Simultaneous quantification of berberine and lysergol by HPLC-UV: evidence that lysergol enhances the oral bioavailability of berberine in rats

Research article

Received: 14 September 2011, Revised: 5 November 2011, Accepted: 5 November 2011 Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/bmc.2674

Simultaneous quantification of berberine andlysergol by HPLC-UV: evidence that lysergolenhances the oral bioavailability of berberinein ratsShrikant Patila, Ranjeet Prasad Dashb, Sheetal Anandjiwalaa andManish Nivsarkarb*

ABSTRACT: A sensitive and simple HPLCmethodwas developed for the simultaneous quantification of berberine and lysergol inrat plasma. The chromatographic separation was achieved on a C18 column using isocratic elution with methanol–acetonitrile–0.1% ortho-phosphoric acid (25:20:55, v/v/v), pH adjusted to 6.5 with triethylamine and detected at a UV wavelength of230nm. The extraction of the berberine and lysergol from the rat plasma with methylene chloride resulted in their highrecoveries (82.62 and 90.17%). HPLC calibration curves for both berberine and lysergol based on the extracts from the rat plasmawere linear over a broad concentration range of 50–1000ng/mL. The limit of quantification was 50ng/mL. Intra- and inter-dayprecisions were <15% and accuracy was 87.12–92.55% for berberine and 87.01–92.26% for lysergol. Stability studies showedthat berberine and lysergol were stable in rat plasma for short- and long-term period for sample preparation and analysis.The described method was successfully applied to study the pharmacokinetics of berberine as well as lysergol following oraladministration in Sprague–Dawley rats. The results of the study inferred that lysergol improved the oral bioavailability ofberberine. Copyright © 2011 John Wiley & Sons, Ltd.

Keywords: berberine; lysergol; HPLC; bioenhancement; pharmacokinetic

* Correspondence to: Manish Nivsarkar, Department of Pharmacology andToxicology, B. V. Patel Pharmaceutical Education and Research Develop-ment Centre, S. G. Highway, Thaltej, Ahmedabad – 380054, Gujarat, India.E-mail: [email protected]

a Department of Natural Products, National Institute of PharmaceuticalEducation and Research-Ahmedabad, S. G. Highway, Thaltej, Ahmedabad-380054, Gujarat, India

b Department of Pharmacology and Toxicology, B. V. Patel PharmaceuticalEducation and Research Development Centre, S. G. Highway, Thaltej,Ahmedabad 380054, Gujarat, India

IntroductionBerberine (Fig. 1), a quarternary protoberberine alkaloid, is foundin the roots, rhizomes and stem bark of Hydrastis canadensis,Coptis chinensis, Berberis aquifolium, Berberis vulgaris, Berberisaristata and Berberis thunbergii (Imanshahidi and Hosseinzadeh,2008). The alkaloid has multiple therapeutic actions that includethe treatment of bacterial infections, diarrhoea, intestinal parasiticinfections and ocular trachoma infections (Birdsall and Kelly, 1997).It is also reported to ameliorate diabetic nephropathy in rats (Liuet al., 2008) and to show hypoglycaemic (Gulfraz et al., 2008) andimmunomodulatory activities (Kim et al., 2003). On intravenousadministration, berberine easily crossed the blood–brain barrier(Wang et al., 2005). Preclinical evidence has suggested its usein various neurodegenerative and neuropsychiatric disorders(Birdsall and Kelly, 1997). Berberine is also available commerciallyas berberine sulfate capsules.

Previous reports on the pharmacokinetic studies of berberineshows that it exhibits extremely low plasma concentrations afteroral administration in humans (Li et al., 2000; Hua et al., 2007)and rats (Zuo et al., 2006). Poor absorption through the gut walland extensive metabolism are the reasons attributed for low oralbioavailability of berberine (Zuo et al., 2006). There is also acurrent report suggesting the role of P-glycoprotein for poorintestinal absorption of berberine (Pan et al., 2002). Thus, therearises a need for improving its bioavailability in order to achievedesired efficacy at a lower dose. Various approaches can be usedfor increasing its oral bioavailability that may be categorized as

