INTRODUCTION:

Berberine chloride (BBC) is herbal isoquinoline alkaloid with manifold promising therapeutic activities and has been widely utilised in Ayurveda and traditional Chinese medicine for decades. Due to cost effectiveness, minimal toxic impact and innumerable therapeutic actions, recently it has gained remarkable curiosity and tremendous attention.

In spite of its significant activities, its oral use has been severely curtailed as it shows extremely low and variable plasma concentrations in humans with an absolute oral bioavailability of less than 1%. In clinical emergencies, it takes a massive dose (up to 1.5g/day) that could cause adverse gastrointestinal consequences¹.

The key factors for poor membrane permeability and subsequently poor bioavailability of drug are the prevalence of cytochrome P450 enzymes in the gut/ liver which are accountable for presystemic drug metabolism, impaired absorption, predominant tissue distribution, drug excretion into the lumen, bile or urine because of the presence of certain efflux transporters such as P- glycoprotein (P-gp) ascribes to very poor absorption and fluctuating plasma concentration following its oral administration. It's a substrate for the intestinal efflux pump P-glycoprotein (P-gp), which acts as a barrier to BBC absorption.¹This problem could be solved with the use of various permeability enhancement techniques to improve drug permeation transiently.

It has been postulated that co-administration or pre-administration of BBC with permeability enhancers might boost its permeability, blood levels, therapeutic activities and lessen its adverse gastrointestinal actions.²

Different studies have been published on improving the bioavailability of BBC such as use of synthetic P-gp inhibitors like cyclosporin A, Verapamil etc.³, use of several absorption enhancers such as TPGS, lysergol, oryzanol, sodium caprate and sodium deoxycholate, beta-cyclodextrin, chitosan and its salt, spray dried mucoadhesive microparticles, nanotechnology-based approach using different techniques and methods of formulation⁴. Thorough literature survey revealed that, bioenhancers use has positive impact on permeability and subsequently bioavailability of drug.

The administration of drug with effective, natural and safe herbal molecules having permeability and bioavailability enhancing actions like piperine, curcumin, naringin, lysergol, quercetine, glycyrrhizin, allicin, sinomenine, genistein, ginger, capsaicin, carum carvi, campul, cow urine distillate, etc. has acquired extensive attentiveness in oral use and creates newer skyline in pharma and healthcare zone⁵.

Utilization of bioenhancers is a medical need to boost per-oral drug delivery. Bioenhancers are entities which, when administered at low doses, boost the permeability, bioavailability and bioefficacy of medicaments. Selective utilization of bioenhancers results in a decrease in drug dosages caused by increased bioavailability, leading to reduced dosing frequency, toxic effects, cost of therapy, adverse effects etc. and increased patient compliance⁵.

As quercetin, curcumin and piperine are natural, safe and effective permeation enhancers having p-gp inhibitory activity which also works through inhibition of drug metabolizing enzymes like CYP 3A4. These bioenhancers (especially piperine) mediated bioavailability enhancing action is partly due to increased blood supply in enteric vessels due to local vasodilatation. It also interacts with small intestinal cells, enhancing their capability of absorbing numerous amino acids. Due to its easy partitioning; it can modulate membrane dynamics, thus helping to efficiently permeate drugs across barriers⁵. Thereby, taking into account the favorable impacts of these bioenhancers, it was deemed worth exploring the pharmacokinetic profile of BBC in the existence of quercetin, curcumin and piperine.

However, in the existence of quercetin, curcumin and piperine separately, neither of the studies has been published to access pharmacokinetic profile of BBC using rabbit model.

The data from our previous study (published^6-9 plus unpublished laboratory data) on optimizing ex-vivo permeability characteristics of BBC across goat intestinal membrane in presence and absence of bioenhancers was effectively adapted for in vivo pharmacokinetic studies in rabbits. Ex-vivo permeability optimized batches showed improved permeability characteristics of BBC upon pre-treatment with bioenhancers. Thus, it stimulated our interest to continue similar work; to investigate bioavailability enhancing potential of natural bioenhancers quercetin, curcumin and piperine for BBC upon oral administration in rabbits.

