High-performance thin-layer chromatography marker-based standardization of Piperine, Asiaticoside, and Withanolide-A in the developed polyherbal formulation and in vitro evaluation of acetylcholinesterase inhibition
Vasudev Pai, KS Chandrashekar, Polu Picheshwara Rao, Manganahalli Manjunath Setty
Department of Pharmacognosy, Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher Education, Manipal, Karnataka, India
|Date of Submission||15-Jan-2019|
|Date of Decision||21-Mar-2019|
|Date of Web Publication||23-Aug-2019|
Manganahalli Manjunath Setty
Department of Pharmacognosy, Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher Education, Manipal - 576 104, Karnataka
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Preparation of highly standardized polyherbal formulation with its chief active chemical constituents supported by therapeutic efficacy in vitro is a valuable approach in the field of pharmaceutical sciences. Objective: The present work aims to develop the high-performance thin-layer chromatography (HPTLC) marker-based standardization of polyherbal formulation using Piperine, Asiaticoside, and Withanolide-A and in vitro acetylcholinesterase inhibition activity. Materials and Methods: For successful standardization, the HPTLC quantification of Piperine, Asiaticoside, and Withanolide-A was carried out. Suitable solvent systems were optimized to achieve the better resolution of the marker compounds, extracts, and sample formulation. The reproducibility of the methods was also confirmed by repeating the procedure twice. The identity of the bands in the sample formulation was confirmed by comparing the Rf value with those of their respective reference standards. In vitro acetylcholinesterase inhibition assay was done on the different crude extracts as well as tablet formulation. Results: HPTLC quantification of formulation for Piperine content showed 1.97% content w/w, whereas Piper longum extract showed 2.44% w/w. Similarly, Asiaticoside showed 0.71% in formulation and in Centella asiatica extract, it was 1.33% w/w. Contrary to the above, Withanolide-A content was 2.12% w/w in formulation and in Withania somnifera extract, it was only 0.81% w/w. In vitro acetylcholinesterase inhibition assay exhibited significant inhibition with IC50 value of 70 μg for tablet formulation. Conclusion: The presence of Piperine, Asiaticoside, and Withanolide-A in formulation was identified by rapid HPTLC quantification method. The method is very precise, reproducible, and accurate. The developed method can be used to quantify the marker compounds in the formulations available in the market. The formulation also evaluated for its acetylcholinesterase inhibition assay and showed significant activity. Hence, it could be used in the treatment of memory-related disorders such as Alzheimer's disease.
Keywords: Acetylcholinesterase inhibition, Alzheimer's disease, Asiaticoside and Withanolide-A, high-performance thin-layer chromatography quantification, Piperine
|How to cite this article:|
Pai V, Chandrashekar K S, Rao PP, Setty MM. High-performance thin-layer chromatography marker-based standardization of Piperine, Asiaticoside, and Withanolide-A in the developed polyherbal formulation and in vitro evaluation of acetylcholinesterase inhibition. Phcog Mag 2019;15, Suppl S2:256-60
|How to cite this URL:|
Pai V, Chandrashekar K S, Rao PP, Setty MM. High-performance thin-layer chromatography marker-based standardization of Piperine, Asiaticoside, and Withanolide-A in the developed polyherbal formulation and in vitro evaluation of acetylcholinesterase inhibition. Phcog Mag [serial online] 2019 [cited 2021 May 14];15, Suppl S2:256-60. Available from: http://www.phcog.com/text.asp?2019/15/64/256/265019
- The therapeutic effect of polyherbal formulation is mainly because of the presence of their chief active constituents, the quantification of the active compounds present in the formulation was performed by rapid HPTLC method using marker compounds like Piperine, Asiaticoside and Withanolide-A. The HPTLC method is very precise, accurate and also reproducible. The formulation was also evaluated by in vitro acetylcholinesterase inhibition assay and showed a promising inhibition. Hence it proves that the formulation could be used in the management of Alzheimer's disease.