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pharmaceutical (development of suitable formulations) orpharmacokinetic (using bioenhancers). However, in this study,an attempt was made to improve the oral bioavailability ofberberine using lysergol (Fig. 1), a natural bioenhancer. Lysergolis an alkaloid present in the seeds of Ipomoea muricata andIpomoea violace belonging to the Convolvulaceae family. It isreported to show hypotensive activity and psychotropic andanalgesic effects as well as uterus- and intestine-stimulatingactivities (Ferrari, 1975). Recently, its bioenhancing potentialhas been explored from in vitro studies, where it facilitated thetransport of the antibiotics across the membrane for betterefficacy on the target site (Khanuja et al., 2003). Its in vivo bioen-hancing potential, however, has not been investigated, whichinspired our study to explore and test whether similar effectswere observed for it in vivo to those in vitro. Analytical methodsfor simultaneous quantification of berberine and lysergol have

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Figure 1. Chemical structures of berberine, lysergol and alfuzosin.

S. Patil et al.

not been reported. Hence, a simple, economic and sensitive HPLC-UV method for the simultaneous plasma quantification ofboth berberine and lysergol was developed and validated. Thedeveloped method was applied for the pharmacokinetic study ofberberine in rats. In this method, alfuzosin (Fig. 1) was selectedas the internal standard, and chromatographic separation oflysergol, alfuzosin and berberine was completed within 14min.

Experimental

Chemicals and reagents

Lysergol (purity 97.38%) and alfuzosin (purity 99.3%) were obtained asgift samples from Chemical Resources, Haryana, India and Hetero DrugsLimited, Andhra Pradesh, India, respectively. Berberine (purity 98.27%)was purchased from Southern Petrochemical Industries Corporation Ltd,Chennai, India. All the solvents and chemicals used for the study were ofchromatographic grade and purchased from Qualigen Fine Chemicals,Mumbai, India. Potassium dihydrogen ortho-phosphate (Qualigen FineChemicals, Mumbai, India) and triethylamine (Spectrochem, Mumbai, India)were of analytical grade. Heparin was purchased from Biological E. Ltd,Hyderabad, India. Deionized water for HPLC was prepared in-house usinga Milli-Q water purifier system (Millipore Elix, Germany).

Isolation of berberine

Roots of Berberis aristata DC. were collected locally and authenticated.These were then shade-dried, stored in an air-tight container andpowdered to 40 mesh whenever required. A specimen of the plantcollected was preserved in the Department of Natural Products, NIPER-Ahmedabad (herbarium specimen no. NIPER-A/NP/1109/08). Berberinewas isolated from powdered root (100g) of Berberis aristata DC. Thepowdered root barks were percolated successively with 2.5% acetic acid(1 L) for 12 h. The filtrate obtained was treated with concentrated hydro-chloric acid until precipitation. The resulting reaction mixture was keptat a temperature of between 2 and 5 �C. Finally, golden yellow precipi-tates of berberine hydrochloride were obtained, which were separatedand dried under vacuum. Crude berberine hydrochloride was thenrecrystallized with the mixture of water and methanol (7:3, v/v). Thefinal yield of berberine was found to be 1.79%. The identity of berberinewas confirmed by co-chromatography with the standard berberine,melting point determination, mass, NMR and UV spectroscopy (datanot presented). The purity of berberine was determined by HPLC-Photo-Diode Array detector (PDA) and was found to be 97.6%.

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Chromatographic conditions

The HPLC system consisted of a PU-980 intelligent HPLC pump (Jasco,Hachioji, Tokyo, Japan) and Intelligent UV-975 UV–vis detector (Jasco,Hachioji, Tokyo, Japan) set at 230 nm and a manual injection port. Thedata were analysed using Borwin version 3.1 software. Chromatographicseparation was achieved using a LiChrosperW 100, RP-18e column(4.6mm i.d. � 250mm, 5mm) maintained at room temperature. Themobile phase consisted of methanol–acetonitrile–0.1% ortho-phosphoricacid (25:20:55, v/v/v) pH adjusted to 6.5 with triethylamine. The mobilephase was prepared daily and degassed before use. The flow-rate wasmaintained at 1.0mL/min. Samples were quantified by determining theresponse (peak areaDrug/peak areaIS).

