a single concentration usually 20µg/ml for extracts or 20µM for pure compounds. The concentration of dimethylsulphoxide (DMSO) should not exceed 0.5% in

Samples for testing were prepared as 10mg/ml or 10mM stock solutions in 100% DMSO. These were diluted with HMI-9 medium to a concentration of 1mg/ml /1mM. 4µl of the test sample was added to the assay well then 96µl HMI-9 medium to give a final assay

**1 2 3 4 5 6 7 8 9 10 11 12 A** Sterility control 1 2 3 4 5 6 7 8 9 10 1.0µM

**B** control 11 12 13 14 15 16 17 18 19 20 0.5µM

**C** control 21 22 23 24 25 26 27 28 29 30 0.25µM

**D** control 31 32 33 34 35 36 37 38 39 40 0.125µM

**E** DMSO control 41 42 43 44 45 46 47 48 49 50 0.062µM

**F** DMSO control 51 52 53 54 55 56 57 58 59 60 0.031µM

**G** DMSO control 61 62 63 64 65 66 67 68 69 70 0.015µM

**H** DMSO control 71 72 73 74 75 76 77 78 79 80 0.008µM

The screening plate was arranged as illustrated in Table 1. Controls including a sterility control and DMSO controls in column 1; test samples in columns 2 to 11 and a concentration range of suramin as a positive control in column12. Trypanosomes were counted using a haemocytometer and diluted to a concentration of 3x104 trypanosomes /ml, 100µl of this suspension was added to each well of the assay plate with the exception of well A1 the

suramin

suramin

suramin

suramin

suramin

suramin

suramin

suramin

dysentery, diabetes, bacterial infections, fevers, pain and snake bites amongst other ailments by the Igede people of Benue State in Nigeria. *Maytenus laevis* is used in traditional medicine in Colombia (Nagakawa et.al., 2004; Gonzalez et al., 1982) against pain and arthritis. Before the introduction of synthetically prepared medicines, herbal remedies were commonly prescribed and were often effective. Thus herbs and spices played very important, sometimes magical, roles in medicine. Hence these plants were collected and their extracts/ compounds isolated were evaluated for anti-trypanosomal and cytotoxic activities. A high throughput (HTS) method was used to screen the extracts and compounds. This has become very useful in modern drug discovery from natural products. It is a form of standardised and replicate screening method which employs 96 well plates which can be used to screen 80 extracts, compounds or fractions at the same time. It can also be adapted to obtain data to plot dose response curves and determine IC50 and MIC values. Using a multilabel counter, suitable indicators, wells with extracts, cells, organisms and controls can be read quickly by examining fluorescence, luminescence and optical density or by radiometry giving exact and replicable values free from errors of visual determinations or measurements. The volumes used in these wells are between 200-250µL and for half well plates 20-40µL such that solutions of extracts or compounds as low as 1mg dissolved in 100µL gives a stock solution of 10mg/ml which can be diluted for all range of concentrations typically from 20- 30µg/ml to ng or µM to nM values using serial dilution methods. This greatly enhances speed and efficacy of screening for hit molecules (Gray et al., 2011). Efforts were made to identify the active compounds contained in the extracts as much as possible. This is because it is the practice today to screen more and more pure compounds rather than crude extracts or fractions. Screening of crude extracts or mixtures/fractions does not allow understanding of the mode of activity of the test compounds against the target cells or tissues. Isolation of the active compounds is required before active concentrations can be determined. We hereby report on these plant extracts, the compounds obtained from them, their *in vitro* activity against the bloodstream form of *Trypanosoma brucei brucei* (*T.b.brucei*) and the cytotoxic activity of the active compounds/extracts against PNT2A cells.

#### **2. Materials and methods**

Standard methods of extraction and isolation or purification of compounds/extracts from plant materials were used while a standardised 96 well based assay was used to screen the extracts for anti-trypanosomal activity and cytotoxicity. The crude extracts as well as pure compounds obtained were screened *in vitro* for trypanosomal activity against T*rypanosoma brucei brucei* (S427) blood stream forms. Active compounds from the preliminary screens were further tested to confirm activity and determine their minimum inhibitory concentration (MIC) values. Cytotoxicity of the active compounds and extracts was then tested against PNT2A cells ("normal" prostatic cells).

