Investigation of Herb-Drug Interactions between Xylopia aethiopica, Its Principal Constituent Xylopic Acid, and Antidepressants

Introduction Depression affects an estimated 350 million people worldwide and is implicated in up to 60% of suicides. Only about 60–70% of patients respond to antidepressant therapy. One of the factors causing patients to not attain therapeutic goals is herb-drug interactions. Objective To investigate any potential herb-drug interaction that might exist between Xylopia aethiopica extract (XAE) or xylopic acid (XA) and selected conventional antidepressants (imipramine, fluoxetine, and venlafaxine) in mice. Methods Dried, powdered fruits of Xylopia aethiopica were cold macerated in 70% ethanol to obtain XAE. XA was isolated by cold macerating dried fruits of Xylopia aethiopica in petroleum ether, crystallising impure XA with ethyl acetate, and purifying XA crystals with 96% ethanol. Pharmacodynamic interaction was assessed via isobolographic analysis of tail suspension tests of the agents individually and in their respective combinations. Pharmacokinetic interaction was assessed by monitoring the effect of coadministrations on the plasma concentration of antidepressants and xylopic acid via HPLC analysis. Results XAE and XA in mice showed significant antidepressant-like activity in the tail suspension test. With interaction indices less than one, synergism of antidepressant effect was observed in the Xylopia aethiopica extract/fluoxetine (γXAE/FL = 0.502), Xylopia aethiopica extract/imipramine (γXAE/IP = 0.322), Xylopia aethiopica extract/venlafaxine (γXAE/VL = 0.601), xylopic acid/imipramine (γXA/IP = 0.556), xylopic acid/venlafaxine (γXA/VL = 0.451), and xylopic acid/fluoxetine (γXA/FL = 0.298) combinations, which may be potentially due to elevation of serotonergic neurotransmission via varying mechanisms. The AUC of imipramine (AUCIP = 1966 ± 58.98 µg/ml.h) was significantly (P < 0.0001) reduced by Xylopia aethiopica extract (AUCIP = 1228 ± 67.40 µg/ml.h) and xylopic acid (AUCIP = 1250 ± 55.95 µg/ml.h), while the AUC of xylopic acid (AUCXA = 968.10 ± 61.22 µg/ml.h) was significantly (P < 0.0001) reduced by venlafaxine (AUCXA = 285.90 ± 51.92 µg/ml.h) and fluoxetine (AUCXA = 510.60 ± 44.74 µg/ml.h), possibly due to the effect of interfering agents on gastric emptying hence reducing oral absorption. Conclusion Xylopia aethiopica extract and xylopic acid interacted synergistically with imipramine, fluoxetine, and venlafaxine and reduced the systemic circulation of imipramine.


Introduction
Depression is a mental disorder characterised primarily by anhedonia and dysphoria, among a host of other symptoms, and afects over 350 million people worldwide [1,2].Depression is a major risk factor for suicide, accounting for as much as 60% of suicidal cases [2][3][4][5][6][7].Te national average prevalence of suicide in Ghana has been estimated at 3.3% of the population [8,9].
Previous research has highlighted that depression may often go unnoticed or untreated, and hence, a signifcant number of adults experiencing depression do not receive the necessary treatment for their symptoms [10].When diagnosed, treatment of depression conventionally employs the use of antidepressants, which have been proven to be efective agents for resolving depression.Te major classes of antidepressants frequently used include selective serotonin reuptake inhibitors (SSRIs), serotonin-noradrenaline reuptake inhibitors (SNRIs), and tricyclic antidepressants (TCAs) [11].
Of the few depression-diagnosed patients who receive treatment, as much as 30-40% do not attain therapeutic goals [12].Antidepressant treatment failure can be due to a heterogeneity of factors.One possible factor responsible for this resistance to antidepressant therapy is the occurrence of drug interactions due to the concomitant administration of other drugs and herbs together with conventional antidepressants [12].With 60% of the global population using herbal medicine, the risk of herb-drug interactions is very high [13][14][15][16][17].Clinically, caution is advised in herb-neuropsychiatric drug coadministrations as they have been revealed to cause harmful side efects and complications through pharmacodynamic and pharmacokinetic interactions [18,19].A herb with a potentially high risk of interacting with antidepressants is the widely used Xylopia aethiopica, as its hydroethanolic extract and isolate, xylopic acid, have been discovered to possess antidepressant-like activities via mechanisms similar to those of classical antidepressants [3,11,20].It is therefore critical to determine whether or not this extract or isolate of Xylopia aethiopica interferes with the antidepressant efect of orthodox antidepressants.
Tis study therefore sought to investigate any potential pharmacological interaction that might exist among the hydroethanolic extract of Xylopia aethiopica or its major constituent, xylopic acid, and selected conventional antidepressants.Te study specifcally evaluates the impact on the antidepressant efcacy of imipramine, fuoxetine, and venlafaxine during concurrent administration of XAE or XA, aiming to elucidate potential interactions and their implications for therapeutic outcomes.Te central research question guiding this study revolves around determining the extent to which the hydroethanolic extract or isolate of Xylopia aethiopica interferes with the antidepressant efects of conventional antidepressants.

