African populations have used herbal medicines for many centuries. Knowledge regarding specific plants and parts of these plants and their function in disease treatment is traditionally passed on from one generation to the next. With the boom in conventional therapeutic drugs, the utilization of herbal remedies sometimes in combination with other medications has increased tremendously over the last decade [1
]. It has been demonstrated over the years that herbal remedies possess great therapeutic potential in managing and treating a number of diseases including malaria, hypertension, HIV/AIDS, and cancer. The use of herbal remedies is especially relevant in developing countries [3
] due to their affordability and accessibility compared to orthodox health interventions. Evidence available shows that there are potential interactions between drugs and herbs, some with fatal clinical outcomes [5
]. Despite these fatal herb-drug clinical outcomes, there is still limited information on monitoring herb–drug interactions [8
(family Bignoniaceae) and Cassia abbreviata
(family Fabaceae) are medicinal herbal plants that are used in Western and Southern African countries for the treatment and management of many conditions. Newbouldia laevis
, also known as the tree of life or boundary tree, is a shrub or small tree that grows in West African countries such as Ghana, Nigeria, and Benin. Local names for this shrub include Faangum in Cameroon, Sasanemasa/Sesemasa or Esisimansa in Ghana, Lifui in Togo, and Akoko in Nigeria. The leaves, stems, and roots are used to treat conditions such as malaria, convulsions, cancer, dyslipidemia, diabetes, hemorrhoids, constipation, peptic ulcer, and cough [9
]. Cassia abbreviata
is a medium-sized tree that grows in most parts of Africa, especially utilized in Southern African countries such as Botswana, South Africa, Uganda, Zambia, and Zimbabwe. Local names for this shrub include Muremberembe in Zimbabwe, Mahemba in Tanzania, and Monepenepe in Botswana and South Africa. The leaves, stems, and roots are used to treat and/or manage a number of diseases such as malaria, diabetes, ulcers, headaches, diarrhoea, constipation, and skin diseases [11
], and are believed to possess anti-HIV activity [13
Despite the advances made in Western medicine, there are many diseases with no cure and so herbal medicines become the default treatment option. However, in many cases, herbal medicines are used concurrently with conventional drugs, raising the risk for adverse drug reactions (ADRs) through the alteration of the pharmacokinetic and pharmacodynamics profiles through herb–drug interactions (HDI). Drug-metabolizing enzymes (DMEs) play a very important role in the metabolism of xenobiotics including therapeutic drugs. The cytochrome P450 (CYP450) family of enzymes especially plays an important role in the biotransformation of about 70% of xenobiotics and other endogenous substances [14
]. The composition of herbal medicines is complicated as they contain a number of different phytochemicals and other substances that give them their therapeutic potential. Notwithstanding the complicated composition of herbal medicines, most of their metabolism is also dependent on DMEs, including the CYP450 enzymes, which might affect the metabolism of other administered drugs, leading to potentially increased risk of HDI [15
]. There are more than 57 cytochrome P-450 genes identified in humans, with the CYP1, CYP2, and CYP3 families contributing to most of the biotransformation of xenobiotics (including therapeutic drugs) [17
]. This study concentrated on investigating the effects of potential interaction between extracts from Newbouldia laevis
and Cassia abbreviata
on CYP1A2, CYP2C9, and CYP2C19 activities.
CYP1A2 is responsible for the metabolism of drugs including theophylline, caffeine, imipramine, paracetamol, and phenacetin [19
]. Among the substrates of CYP2C9 is the anticoagulant warfarin, non-steroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen, diclofenac, and naproxen, the hypoglycemic agent tolbutamide, phenytoin, and the angiotensin-II receptor antagonist losartan. CYP2C19 metabolizes a number of commonly used drugs including benzodiazepine diazepam, the proton pump inhibitor omeprazole, propranolol, and the antidepressant amitriptyline [19
Despite the long-term use of Newbouldia laevis and Cassia abbreviata by several African populations for various ailments, their effects on cytochrome P450 and information on their potential to cause herb-drug interaction (HDI) by inhibition of cytochrome P450 has not been reported. This study therefore investigated the inhibition profiles of extracts from two medicinal plants, Newbouldia laevis and Cassia abbreviata, on three major CYP450 enzymes, CYP1A2, CYP2C9, and CYP2C19, using recombinant human CYP450 enzymes. Enzyme kinetic studies were carried out to determine the inhibitory mechanisms of Newbouldia laevis and Cassia abbreviata. The potential of these two herbal plants to cause HDI was also evaluated. Phytofingerprinting was performed in order to evaluate the profile of possible phytocompounds in both Newbouldia laevis and Cassia abbreviata.