#### **2.1 General bioassay**

The anti-trypanosomal and cytotoxicity assays were performed using the REDOX indicator Alamar blue™. The active component of which is resazurin (blue in colour) which in the presence of live parasites or cells is reduced to the bright pink fluorescent resorufin. The fluorescence values for the test plates are measured using a microplate reader in fluorescence mode with excitation and emission wavelengths of 560 and 590nm respectively. Wells containing active compounds are easily identified as they remain blue in colour and have background levels of fluorescence. The test samples are initially screened at a single concentration and then MIC or IC 50 values are determined for the active compounds at n=2 or n=3. Positive and negative controls and a sterility checks are included in all assays. The incubation and treatment times, incubation conditions and seeding densities are optimised for each test species and cell line.

#### **2.1.1 Anti-trypanosomal activity**

376 Phytochemicals – Bioactivities and Impact on Health

dysentery, diabetes, bacterial infections, fevers, pain and snake bites amongst other ailments by the Igede people of Benue State in Nigeria. *Maytenus laevis* is used in traditional medicine in Colombia (Nagakawa et.al., 2004; Gonzalez et al., 1982) against pain and arthritis. Before the introduction of synthetically prepared medicines, herbal remedies were commonly prescribed and were often effective. Thus herbs and spices played very important, sometimes magical, roles in medicine. Hence these plants were collected and their extracts/ compounds isolated were evaluated for anti-trypanosomal and cytotoxic activities. A high throughput (HTS) method was used to screen the extracts and compounds. This has become very useful in modern drug discovery from natural products. It is a form of standardised and replicate screening method which employs 96 well plates which can be used to screen 80 extracts, compounds or fractions at the same time. It can also be adapted to obtain data to plot dose response curves and determine IC50 and MIC values. Using a multilabel counter, suitable indicators, wells with extracts, cells, organisms and controls can be read quickly by examining fluorescence, luminescence and optical density or by radiometry giving exact and replicable values free from errors of visual determinations or measurements. The volumes used in these wells are between 200-250µL and for half well plates 20-40µL such that solutions of extracts or compounds as low as 1mg dissolved in 100µL gives a stock solution of 10mg/ml which can be diluted for all range of concentrations typically from 20- 30µg/ml to ng or µM to nM values using serial dilution methods. This greatly enhances speed and efficacy of screening for hit molecules (Gray et al., 2011). Efforts were made to identify the active compounds contained in the extracts as much as possible. This is because it is the practice today to screen more and more pure compounds rather than crude extracts or fractions. Screening of crude extracts or mixtures/fractions does not allow understanding of the mode of activity of the test compounds against the target cells or tissues. Isolation of the active compounds is required before active concentrations can be determined. We hereby report on these plant extracts, the compounds obtained from them, their *in vitro* activity against the bloodstream form of *Trypanosoma brucei brucei* (*T.b.brucei*) and the

cytotoxic activity of the active compounds/extracts against PNT2A cells.

tested against PNT2A cells ("normal" prostatic cells).

Standard methods of extraction and isolation or purification of compounds/extracts from plant materials were used while a standardised 96 well based assay was used to screen the extracts for anti-trypanosomal activity and cytotoxicity. The crude extracts as well as pure compounds obtained were screened *in vitro* for trypanosomal activity against T*rypanosoma brucei brucei* (S427) blood stream forms. Active compounds from the preliminary screens were further tested to confirm activity and determine their minimum inhibitory concentration (MIC) values. Cytotoxicity of the active compounds and extracts was then

The anti-trypanosomal and cytotoxicity assays were performed using the REDOX indicator Alamar blue™. The active component of which is resazurin (blue in colour) which in the presence of live parasites or cells is reduced to the bright pink fluorescent resorufin. The fluorescence values for the test plates are measured using a microplate reader in fluorescence mode with excitation and emission wavelengths of 560 and 590nm respectively.