Materials and Method
2.1.Animals.Institute of Cancer Research (ICR) mice (26 ± 10 g) were obtained from Noguchi Memorial Institute for Medical Research, University of Ghana, Accra.Te mice were housed in the vivarium of the Department of Pharmacology, Kwame Nkrumah University of Science and Technology (KNUST), Kumasi, to acclimatise to the laboratory conditions.Te animals were housed 10 mice per stainless steel cage, with softwood shavings as bedding, and fed with a commercially available pellet diet, and given water ad libitum.Experiments were conducted in accordance with internationally accepted principles for laboratory animal use and care, and ethical approval was obtained from the Animal Research Ethics Committee of KNUST (KNUST 0039).
Te sample size of seven animals for this pharmacological study was determined through a meticulous power analysis, ensuring a balance between statistical robustness and ethical considerations.Our power analysis indicated that with this sample size, we could achieve a statistically meaningful level of power to detect expected efect sizes while minimising the likelihood of type II errors.Anticipated efect sizes, drawn from preliminary studies and historical data, validated the feasibility of detecting these efects within the chosen sample.Upholding ethical guidelines advocating for minimal animal use, we selected a sample size that aligned with scientifc integrity without unnecessary animal burden.Te simplicity of our experimental design allowed for a focused assessment, optimising the sample size to efciently capture variations and trends.Contingencies for potential attrition and unforeseen circumstances were accounted for, ensuring the reliability of our statistical analyses.

Drugs.
Te antidepressants (imipramine (IP), venlafaxine (VL), and fuoxetine (FL)) were selected as representatives of the three major classes of antidepressants that are currently used in clinical practice.Imipramine is a tricyclic antidepressant (TCA); venlafaxine, a serotoninnoradrenaline reuptake inhibitor (SNRI); and fuoxetine, a selective serotonin reuptake inhibitor (SSRI).Doses of antidepressants used were selected from literature [3,20].Tey served as standard antidepressants for the validation of the model.

