|Year : 2015 | Volume
| Issue : 4 | Page : 159-164
Evaluation of the bioavailability of major withanolides of Withania somnifera using an in vitro absorption model system
Santosh T Devkar1, Amit D Kandhare2, Brian D Sloley3, Suresh D Jagtap4, James Lin3, Yun K Tam3, Surendra S Katyare1, Subhash L Bodhankar2, Mahabaleshwar V Hegde1
1 Centre for Innovation Nutrition Health Disease Interactive Research School for Health Affairs, Bharati Vidyapeeth University, Medical College Campus, Pune, Maharashtra, India
2 Department of Pharmacology, Poona College of Pharmacy, Bharati Vidyapeeth Deemed University, Pune, Maharashtra, India
3 SinoVeda Canada Inc. Suite 100, BBDC 2011 94th Street Edmonton, AB T6N 1H1 Canada, India
4 Interactive Research School for Health Affairs, Herbal Biotechnology Research Laboratory, Bharati Vidyapeeth University, Medical College Campus, Pune, Maharashtra, India
|Date of Web Publication||5-Oct-2015|
Subhash L Bodhankar
Department of Pharmacology, Poona College of Pharmacy, Bharati Vidyapeeth Deemed University, Erandwane, Paud Road, Pune - 411 038, Maharashtra
Source of Support: None, Conflict of Interest: None
Withania somnifera (L.) Dunal, shows several pharmacological properties which are attributed mainly to the withanolides present in the root. The efficacy of medicinally active withanolides constituents depends on the absorption and transportation through the intestinal epithelium. We examined these characteristics by employing the Sino-Veda Madin-Darby canine kidney cells culture system, which under in vitro condition shows the absorption characteristics similar to the human intestinal epithelium. Thus, the aim of the present investigation was to assess the bioavailability of individual withanolides. Withanolides were diluted in Hank's buffered saline at a concentration of 2 μg/ml were tested for permeability studies carried out for 1 h duration. Permeability was measured in terms of efflux pump (Peff ) in cm/s. Peff values of withanolide A (WN A), withanone (WNN), 1,2-deoxywithastramonolide (1,2 DWM), withanolide B (WN B), withanoside IV-V (WS IV-V), and withaferin A were 4.05 × 10−5 , 2.06 × 10−5 , 1.97 × 10−5 , 1.80 × 10−5 , 3.19 × 10−6 , 3.03 × 10−6 and 3.30 × 10−7 respectively. In conclusion, the nonpolar and low molecular weight compounds (WN A, WNN, 1,2 DWM, and WN B) were highly permeable. As against this, the glycosylated and polar WS IV and WS V showed low permeability. Surprisingly and paradoxically, the highly biologically active withaferin A was completely impermeable, suggesting that further studies possibly using human epithelial colorectal adenocarcinoma (Caco-2) cells may be needed to delineate the absorption characteristics of withanolides, especially withaferin A.
Keywords: Absorption model, ashwagandha, bioavailability, Caco-2 cells, Madin-Darby canine kidney cells, withanolides
|How to cite this article:|
Devkar ST, Kandhare AD, Sloley BD, Jagtap SD, Lin J, Tam YK, Katyare SS, Bodhankar SL, Hegde MV. Evaluation of the bioavailability of major withanolides of Withania somnifera using an in vitro absorption model system. J Adv Pharm Technol Res 2015;6:159-64
|How to cite this URL:|
Devkar ST, Kandhare AD, Sloley BD, Jagtap SD, Lin J, Tam YK, Katyare SS, Bodhankar SL, Hegde MV. Evaluation of the bioavailability of major withanolides of Withania somnifera using an in vitro absorption model system. J Adv Pharm Technol Res [serial online] 2015 [cited 2020 Sep 29];6:159-64. Available from: http://www.japtr.org/text.asp?2015/6/4/159/165023
| Introduction|| |
Withania somnifera, commonly known as Ashwagandha, Winter Cherry, and Indian ginseng, is one of the most important plants used for over 3000 years in Indian Ayurvedic medicinal system. W. somnifera shows several pharmacological activities which are mainly attributed to withanolides present in the roots. ,,, Withanolides are the naturally occurring C 28 steroidal lactones built on ergostane framework in which C 22 and C 26 are oxidized to form a six-member lactone ring [Figure 1]. About 35 withanolides have been isolated from the roots of W. somnifera of which withanoside V (WS V), withaferin A, withanolide A (WN A) and withanolide B (WN B) are the major components. ,, Withaferin A has anti-tumor, apoptotic, anti-angiogenesis, radiosensitizing and anti-inflammatory activities. ,,, WN A is effective as a neurological, immunological and anti-stress agent. ,, WS IV and V play an important neuro-regenerative role. Thus in spinal cord injury WS IV and V improve hindlimb function and increase the myelin layer in the peripheral nervous system.  In the light of this, it is important to find out the bioavailability of the individual withanolide compounds to ascertain their therapeutic efficacy. 
