Herb-drug Interaction using the seed extract of Hunteria umbellata and Metronidazole in Plasma of Rabbit

Omotoso et al., ISSN: 2814-1423 45 Nigerian Journal of Pharmaceutical and Biomedical Research Vol. 8 Issue.1 April, 2024. p-ISSN: 2579-1419 e-ISSN: 2814-1423 Herb-drug Interaction using the seed extract of Hunteria umbellata and Metronidazole in Plasma of Rabbit Omotoso, Abayomi E, Chinaka, Chioma N1*, Ugwunweze, Ikenna J Department of Medicinal and Pharmaceutical Chemistry, University of Port-Harcourt, PortHarcourt, Nigeria. *Corresponding Author Email: [email protected], doi.org.10.55639/njpbr.1041.004 Abstract Herbal medicines contain multiple bioactive constituents which are capable of interacting with various bio-molecular targets and therefore can interfere with the pharmacokinetics of most orthodox medicine. This work studies the pharmacokinetic profile of metronidazole administered concomitantly with Hunteria umbellata using rabbits. Single oral dose of metronidazole was administered to the rabbits (5.0 mg/kg) after which 2 mL of blood samples were serially withdrawn at 0, 0.5, 1.0, 2.0, 4.0, 8.0, 12.0 and 24 h. After a washout period of 14 days, the aqueous extract of Hunteria umbellata (50 mg/ml) was co-administered with metronidazole (5mg/kg) as a single oral dose. Thereafter, blood samples (2ml) were withdrawn at time interval indicated in the first phase. The samples were analyzed using a validated UV spectrophotometric assay method. The following pharmacokinetic parameters were obtained: for metronidazole only were Cmax (4.858 µgml-1), Tmax (2.0 h), T1/2 (3.111 h), Ka (1.647 h-1), AUC0-∞ (40.710 µgml-1h-1), Vd (0.551mlKg-1), Cl (0.123 ml h-1) and Kel (0.223 h-1) while the pharmacokinetic parameters after co-administration are Cmax (4.658 h), Tmax (2.0 h), T1/2 (2.560 h), Ka (1.850 h-1), AUC0-∞ (28.842 µgml-1h-1), Vd (0.693 mlKg-1), Cl (0.138 ml h-1) and Kel (0.223 h-1). The following parameters: AUC0-24, AUC0-∞, Kel, Te1/2 and Cl of metronidazole were significantly decreased when administered concurrently with the extract, there was no statistical difference (p < 0.05) in the Ka, T1/2, Cmax and Tmax of metronidazole when administered alone or coadministration with H. umbellata. There is therefore need for proper counseling of patients on the concomitant use of metronidazole with aqueous extract of Hunteria umbellata seed. Keyword: herb-drug interaction, Hunteria umbellata, metronidazole, Pharmacokinetics Introduction: The use of herbal medicine is widely practiced and accepted in most developing countries like Nigeria where most of the rural populace are poor and without access to good health care facilities (Oreagba et al, 2011). This has resulted in increasing dependence on herbal medicine which is believed to be affordable, natural and therefore with less toxicity (Osemene et al, 2011; Ameade et al., 2018). Herbs have been used in many chronic illnesses (e.g. diabetes mellitus, ulcers, cardiovascular diseases, cancers and depression) because of their multiple active components (e.g. alkaloids, flavonoids, terpenoids, tannins, saponins and cardiac glycosides) and are therefore beneficial especially in such diseases with many pathological pathways (Liu et al., 2015). The concomitant use of herbal medicine and prescription drugs for desired therapeutic benefit in treating diseases have oftentimes resulted in herbdrug pharmacokinetic interactions (Choi et al, 2011). These interactions are of safety concern especially for drugs with narrow therapeutic index (e.g. Digoxin, Phenytoin and Warfarin etc.) and for chronic illnesses (such as cancer) which involves long-term Omotoso et al., ISSN: 2814-1423 46 use of drugs (Yang et al., 2014). These interactions are of special clinical significance because it could alter the pharmacokinetic parameters of the drug used concomitantly with the herb (Tarirai et al, 2010; Meng and Liu, 2014). The process of drug absorption at the site of absorption into the circulatory system can be altered due to modulation of efflux and uptake transporters, complex formation, changes in gastrointestinal motility or the fluids physiological pH (Rosenkranz et al, 2012). While the effect on the drug biotransformation could be due to either inhibition or induction of Cytochrome P450 metabolizing enzymes (Zhou et al., 2003). These factors not only alter the bioavailability and plasma clearance of the administered therapeutic drug but also affect their effectiveness and toxicity. Some of the pharmacokinetic parameters which may