MATERIALS AND METHODS:

Materials:

Sample of BBC was gladly bestowed by Indo German Alkaloids, Mumbai, India. Quercetin and curcumin were purchased from High Media Laboratories Pvt. Ltd. India. Piperine was bought from Loba Chemie Pvt. Ltd. India. All the solvents and chemicals were of HPLC grade and purchased from Qualigen Fine Chemicals, Mumbai, India. The research experiment was carried out using Milli-Q water.

Development and validation of bioanalytical HPLC-UV method for estimation of BBC from Rabbit Plasma¹⁰:

In the present investigation, reverse phase HPLC method was developed and used for the estimation of BBC in plasma.

Instrumentation:

The chromatographic analysis was performed on a HPLC system (Shimadzu Prominence – i series LC-2030c 3D plus, Kyoto, Japan). Lab solutions Version 5.97 SP1 software was used for data collection and analysis.

Chromatographic Conditions:

BBC was separated chromatographically using a Shim-pack GIST C18 column (250 x 4.5mm i.d., 5m,) equipped with a C18 guard column (ODS; 4 x 3.0mm i.d., Phenomenex, USA). The mobile phase is a 45:55 v/v mixture of Methanol and 0.2 percent orthophosphoric acid (OPA) eluted at 1.0mL/min flow rate. The UV detection was done at 346nm, the column temperature was kept at 40°C, and each sample was injected at a volume of 20µL. The total chromatographic run time was 20min. Samples were quantified by determining the response (peak area Drug/peak area Internal Standard). p-Dimethyl amino benzaldehyde was used as internal standard in the present study.

Preparation of standard solutions:

A stock solution of BBC (1mg/mL) and IS (1mg/ml) were prepared by dissolving 10mg of each sample in methanol and stored at 4°C. The working solutions of BBC with concentrations of 1, 2, 4, 8, 12, 16 and 20 µg/mL were obtained by dilution of the stock solution with methanol. A solution containing 10µg/mL IS was also prepared in the methanol.

Calibration curve and quality control samples:

The samples for the standard calibration curve were prepared by spiking the blank rabbit plasma samples (180µL) with 10µL of the appropriate working solution of BBC to yield following concentrations of 50, 100, 200, 400, 600, 800 and 1000ng/ml. Finally 10µL of internal standard (IS) was added to each sample. Quality control samples were prepared from the blank rabbit plasma samples at concentrations of 100, 400 and 800 ng/ml.

Accuracy and Precision:

Intra-day precision and accuracy were calculated using six replicates of QC samples with BBC (100, 400, and 800ng/mL), while inter-day precision and accuracy were calculated using six replicates of concentrations 100, 400, and 800ng/mL from QC samples over three days, along with the standard calibration curve. The samples were extracted and analysed using HPLC-UV, as stated in the sample preparation section. The calibration curve was used to compute the analyte concentrations.

Recovery:

The recovery of the extraction method was estimated by comparing the peak area ratios of six extracted samples of 100, 400 and 800ng/mL with those of unextracted BBC samples.

Sample preparations:

A liquid–liquid extraction method was used to extract plasma samples, calibration standards, and QC samples. Plasma samples were thawed at room temperature after being stored at -80°C. An aliquot of 200µL of blank rabbit plasma samples were mixed with 10µL of IS working solution (10µg/mL). The mixture was then vortex blended for 3 minutes after adding 600µL of acetonitrile. At 4°C, the sample was centrifuged for 10 minutes at 4000rpm. The supernatant was separated and filtered through nylon filters with a mesh size of 0.45 µm. Under nitrogen stream, the filtrate was evaporated to dryness. An aliquot of 20µL was injected into the HPLC system after the residue was reconstituted with 200µL of mobile phase.

In-vivo pharmacokinetic studies of BBC using rabbit as an animal model:

IAEC approval:

The institutional animal ethical committee (IAEC) of Maratha Mandal’s college of Pharmacy, Belgaum, Karnataka, India agreed the proposed protocol of present animal studies. The approval was recorded and protocol approval number is MMCP/2019-20/B. Pharm/260.