|Figure 1: The major withanolides of Withania somnifera and their high performance liquid chromatography elution profiles|
Click here to view
The intestinal epithelium plays an important role in absorption and transportation of drugs. It has been suggested that in vitro cell culture system may be a useful model system to quickly assess the bioavailability of a given drug. ,, Sino-Veda Canada has developed and patented a system employing Madin-Darby canine kidney (MDCK) cells which were found to be useful in assessing the bioavailability of various drugs. ,, In the present investigation, we report the bioavailability profiles of major withanolides of W. somnifera employing the MDCK cell culture system.
| Materials and methods|| |
Hanks' buffered saline, 4-(2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES) and Lucifer Yellow were parched form Sigma-Aldrich, St. Louis, MO, USA. WS IV, WS V, withaferin A (WF A), 1,2 deoxywithastramonolide (1,2 DWM), withanone (WNN), WN A and WN B were obtained from Natural Remedies Pvt. Ltd., Bengaluru, India. The purity of the standards was established by high-performance liquid chromatography (HPLC) analysis [Figure 1].
The individual withanolides were diluted in Hank's buffered saline and tested for permeability by the Sino-Veda's cell culture system [Figure 2]. The concentrations of withanolides in epical and basal chambers were determined by HPLC coupled to diode array absorbance detection (diode array detector [DAD]) and positive mode electrospray ionization mass spectroscopy (MS). A Phenomenex Luna 3 μ C18 (2) 100A 15 cm × 4.60 mm column equipped with a guard column (security guard C18) and column heater set at 40°C was used. For HPLC-MS analysis, mobile phase A was 5 mM ammonium acetate (pH 3.0 adjusted with formic acid) in 18 mega Ω water, and mobile phase B was HPLC grade acetonitrile. Based on the behavior of withanolides in the cell culture system the mobile phase gradient program was modified to obtain better resolution. Flow rate was 0.7 ml/min and solvent gradient program for WF A, WN A, WN B, WNN and 1,2 DWM was 0-15 min A: B (60:40%), 15-20 min A: B (15:85%) and 20-25 min A: B (60:40%). For WS IV 0-15 min A: B (70:30%), 15-20 min A: B (15:85%) and 20-25 min A: B (70:30%). Mobile phase program for WS V was 0-15 min A: B (70:30%), 15-20 min A: B (75:25%) and 20-25 min A: B (70:30%). DAD condition specific signals were collected at 205 nm (bandwidth 16), 210 nm (bandwidth 8), 254 nm (bandwidth 16), 270 nm (bandwidth 16) and 280 nm (bandwidth 16). All spectra were scanned from 190 nm to 400 nm with 2 nm step. Electrospray mass spectrometer conditions were in positive mode, gas temperature 350°C, drying gas 13 L/min, neb pressure 60 psi, vaporizer 350°C and capillary voltage 3000 V. Scan conditions were low mass 150, high mass 600, fragmentor 70, gain 1, threshold 150, stepsize 0.20. Selected ion mode signals were monitored at various ranges. Electrospray mass spectrometer selected ion mode signals were monitored at 471.3, 488.4 (WN A), 455.4, 472.4 477.4 (WN B), 471.2, 453.2, 493.2 (WNN), 471.3, 493.2 (WF A, 1,2 DWM), 411.2, 441.4, 459.4, 805.4 (WS IV) and 407.4, 425.4, 443.4, 767.4, 784.6, 789.4 (WS V) except for WS IV and WS V where the high mass is 850.