be altered as a result of herbdrug interaction include: the area under the curve (AUC), the maximum plasma concentration (Cmax), time to reach the maximum plasma concentration (Tmax), elimination half-life and clearance etc. In developing nations, the populaces often get involved in self-medication with common drugs such as analgesic, antimicrobial, haematinics among others. Among the antimicrobial agents, metronidazole is readily available, and due the level of hygiene and the eating habit of the populace, gastrointestinal problems regularly occurred. Metronidazole is a synthetic antiprotozoal drug. It is effective against obligate anaerobic bacteria such as Protococcus, Peptostreptococcus, Clostridium bacteroides, and Fusobacterium. It is also active against few non-spores forming gram-positive organisms. Metronidazole is highly effective against anaerobic protozoa infections including Trichomonas vaginalis. Entamoeba hystolytical, Giardia lamblia and Balantidium coli. Metronidazole is the drug of choice for amoeabiasis and Trichomonadic urethritis in men, lambliosis, amoebic dysentery and other sensitive anaerobic infection (Vardanyan and Hruby, 2006; Andrews et al, 2014) 2Methyl -5nitroimidazol-1-ethanol The plant Hunteria umbellata (K. Shum) is a tropical rain forest tree which belongs to the family Apocynaceae, is widely used in traditional medicine as an antiobesity, antihyperlipidaemia, antihyperglycemia, infertility, aphrodisiac, stomachache and ulcers(Ajiboye et al., 2017; Adeneye et al, 2019). It is known by names such as “Demouain” (French), “Madaci” (Hausa), “Mkpokiri” (Igbo) and “Abere” (Yoruba) (Adejuwon et al, 2011; Adeneye et al, 2011) . Some herbal medicines are known to induce CYP3A4; an enzyme which is normally responsible for the oxidative metabolism of most drugs. It is therefore important to investigate the potential outcome of most herbal-drug interactions so as to avoid their adverse effects. This effort will encourage rational and safe use of herbal medicine and therapeutic drugs as there have been an increase in their coadministration especially among the elderly patients due to the presence of two or more co-existing health challenge. To further this knowledge, we undertook this study on the pharmacokinetic interaction between the plant Hunteria umbellata and N N CH2CH2OH H3CNO2 Omotoso et al., ISSN: 2814-1423 47 the drug metronidazole using animal model. Materials and Methods Collection of Plant materials and authentication Fresh mature pods of the Hunteria umbellata plant were collected from the deciduous forest of Offa (Kwara state, Nigeria), in April, 2018. Plant authentication was done by Mr. Suleiman, (Department of Pharmacognosy & Phytotherapy, University of Port-Harcourt) and the Voucher specimen deposited in Forest Research Institute of Nigeria, (FRIN) Ibadan, Oyo State, Nigeria with the reference no. FHI 109457, allotted. Experimental animals Four healthy white male New Zealand rabbits weighing between 1.5 -1.7 kg and aged between 4 - 6 months were purchased from the University of Port Harcourt animal house were used for the study. They were acclimatized in an environmentally controlled room (temperature about 22 ◦C, photo period of 12 h light and 12 h darkness with relative humidity of 60 %) for two weeks prior to the study. Prescribed feed and water were provided ad libitum during the period. The guideline of the Institutional Animal Ethics Committee was observed throughout the study. The reagents used in the course of the research work were of analytical grade and these include; methanol (Guangzhou Jinhuadu Chemical Reagent Co., Ltd., China), sulphuric acid (Sigma Aldrich, Germany), sodium hydroxide (Sigma Aldrich, Germany), hydrochloric acid (Sigma Aldrich, Germany), Molisch’s reagent, ferric chloride, chloroform, Pure sample of metronidazole, Normal saline (Juhel Pharma, Nigeria), Water bath (HH6 Techmel and Techmel, USA) UV/Visible spectrophotometer (Techmel and Techmel, USA); Sample bottles (Skytec medical, Nigeria); Lithium heparinized sample bottle (Skytec instruments); Vortex mixer (XH-B Vortex, SearchTech China). Methods Extraction of Plant Materials The de-coated seeds of H. umbellata were gently washed with distilled water, dried and then grinded to a coarse powder using domestic blender. A kilogram of coarsely powdered seeds of H. umbellata was intermittently shaken and exhaustively extracted with distilled water (1:10) for a period of 72 hours. Thereafter, the marc obtained was oven dried at 100ºC. The