The therapeutic efficacy of medicinal herbal plants is well acknowledged in both developed and developing countries. Medicinal plants from different countries mostly lack valid documentation on their safety, efficacy, manufacturing standards, and quality control. Given the varied amounts of compounds found in medicinal herbal plants, standardization parameters and development of marker profiles of commonly used medicinal plants are very important for maintaining the quality of herbal remedies and also provide knowledge on the optimal concentrations of the various bio-constituents [25
]. Despite being natural, medicinal plant extracts are not free from risks and interactions with other drugs and herbs, which may lead to significant public health consequences including the possibility of being fatal [5
]. Traditionally, whole plants or mixtures of medicinal plants are used rather than isolated compounds from these plants. Evidence indicates that crude plant extracts often have greater in vitro and/or in vivo therapeutic activity than isolated constituents at an equivalent dose. The aim of this study therefore was to evaluate the CYP450 inhibition activities of Newbouldia laevis
and Cassia abbreviata
extracts commonly used in West and Southern Africa for the treatment and/or management of common communicable and non-communicable diseases. It was observed that extracts from these two plants had differential inhibitory effects on CYP1A2, 2C9, and 2C19. This could be used for initial assessment of the potential risk of HDI, which would bring awareness and educate the public and/or patients on the dangers of co-use of herbal medicinal remedies and conventional medications.
Drug interactions can result in clinical fatalities; thus, in the drug discovery process, guidelines and opinions have been published by the FDA, EMA, and pharmaceutical industries [27
] for the conducting of drug–drug interaction (DDI) studies to help target drugs that are likely to cause interactions at the early stages so that such entities can be either discontinued or modified before further processing. The potential interactions of herbal remedies with prescribed or over-the-counter drugs has been a major safety concern for clinicians and public health practitioners, as such interactions are difficult to predict and also, generally, there is a lack of available information on the composition of herbs and their pharmacological actions [29
]. With the tremendous surge in the acceptance of and public interest in herbal medicinal remedies, researchers are adopting the guidelines and opinions proposed for DDI studies to study HDI. This is helpful with labelling purposes for commercially available herbal products and also to caution patients from combining herbal remedies with known conventional medications [30
]. There is limited information currently available regarding the potential of Newbouldia laevis
and Cassia abbreviata
to interact with clinically prescribed drugs. This is a safety issue as these herbal plant remedies are commonly used by certain populations. This study therefore looked at the drug interaction potential of N. laevis
and C. abbreviata
extracts on three major cytochrome P450 enzymes, CYP1A2, CYP2C9, and CYP2C19. Typically, new chemical entities (NCE) are assessed for their potential to cause CYP-mediated drug interaction based on a single concentration screening and later a detailed classification involving IC50
, TDI, and inactivation constants like Ki
Crude N. laevis and C. abbreviata extracts were used in this study to mimic the way patients take these herbal remedies and also the mode of extraction simulated to show the indigenous mode of extraction. Newbouldia laevis extract showed a strong reversible inhibitory effect on CYP1A2 and CYP2C9 enzyme activities whilst exhibiting a moderate reversible inhibition on CYP2C19. Cassia abbreviata extract showed a strong reversible inhibitory effect on CYP1A2, CYP2C9, and CYP2C19 enzyme activities. The implication is that there is a possibility of enzyme recovery should the herbal extracts inactivate these CYPs.
Time-dependent inhibition of CYP450 enzymes that is caused by NCEs is of great concern because such compounds can cause clinically relevant DDI or HDI [31
]. The TDI of a CYP enzyme refers to the change in inhibition potency of a NCE as a result of a dosing period that leads to formation of inhibitory metabolites and mechanism-based inhibition [32
]. The use of the IC50
-shift approach as an initial step for TDI assessment has been recommended by the Pharmaceutical Research and Manufacturers Association (PhRMA) [27
], where HLMs or recombinant CYPs are used to evaluate the increase in potency of a NCE to cause CYP-inhibition when the chemical entity is pre-incubated with NADPH [20
]. This approach was used to assess the TDI potency of Newbouldia laevis
and Cassia abbreviata
extracts. Though the IC50
-shift approach is recommended, there is a challenge with the interpretation of the criterion used to define a chemical as a potential TDI. PhRMA recommends the criterion of 1.2- to 3-fold decrease [32
] for classification of potential TDI candidates, whilst Berry and Zhao [20
] have adopted a criterion of ≥1.5 as being significant for classification for TDI candidates. This study therefore adopted the Berry and Zhao approach of setting the TDI criterion at ≥1.5. As seen in Figure 4
, Newbouldia laevis
and Cassia abbreviata
showed TDI potencies of 4.08 and 1.64, respectively, on CYP1A2. TDI potency of 0.15 and 2.01 was observed for α-naphthoflavone and furafylline known non-TDI and TDI compounds respectively. Newbouldia laevis
and Cassia abbreviata
extracts did not exhibit any TDI potency on CYP2C9 enzyme with values of 1.18 and 1.41, respectively, implying they were not potential CYP2C9 TDI candidates. Although Newbouldia laevis
extract exhibited a non-TDI effect (1.03) on the CYP2C19 enzyme, Cassia abbreviata
extract exhibited a TDI effect on the CYP2C19 enzyme with a ratio of 1.72. Sulphaphenazole and miconazole, which are non-TDI compounds of CYP2C9 and CYP2C19, were used to validate the assay, respectively.