|Figure 2: Illustrates the cell culture chamber, the apical medium, to which the apical surface of a cell monolayer is exposed, and basal medium, to which the basal surface of a cell monolayer is exposed are provided to the cell culture chamber via the apical medium flow inlet and the basal media flow inlet. Samples to be tested for permeability are incubated under control conditions and allowed to pass through the cell monolayer. Samples collected from apical and basal chambers are analyzed by high performance liquid chromatography-mass spectroscopy for ascertaining permeability of different withanolides|
Click here to view
MDCK cells were cultured for 3 days and monolayers with Trans Epithelial Electric Resistance between 80 and 120 Ωcm 2 were used in the study. Standard stock solutions were prepared in methanol and further diluted with Hanks' buffered saline containing 20 mM HEPES buffer pH 7.4 to desired concentrations. Lucifer yellow was added to the test solution as an indicator for monitoring the integrity of membrane monolayer. Incubation was carried out in a shaker water bath (50-70 rpm) at 37°C for 1 h. Samples were collected from apical donor side before incubation and form basal receiver side after incubation [Figure 2]. All withanolides were tested at the concentration of 2 μg/ml which ascertained that the withanolides did not disrupt membrane integrity and the concentrations at the receiver side were within the permissible range of their quantification limits; it has been reported that the IC 50 for withanolides in cytotoxicity assay ranged from 0.067-9.3 μM (0.7-9.8 μg/ml). Thus, the concentrations used for the present studies were well within the permissible range.  Samples from the apical chamber were diluted 1:20 and the injection volume was 20 μL for LC-MS analysis. For samples from basal chamber, no dilution was necessary, and 40 μL was injected.
| Results|| |
HPLC studies revealed that the mean retention time (Rt ) for withanolides WS IV, WF A, 1,2 DWM, WN A, WS V, WNN, and WN B, respectively, was 2.25, 6.8, 7.75, 9.01, 9.2, 9.31, and 13.1 min. Representative mass spectrometric selected ion mode chromatograms for the individual withanolides which were collected from apical and basal chambers are shown in [Figure 3] and [Figure 4]. A single peak was observed for all withanolides except for WNN and WNB which showed an extra peak possibly representing a modified form or an adduct formation in the buffer system. WNN and WNB and the unidentified products were highly permeable. By contrast, WS IV and WS V seem to be partially permeable. The amount of individual withanolides in apical and basal chambers was quantified based on the standard curve. Permeability was measured in terms of efflux pump (cm/s) and was calculated as follows:
Peff values of WN A, WNN, 1,2 DWM, WN B, WS IV, WS V and WF A were 4.05 × 10−5 , 2.06 × 10−5 , 1.97 × 10−5 , 1.80 × 10−5 , 3.19 × 10−6 , 3.03 × 10−6 and 3.30 × 10−7 respectively [Table 1].
|Figure 3: Apical and basal distribution pattern for highly permeable withanolides. Unidentified product is denoted by D|
Click here to view
|Figure 4: Apical and basal distribution pattern for low and impermeable withanolides|
Click here to view
| Discussion|| |
The biological activity of some individual withanolides has been evaluated in in vivo assay system. ,,,,,,, However, as far as we are aware in vitro assay for evaluation of bioavailability of withanolides have not been carried out. Assessment of the bioavailability of a compound is a most important primary step in drug development. The intestinal epithelium plays an important role in the absorption and transportation of a given drug. It has been suggested that the in vitro cell culture may be useful systems for rapidly monitoring the absorption behavior of withanolides. , In view of this we decided to estimate the bioavailability of withanolides by using the Sino-Veda MDCK cell culture system. ,
It has been reported that WNA is the most stable and bioavailable withanolide under in vivo condition; ,,,, results of our previous studies are in conformity of this observation.  MDCK cells grown under the specified conditions have a narrow range of paracellular permeability (1 × 10−6 to 1 × 10−5 cm / s). We observed that the Peff value of WN A was 4.05 × 10−5 which indicates high permeability. The permeability observed in these cells correlates directly to the permeability in the human system.  Based on our results, we observe that the WNN, 1,2 DWM, and WN B are highly permeable [Figure 3]. [Figure 3] also shows that an unidentified product appeared in WNN and WN B. Apparently, only WNN and WNB seem to be susceptible under the experimental conditions. These products of WNN and WNB are unidentified and need further investigations. We observed that Peff values for the WS IV and WS V were 3.19 × 10−6 , 3.03 × 10−6 respectively signifying their low permeability as compared to other above mentioned withanolides. WS IV and WS V have glucose moiety at C 3 position [Figure 1]. Thus they are polar and also have higher molecular weight. It is known that hydrophobic interior of the cell membrane-the lipid bilayer-serves as a barrier to the passage of polar and high molecular weight molecules.  The gut has glucosidases which could hydrolyze the glucose moiety, thus facilitating the absorption of such compounds. In view of this it may be suggested that further studies on these lines may be warranted.