Method validation

Calibration curve. One milligram each of berberine and lysergol weredissolved in methanol (final adjusted volume 100mL) to obtain a stocksolution with concentration of 10mg/mL. The working solutions wereprepared from the stock solution by dilution with methanol. Spikedplasma samples containing berberine and lysergol at concentrations of50, 100, 200, 400, 600, 800 and 1000 ng/mL were prepared using thestock and working solutions in order to plot calibration curve. The lowerlimit of quantification was determined as the lowest concentration of theanalytes in plasma that could be quantified with acceptable precisionand accuracy under the experimental conditions (less than 20% variationin precision). The analyte response at the lower limit of quantitation(LLOQ) was found to be 5 times the response of the blank response.The limit of detection was determined by injecting the serial dilutedstandard solutions to obtain a signal-to noise ratio of 3. The stockconcentration of internal standard was 2.5mg/mL, from which 20 mL wasadded to each sample. Liquid–liquid extraction of the plasma sampleswas carried out as per the procedure described below. Standard curveswere constructed by plotting ratio of the peak areas of berberine tointernal standard and lysergol to internal standard vs concentration.The calibration curves were obtained by least square linear regressionanalysis using a weighting scheme as 1/c (c= concentration) usingBorwin software version 1.3.

Preparation of quality control samples. The concentrations ofberberine and lysergol in rat plasma were 150, 500 and 900ng/mL torepresent low, medium and high quality control (QC) samples, respectively.To prepare QC samples, appropriate volumes of berberine and lysergolfrom the stock solution were transferred to 10mL stoppered centrifugetubes and solvent was evaporated under a gentle stream of nitrogen. Thenrequired amount of rat blank plasma was added and mixed. These QCsamples were stored at �80 �C and used for validation and application ofthe specific method.

Precision and accuracy. Intra-day precision and accuracy were calcu-lated by taking six replicates of QC samples with berberine and lysergol(150, 500 and 900 ng/mL) whereas inter-day accuracy and precisionwere calculated by taking six replicates of concentrations 150, 500 and900 ng/mL from QC samples for three consecutive days along with thestandard calibration curve. The samples were extracted as per theprocedure described below and analysed using HPLC-UV. Concentra-tions of the analytes were calculated from the calibration curve.

Extraction procedure. Twenty microlitres of alfuzosin (internal stan-dard; 2.5mg/mL) was added to 100 mL of rat plasma, and vortexed for1min. A 50 mL aliquot of 0.1 M sodium hydroxide solution was addedto the above sample. The resulting sample was vortexed for 1min,followed by addition of 1mL of methylene chloride. The mixture wasvortexed for 1min and centrifuged at 2000 rpm for 10min at 4 �C. Theorganic layer (lower phase) was transferred into a 10mL conical glasstube after discarding the supernatant and evaporated under the gentlestream of nitrogen gas. The residue was reconstituted in 50 mL of mobilephase, of which 20 mL was then injected on the HPLC column.

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Oral bioavailability enhancement of berberine by lysergol

Recovery. Recovery of the extraction procedure was calculated byanalyzing six extracted samples of 150, 500 and 900ng/mL and then com-paring the peak area ratio of these samples with those of unextractedberberine and lysergol samples.

Stability studies of plasma samples. Stability of berberine and lysergolin rat plasma during storage and processing were checked using QCsamples. Bench-top stability was determined for the aliquots of each ofthe low and high QC samples, which were spiked into rat plasma and keptat room temperature for 6 h prior to their analysis. For freeze–thaw stability,six replicates of high and low control samples were frozen at �80 �C andanalysed for three freeze–thaw cycles. A dry extract stability study wasperformed for six replicates of high and low controls after storing it at�80 �C for 24 h. The samples were processed and kept at room temper-ature for 6 h and then analysed after reconstitution with the mobilephase. Long-term stability of berberine and lysergol were checked forsix replicates of the high and low control samples after storing themfor 30 days at �80 �C. The samples were then processed and analysedas per the method described above.