Extract Preparation. Dried fruits of Xylopia aethiopica
were obtained from Kwahu Asakraka (6 °37′45″N 0 °41′11″W) in the Eastern Region of Ghana.Te fruits were authenticated at the Department of Pharmacognosy, Faculty of Pharmacy and Pharmaceutical Sciences, KNUST.A voucher specimen (KNUST/HM1/2015/FR001) was deposited at the herbarium of the faculty.Te fruits were coarsely milled, and 2.5 kg was cold macerated in 70% (w/v) ethanol for 72 h.Te extract obtained was then concentrated into a dark-brown, oily sludge (yield 13.34% w/v).Te extract, XAE, was subsequently used in the experiments at selected doses 30, 100, or 300 mgkg −1 .Dosing was based on preliminary toxicity studies, which are not reported in this current study.An HPLC fngerprint of the extract was obtained as described earlier [3,6].Te column employed was Phenomenex Luna 5µ C8 (2) 150 × 4.60 mm.Te mobile phase contained water (10%) and methanol (90%).Te eluent was monitored at 206 nm.
2.4.Xylopic Acid Isolation.Xylopic acid (XA) was isolated as described by Osafo et al. [6].A mass of 2.5 kg of powdered dried fruits of Xylopia aethiopica was cold macerated in petroleum ether for 72 h.Te extract obtained was fltered and concentrated via rotary evaporation at 45 °C.Xylopic acid crystals were precipitated from the concentrate by the addition of ethyl acetate, and the crystals obtained were purifed via recrystallisation with 96% ethanol.A yield of ).Te vehicle served as the negative control.At the time of maximum efect (1 h), the mice were individually suspended by their tail, 1 cm from the tip, with an adhesive tape on a horizontal suspension bar that was elevated 52 cm from the base.Te duration of escape-oriented behaviours and immobility were recorded with a video camera for 6 minutes and quantifed with JWatcher ™ by an experienced observer blinded to all treatment groups.Mice that climbed on their tails were gently pulled down, and the test was continued.Scored behaviour was defned as mobility (struggling) and immobility (lack of movement).Tis protocol was again employed in randomly grouped mice (n � 7) treated with coadministration of extract/antidepressants and XA/antidepressants.2.6.Isobolographic Analysis.Te isobolographic analysis employed was similarly described by Woode et al. and Boakye-Gyasi et al. [4,5].Te ED 50 s of XAE, XA, imipramine, venlafaxine, and fuoxetine were determined from dose-response curves obtained after subjecting each agent to the tail suspension test.
Another dose-response curve was subsequently obtained and analysed upon coadministration of antidepressants with XAE or with XA in a fxed-ratio (1 : 1) combination based on fractions 1/2, 1/4, and 1/8 of their respective ED 50 s as obtained from the tail suspension test.
An isobologram was then constructed by connecting the plot of the theoretical ED 50 s of the antidepressants (plotted on the ordinate) with those of the XAE or XA (plotted on the abscissa) so as to attain an additivity line.Te experimental ED 50 and its associated 95% confdence interval for each drug mixture were determined by linear regression analysis of the log dose-response curve and compared by a t-test to a theoretical additive ED 50 obtained via calculation using the formula: ) of extract or isolate, where f is the fraction of each component in the mixture and the variance (Var) of Z add was also calculated using the following formula: VarZ add � f 2 (Var ED 50 of antidepressant) + (1 − f ) 2 ED 50 of extract or isolate.
Te S.E.M.s were determined from the variances and resolved based on the ratio of the individual drugs present in each combination.

Plasma Concentration.
Mice were well-fed prior to the experiment, and they were provided water ad libitum.Te mice (n � 5) were grouped and given a single oral dose of ED 50 of each antidepressant and xylopic acid, a combination of ED 50 of antidepressant and XA/XAE, and 10 mLkg −1 of vehicle as control.

Data Analysis.
Te data were presented as mean-± S.E.M. Te dose-response curves plotted were subjected to a two-way (treatment x time) repeated-measure analysis of variance (ANOVA) followed by Tukey's multiple comparisons test.An iterative nonlinear regression (3-parameter logistic) equation was used in the determining ED 50 of all agents alone and in their various combinations from the respective dose-response curves.
where X is the logarithm of dose and Y is the response.Y starts at a (bottom) and goes to b (top) with a sigmoid shape.Te ftted midpoints (ED 50 s) of the curves were compared statistically using the F test.Microsoft ® Excel ® and GraphPad Prism for Windows version 8 (GraphPad Software, San Diego, CA, USA) were used in performing isobolographic calculations, and the corresponding isoboles were subsequently plotted using GraphPad Prism for Windows version 8 (GraphPad Software, San Diego, CA, USA).Noncompartmental analysis was employed in the monitoring of drug plasma concentration.Statistical difference between AUC of antidepressants alone and AUC of antidepressants after the administration of combination treatment was determined by Dunnett's T3 multiple comparison test using GraphPad Prism for Windows version 8 (GraphPad Software, San Diego, CA, USA).

HPLC Analysis. Te HPLC-UV fngerprinting of
Xylopia aethiopica extract produced a chromatogram, which showed twelve peaks with varying retention times, which was indicative of twelve diferent components being present in the Xylopia aethiopica extract (Figure 1).Te isolated Advances in Pharmacological and Pharmaceutical Sciences xylopic acid had a fairly prominent peak, indicative of a very pure isolate.Te HPLC retention time of isolated xylopic acid was 3.368 (Figure 1).

Plasma Concentration.
Upon monitoring the plasma concentration of antidepressants and xylopic acid after administration of drug combinations to test mice via noncompartmental analysis, it was observed that Xylopia aethiopica extract and xylopic acid signifcantly altered the amount of imipramine in systemic circulation, and venlafaxine and fuoxetine signifcantly altered the amount of xylopic acid in systemic circulation.Te other combinations, however, did not cause any signifcant changes in the amount of antidepressants or xylopic acid getting into systemic circulation.