Several researchers have reported that WF A is a biologically highly active compound. ,,, In our earlier studies, we noted that the anti-oxidant potential of WF A is the highest.  After oral administration of aqueous extract of W. somnifera bioavailability of WF A was greater compared to WN A.  Surprisingly and paradoxically we observed that the Peff value of WF A was the lowest (3.30 × 10−7 ) implying that it may be impermeable under in vitro system, that is, in the MDCK cells or that it may be metabolized as it passes through the cell layer and we are unable to measure the metabolite. However, as mentioned above, after oral administration of aqueous extract of W. somnifera to mice there is significant absorption resulting in a high concentration of WF A in the plasma.  It seems that the process of WF A absorption is more complex, and the MDCK in vitro model possibly does not provide the exact in vivo environment. However, this possibility needs to be verified further by more direct experiments using different cell culture system such as human epithelial colorectal adenocarcinoma (Caco-2) cell monolayer.  Although both CaCo2 cells and MDCK cells are predictors for passive properties and lack of active transporters, the discrepancy seen in the present studies could likely be attributed to the lack of active transporters or species difference.
| Conclusions|| |
Based on the present studies on the absorption characteristics of the tested withanolides it may be concluded that WN A, WNN, 1,2 DWM and WN B were highly permeable; whereas WS IV, and WS V showed low permeable. Surprisingly WF A, the highly biologically active withanolide was found to be either impermeable or metabolized on passing through the cell layer. It is likely that absorption of WFA in vivo is a complex process and possibly a system employing Caco-2 cells could provide better insight in the absorption characteristics of WF A.
We thank Dr. Shivajirao Kadam, Vice Chancellor, Bharati Vidyapeeth Deemed University for his keen interest and encouragement. Dr. M. V. Hegde thankfully acknowledges the award of Research Grant by Indian Council of Agriculture Research under the National Agriculture Innovation Project.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Kulkarni SK, Dhir A. Withania somnifera
: An Indian ginseng. Prog Neuropsychopharmacol Biol Psychiatry 2008;32:1093-105.
Mishra LC, Singh BB, Dagenais S. Scientific basis for the therapeutic use of Withania somnifera
(ashwagandha): A review. Altern Med Rev 2000;5:334-46.
Devkar ST, Suryapujary SM, Jagtap SD, Katyare SS, Hegde MV. Effect of macronutrient deficiency on withanolides content in the roots of Withania somnifera and its correlationship with molybdenum content. Pharm Biol 2015;53:518-23.
Ali M, Shuaib M, Ansari SH. Withanolides from the stem bark of Withania somnifera
. Phytochemistry 1997;44:1163-8.
Sangwan RS, Das Chaurasiya N, Lal P, Misra L, Tuli R, Sangwan NS. Withanolide A is inherently de novo biosynthesized in roots of the medicinal plant ashwagandha (Withania somnifera
). Physiol Plant 2008;133:278-87.
Chatterjee S, Srivastava S, Khalid A, Singh N, Sangwan RS, Sidhu OP, et al.
Comprehensive metabolic fingerprinting of Withania somnifera
leaf and root extracts. Phytochemistry 2010;71:1085-94.
Devkar ST, Badhe YS, Jagtap SD, Hegde MV. Quantification of major bioactive withanolides in Withania somnifera
(ashwagandha) roots by HPTLC for rapid validation of ayurvedic products. J Planar Chromatogr Mod TLC 2012;25:290-4.