Animals:

Sri Venkateshwara Enterprises, Bangalore, Karnataka, India, provided male New Zealand healthy rabbits (weight range: 1.5 to 2.0kg). All rabbits were housed under standard laboratory conditions. Prior to each experiment, the animals were acclimatised to laboratory conditions for at least seven days and then fasted for 12 h with free access to water. Prior to the beginning of the experiment, the Institutional Animal Ethics Committee evaluated and approved all experimental protocols.

Application to Pharmacokinetic studies:

Rabbits were divided into four groups (group I – BBC, group II - quercetin pre-treatment prior to BBC, group III - curcumin pre-treatment prior to BBC and group IV - piperine pre-treatment prior to BBC)randomly (n=6 in each group). The first group was orally dosed with 200 mg per kg body weight of BBC, suspended in 1% gum acacia aqueous solution just before each experiment. Group II, III and IV had received pre-treatment with the respective bioenhancer, followed by BBC 200mg/kg body weight given orally as mentioned in Table 1.

Table 1: Subjects and Study Design for in-vivo pharmacokinetic studies in rabbits.

Group	Combination	Dose (mg/kg)	Pre-treatment time with Quercetin / Curcumin/ Piperine (min)
Control	BBC	200	---------
I	BBC+ Quercetin	200+200	30
II	BBC+ Curcumin	200+40	60
III	BBC+ Piperine	200+40	30

BBC- Berberine chloride

At 0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, and 10 hours after administering the dose, blood samples (1ml) were collected in a heparinized Eppendorf tube via the marginal ear vein. The plasma samples were collected after centrifuging at 6,000 rpm for 10 minutes and then frozen at -80°C until analysis. The role of bioenhancer (quercetin/curcumin/piperine/) for bioavailability enhancement of BBC was evaluated at previously optimized dose and pre-treatment time (as mentioned in above Table 1).

Extraction of BBC from Plasma^10,11:

Protein precipitation method:

Plasma samples were removed from - 80°C storage and allowed to thaw at room temperature. An aliquot of 200 µL of rabbit plasma samples were combined with 10µL of IS working solution (10µg/mL). The mixture was then vortex blended for 3 minutes after adding 600µL of acetonitrile. At 4°C, the sample was centrifuged for 10 minutes at 4000rpm. The supernatant was separated and filtered through nylon filters with a mesh size of 0.45 µm. Under nitrogen stream, the filtrate was evaporated to dryness. An aliquot of 20µL was injected into the HPLC system after the residue was reconstituted with 200µL of mobile phase.

HPLC analysis of BBC plasma sample:

To measure the BBC plasma concentration, a bioanalytical HPLC method was developed and used. By introducing the supernatant liquid onto the HPLC column (loop volume 20μl and flow rate 1ml/min), the quantitative analysis of BBC in plasma was performed. The analysis was carried out at ambient temperature with a 20-minute run time, and the eluents were monitored using a UV detector at 246nm.

Data Analysis and Statistical analysis:¹⁰

The Microsoft Excel Add-in Program PK Solver was used to calculate the pharmacokinetic parameters. Non-compartmental analysis was used for the same. Graph Pad Prism software version 5.1 (GraphPad Software Inc., CA, USA) was used for statistical analysis utilizing appropriate statistical tests wherever applicable (ANOVA). Values of *p < 0.05, **p < 0.01, and ***p < 0.0001 were considered statistically significant.

RESULT:

Bioanalytical HPLC Method Development:

A new bioanalytical method was developed and validated for estimation of BBC in rabbit plasma. Under the mentioned chromatographic conditions, BBC and internal standard (IS) eluted at a retention time of 6.99 min and 14.72min respectively.