Animals

Male Sprague–Dawley rats weighing 280–300g were obtained from theanimal house of B. V. Patel PERD Centre, Ahmedabad. The animals werehoused singly per cage in polypropylene cages and were placed in theexperimental room where they were allowed to acclimatize for a weekbefore experiment. A 10% air exhaust conditioning unit was maintainedalong with a relative humidity of 60� 5% and a temperature of 25� 3 �Cin the animal house facility. A 10:14 h light:dark cycle was also regulatedfor the experimental animals. Amrut certified rodent diet (MaharashtraChakan Oil Mill Ltd) and tap water (boiling hot water cooled to roomtemperature) were provided ad libitum to the experimental animals. Allexperimental protocols were reviewed and accepted by the InstitutionalAnimal Ethics Committee prior to initiation of the experiment.

Pharmacokinetic study of berberine and lysergol

Eighteen animals (six animals per group) were used in the study. The firstgroup was dosed with 200mg/kg body weight of berberine; the secondwith both 200mg/kg body weight of berberine and 20mg/kg bodyweight of lysergol; and the third with 20mg/kg body weight of lysergol.The dose of berberine was selected based on a previous report (Lu et al.,2006). However, the dose was finalized after analysing the results of a pi-lot study (study in small group of animals, n=3) which was based on thelevel of analyte detection in plasma. The role of lysergol for bioavailabil-ity enhancement was evaluated at different dose levels (20, 50 and100mg/kg body weight). The initial doses were selected based on thesuitability for unambiguous detection of lysergol in plasma. However,no significant difference was observed in the pharmacokinetic profileof berberine upon its coadministration with lysergol at the three differ-ent dose levels (data not presented). Thus, based on the pharmacoki-netic data of the dose titration studies and appropriate plasma detectionlevel, the dose of lysergol was finalized for 20mg/kg body weight. All thecompounds were orally administered as a suspension in 0.2% agar. Thejugular veins of all the animals were cannulated for collection of theblood at different sampling time points. The blood samples of 0.3mLwere withdrawn from each rat at 0 and 30min, and 1, 2, 2.25, 2.5, 2.75,3, 5, 7, 9 and 11 h, post dose, collected into heparinized microcentrifugetubes and centrifuged at 4000 rpm for 7min at 4 �C. The resulting plasmasamples were kept frozen at �80 �C prior to HPLC analysis.

Data analysis

The maximum plasma concentration (Cmax) and the time to reach themaximum concentration (Tmax) were directly determined from theplasma concentration vs time curves. The area under the curve from 0to t (AUC0�t) was calculated following linear trapezoidal rule by


summing the area from 0 to th. Elimination rate constant (Kel) was deter-mined by taking the absolute value of the slope of any three points lyingon a straight line of the curve after the Cmax, i.e. during the eliminationphase. Elimination half life (t1/2) was determined using the relationshipt1/2= 0.693/Kel. The volume of distribution (Vd) was calculated by dividingamount of drug dosed by the total plasma concentration. The total clear-ance (ClT) was calculated using the relationship ClT = 0.693Vd/t1/2.

Results and discussionA new analytical method for simultaneous estimation of berber-ine and lysergol in rat plasma was developed and validated. Thecurrent method uses liquid–liquid extraction procedure bymethylene chloride. The method is reproducible and specific.The chromatograms of blank rat plasma, unextracted pure stan-dards of berberine and lysergol, plasma spiked with mixed stan-dards of berberine and lysergol at a concentration of 200 ng/mL,and a plasma sample from a rat 2.5 h after administration of ber-berine along with lysergol are shown in Fig. 2(a–d), respectively.A well-resolved chromatogram of berberine and lysergol wasobtained following the use of the present HPLC-UV conditions.Retention times of lysergol, alfuzosin (internal standard) and ber-berine were 5.70, 7.13 and 11.62min, respectively. Total HPLCrun time was 14min. Berberine and lysergol were unambigu-ously identified in the plasma upon comparison of the retentiontimes with those of their respective standards.

Linearity and lower limit of quantification

Standard curves were constructed by plotting ratio of peakareas of berberine and lysergol to internal standard vs theirrespective concentration, which were linear in the range of50–1000 ng/mL. The correlation coefficient was found to bemore than 0.998 and 0.997 (n = 3) for extracted berberine andlysergol samples and 0.999 and 0.998 (n = 3) for unextractedberberine and lysergol samples, respectively. The lower limitof quantification was found to be 50 ng/mL; however, the lowerlimit of detection was 10 ng/mL for both berberine and lysergol.