Abbreviations used: HPTLC: High-performance thin-layer chromatography; AD: Alzheimer's disease; DC: Direct compression; AchE: Acetylcholinesterase enzyme; RS: Reference standard; PE: Piper extract, SF: Sample formulation; CE: Centella extract; WE: Withania extract; ICH: International Council for Harmonization.
| Introduction|| |
Alzheimer's disease (AD) is now selected as a major health problem which is affecting thousands of elderly people and their families worldwide. The incidence rates range from 1% to 5% of the population every year and are adding to the group of elderly people. In the USA, AD is the sixth leading cause of death, and about 5 million elderly people are suffering and living with AD. One in three seniors lose their life because of AD and other forms of dementia. Alzheimer's organization 2016 shows that about 5.4 millions of Americans are having AD and was estimated that around 5.2 million elderly people are of the 65 years' age group. Approximately 0.2 million individuals are under age group 65 who are having high risk of young-onset Alzheimer's. These numbers are rapidly increasing in the upcoming years. By 2050, the number of people in the 65 years' age group having AD may be nearly 5.3 million to 13.9 million, respectively.,,
Herbal product standardization supports and encourages marketing opportunities for polyherbal formulations. Standardization includes many steps and is starting from raw material to its end products. The main chemical composition present in the formulation in optimum level is responsible for biopotency. Hence, it is very important to estimate such active constituents using marker compounds and developing the analytical methodology for its identification. The marker-based standardization using high-performance thin-layer chromatography (HPTLC) is one of the best analytical methods to standardize most of the herbal formulation, which gives an idea about the required constituent in that formulation.,
The polyherbal formulation consists of hydroalcoholic extracts of Piper longum, Centella asiatica, and Withania somnifera. The polyherbal formulation is developed in the form of tablet formulation and is evaluated for its acetylcholinesterase inhibition assay. The present work was aimed to standardize the formulation using marker compound Piperine, Asiaticoside, and Withanolide-A by the HPTLC method.
| Materials and Methods|| |
The hydroalcoholic extracts were used to make tablet formulation with DC grade excipients. The developed tablet is used for quantification of Piperine, Asiaticoside, and Withanolide– A. Extracts used in the formulation are given in [Table 1].
Extraction and preparation of polyherbal formulation
The compressed tablet was powdered and extracted in methanol, and the individual drug extracts were also dissolved in methanol and dried. The dried powder in used for HPTLC quantification.
Chemicals and solvents
Asiaticoside and Withanolide-A were procured from Natural Remedies Pvt. Ltd, Bengaluru, and Piperine was procured from Sigma Aldrich Pvt. Ltd. (Steinheim, Germany; ≥97.0%). All the other solvents and chemicals were of analytical grade and were purchased from Merck, Ltd., Mumbai, and SD Fine Chemicals, Mumbai.
Development of solvent system by thin-layer chromatography study
Standard marker compound, individual crude extracts, and sample formulation were subjected for TLC study using different solvent systems, and optimization was carried out for each marker compound. The solvent system optimization was carried out to get maximum separation of phytoconstituents and the same solvent system was used for HPTLC studies.
Development of high-performance thin-layer chromatography method
High-performance thin-layer chromatography instrumentation
The sample solution is applied in the pattern of bands of 6 mm with sample applicator 100 μl syringe on precoated TLC plate Silica gel 60F254 of E. Merck (20 cm × 10 cm with 250 μm thickness) using Linomat applicator. The slot dimension was kept about 10 cm × 10 cm, each track was scanned three times and baseline correction was performed. The mobile phase for piperine consists of toluene: ethyl acetate (9:1), the mobile phase for Asiaticoside was ethyl acetate: methanol: water (10:2.5:1), and the mobile phase for Withanolide-A consists of toluene: ethyl acetate: formic acid (5:5:1). The Camag twin trough glass chamber saturated with the respective mobile phases is used for each marker compound. The optimum time for saturation of the glass chamber was about 25–30 min at room temperature around 25°C–27°C. The migration distance was about 80 mm. The precoated TLC plates were dried using drier with hot air. Densitometric scanning is also done using Camag TLC Scanner at different wavelengths.
Preparation of solution
Preparation of standard solution of marker compounds
- Piperine 100μg/ml in methanol (Sigma Aldrich with purity 97%).
- Asiaticoside 100μg/ml in methanol (Natural Remedies with purity 95%).
- Withanolide – A 100μg/ml in methanol (Natural Remedies with purity 95%).
Preparation of sample solutions
5 mg/ml of Piper longum, Centella asiatica and Withania somnifera extracts were taken in methanol. Similarly, sample formulation was also prepared as 5mg/ml in methanol.