**2. Materials and methods**

**2.1 General bioassay**

Samples were tested against the bloodstream form of *Trypanosoma brucei brucei* (*T.b.brucei*) S427. The activity of the plant extracts and isolated compounds was determined using the well-established Alamar blue™ 96 well microplate assay. Samples were initially screened at a single concentration usually 20µg/ml for extracts or 20µM for pure compounds. The concentration of dimethylsulphoxide (DMSO) should not exceed 0.5% in the initial screen.

Samples for testing were prepared as 10mg/ml or 10mM stock solutions in 100% DMSO. These were diluted with HMI-9 medium to a concentration of 1mg/ml /1mM. 4µl of the test sample was added to the assay well then 96µl HMI-9 medium to give a final assay concentration after a 1:1 dilution of 20µg/ml / 20µM.

Table 1. Anti-trypanosomiasis assay plate layout.

The screening plate was arranged as illustrated in Table 1. Controls including a sterility control and DMSO controls in column 1; test samples in columns 2 to 11 and a concentration range of suramin as a positive control in column12. Trypanosomes were counted using a haemocytometer and diluted to a concentration of 3x104 trypanosomes /ml, 100µl of this suspension was added to each well of the assay plate with the exception of well A1 the

Anti-Trypanosomal Activity and Cytotoxicity

using Graphpad prism software.

spectrophotometer.

**2.2.1 Plant materials**

in open air and ground using a mill.

**2.2 Isolation and structure elucidation of compounds**

of Some Compounds and Extracts from Nigerian Medicinal Plants 379

Cells were seeded into 96 well microplates at a density of 0.5 x 104 cells/well in 100µl Dulbecco's modified Eagle's medium (DMEM; Invitrogen) and incubated at 37ºC, 5% CO2 with a humidified atmosphere for 24 hours. After which DMEM solutions of test compounds at the desired concentrations and DMSO as a negative control and 0.1% Triton X as a positive control were added to give a total well volume of 200µl. The microplates were incubated for 24 hours before the addition of 10µl of alamar blue. After a further 20 hours incubation fluorescence was determined using a Wallac Victor microplate reader in fluorescence mode (Excitation 530nm; Emission 590nm). The results were calculated as % of the DMSO control values. IC50 values were calculated for the concentration response data

Melting points (uncorr.) were determined on a Buchi B-540 melting point apparatus. The 1H NMR and 13C NMR spectra were run on a Bruker AV 300 (400MHz) and DRX 500 spectrometers using CDCl3 or DMSO-d6 as solvents and TMS as internal standard. HRESI-MS were run using Thermo LTQ Orbitrap or Thermo Exactive orbitrap mass spectrometer. A capillary voltage of 46.00V for the positive mode and -48.00V for the negative mode was used for HRESI-MS. Column chromatography were performed using silica gel MN-60 (Macherey-Nagel GmbH & Co. KG) and Gel filtration chromatography using Sephadex LH-20 (GE Health Lifescience UK) were performed in glass columns. Spots on TLC were visualised using Anisaldehyde-H2SO4 reagent or Dragendorff's reagent for the alkaloids. Developed TLC plates were also observed under UV lamp using short (λ= 254 nm) and long (λ= 366 nm) wavelengths. The structures of the pure compounds were determined using spectroscopic methods (1D/2D NMR and mass spectrometry). Column chromatography (CC) were carried out on wet packed silica gel in glass columns eluting gradient wise with hexane, hexane-ethyl acetate, ethyl acetate and ethyl acetate-methanol mixtures or with Sephadex LH-20 pre-soaked and wet packed in methanol. After the introduction of the extracts or fractions in methanol, the Sephadex columns were also eluted with methanol. TLC analysis was performed on pre-coated silica gel aluminum plates cut to desired size. NMR experiments were run in CDCl3 or DMSO-d6 with TMS as internal standard on Bruker AV 300 (400MHz) or DRX-500