filtrate was concentrated using rotary evaporator before it was freeze dried. The extract obtained was stored in a moisture tight clean vessel and kept in a refrigerator (4°C). Phytochemical screening of the aqueous seed extract was carried out according to the standard methods (Trease and Evans, 2009). Invivo Pharmacokinetic Study The study was a simple cross-over design comprising of phase 1 and phase 2 sections with a washout period of 2 weeks inbetween. All the animals (rabbits) were made to fast overnight and remained fasted until 4 h after the drug intake. Water was given ad libitum during the study. In the Phase 1 of the study, the animals, were divided into: Group I; the control group animals received only water (2 ml/kg)), while the Group II animals were orally administered metronidazole (5 mg/kg) by gavage. In the phase 2: the Group I Omotoso et al., ISSN: 2814-1423 48 animals were given water (2 ml/kg) while Group II animals were administered H. umbellata extract (50 mg/kg) by gavage every 24 h for 72 h and immediately after the last dose, 5 mg/kg metronidazole was administered. Blood samples, (2.0 mL) were collected from the ear lobe vein of the rabbits at the following time intervals: 0.0, 1.0, 2.0, 4.0, 8.0, 12.0 and 24.0 h via a modified cannula and transferred into a heparinized tube in both phases, centrifuged immediately at 3,500 rpm for 10 minutes and the plasma obtained were stored at -4oC until analysis. Extraction of metronidazole from plasma The extraction of metronidazole from the blood samples was carried out using the Ofoefule et al. method: Briefly: Blood samples (0.5 mL) were withdrawn from the ear lobe vein of the rabbit using a modified needle cannula syringes into a heparinized tube. This was mixed with 5 mL of 0.05M H2SO4 in methanol, vortex mixed and centrifuged for 10 min at 3000 rpm. The supernatant layer containing metronidazole was allowed to evaporate on water bath to a final volume of 1.0 ml and analyzed using a UV/ Visible spectrophotometer at λmax of 277 nm for metronidazole. Preparation of Standard Solution of Metronidazole To prepare the stock solution (100 µg/ml), 50 mg of pure metronidazole powder was accurately weighed and dissolve in 300 ml of 0.05M H2SO4 in methanol and the solution made to mark in a 500 ml volumetric flask. Then, 2, 4, 6, 8 and 10 ml aliquot of the stock solution were separately made up to 100 ml with 0.05M H2SO4 in methanol to prepare 2, 4, 6, 8 and 10 µg/ml solutions respectively. Determination of λmax and calibration curve A milliliter of the stock solution (100 µg/ml) was diluted to 10 ml with 0.05M H2SO4 in methanol to obtain a 10 µg/ml solution. The absorbance of resulting solution was scanned in the UV spectrometer in the range 200 – 400 nm. The maximum absorbance value was found at a wavelength of 277 nm (λmax). The absorbance values of the different standard solutions prepared were determined at this wavelength in order to prepare the calibration curve. Pharmacokinetic Data Analysis The plot of plasma concentration (C) against time (t) of metronidazole alone and after co-administration with the H. umbellata extract was carried out using PK solver (version 2) software. The data were analyzed to obtain the pharmacokinetic parameters such as peak plasma concentration (Cmax), time to peak plasma concentration (Tmax), elimination rate constant (Kel), elimination half-life (Tel1/2); apparent volume of distribution (Vd); area under the plasma concentration– time curve (AUC) and drug clearance (Cl) were determined using non-compartmental analyses. The absorption rate constant was calculated by logarithmic linear regression of the plasma concentration–time curve. The absorption half-life was calculated as 0.693 divided by the elimination constant. The AUC to the final measurable sample was determined using the trapezoidal rule and extrapolated to infinity with the final plasma concentration being divided by the elimination constant, calculated from the Omotoso et al., ISSN: 2814-1423 49 apparently linear portion of the log plasma concentration–time curve. Statistical analysis The results from independent experiments were analyzed statistically using Microsoft Excel (Student’s t-test) and Graph pad Prism 6 software for windows (Graph pad software, San Diego CA www.graphpad.com). The data obtained were expressed as mean ± SD. The differences between mean values were analyzed for significance using one way analysis of variance (ANOVA). p ˂ 0.05 were considered to be statistically significant. Results