Estimation of marker compounds in sample formulation
A volume of 40 μL of standard Piperine solution, 40 μL of P. longum extract, and 40 μL of sample formulation were used for spotting. The plates were developed and dried. For Piperine, the plate was derivatized using 5% aqueous H2 SO4 and heated at 110°C. The developed plates were scanned at 254 nm, 366 nm, and visible light.,,
A volume of 40 μL of standard Asiaticoside solution, 40 μL of C. asiatica extract, and 40 μL of sample formulation were used for spotting. The plates were developed and dried. For Asiaticoside, the plate was derivatized using 5% aqueous H2 SO4 and heated at 110°C. The developed plates were scanned in visible light.,
A volume of 40 μL of standard Withanoilde solution, 40 μL of W. somnifera extract, and 40 μL of sample formulation were used for spotting. The plates were developed and dried. For Withanolide-A, the plate was derivatized using 5% aqueous H2 SO4 and heated at 110°C. The developed plates were scanned in 254 nm and visible light.
In vitro acetylcholinesterase inhibition assay
Approaches to enhance cholinergic function in AD have included stimulation of cholinergic receptors or prolonging the availability of acetylcholinesterase (Ach) released into the neuronal synaptic cleft by use of agents which restore the level of acetylcholine through inhibition of ACh enzyme (AChE). AChE holds a key role not only to enhance cholinergic transmission in the brain but also to reduce the aggregation of β-amyloid and the formation of the neurotoxic fibrils in AD. Therefore, AChE inhibitors have become remarkable alternatives in treatment of AD. Inhibiting the activity of AChE increases the concentration of the neurotransmitter with positive effect on cognitive function. Inhibition of AChE also serves as a strategy for the treatment of not only AD but also senile dementia.
The colorimetric method of Ellman et al. (1961) which is based on determining the amount of thiocholine released when acetylthiocholine is hydrolyzed by AChE is widely used. The thiocholine released is quantified by its reaction with 5,5'-bisdithionitrobenzoic acid (DTNB), which produces a yellow 5-thio-2-nitrobenzoate anion.
Extracts used for assay
Hydroalcoholic extracts of P. longum, C. asiatica, and W. somnifera and punched tablet was dissolved in 90% alcohol and extract was used for the assay.
Acetylcholinesterase inhibition assay
Cholinesterase inhibition assay was performed in flat-bottom 96-well microplates using colorimetric method by Ellman et al. and was adapted by Okello et al. The run volume consisted of 5μL of bovine AChE solution, 200 μL of 0.1 M phosphate buffer (pH 8), 5 μL of DTNB in 0.1M phosphate buffer (pH 7) and 5μL of test extract. All the reactants were mixed and preincubated for about 15 min at 30°C. The reaction was started by adding 5 μL of ATChI at a final concentration of 0.5 mM. As a control, the inhibitor solution was replaced with buffer. The control was assayed three times. To check any nonenzymatic hydrolysis in the final reaction mixture, two blanks for each run were prepared in triplicate. One blank consisted of buffer replacing enzyme and a second blank had buffer replacing substrate. Change in absorbance at 405 nm was measured on a TECAN Sunrise Microplate 96-well plate reader for a period of 6 min at 30°C.,
| Results and Discussion|| |
Piperine is one of the important therapeutic phytoconstituents, and in this study the quantification of Piperine in the formulation and extract was determined by rapid HPTLC method. The solvent system used to develop chromatogram was toluene:ethyl acetate (9:1) and Rf value 0.24 and the details are given in [Table 2]. The developed plates were scanned at 254, 366, and in visible light [Figure 1]. Three-dimensional (3D) chromatogram of Piperine is shown in [Figure 2].
|Table 2: Details of quantification of Piperine in the formulation and Piper longum extract|
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|Figure 1: Thin-layer chromatography of Piperine at 254 nm, 366 nm, and in visible light|
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C. asiatica is also popularly known as brahmi and is one of the traditional herbs used in Ayurveda for memory-related disorders. The chief important constituent is Asiaticoside. Solvent system used for quantification of Asiaticoside was ethylacetate:methanol:water (10:2.5:1) and the Rf value was 0.71. The details are given in [Table 3]. The developed plates were scanned in visible light only [Figure 3]. 3D chromatogram of Asiaticoside is shown in [Figure 4].