All plant materials were collected from Oju LGA Benue State, Nigeria between July and September 2008. The plants were authenticated by the Department of Forestry and Wildlife of the University where voucher specimens were deposited in the University of Agriculture Forest Herbarium. *Alcornea cordifolia (*bark), *Euphorbia poisonii* (stem), *Monodora myristica*  (seeds), *Prosopis Africana* (bark), *Spondias mombim* (root)*, Nauclea pobeguinii* (bark)*, Nauclea latifolia* (bark)*, Cochlospermum planchonii(*root*)* and *Withania somnifera* (whole plant), *Terminalia avicennioides* (bark). *Maytenus laevis* (root) was from samples already housed at the Strathclyde Institute of Pharmacy and Biomedical Sciences (SIPBS). They were all sun dried

sterility check. The assay plate was incubated at 37ºC, 5% CO2 with a humidified atmosphere for 48 hours. After which 20µl of Alamar blue was added and the incubation continued for a further 24 hours. Fluorescence was then determined using the Wallac Victor microplate reader (Excitation 530nm Emission 590nm). The results were calculated as % of the DMSO control values. Minimum inhibitory concentration values (MICs) were determined for samples with less than 10% of control values. MIC determinations were carried out in duplicate. 200µg/ml test solutions were prepared in column 2 by pipetting 4µl of (10mg/ml) test stock solution and 196µl HMI-9 medium into each well. 100µl HMI-9 medium was pipetted into columns 1 and 3-12 and 1:1 serial dilutions were carried out from columns 2 to 11. 80µl of HMI-9 medium was added to column 12 and 20µl of x10 concentrations of suramin to give a final concentration range of 0.008 to 1.0µM. An inoculum of 100µl of trypanosomes at a concentration of 3x104/ml was added to each well except A1, and the procedure continued as previously described.

Table 2. Final assay concentrations µg/ml

MIC values were determined as the concentration calculated to have < 5% of control values.

#### **2.1.2 Cytotoxicity determinations**

Cytotoxicity determinations were carried out using a modification the Alamar blue™ redoxbased microplate assay (O'Brien *et al.*, 2000). The cytotoxic activity of the fractions and compounds was initially determined against PNT2A cells a "human" normal prostatic epithelial cell line. Initial screening was carried out at a concentration of 100µg/ml. Concentration response studies (300 to 0.3µg/ml) were then carried out in duplicate for samples with less than 60% of control values in the initial screen.

sterility check. The assay plate was incubated at 37ºC, 5% CO2 with a humidified atmosphere for 48 hours. After which 20µl of Alamar blue was added and the incubation continued for a further 24 hours. Fluorescence was then determined using the Wallac Victor microplate reader (Excitation 530nm Emission 590nm). The results were calculated as % of the DMSO control values. Minimum inhibitory concentration values (MICs) were determined for samples with less than 10% of control values. MIC determinations were carried out in duplicate. 200µg/ml test solutions were prepared in column 2 by pipetting 4µl of (10mg/ml) test stock solution and 196µl HMI-9 medium into each well. 100µl HMI-9 medium was pipetted into columns 1 and 3-12 and 1:1 serial dilutions were carried out from columns 2 to 11. 80µl of HMI-9 medium was added to column 12 and 20µl of x10 concentrations of suramin to give a final concentration range of 0.008 to 1.0µM. An inoculum of 100µl of trypanosomes at a concentration of 3x104/ml was added to each well