The crude aqueous extract of H. umbellata was orange colored, which changed to chocolate brown after freeze drying. The percentage yield of the plant extract is 24.36%. Phytochemical analysis The phytochemical screening of the aqueous seed extract of H. umbellata revealed the presence of the following phytochemical compounds: saponins, alkaloids, flavonoid, anthraquinones, tannins, and cardiac glycoside (Table 1). Table 1. The phytochemical screening of the aqueous seed extract of H. umbellata Test Observation Inferences Saponins Frothing test Persistent frothing Saponins present Alkaloids Mayer’s test Cream precipitate Alkaloids present Dragendorff’s test Red precipitate present Hager’s test Yellow precipitate present Flavonoids Shinoda’s reduction test Orange colouration Flavonoid present Anthraquinones Borntrager’s test Violet coloration in ammonia phase Anthraquinones present Tannins Ferric chloride A blue black colouration Tannins present Cardiac glycosides Lieberman test A pink colouration Triterpenoid nucleus Burchard’s test A reddish brown colouration at the interface Steroidal nucleus present Salkowski’s test A reddish brown colouration Unsaturated lactone ring Omotoso et al., ISSN: 2814-1423 50 In vivo Pharmacokinetic Study The pharmacokinetic parameters of metronidazole when administered alone and after co-administration with H. umbellata were calculated using the noncompartmental model with PK solver (version 2) software and the result is as shown in Table 2. The graph of mean plasma concentration –time curves (AUC) of metronidazole when administered alone and after co-administration with H. umbellata are shown in Figure 2. Table 2: Pharmacokinetic parameters of metronidazole alone and after coadministration of metronidazole and Hunteria umbellata extract. Pharmacokinetic parameter Mean values Metronidazole Metronidazole + H. umbellata extract Kel (h-1) 0.223 0.350 T1/2 (h) 3.111 3.771 Ka (h-1) 1.647 1.850 Ta1/2 (h) 0.421 0.385 Tmax( h) 2.000 ± 0.002 2.000 ± 0.001 Cmax (μg/ml) 4.858 ± 0.005 4.658 ± 0.004 Tlag (h) 0 0 Clast_obs/Cmax 0.015 0.004 AUC 0-24h (μg/ml*h) 40.382 28.763 AUC 0-inf_obs (μg/ml*h) 40.710 28.842 AUMC 0-inf_obs (μg/ml*h^2) 277.778 152.475 MRT 0-inf_obs(h) 6.823 5.287 Vd_obs (ml/kg) 0.551 0.693 Cl_obs (ml/kg* h) 0.123 0.253 AUC: Area under the curve, Tmax: time taken by the drug to reach the maximum concentration, Cmax: maximum concentration of the drug, T1/2: half-life (the period of time required for the concentration of drug in the body to be reduced by one=half), CL: Clearance (the estimate of the plasma cleared by the body per unit time: MRT: mean residence time of the drug, Vd: volume of distribution (the volume of plasma that would be necessary to account for the total amount of drug in the animal) Determination of λmax and calibration curve The calibration curve for the determination of metronidazole (Figure 1.0) in the plasma was obtained plotting the plasma concentration against time, using UVVisible spectrophotometer which shows linearity and sensitivity between 0 - 10 μg/ml. Omotoso et al., ISSN: 2814-1423 51 Figure 1.0: Calibration curve of metronidazole in 0.05M H2SO4 in methanol solution Discussion The use of herbal medicine as complementary medicine for most diseases has become a worldwide practice and because herbs are known to interact with some enzymes responsible for drug metabolism (CYP3A4) thereby leading to both safety issues and treatment failures. This has led to the continued research on interactions between herbs and orthodox medicine (Zhou et al., 2007). From our study the phytochemical screening of the plant H. umbellata reveals the presence of an alkaloids, anthraquinones, cardiac glycosides, flavonoids, saponins and tannins, which could be responsible for the plants pharmacological actions. The metronidazole absorption rate when administered alone compared to when it was administered concomitantly with H. umbellata shows a slight insignificant increase from 1.647 h-1 to 1.850 h-1. The time taken for half of the singly administered metronidazole dose to be absorbed and after its co-administration with H. umbellata was significantly different (p >0.05): 0.421 h and 0.347 h respectively. The implication of the above y = 0.0089x + 0.0175 R² = 0.9756 0 0.02 0.04 0.06 0.08 0.1 0.12 0 2 4 6 8 10 12 ABSORBANCE CONCENTRATION(μg/ml) tim e (h ) plasm a con c. (g/m l) 0 10 20 30 0 2 4 6 M etronidazole only M etronidazole + H . um bellata F igure 2. P lasm a concentrationtim e cu rve of m etron idazole an d after