The differential reversible and irreversible inhibition of CYP1A2, CYP2C9, and CYP2C19 enzymes by Newbouldia laevis and Cassia abbreviata extracts implies that patients who are on prescribed drugs that have narrow therapeutic ranges (for example, theophylline, caffeine, imipramine, paracetamol, phenacetin-CYP1A2, warfarin, non-steroidal anti-inflammatory drugs (NSAIDs), tolbutamide, phenytoin, losartan-CYP2C9, diazepam, omeprazole, propranolol, amitriptyline-CYP2C19, or over-the-counter drugs that are metabolized by similar CYP450 enzymes) stand a risk of having HDI, which can lead to clinical consequences. The TDI effects of these herbals is felt when patients chronically use them because sufficient intermediary metabolites may be generated and this can be deleterious to consumers concomitantly taking conventional medications, as stated earlier.
Single point screening for the effects of pre-incubation with NADPH was performed on CYP1A2, CYP2C9, and CYP2C19. Data showed significant effects of pre-incubation of Newbouldia laevis extracts on CYP1A2 enzyme activity but not CYP2C9 and CYP2C19 enzyme activities. Cassia abbreviata extracts, however, exhibited significant effects on CYP1A2 and CYP2C19 enzyme activities after pre-incubation with NADPH but not CYP2C9 enzyme activity. These results combined with the TDI potencies imply that these herbs’ inhibitory effects may either be increased or reduced after pre-incubation. The weakening inhibition potency could be due to recovery of enzyme activity since the inhibition potency of the herbs weakens with time regardless of the concentration applied, whilst the increase in inhibitory potency could be due to the interaction of the herb with CYP450 enzymes during pre-incubation.
Following the inactivation of CYP1A2, CYP2C9, and CYP2C19, the inactivation parameters were determined. The kinetic parameters for the inactivation of CYP1A2 by Newbouldia laevis and Cassia abbreviata extracts were: Ki; 2.84 µg/mL and 4.86 µg/mL, Kinact; 0.024 min−1 and 0.033 min−1, respectively. Inactivation kinetic parameters of CYP2C9 were: Ki; 1.55 µg/mL and 5.98 µg/mL, Kinact; 0.027 min−1 and 0.067 min−1, respectively. Kinetics parameters of inactivation for CYP2C19 were Ki; 1.23 µg/mL and 1.58 µg/mL, Kinact; 0.031 min−1 and 0.029 min−1, respectively. Determination of inactivation parameters gives more information on the type of inactivation, thus allowing patients taking these herbs concomitantly with conventional medication to be aware of the implications.
Herbal medicines consist of multi-phytochemical constituents with different physicochemical properties. The risk of HDI of orally administered herbal remedies depends on the bioavailable fraction of the phyto-constituents [34
], which may or may not permeate through membrane barriers. Due to the multi-phyto nature of herbals, their bio-availabilities are variable depending on the constituents [24
]. Prediction of in vivo HDI using in vitro results [21
] gives preliminary information that can caution patients to be wary of combination therapies of herbals with conventional medications and also for pharmaceutical companies and researchers to get baseline information guiding further investigation. For herbal extracts that are already in use, HDI studies allow assessment of risk, helping with the design of in vivo HDI studies and revision of product labels to highlight the risk of co-use of these herbs with conventional medication, as in the case of Saint John′s Wort and the protease inhibitor indinavir [37
]. This study assessed the risk of HDI on CYP1A2, CYP2C9, and CY2C19 activities and predicted these herbals can cause HDIs in vivo to a significant extent, with the assumption that at least one phyto-constituent is absorbed to cause HDI.
Profiling of herbal extracts for the phyto-constituents is a critical step in elucidating the possible mechanisms for HDI potential. Based on the UPLC-MS data obtained, the crude extracts of Newbouldia laevis
and Cassia abbreviata
are composed of several different phenolic compounds. The UPLC-MS profiles indicated the presence of at least 10 polyphenolic candidates including catechins and potent antioxidant flavonoids, which likely give these herbs their therapeutic effects. Epidemiological studies of polyphenols suggest that consumption of polyphenol-rich herbs and beverages may contribute to the prevention of diseases including cancers, cardiovascular diseases, osteoporosis, neurodegenerative diseases, and diabetes [38
]. However, some of these polyphenols are known to inhibit CYP450 [40
]. It is therefore possible that the interactions observed in this study may be related to some of the constituents that are in these herbs.