Linearity:

Standard curves were constructed by plotting ratio of peak areas of BBC to internal standard vs their respective concentration. The standard calibration curve for BBC (Figure 1) was found to be linear in the concentration range of 50-1000ng/mL with the regression equation of y = 0.0007x + 0.0009 and a correlation coefficient (r²) of 0.9915.Calibration curve of BBC in plasma samples, established within the range of 50 –1000ng/mL as shown in Table 2. Data are represented as the mean ± SEM of three determinations.

Table 2: Parameters for estimation of BBC in rabbit plasma by HPLC.

λmax	346 nm
Linearity range	50-1000 ng/mL
Regression equation	y = 0.0007x + 0.0009
Coefficient of correlation (r²)	0.9915

Figure 1: Calibration curve of BBC in plasma sample.

Accuracy and precision:

Table 3 presents intra- and inter-day precision and accuracy of the method for BBC determination. All accuracy and precision values were within the specified limits. For both intra-day and inter-day precisions, the coefficient of variation (CV) was less than 10%. BBC's intra-day and inter-day accuracies ranged between 88.71% and 92.54%. The result demonstrated that the developed procedure was precise and accurate.

Table 3: Intraday and interday precision and accuracy of BBC.

Sample	Nominal Conc. (ng/ml)	Calculated Conc. (ng/ml)	% CV	Accuracy %
Intraday (n=6)	100	90.76	3.39	92.54
	400	381.30	4.34	91.24
	800	782.43	2.93	88.71
Interday (n=18)	100	86.20	6.52	90.26
	400	376.52	3.84	89.54
	800	767.01	2.54	91.54

% CV- percentage coefficient of variation

Recovery:

Recovery of BBC ranged between 85.74 and 92.57%. The recovery of internal standard was 87.40%.

Figure 2 shows a typical chromatogram of blank plasma and Figure 3 shows chromatogram of plasma spiked with standard BBC (400 ng/ml) and IS. Figure 4, 5 and 6 indicates chromatogram of plasma sample from a quercetin pre-treated rabbit 2 h after administration of BBC, from a curcumin pre-treated rabbit 1 h after administration of BBC and from a piperine pre-treated rabbit 2 h after administration of BBC respectively.

Table 4: Pharmacokinetic parameters of BBC in presence and absence of all bioenhancers (Quercetin, Curcumin and Piperine) (optimized batch).

Parameter/ Animal no.

C_max (ng/ml)

T_max (h)

AUC_o-t (ng*h/ml)

Kel

(1/h)

t _1/2

(h)

V_d

(L)

Cl_T

(L/h)

MRT

(h)

BBC Control

119.27±

8.55

2.5

767.06

±102.33

0.130

±0.02

5.48±

0.96

1.46±

0.10

0.189±

0.04

8.84±

1.22

BBC+ Quercetin

598.57 ±

7.73

2336.34 ±

85.99

0.066

± 0.01

10.41±

0.54

0.665±

0.018

0.044±

0.01

14.27±

0.79

BBC+ Curcumin

320.36 ± 13.28

1152.80

±117.03

0.065

±0.01

10.75±

1.05

1.35±

0.13

0.09±

0.01

14.88±

1.48

BBC+ Piperine

866.53±

9.13

2471.13

±94.16

0.079

±0.01

8.82±

1.04

0.48±

0.21

0.045±

0.01

12.42±

1.34

Mean ±SD (n = 6); C_max-maximum plasma concentration, t_max -time to reach maximum concentration ; AUC - area under the curve; K_el - elimination rate constant; t_1/2-elimination half-life; V_d- volume of distribution; Cl_T - total clearance; MRT- mean residence time. PK- Pharmacokinetic parameters; BBC- Berberine Chloride.

Table 5: The statistical analysis test for pharmacokinetic parameters of control and bioenhancer optimized batch.