**1 2 3 4 5 6 7 8 9 10 11 12**

**A** control 100 50 25 12.5 6.25 3.1 1.55 0.78 0.34 0.17 1.0µM

**B** control 100 50 25 12.5 6.25 3.1 1.55 0.78 0.34 0.17 0.5µM

**C** control 100 50 25 12.5 6.25 3.1 1.55 0.78 0.34 0.17 0.25µM

**D** control 100 50 25 12.5 6.25 3.1 1.55 0.78 0.34 0.17 0.125µM

**E** control 100 50 25 12.5 6.25 3.1 1.55 0.78 0.34 0.17 0.062µM

**F** control 100 50 25 12.5 6.25 3.1 1.55 0.78 0.34 0.17 0.031µM

0.03% DMSO

0.03% DMSO 0.01% DMSO

0.01% DMSO 0.00% DMSO

0.00% DMSO 0.00% DMSO

0.00% DMSO 0.00% DMSO

0.00% DMSO

0.06% DMSO

0.06% DMSO

MIC values were determined as the concentration calculated to have < 5% of control values.

Cytotoxicity determinations were carried out using a modification the Alamar blue™ redoxbased microplate assay (O'Brien *et al.*, 2000). The cytotoxic activity of the fractions and compounds was initially determined against PNT2A cells a "human" normal prostatic epithelial cell line. Initial screening was carried out at a concentration of 100µg/ml. Concentration response studies (300 to 0.3µg/ml) were then carried out in duplicate for

suramin

suramin

suramin

suramin

suramin

suramin

0.015µM suramin

0.008µM suramin

except A1, and the procedure continued as previously described.

**G** control 1%

**H** control 1%

DMSO

DMSO

0.5% DMSO

0.5% DMSO

Table 2. Final assay concentrations µg/ml

**2.1.2 Cytotoxicity determinations**

0.25% DMSO

0.25% DMSO

samples with less than 60% of control values in the initial screen.

0.12% DMSO

0.12% DMSO Cells were seeded into 96 well microplates at a density of 0.5 x 104 cells/well in 100µl Dulbecco's modified Eagle's medium (DMEM; Invitrogen) and incubated at 37ºC, 5% CO2 with a humidified atmosphere for 24 hours. After which DMEM solutions of test compounds at the desired concentrations and DMSO as a negative control and 0.1% Triton X as a positive control were added to give a total well volume of 200µl. The microplates were incubated for 24 hours before the addition of 10µl of alamar blue. After a further 20 hours incubation fluorescence was determined using a Wallac Victor microplate reader in fluorescence mode (Excitation 530nm; Emission 590nm). The results were calculated as % of the DMSO control values. IC50 values were calculated for the concentration response data using Graphpad prism software.

#### **2.2 Isolation and structure elucidation of compounds**

Melting points (uncorr.) were determined on a Buchi B-540 melting point apparatus. The 1H NMR and 13C NMR spectra were run on a Bruker AV 300 (400MHz) and DRX 500 spectrometers using CDCl3 or DMSO-d6 as solvents and TMS as internal standard. HRESI-MS were run using Thermo LTQ Orbitrap or Thermo Exactive orbitrap mass spectrometer. A capillary voltage of 46.00V for the positive mode and -48.00V for the negative mode was used for HRESI-MS. Column chromatography were performed using silica gel MN-60 (Macherey-Nagel GmbH & Co. KG) and Gel filtration chromatography using Sephadex LH-20 (GE Health Lifescience UK) were performed in glass columns. Spots on TLC were visualised using Anisaldehyde-H2SO4 reagent or Dragendorff's reagent for the alkaloids. Developed TLC plates were also observed under UV lamp using short (λ= 254 nm) and long (λ= 366 nm) wavelengths. The structures of the pure compounds were determined using spectroscopic methods (1D/2D NMR and mass spectrometry). Column chromatography (CC) were carried out on wet packed silica gel in glass columns eluting gradient wise with hexane, hexane-ethyl acetate, ethyl acetate and ethyl acetate-methanol mixtures or with Sephadex LH-20 pre-soaked and wet packed in methanol. After the introduction of the extracts or fractions in methanol, the Sephadex columns were also eluted with methanol. TLC analysis was performed on pre-coated silica gel aluminum plates cut to desired size. NMR experiments were run in CDCl3 or DMSO-d6 with TMS as internal standard on Bruker AV 300 (400MHz) or DRX-500 spectrophotometer.