|Table 3: Details of quantification of Asiaticoside in the formulation and Centella asiatica extract|
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Ashwagandha is one of the oldest medicinal herbs widely used in the Ayurvedic system of medicine for various health complications. It contains various constituents and Withanolide-A is one among having wide therapeutic actions on memory-related deficits. Solvent system used for quantification of Withanolide-A was toluene:ethylacetate:formic acid (5:5:1) and the Rf value was 0.77; the details are given in [Table 4]. The developed plates were scanned at 254 and in visible light [Figure 5]. 3D chromatogram of Withanolide-A is shown in [Figure 6].
|Table 4: Details of quantification of Withanolide-A in the formulation and Withania extract|
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|Figure 5: Thin-layer chromatography of Withanolide-A at 254 nm and in visible light|
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Acetylcholinesterase inhibition assay
The assessment of cholinesterase inhibition was done with hydroalcoholic extracts of P. longum, C. asiatica, and W. somnifera and punched tablet formulation. The results of the assay are more promising by inhibiting the enzyme acetylcholinesterase that is one of the hallmarks in controlling the disease symptom. [Table 5] and [Figure 7] show the results of acetylcholinesterase inhibition assay.
| Summary and Conclusion|| |
The proposed HPTLC method for standardizing and validating the formulation as per the ICH guidelines and was very rapid and accurate for quantitative estimation of Piperine, Asiaticoside, and Withanolide-A in the extracts and tablet formulation. The method is very reliable and suitable for estimation of marker compound present in the formulation which is having most effective therapeutic efficacy and found to be very helpful in detecting the quality of the extracts and formulation made out of it. The formulation was also evaluated for its efficacy by in vitro acetylcholinesterase inhibition and was found to be more promising in controlling symptoms of memory-related deficits.
The authors are thankful to Department of Pharmacognosy, Manipal College of Pharmaceutical Sciences and Manipal Academy of Higher Education for providing laboratory facility and other infrastructure to carry out the work.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Adsersen A, Gauguin B, Gudiksen L, Jäger AK. Screening of plants used in Danish folk medicine to treat memory dysfunction for acetylcholinesterase inhibitory activity. J Ethnopharmacol 2006;104:418-22.
Adewusi EA, Moodley N, Steenkamp V. Antioxidant and acetylcholinesterase inhibitory activity of selected Southern African medicinal plants. S Afr J Bot 2011;77:638-44.
Machado LP, Carvalho LR, Young MC, Cardoso-Lopes EM, Centeno DC, Zambotti-Villela L, et al
. Evaluation of acetylcholinesterase inhibitory activity of Brazilian red macroalgae organic extracts. Rev Bras Farmacogn 2015;25:657-62.
Ladva BJ, Mahida VM, Kantaria UD, Gokani RH. Marker based standardization of polyherbal formulation (SJT-DI-02) by high performance thin layer chromatography method. J Pharm Bioallied Sci 2014;6:213-9.
Wagner H, Bladt S. Plant drug analysis: A thin layer chromatography atlas. Springer-Verlag Berlin Heidelberg 1996.
Jeganathan NS, Kannan K. Quantitative estimation of piperine in Trikatuku curanam. Asian J Chem 2008;20:1621.
Rajopadhye A, Upadhye A, Mujumdar A. HPTLC method for analysis of piperine in fruits of Piper
species. J Planar Chromatogr Mod TLC 2011;24:57-9.
Parmar VS, Jain SC, Bisht KS, Jain R, Taneja P, Jha A, et al
. Phytochemistry of the genus Piper
. Phytochemistry 1997;46:597-673.
Gupta A, Verma S, Kushwaha P, Srivastava S, Aks R. Quantitative estimation of asiatic acid, asiaticoside and madecassoside in two accessions of Centella asiatica
(L) urban for morpho-chemotypic variation. Indian J Pharm Educ 2014;48:75-9.
Brinkhaus B, Lindner M, Schuppan D, Hahn EG. Chemical, pharmacological and clinical profile of the East Asian medical plant Centella asiatica
. Phytomedicine 2000;7:427-48.
Ellman GL, Courtney KD, Andres V Jr., Feather-Stone RM. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 1961;7:88-95.
Okello EJ, Savelev SU, Perry EK. In vitro
anti-beta-secretase and dual anti-cholinesterase activities of Camellia sinensis
L. (tea) relevant to treatment of dementia. Phytother Res 2004;18:624-7.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]