Discussion
Depression is a psychiatric disorder afecting an estimated Advances in Pharmacological and Pharmaceutical Sciences          Tis study investigated potential herb-drug interactions that might occur between antidepressants and the hydroethanolic extract of the popularly used herb, Xylopia aethiopica, or its isolate, xylopic acid.Te study found a synergistic pharmacodynamic herb-drug interaction between the extract and all the standard antidepressants, and between the isolate and all the standard antidepressants.Te pharmacokinetic dispositions of imipramine and xylopic acid were altered.
Xylopia aethiopica extract, xylopic acid, and antidepressants (imipramine, fuoxetine, and venlafaxine) all showed signifcant antidepressant-like and antidepressant activity when subjected to the tail suspension test-a model for detecting potential antidepressants [23].All the agents elongated the duration spent by tail-suspended mice engaging in escape-oriented behaviour (pedalling, curling, and swinging), while correspondingly reducing the duration spent being immobile.Advances in Pharmacological and Pharmaceutical Sciences Tis antidepressant-like activity of Xylopia aethiopica extract has been previously established to be due to the ability of the extract to interact with serotonergic neurotransmission, with a possible glutamatergic efect via glycine B co-binding site and nitric oxide synthase inhibition [3].Additionally, the antidepressant-like activity of the extract was likely due to the presence of some secondary metabolites (sterols, favonoids, and xylopic acid), which are known to possess antidepressant-like properties [20,24,25].UV fngerprinting of the extract, as shown in this study, revealed the presence of several components.Similarly, xylopic acid owes its antidepressant-like activity to its efect on serotonergic mechanisms, as well as neuroprotective mechanisms, involving brain-derived neurotrophic factor (BDNF) and antioxidant enzymes [20].Te exact mechanisms by which the extract and isolate afect serotonergic neurotransmission, however, remain unknown.Te standard antidepressants inhibit the reuptake of neurotransmitters, resulting in elevated levels of these neurotransmitters.Imipramine inhibits the reuptake of serotonin, dopamine, and noradrenaline; fuoxetine inhibits the reuptake of serotonin; and venlafaxine inhibits the reuptake of serotonin and noradrenaline [11].
Isobolographic analysis of drug combinations of Xylopia aethiopica extract with antidepressants and xylopic acid with antidepressants revealed that the experimental ED 50 s were    12 Advances in Pharmacological and Pharmaceutical Sciences signifcantly less than the theoretical additive ED 50 s for all the drug combinations studied.Tese were indicative of synergistic pharmacodynamic interactions due to increased potency [26][27][28].Tese synergistic interactions serve as a tool to examine drug mechanisms, as the synergistic efects observed suggest that the agents in each combination either act simultaneously at distinct sites or activate diferent pathways [29].It can therefore be inferred that both Xylopia aethiopica extract and xylopic acid potentiated the antidepressant activities of imipramine, fuoxetine, and venlafaxine, possibly by elevating serotonergic neurotransmission via mechanisms that vary from those employed by the antidepressants.Te observed synergistic efects could have also been contributed by the other nonserotonergic mechanisms of Xylopia aethiopica extract and xylopic acid as established in previous studies [3,20].
Pharmacokinetically, Xylopia aethiopica extract and xylopic acid interfered with the oral absorption of imipramine, resulting in a signifcant reduction in the amount of imipramine the body of the mice was exposed to.A previous study found that Xylopia aethiopica extract had smooth muscle relaxant properties via the serotonergic pathway, hence hindering gastric emptying [30].
Gastric emptying is an important factor that afects drug absorption and is usually the rate-limiting step in the absorption of xenobiotics, as it defnes how quickly a xenobiotic gets to the upper small intestine, where absorption is greatest [30,31].
By impeding gastric emptying, Xylopia aethiopica extract was able to cause a decline in the amount of imipramine to get into systemic circulation.Previous studies have shown that Xylopia aethiopica extract and xylopic acid share a similarity in their mechanism of action via the serotonergic pathway [3,20].Hence, xylopic acid is likely to have a similar efect on gastric emptying as Xylopia aethiopica extract, resulting in the reduced amount of imipramine getting into systemic circulation that was observed.Venlafaxine and fuoxetine also reduced the amount of xylopic acid getting into systemic circulation after oral administration by possibly slowing down gastric emptying [32,33].
Xylopic acid also decreased the elimination rate of imipramine due to its substrate activity on p-glycoprotein, hence competing with imipramine-which is also a substrate of p-glycoprotein-for binding site [34][35][36][37].Tis resulted in the inefcient efux of imipramine from systemic circulation and therefore slowing down the elimination process [38].Venlafaxine and fuoxetine also likely had a similar efect on xylopic acid as they are both substrates of pglycoprotein [35,36].
Te inhibitory efect of xylopic acid on CYP 3A4 could have contributed to the decrease in the elimination rate of imipramine, as the enzyme is responsible for metabolising imipramine [34,39,40].Venlafaxine and fuoxetine possibly had a similar efect on xylopic acid as they are also potent inhibitors of CYP 2D6, a metaboliser of xylopic acid, hence, decreasing the elimination rate of xylopic acid [34,39,41,42].
With Xylopia aethiopica extract and xylopic acid both having the ability to alter the pharmacodynamic and pharmacokinetic disposition of antidepressant, this could impact the clinical management of depression in patients who coadminister the extract or isolate with their antidepressants.Herb-drug coadministration could improve clinical therapeutic outcomes, as the potential elevated antidepressant activity made possible due to the synergistic relationship between herb and antidepressant would provide a greater resolution of depression as compared to the administration of antidepressants alone.Prescribers might therefore encourage coadministration of the extract or isolate with antidepressants.Te dose of imipramine being administered may need to be adjusted higher as the amount of imipramine getting into systemic circulation could be signifcantly reduced by either the extract or isolate.Tis may require the plasma concentration of imipramine in patients to be constantly monitored to ensure that therapeutic range and steady-state concentration of imipramine are attained.
Xylopic acid and Xylopia aethiopica extract reduced the amount of imipramine in systemic circulation, and venlafaxine and fuoxetine reduced the amount of xylopic acid in systemic circulation, likely via decreasing the rate of gastric emptying.