#### **2.2.1 Plant materials**

All plant materials were collected from Oju LGA Benue State, Nigeria between July and September 2008. The plants were authenticated by the Department of Forestry and Wildlife of the University where voucher specimens were deposited in the University of Agriculture Forest Herbarium. *Alcornea cordifolia (*bark), *Euphorbia poisonii* (stem), *Monodora myristica*  (seeds), *Prosopis Africana* (bark), *Spondias mombim* (root)*, Nauclea pobeguinii* (bark)*, Nauclea latifolia* (bark)*, Cochlospermum planchonii(*root*)* and *Withania somnifera* (whole plant), *Terminalia avicennioides* (bark). *Maytenus laevis* (root) was from samples already housed at the Strathclyde Institute of Pharmacy and Biomedical Sciences (SIPBS). They were all sun dried in open air and ground using a mill.

Anti-Trypanosomal Activity and Cytotoxicity

2 *Cochlospermum planchonii*

5 *Monodora myristica*

10 *Terminalia*

11 *Withania somnifera*

*avicennioides*

S/No Plant Part Extract codes

Whole plant

Table 3. Extracts produced from the plant materials

Plate 1. Photo showing MIC of compounds **11** and **12**.

of Some Compounds and Extracts from Nigerian Medicinal Plants 381

Ethyl acetate (E)

Root (R) CPH CPE CPHE CPM

Seed MMH MME MMHE MMM

Bark (B) TAH TAE TAHE TAM

WSH WSE WSHE WSM

Combined hexane + ethyl acetate (HE)

Methanol (M)

Hexane (H)

1 *Alcornea cordifolia* Bark (B) ACBH ACBE ACBHE ACBM

3 *Euphorbia poisonii* Stem EPH EPE EPHE EPM 4 *Maytenus laevis* Root (R) MLH MLE MLHE MLM

*Nauclea latifolia* Bark (B) NLH NLE NLHE NLM *Nauclea pobeguinii* Bark (B) NPH NPE NPHE NPM *Prosopis Africana* Bark (B) PAH PAE PAHE PAM *Spondias mombim* Root (R) SMH SME SMHE SMM

#### **2.2.2 Extraction and identification of the components**

The plant materials (500g – 1.0kg) were air dried and extracted using hexane, ethyl acetate and methanol in a Soxhlet apparatus. The crude extracts were allowed to stand and sometimes cooled to allow for the precipitation of compounds. Where all precipitates have been produced or no (further) precipitation, the crude extracts or precipitates (where not a pure compound) were subjected to column chromatography (CC) eluting gradient- wise using hexane-ethyl acetate and subsequently ethyl acetate-methanol solvent mixtures. Using TLC as a guide, similar fractions were combined and allowed to evaporate under the fume hood to allow for the crystallisation of pure compounds. Where no pure compounds were separated and the fractions were active, such fractions were further purified via preparative thin layer chromatography or Gel filtration (GF) using Sephadex LH-20 to obtain the pure compounds. Methanol extracts which were usually viscous and complex were subjected to an initial vacuum liquid chromatography (VLC) to fractionate the extracts. Similar fractions (TLC) were also combined and further purified via column chromatography or Gel filtration. Flash chromatography using an iES Reveleris Automated Flash Chromatography system was used for the isolation of certain compounds. The column was dry packed after taking up the extracts with silica gel and then loaded onto the pre-packed flash column. The compounds were eluted starting with hexane with incremental additions of ethyl acetate, then with ethyl acetate with additions of methanol and finally with methanol. Using two variable length UV detectors and one ELSD detector, similar eluates were combined and confirmed by TLC and 1H NMR. Pure compounds were then allowed to settle out from the combined fractions. All columns were exhaustively eluted and washed with methanol. Washings were allowed to stand to allow for the precipitation of compounds. The identification of pure compounds was carried out using one dimensional 1H and 13C NMR spectroscopy (including *J*-mod, DEPT-q and DEPT 135 or 90). Spectral data of known compounds were compared with published spectral data and were thereby identified. Further 2D experiments (including correlation spectroscopy (COSY), heteronuclear multiple quantum coherence (HMQC), heteronuclear multiple bond correlation (HMBC) and nuclear overhauser enhancement spectroscopy (NOESY)) were carried out for more complex molecules for accurate assignments of proton and carbon chemical shifts. Assigned structures were confirmed using ESI-MS and HRESI-MS to confirm their mass and molecular formula. The various extracts produced are listed in table 3 below. Extract code descriptions or extensions are: precipitate (P), latex (L), Sephadex (S), methanol wash (w), VLC fraction (V), flash chromatography fraction (F), root (R), bark (B).