Precision and accuracy

Table 1 shows intra-day and inter-day precision and accuracy.The intra-day precisions (coefficient of variation, CV) of low,medium and high QC samples of berberine were 6.91, 4.82, and7.16%, respectively, and of lysergol were 10.81, 3.85 and 2.91%,respectively. Inter-day precisions (CV) of low, medium and highQC samples of berberine were 9.23, 8.19 and 6.99% respectivelyand of lysergol were 10.53, 6.80 and 3.16%, respectively. Intra-dayand inter-day accuracies for berberine ranged between 87.12 and92.55% and for lysergol ranged from 87.01 to 92.26%. The resultsinferred that the developed method was accurate and precise.

Recovery

Recovery of berberine ranged between 83.44 and 89.91% andthat for lysergol was 82.62–90.17%. The recovery of internalstandard was 85.50%.

Stability study of plasma samples

Table 2 shows the results for bench-top stability, freeze–thawstability, dry extract stability and long-term stability. The results

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Figure 2. HPLC chromatograms of (a) blank rat plasma, (b) unextracted pure standards of berberine and lysergol, (c) plasma spiked with mixed standardsof berberine and lysergol at a concentration of 200ng/mL, and (d) plasma sample from a rat 2.5h after administration of berberine along with lysergol.

Table 1. Intra-day and inter-day precision and accuracy for berberine and lysergol

Nominalconcentration(ng/mL)

Calculated concentration (ng/mL) Precision (% CV) Accuracy (%)

Berberine Lysergol Berberine Lysergol Berberine Lysergol

Intra-day (n = 6)a

900 832.96 807.8 7.16 2.91 92.55 89.76500 436.7 461.3 4.82 3.85 87.34 92.26150 132.08 130.51 6.91 10.81 88.05 87.01Inter-day (n = 18)b

900 824.50 794.02 6.99 3.16 91.61 88.22500 435.60 442.60 8.19 6.80 87.12 88.52150 138.47 137.43 9.23 10.53 92.31 91.62aIntra-day precision: data expressed as means (n=6).bInter-day precision: data expressed as means (n= 18).

S. Patil et al.

showed that berberine and lysergol were stable during proces-sing and storage for up to one month.

Pharmacokinetic parameters

Pharmacokinetic parameters of the different animal groups areshown in Table 3. The mean plasma concentration vs timeprofile of berberine when administered alone and along withlysergol is shown in Fig. 3(a), whereas Fig. 3(b) shows the meanplasma concentration vs time profile of lysergol (single) andlysergol when co-administered with berberine in rats. The results


inferred that lysergol significantly improved the oral bioavailabil-ity of berberine.

Statistical analysis

One-way analysis of variance was applied to determine thedifference in bioavailability of berberine alone and with lysergol.A significant difference was found between the bioavailability ofberberine upon administration as a single compound and incombination with lysergol with p< 0.05 as calculated from AUCsof the respective groups. However, no significant difference was


Table 2. Stability data of berberine and lysergol in rat plasma

QCsamples

Mean concentration observed at 0 h Mean concentration observed at last h Deviation (%)

Berberine Lysergol Berberine Lysergol Berberine Lysergol

Bench-top stability (n = 6; after 6 h)High 827.00� 14.18 806.60� 19.49 822.00� 14.09 803.50� 19.42 4.20 5.92Low 136.68� 0.69 134.82� 0.21 133.88� 0.68 132.60� 0.20 1.24 0.38Freeze–thaw stability (n = 6; three cycles)High 831.47� 15.48 803.27� 21.44 827.10� 15.39 800.72� 21.37 4.56 6.54Low 132.55� 1.83 134.51� 2.55 130.80� 1.80 131.45� 2.50 3.38 4.65Dry extract stability (n = 6; 24 h)High 825.47� 15.87 807.45� 11.27 822.68� 15.82 803.25� 11.21 4.71 3.42Low 135.53� 1.73 132.46� 2.81 133.93� 1.71 130.23� 2.76 3.12 5.19Long-term stability (n = 6; 30 days)High 825.44� 17.32 805.57� 13.71 822.76� 17.26 799.66� 13.61 5.14 4.17Low 135.57� 2.18 132.54� 2.69 133.27� 2.14 130.14� 2.64 3.93 4.98

Data are expressed as means � SEM.