Study Limitations.
In assessing potential sources of bias or confounding factors, eforts to minimise bias through blinding techniques during behavioural assessments were carried out.With regard to external validity of the study's fndings, the limitations inherent in animal models should be acknowledged.Tese models, while valuable, might restrict direct extrapolation to human responses due to variances in pharmacokinetics, physiology, and behavioural nuances between species.Furthermore, while the chosen antidepressants mirror major clinical classes, the exclusive

Figure 6 :
Figure 6: Isobolograms for oral coadministration of Xylopia aethiopica extract with (a) imipramine, (b) fuoxetine, (c) venlafaxine in the tail suspension test.Te theoretical ED 50 for an additive efect and the experimental ED 50 values are represented in the graphs.

Figure 7 :
Figure 7: Isobolograms for oral coadministration of xylopic acid with (a) imipramine, (b) fuoxetine, and (c) venlafaxine.Te theoretical ED 50 for an additive efect and the experimental ED 50 values are represented in the graphs.

Table 1 :
Te potencies and peak efects of the various agents used in the tail suspension test.

Table 2 :
Teoretical and experimental ED 50 ± S.E.M. of Xylopia aethiopica extract and antidepressant combinations in the tail suspension test and the corresponding computed indices.

Table 4 :
Efect of Xylopia aethiopica extract and xylopic acid on the pharmacokinetic disposition of imipramine.

Table 5 :
Efect of Xylopia aethiopica extract and xylopic acid on the pharmacokinetic disposition of fuoxetine.P ≤ 0.05 (Dunnett's T3 multiple comparisons test) compared AUC of coadministrations to AUC of single-agent administration.Values are expressed as mean ± S.E.M (n � 5). *

Table 6 :
Efect of Xylopia aethiopica extract and xylopic acid on the pharmacokinetic disposition of venlafaxine.

Table 7 :
14ect of antidepressants on the pharmacokinetic disposition of xylopic acid.51.92 * * * 510.60 ± 44.74 * * * P ≤ 0.05, * * P < 0.01, and * * * P < 0.001 (Dunnett's T3 multiple comparisons test) compared AUC of coadministrations to AUC of single-agent administration.Values are expressed as mean ± S.E.M (n � 5).14Advances in Pharmacological and Pharmaceutical Sciences focus on these agents might overlook diferences in response patterns with other antidepressants when combined with the herbal extract or isolated compound under investigation.Tese factors collectively highlight the necessity for cautious interpretation and the potential limitations in directly applying fndings from this animal study to clinical scenarios in human populations.