#### **3. Results**

#### **3.1 Anti-trypanosomal screening**

The photographs below show the results from the concentration response studies to determine the MIC of the triterpenoids (compounds 11 and 12) from *M.laevis.* Well A1 is the sterility check, wells B1 to H1 negative controls and wells G2 to G11 and H2 to H11 are the DMSO 1:1 dilution controls. Column 12 contains a concentration range of 1 to 0.008µM of the positive control suramin. In plate 1 compounds 11 (rows C and D) and 12 (rows E and F) are tested n=2 over a concentration range of 100 to 0.17µM. In plate 2 compounds 11 (rows A, B and C) and 12 (rows D,E and F) are tested n=3 over a concentration range of 10 to 0.02µM.

The plant materials (500g – 1.0kg) were air dried and extracted using hexane, ethyl acetate and methanol in a Soxhlet apparatus. The crude extracts were allowed to stand and sometimes cooled to allow for the precipitation of compounds. Where all precipitates have been produced or no (further) precipitation, the crude extracts or precipitates (where not a pure compound) were subjected to column chromatography (CC) eluting gradient- wise using hexane-ethyl acetate and subsequently ethyl acetate-methanol solvent mixtures. Using TLC as a guide, similar fractions were combined and allowed to evaporate under the fume hood to allow for the crystallisation of pure compounds. Where no pure compounds were separated and the fractions were active, such fractions were further purified via preparative thin layer chromatography or Gel filtration (GF) using Sephadex LH-20 to obtain the pure compounds. Methanol extracts which were usually viscous and complex were subjected to an initial vacuum liquid chromatography (VLC) to fractionate the extracts. Similar fractions (TLC) were also combined and further purified via column chromatography or Gel filtration. Flash chromatography using an iES Reveleris Automated Flash Chromatography system was used for the isolation of certain compounds. The column was dry packed after taking up the extracts with silica gel and then loaded onto the pre-packed flash column. The compounds were eluted starting with hexane with incremental additions of ethyl acetate, then with ethyl acetate with additions of methanol and finally with methanol. Using two variable length UV detectors and one ELSD detector, similar eluates were combined and confirmed by TLC and 1H NMR. Pure compounds were then allowed to settle out from the combined fractions. All columns were exhaustively eluted and washed with methanol. Washings were allowed to stand to allow for the precipitation of compounds. The identification of pure compounds was carried out using one dimensional 1H and 13C NMR spectroscopy (including *J*-mod, DEPT-q and DEPT 135 or 90). Spectral data of known compounds were compared with published spectral data and were thereby identified. Further 2D experiments (including correlation spectroscopy (COSY), heteronuclear multiple quantum coherence (HMQC), heteronuclear multiple bond correlation (HMBC) and nuclear overhauser enhancement spectroscopy (NOESY)) were carried out for more complex molecules for accurate assignments of proton and carbon chemical shifts. Assigned structures were confirmed using ESI-MS and HRESI-MS to confirm their mass and molecular formula. The various extracts produced are listed in table 3 below. Extract code descriptions or extensions are: precipitate (P), latex (L), Sephadex (S), methanol wash (w),

**2.2.2 Extraction and identification of the components**

VLC fraction (V), flash chromatography fraction (F), root (R), bark (B).

12 (rows D,E and F) are tested n=3 over a concentration range of 10 to 0.02µM.