Yang H, Shi G, Dou QP. The tumor proteasome is a primary target for the natural anticancer compound withaferin A isolated from Indian winter cherry. Mol Pharmacol 2007;71:426-37.
Sen N, Banerjee B, Das BB, Ganguly A, Sen T, Pramanik S, et al.
Apoptosis is induced in leishmanial cells by a novel protein kinase inhibitor withaferin A and is facilitated by apoptotic topoisomerase I-DNA complex. Cell Death Differ 2007;14:358-67.
Mohan R, Hammers HJ, Bargagna-Mohan P, Zhan XH, Herbstritt CJ, Ruiz A, et al.
withaferin A is a potent inhibitor of angiogenesis. Angiogenesis 2004;7:115-22.
Devi PU, Sharada AC, Solomon FE. In vivo
growth inhibitory and radiosensitizing effects of withaferin A on mouse Ehrlich ascites carcinoma. Cancer Lett 1995;95:189-93.
Kuboyama T, Tohda C, Komatsu K. Neuritic regeneration and synaptic reconstruction induced by withanolide A. Br J Pharmacol 2005;144:961-71.
Malik T, Pandey DK, Dogra N. Ameliorative potential of aqueous root extract of Withania somnifera
against paracetamol induced liver damage in mice. Pharmacologia 2013;4:89-94.
Kour K, Pandey A, Suri KA, Satti NK, Gupta KK, Bani S. Restoration of stress-induced altered T cell function and corresponding cytokines patterns by withanolide A. Int Immunopharmacol 2009;9:1137-44.
Nakayama N, Tohda C. Withanoside IV improves hindlimb function by facilitating axonal growth and increase in peripheral nervous system myelin level after spinal cord injury. Neurosci Res 2007;58:176-82.
Kohli K, Chopra S, Dhar D, Arora S, Khar RK. Self-emulsifying drug delivery systems: An approach to enhance oral bioavailability. Drug Discov Today 2010;15:958-65.
Pang G, Buret A, O′Loughlin E, Smith A, Batey R, Clancy R. Immunologic, functional, and morphological characterization of three new human small intestinal epithelial cell lines. Gastroenterology 1996;111:8-18.
Engman HA, Lennernäs H, Taipalensuu J, Otter C, Leidvik B, Artursson P. CYP3A4, CYP3A5, and MDR1 in human small and large intestinal cell lines suitable for drug transport studies. J Pharm Sci 2001;90:1736-51.
Owens RB, Smith HS, Nelson-Rees WA, Springer EL. Epithelial cell cultures from normal and cancerous human tissues. J Natl Cancer Inst 1976;56:843-9.
Irvine JD, Takahashi L, Lockhart K, Cheong J, Tolan JW, Selick HE, et al.
MDCK (Madin-Darby canine kidney) cells: A tool for membrane permeability screening. J Pharm Sci 1999;88:28-33.
Lin YC, Tam Y, Semple H, Sloley B. Model epithelial cell cultures. 2003. Publication number WO2004018657 A1.
Hugger ED, Novak BL, Burton PS, Audus KL, Borchardt RT. A comparison of commonly used polyethoxylated pharmaceutical excipients on their ability to inhibit P-glycoprotein activity in vitro
. J Pharm Sci 2002;91:1991-2002.
Zhang H, Samadi AK, Gallagher RJ, Araya JJ, Tong X, Day VW, et al.
Cytotoxic withanolide constituents of Physalis longifolia
. J Nat Prod 2011;74:2532-44.
Anderson BJ, Holford NH, Armishaw JC, Aicken R. Predicting concentrations in children presenting with acetaminophen overdose. J Pediatr 1999;135:290-5.
Patil D, Gautam M, Jadhav U, Mishra S, Karupothula S, Gairola S, et al.
Physicochemical stability and biological activity of Withania somnifera
extract under real-time and accelerated storage conditions. Planta Med 2010;76:481-8.
Patil D, Gautam M, Mishra S, Karupothula S, Gairola S, Jadhav S, et al.
Determination of withaferin A and withanolide A in mice plasma using high-performance liquid chromatography-tandem mass spectrometry: Application to pharmacokinetics after oral administration of Withania somnifera
aqueous extract. J Pharm Biomed Anal 2013;80:203-12.