Table 3. Pharmacokinetic parameters of berberine and lysergol in rats upon oral administration of berberine, berberine withlysergol and lysergol

Groups Cmax

(ng/mL)Tmax (h) AUC0�t

(ng h/mL)Kel (1/h) t1/2 (h) Vd (L) ClT (L/h)

Group 1 (berberine) 112.30� 12.07 2.5� 0.00 655.03� 81.34* 0.136� 0.013 5.23� 0.41 1.14� 0.07 0.154� 0.015Group 2 (berberineand lysergol)Berberine

191.38� 15.42 2.44� 0.06 975.95� 44.11* 0.071� 0.004 9.88� 0.62 1.22� 0.04 0.086� 0.004

Group 2 (berberineand lysergol)Lysergol

356.43� 27.25 2.38� 0.07 1250.29� 68.12 0.024� 0.002 29.82� 3.71 1.48� 0.03 0.036� 0.004

Group 3 (lysergol) 316.54� 26.83 2.25� 0.00 1383.22� 68.57 0.032� 0.002 21.93� 1.75 1.40� 0.02 0.045� 0.004

Data are expressed as means � SEM, (n = 6); * p< 0.05.

Figure 3. Mean (� SEM) plasma concentration of (a) berberine (single) and berberine upon co-administration with lysergol and (b) lysergol (single)and lysergol upon co-administration with berberine in rats.

Oral bioavailability enhancement of berberine by lysergol

found in the bioavailability of lysergol when administered aloneand in combination with berberine. The results are expressed asmeans � SEM.

The results of the pharmacokinetic studies showed thatlysergol at a dose of 20mg/kg body weight improved the oral


bioavailability of berberine. However, it did not alter the absorp-tion and elimination pattern. The pharmacokinetic data showedthat berberine did not affect the pharmacokinetic profile oflysergol, because the pharmacokinetic profile of lysergol admin-istered alone was found to be same as that of the lysergol

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S. Patil et al.

administered along with berberine. Various studies have reportedthat the low bioavailability of berberine is due to extensivemetabolism in the liver (Zuo et al., 2006) or due to efflux byP-glycoprotein (Pan et al., 2002). Hence any compound thatcan inhibit the metabolism of berberine or alter the membranetransport via P-glycoprotein may prove helpful in enhancing itsoral bioavailability. Although lysergol is reported to be a bioenhan-cer from in vitro studies (Khanuja et al., 2003), no mechanisticstudies have been done yet to elucidate its bioenhancing prop-erty. Thus, considering the reasons for low oral bioavailability ofberberine, it may be inferred that lysergol might have the potentialto inhibit the metabolism or modulate the membrane transport.However, further studies are required to confirm the mechanisticapproach underlying the bioenhancing property of lysergol.

ConclusionA simple, precise, economical, specific and sensitive method forsimultaneous quantification of berberine and lysergol in ratplasma was developed. This is the first reported HPLC methodwith UV detection for simultaneous quantification of berberineand lysergol in rat plasma using liquid–liquid extraction. Stabilitystudies showed that berberine and lysergol were stable for short-and long-term periods (30 days). To improve the selectivity andspecificity of analytes, chromatographic separation was carriedout at a UV wavelength 230nm, using a C18 (4.6� 250mm,5mm) column. This analytical procedure was successfully appliedto the pharmacokinetic study of berberine alone and in combina-tion with lysergol in male Sprague–Dawley rats. The difference inthe pharmacokinetic profile of berberine upon administration asa single compound and along with lysergol was determined. Theresults showed that lysergol enhanced the bioavailability of orallyadministered berberine in rats.

AcknowledgementsThe authors wish to acknowledge NIPER – Ahmedabad for provid-ing all the facilities and their grant of a Junior Research ScholarshiptoMr Shrikant Patil to carry out this work; also Chemical Resources,India as well as Hetero Drugs Limited, India for providing lysergoland alfuzosin, respectively, as gift samples.


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Simultaneous quantification of berberine and lysergol by HPLC-UV: evidence that lysergol enhances the oral bioavailability of berberine in rats