At present, although several drugs and therapies are in use to mitigate or to prevent from illness caused by SARS-CoV-2, still the urge towards developing the new medical strategies is needful (33, 34). The use of integrative medicine (IM) has become a trend either as a primary (to treat disease/illness) or secondary (to downgrade the prevalence of disease/illness) prevention (35). Moreover, the ideal therapeutic regime to treat COVID-19 should possess multi-targetability for immunomodulatory, adaptogenic, rejuvenating, anti-stress, anti-inflammatory, antiviral activity, etc (11, 12). It is also important for such IM pharmacotherapeutic regimen to be safe and efficacious to prevent or control the associated comorbidities. From the previous literature the identified phytocompounds from our study are already profound to elicit immune defence and anti-viral properties by which they might act as an adjuvant to treat COVID-19 (36). The usage of herbal extracts/formulations which are the mixture of large number of phytoconstituents renders to show multiple pharmacological activities as well untoward interactions when administered along with conventional medicines (herb-drug interactions)
(37). Such HDIs can be beneficial, harmful or even fatal. Aswagandha well-known Rasayana
(~rejuvenator) also referred as “Indian Ginseng” is well established for its immunomodulatory, adaptogenic, anticancer, anti-diabetic, anti-COVID-19 activities, etc (28).
In the present study we have characterized the aqueous extract of Aswagandha and found 11 important withanolide and withanoside glycosides were charecterised based on their MS/MS spectra. These glycosides like withanoside VII, V, X are known to elicit immunomodulatory activity and antiviral activity (38). Similarly, AYUSH-64 a polyherbal formulation, AYUSH64 contains the aqueous extracts of rasayana botanicals namely, Saptaparna bark (Alstonia scholaris R. Br.), Katuki roots (Picrorhiza kurroa Royle ex. Benth), Kiratatikta whole-plant (Swertia Chirata Pexbex. Karst), and Kuberaksha seed (Caesalpinia crista L.) (39) was characterized with the help of MS/MS fragmentation pattern and 24 plant phytoconstituents were identified.
The in-silico pharmacokinetics of Ashwagandha and AYUSH-64 revealed that most of the key phytoconstituents of Aswagandha and AYUSH-64 showed good oral bioavailability, drug like properties and GI affinity. Whilst some of the bioactive compounds showed inhibitory affinity towards cytochrome P450 enzymes (CYP3A4, 2C8, 2C9, 2D6, 1A2). However, the low relative abundance of these molecules might be the reason for the IC50 value below 100 ug/mL. Thereby, the dose reduction may be required to have safe and beneficial HDIs. Whereas, opposite will be applicable for the prodrugs (39). Also, some of the docking studied also revealed the key phytoconstituents like withaferin A, viscosalactone B, withanolide A, vallesamine, neocaesalpin B, sweroside, picrine, β-Caesalpin were profound to show good docking core against COVID-19 (15, 39–41). Furthermore, to assure its safe usage in combination with other conventional drugs used in COVID-19 management and also to prescribe for the patients with other comorbidities, we performed the in-vitro herb drug interactions of aqueous extract of Aswagandha and AYUSH-64 by using human liver microsomes.
For instance, the usage of herbal medicines alone and also in combination of other conventional drugs were profoundly increased in the recent times (30). Hence, it is necessary to address their impact on cytochrome P450 enzymes (which contributes to the major hepatic metabolism of drugs) to understand their plausible metabolic interactions (42). In the present study, to simulate clinical context we incorporated to study the activity of herbs alone, as the selected herbs were already profound to be effective towards SARS-CoV-2. Besides, we also evaluated these extracts in combination with most commonly encountered drug remdesvir as a case example to understand their interaction with common CYP isozymes. For the same, the study design was focused on CYP enzymes that metabolise 60-80% of xenobiotic spectrum. CYP3A4 alone is responsible for the metabolism of more than 50% of all xenobiotics prescribed during various illnesses (43). Whereas CYP2C8 and CYP2D6 are responsible for 5% and 20% respectively (44, 45). Thereby, the prediction for CYP mediated inhibitory kind of HDIs for the drugs that are substrate of these CYPs can be done for the clinical translational purpose. The remdesivir was the most commonly prescribed for the COVID-19 pharmacotherapeutic management during the second wave of ongoing pandemic (46). It was therefore used as a case example for the in-vitro CYP based HDI studies. As individuals are mainly prone for the concomitant consumption of such herbal formulations with allopathic drugs, hence we also concomitantly subjected HLM to the remdesivir and herbal formulations at the same time.
Remdesivir is a substrate of CYP3A4, 2D6, 2C8. Results from our study indicated that [I]/Ki ratio of remdesivir towards CYP3A4, 2D6, 2C8 was 38.07, 27.04, 16.62 respectively indicating that the specified case molecule showed poor inhibitory kind of potential which consistently supports the literature (47, 48). However, the aqueous extract of Aswagandha did not show any inhibitory potential, as the corresponding IC50 values was >100 µg/ml towards all the three specified CYP isoenzymes (49). Interestingly this matches with the already published literature for Remdesivir, showing it as a weak inhibitor of CYP3A4 (50). Whereas for the Aswagandha our results match with the observations published by Patil et. al. showing aqueous extracts prepared as per Ayurvedic procedure does not show inhibitory effects on CYP3A4 (12, 24, 32). In addition, Aswagandha in combination with remdesivir exhibited IC50 > 100 µg/ml which confirmed the weak inhibitory potential of remdesivir also in the presence aqueous extract of Aswagandha. This indicates that the combination of Withania somnifera and remdesivir, or any other drug which is the substrate of CYP3A4, 2D6, 2C8 might be safe to use for better pharmacotherapy.
Furthermore, we also identified the HDIs of polyherbal formulation (AYUSH-64) alone and in combination with the case control drug remdesivir. Results obtained signify that AYUSH-64 had no inhibitory interaction with CYP3A4 and CYP2D6 as IC50 values were found be >100µg/ml. While, it showed a weak or moderate inhibitory interaction with CYP2C8 with an IC50 of 63.44±14.9. It is therefore inferred that AYUSH-64 will be safe to administer with the substrates of CYP3A4 and 2D6, while a caution should be observed with drugs that are substrates of CYP2C8. Moreover, the action of AYUSH-64 in combination with remdesivir showed no inhibitory kind of interaction towards CYP3A4 and 2D6 with which IC50 was >100µg/ml. As specified before both the AYUSH-64 and remdesivir showed weak or moderate interactions towards CYP2C8, the IC50 of the combination showed 85.21±18.3. Indeed, this fall of IC50 within the inhibitory limit confer that of these two together may competitively binds to substrate and showed moderate inhibition towards CYP2C8. The HDIs and pharmacokinetic parameters presented in the current study may provide insights to develop new and safe therapeutic regimen to mitigate SARS-CoV-2 and also provides safety profile to use concomitantly with other drugs. However, the study could not did focused on time dependant inhibition and induction type of Pharmacokinetic HDIs. Therefore, further, in-vivo and clinical HDIs studies should be performed in future for the translational understanding and better outcomes.