Group

C_max*

(ng/ml)

T_max

(h)

AUC (0-t)*

(ng*h/ml)

K_el*

(1/h)

t_1/2*

(h)

V_d*

(L)

Cl_T*

(L/h)

MRT*

(h)

BBC Control

119.27±

8.55

2.5

767.06±

102.33

0.130±

0.02

5.48±

0.96

1.46±

0.10

0.189±

0.04

8.84±

1.22

BBC+ Quercetin

598.57±

7.73^a,c

2336.34±

85.99^d,e,f

0.066±

0.01

10.41±

0.54

0.665±

0.018

0.044±

0.01

14.27±

0.79

BBC+ Curcumin

320.36±

13.28^a

1152.80±

117.03^d

0.065±

0.01

10.75±

1.05

1.35±

0.13

0.09±

0.01

14.88±

1.48

BBC+ Piperine

866.53±

9.13^a,b

2471.13±

94.16^d,e

0.079±

0.01

8.82±

1.04

0.48±

0.21

0.045±

0.01

12.42±

1.34

Table 4 depicts pharmacokinetic parameters of BBC (Mean±SD, n=6) in presence and absence of three different bioenhancers (optimized batch). The mean plasma concentration vs. time profile of BBC when administered alone and on pre-treatment with different bioenhancers (quercetin, curcumin and piperine) has been shown in Figure 7.

Pharmacokinetic parameters of BBC and all three optimized batches were compared. The C_max of piperine optimized batch (pre-treatment with 40mg piperine for 30min) was outstandingly increased by 626.53% (7.27-fold) than quercetin optimized batch (pre-treatment with 200mg quercetin for 30min) (401.86%; 5.02 – fold) than curcumin optimized batch (pre-treatment with 40mg curcumin for 60min) (168.60%; 2.69-fold) as compared to BBC (Control). Enhancement in the C_max of optimized batches showed enhancement in extent of drug absorption. The T_max was also notably minimized (1 h) for curcumin optimized batch and 2h for quercetin and piperine optimized batch as compared with BBC (control sample 2.5h) that demonstrated enhancement in rate of drug absorption. The AUC_o-t of all optimized batches were also boosted upto 204.58% (3.045-fold) for quercetin optimized batch, 50.29% (1.50-fold) for curcumin optimized batch and 222.16% (3.22-fold) for piperine optimized batch than BBC, that revealed remarkable improvement in the relative bioavailability of optimized batch compared with the BBC alone (control). Increase in peak plasma concentration of optimized batches, suggested enhancement in drug absorption. Increase in AUC of optimized batches demonstrated, improvement of bioavailability.

Piperine was also found to be significantly effective in enhancing pharmacokinetic parameters of BBC as compared to quercetin and curcumin, that revealed significant improvement in the relative bioavailability of all optimized batches compared with the BBC alone (control).These results suggested enhancement in drug absorption and improvement of drug bioavailability in bioenhancer optimized batches. Data represented in Table 4 illustrates marked correlation in pharmacokinetic parameters of BBC in presence and absence of bioenhancers.

Decreased worth of K_elwas observed in all permeability optimized batches when compared to control sample that showed reduced elimination rate.

Reduced value of V_d of all three optimized batches over control suggested enhancement of plasma concentration of BBC in optimized batch. Drug clearance was found to be reduced in all optimized batches over control that revealed greater plasma drug concentration. Half life (t_1/2) and mean residence time of all optimized batches were found to be increased as compared to control that indicated less of the drug being eliminated.

The results proposed that the optimized batch could promote the rate and extent of drug absorption and enhances drug half life with decrease in drug elimination rate and volume of distribution. The results inferred that bioenhancers significantly improved the oral bioavailability of BBC. As demonstrated in Table 4, there was a significant difference in C_max and AUC_0-tbetween the control and optimized batches.

The statistical analysis test for pharmacokinetic parameters of control and bioenhancer optimized batch was mentioned in Table 5. The pharmacokinetic parameters of the control and optimized batches were found to be significantly different.

DISCUSSION:

The limited oral bioavailability of BBC has been attributed to significant hepatic metabolism¹² or efflux by P-glycoprotein ¹³. As a result, any substance that inhibits BBC metabolism or alters the membrane transport via P-glycoprotein could actually boost its oral bioavailability.