Devkar S, Jagtap S, Katyare S, Hegde M. Estimation of antioxidant potential of individual components present in complex mixture of Withania somnifera
(Ashwagandha) root fraction by thin-layer chromatography-2, 2-diphenyl-1-picrylhdrazyl method. J Planar Chroma-Modern TLC 2014;27:157-161.
Sun D, Lennernas H, Welage LS, Barnett JL, Landowski CP, Foster D, et al.
Comparison of human duodenum and Caco-2 gene expression profiles for 12,000 gene sequences tags and correlation with permeability of 26 drugs. Pharm Res 2002;19:1400-16.
Chaffey N, Alberts B, Johnson A, Lewis J, Raff M, Roberts K, et al
. Molecular biology of the cell. Ann Bot 2003;91:401.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
|This article has been cited by|
||Withanolides from the genus Physalis
: a review on their phytochemical and pharmacological aspects
| ||Min Huang,Ji-Xiang He,Hui-Xin Hu,Kan Zhang,Xiao-Ning Wang,Bao-Bing Zhao,Hong-Xiang Lou,Dong-Mei Ren,Tao Shen |
| ||Journal of Pharmacy and Pharmacology. 2019; |
|[Pubmed] | [DOI]|
||Assessment of the Bioaccessibility and Bioavailability of the Phenolic Compounds of Prunus avium L. by in Vitro Digestion and Cell Model
| ||Joana Gonçalves,Rodrigo Ramos,Ângelo Luís,Sandra Rocha,Tiago Rosado,Eugenia Gallardo,Ana Paula Duarte |
| ||ACS Omega. 2019; 4(4): 7605 |
|[Pubmed] | [DOI]|
||Studies on oral bioavailability and first-pass metabolism of withaferin A in rats using LC–MS/MS and Q-TRAP
| ||Tianming Dai,Weifan Jiang,Zizheng Guo,Zhenyu Wang,Mingping Huang,Guorui Zhong,Chuxin Liang,Xuzhe Pei,Renke Dai |
| ||Biomedical Chromatography. 2019; : e4573 |
|[Pubmed] | [DOI]|
||Exploring Medicinal Plant Legacy for Drug Discovery in Post-genomic Era
| ||Satendra Singh,Dev Bukhsh Singh,Shivani Singh,Rohit Shukla,Pramod W. Ramteke,Krishna Misra |
| ||Proceedings of the National Academy of Sciences, India Section B: Biological Sciences. 2018; |
|[Pubmed] | [DOI]|
||Determination of permeability, plasma protein binding, blood partitioning, pharmacokinetics and tissue distribution of Withanolide A in rats: A neuroprotective steroidal lactone
| ||Sandeep K. Singh,Guru R. Valicherla,Pankaj Joshi,Sudhir Shahi,Anees A. Syed,Anand P. Gupta,Zakir Hossain,Kishan Italiya,Vishal Makadia,Shio K. Singh,Mohammad Wahajuddin,Jiaur R. Gayen |
| ||Drug Development Research. 2018; |
|[Pubmed] | [DOI]|
||Ameliorative effect of berberine against gentamicin-induced nephrotoxicity in rats via attenuation of oxidative stress, inflammation, apoptosis and mitochondrial dysfunction
| ||Mohammad Adil,Amit D. Kandhare,Gautami Dalvi,Pinaki Ghosh,Shivakumar Venkata,Kiran S. Raygude,Subhash L. Bodhankar |
| ||Renal Failure. 2016; 38(6): 996 |
|[Pubmed] | [DOI]|
||Hepatoprotective effect of withanolide-rich fraction in acetaminophen-intoxicated rat: decisive role of TNF-a, IL-1ß, COX-II and iNOS
| ||Santosh T. Devkar,Amit D. Kandhare,Anand A. Zanwar,Suresh D. Jagtap,Surendra S. Katyare,Subhash L. Bodhankar,Mahabaleshwar V. Hegde |
| ||Pharmaceutical Biology. 2016; : 1 |
|[Pubmed] | [DOI]|
||Withania somnifera: From prevention to treatment of cancer
| ||Dushani L. Palliyaguru,Shivendra V. Singh,Thomas W. Kensler |
| ||Molecular Nutrition & Food Research. 2016; 60(6): 1342 |
|[Pubmed] | [DOI]|