co-ad m in istatio n w ith ex tract H . u m be lla ta Omotoso et al., ISSN: 2814-1423 52 findings is that H. umbellata does not significantly interfere with the oral absorption of metronidazole. The time taken to achieve the maximum plasma concentration of metronidazole when singly administered and when administered concomitantly with H. umbellata were the same (2 h), although their peak plasma concentration differs but not significantly (p>0.05): metronidazole alone is 4.858 μg/ml while metronidazole with H. umbellata is 4.658 μg/ml., the above findings is in agreement with the findings of Zhou et al., (2007) However, there was a significant difference in the area under the curve of plasma drug concentration versus time curve (Figure 2) and the area under the first moment curve (AUMC0-24) which is a reflection of the body’s exposure to the drug. The AUC and AUMC values when metronidazole was administered alone were 40.380 and 277.778 µg/ml/h respectively while the values when metronidazole was coadministered with H. umbellata were 28.841 and 152.475 µg/ml/h respectively. The above data indicates that H. umbellata significantly reduced the amount of drug that reaches the rats’ bloodstream in a given period of time after a dose is given and also reduces quantitatively the area under the curve formed by time and the product of concentration and time. This is in tandem with the value of mean residence time (MRT) obtained in this study. The mean residence time (MRT) for metronidazole and H. umbellata when concomitantly administered (5.287 h) was lower than when metronidazole was administered alone (6.823 h). This could be supported by the result of the mean residence time (MRT) and volume of distribution (Vd) of 6.819 h and 0.549 mL/kg respectively for metronidazole alone while the values for coadministration with H. umbellata were 6.287 h and 0.693 mL/kg respectively. This implies that metronidazole is more available in the tissues when administered concomitantly with H. umbellata than when administered alone. This could probably account for two compartmental model observed in this study (Figure 2). The elimination rate of metronidazole coadministered with H. umbellata (0.351 h-1) was significantly higher than that of metronidazole administered alone (0.224 h-1). Therefore it takes less time to eliminate metronidazole from the systemic circulation when it is administered concomitantly with the herb than when it was administered alone. The parameter which relate the amount of drug in the body to the concentration of drug in the plasma (Vd) is higher when metronidazole was administered concomitantly with H. umbellata (0.693 ml/kg) than when administered alone (0.551 ml/kg). This implies that H. umbellata either increases the lipid solubility of metronidazole through the surfactant activity of the saponins possessed by H. umbellata (Liao et al., 2021; Rai et al., 2021), increase in the tissue binding ability of metronidazole through the tissue shrinking activity of tannin possessed by H. umbellata) (Ashok and Upadhyaya, 2012) or an increase in metronidazole plasma protein binding (Sanvordeker et al., 1975). The limitation of this study include the number of rabbits, the inter-specie variation which may make interpolation to human difficult, though the study established a relationship between H. umbellata and metronidazole, but the study was unable to establish the mechanism of the interaction. This study revealed that H. umbellata has significant effect on the amount of Omotoso et al., ISSN: 2814-1423 53 metronidazole absorbed, the mean residence time, and the rate at which it is eliminated. Therefore, co-administration of H. umbellata and metronidazole should not be encourage rather patients should be encouraged to reveal their usage of herbal medicine together with orthodox medicine to prevent any advent of toxicities. Herbdrug interaction can have a lethal effect especially where poly-pharmacy is a common practice. Conflict-of-Interest The authors declare no conflict of interest. Authors’ Declaration The authors hereby declare that the work presented in this article is original and that any liability for claims relating to the content of this article will be borne by them. References Adejuwon, A. A., Oluwatoyin, S. M. and Sunday, A. O. (2011) ‘Antiinflammatory and Antioxidant activities of Hunteria umbellata Seed Fractions’, Pharmacologia, 2(6), pp. 165–71. Adeneye, A. A., Olagunju, J. A. and Murtala, B. A. 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