The results of the pharmacokinetic studies showed that quercetin at a dose of 200 mg/kg body weight pre-treated for 30 min, curcumin at a dose of 40 mg/kg body weight pre-treated for 60 min and piperine at a dose of 40 mg/kg body weight pre-treated for 30 min, improved the oral bioavailability of BBC.

Quercetin has been shown to be a modulator of P-gp^6,7 and to inhibit the gastro-intestinal P-gp efflux pump and metabolising enzyme, CYP3A4 in vitro^6,7. It was previously observed in investigations of diltiazem oral bioavailability enhancement in rabbits pre-treated with quercetin that the bioavailability of diltiazem pre-treated with quercetin was greatly increased as compared to the control. Early gastro-intestinal absorption of quercetin inhibited diltiazem metabolising enzyme CYP3A4 and efflux pump P-gp, resulting in these effects¹⁴.

Similarly, in our in-vivo permeability studies in rabbits increase in the bioavailability of BBC during pre-treatment with quercetin was found.

In addition a detailed literature review revealed that, use of quercetin as a bioavailability enhancer was found to be very fruitful for enhancement in the bioavailability and pharmacokinetic parameters of therapeutically important drugs like losartan, chlorzoxazone, ranolazine, valsartan, doxorubicin^, epigallocatechin gallate, fexofenadine, etoposide, irinotecan, pioglitazone, tamoxifen, verapamil, paclitaxel, moxidectin. All these investigations concluded that quercetin significantly improved pharmacokinetic profile of drug. Because quercetin inhibits CYP 3A and the P-gp efflux pump, it has the ability to limit drug metabolism or modulate drug membrane transport respectively¹⁵.

In consonance with literature reports, co-administration or pre-treatment of numerous therapeutic molecules together with natural bioenhancer curcumin has really shown marked improvement in bioavailability profile of drug candidates. As curcumin was found to be a modulator of P-gp, it inhibits its function and expression⁸ therefore; the presence of curcumin might decrease the efflux of BBC due to inhibition of P-gp in the intestine.

Curcumin, a flavonoid found in turmeric, inhibits drug metabolizing enzymes such as CYP3A4 in the liver. It also reduces the amount of UDP-glucuronyl transferase in intestine and liver tissues. It has shown enhancement in the bioavailability of celiprolol and midazolam in rats by inducing change in the transporter P-gp. It also modifies the physiological activity in the GIT leading to better absorption of drugs⁸.

This observation was consistent with previous studies reporting that a single oral administration of curcumin by pre-treatment for 30 min with drugs like loratadine¹⁶, etoposide¹⁷ etc. resulting into its enhanced pharmacokinetic parameters.

It was also reported that, curcumin on pre-treatment or co-administration with therapeutically important drugs like marbofloxacin, rosuvastatin, docetaxel, ciprofloxacin, phospho-sulindac, warfarin and clopidogrel, losartan, norfloxacin, talinolol¹⁸ etc. resulting into its enhanced pharmacokinetic parameters.

Similarly, in our in-vivo permeability studies in rabbits increase in the bioavailability of BBC during pre-treatment with curcumin was found.

Piperine is one of the natural, safe and effective permeation enhancer having p-gp inhibitory activity which works through inhibition of drug metabolizing enzymes like CYP 3A4. Piperine mediated bioavailability enhancing action is partly due to increased blood supply in enteric vessels due to local vasodilatation. It also interacts with small intestinal cells and enhancing their capacity to absorb more amino acids. Due to its easy partitioning, it can modulate membrane dynamics, thus helping to efficiently permeate drugs across barriers^15.

As per literature review piperine inhibits human P-gp and CYP3A4 proteins that leads to ﬁrst-pass elimination of many drugs and that makes it an important enhancer for enhancing the bioavailability of a variety of drugs against different diseases. Literature review reveals that, co-administration of piperine (10mg/kg orally) along with several drug molecules, such as ciprofloxacin, gatifloxacin, ginsenoside Rh2, atenolol, fexofenadiene, phenytoin etc., has shown notable enhancement in pharmacokinetic profile of drug⁹. Also, pre-treatment studies of mice and healthy human volunteers with piperine (present as a food constituent) for 30min has shown significant enhancement in the pharmacokinetic profile of phenytoin¹⁹. It was also reported that, pre-treatment of piperine along with certain drug candidates like domperidone, diclofenac²⁰ etc. showed marked improvement in the pharmacokinetic profile of drug. Piperine has been shown to affect plasma concentrations of P-gp substrates and CYP3A4 substrates in humans, particularly when these drugs are taken orally. Drug metabolizing enzymes like CYP1A1, CYP1B1, CYP1B2, CYP2E1, and CYP3A4 etc get inhibited by piperine thus improves bioavailability of various drugs through its action on drug metabolism. It was noticed that, enhancement in the pharmacokinetics of drug was due to the cumulative impact of piperine on drug absorption kinetics and drug metabolism inhibitory effect. They recommended that piperine mediated P-gp efflux pump inhibition was predominantly attributed to intensified oral drug permeability and consequently bioavailability⁹.

Similarly, in the current pharmacokinetic investigation in rabbits, extremely significant improvement in the pharmacokinetic profile of BBC during pre-treatment with 40mg piperine for 30min was found.

All three bioenhancers were significantly effective in improving pharmacokinetic parameters such as C_max, T_max, AUC0_0-tand t_½ of BBC in their presence. When bioenhancing effect of these bioenhancers was statistically compared in terms of pharmacokinetic parameters, it was revealed that piperine exhibited strongest bioenhancing effect (rate and extent) on BBC absorption as compared to quercetin and curcumin. However, no significant difference was found in improving the extent of absorption between quercetin and piperine. The C_max and AUC_0-t of piperine optimized batch was significantly improved as compared to control, quercetin and curcumin optimized batches.

The results inferred that, all three bioenhancers shown significant improvement in all the pharmacokinetic parameters as compared to control. Therefore, taking into account the reasons for BBC's limited oral bioavailability, it can be interpreted that these bioenhancers may have the potential to block metabolism or modify membrane transport, since no mechanistic research have yet been performed to explicate its bioenhancing capacity. This could be ascribed to bioenhancer - mediated suppression of the P-gp efflux pump and drug metabolizing enzyme inhibition contributing to an improvement in the BBC pharmacokinetic profile. Amongst three different bioenhancers, piperine has shown extremely remarkable enhancement in all the pharmacokinetic parameters of BBC due to its virtue of multiples of action on the drug absorption kinetics and drug metabolism inhibitory effect that attributed for enhancement in oral drug bioavailability^5,21,22,23.

At the end it could be summarized that, pre-treatment of natural bioenhancers could be an effective approach to improve the biopharmaceutical characteristics of poorly permeable drug like molecules. In addition, an ultimate combination of Ayurveda and Allopathy could give multidirectional benefits in the way of therapeutics. Natural spices ingredients found to have a beneficiary influence on absorption of poorly absorbable drugs.

CONCLUSION:

The limitation of poor intestinal permeability and bioavailability of BBC could be successfully overcome by pre-treatment with bioenhancers. These effects might be due to bioenhancer mediated inhibition of the P-gp efflux pump and drug metabolizing enzymes which might be solely accountable for bioavailability improvement of BBC.

It would be expected that, improvement in the bioavailability of BBC in presence of bioenhancers would be expected to reduce dose, dosing frequency of BBC and thereby contributing for reduced toxicity, cost and improved patient compliance. However further clinical trials are necessary to be done in optimizing performance of this work.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

Authors are thankful to Principal, Government College of Pharmacy, Karad, Maharashtra, India for providing laboratory facilities and constant encouragement. Authors are grateful to Maratha Mandal’s NGH Institute of Dental Sciences and Research Centre, Belgaum, Karnataka, India for performing in-vivo pharmacokinetic studies on rabbit model.

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Received on 28.01.2022 Modified on 31.12.2022

Research J. Pharm. and Tech 2023; 16(11):5121-5129.

DOI: 10.52711/0974-360X.2023.00830