Introduction

MedhyaRasayan or nootropic agents are assets of Ayurvedic pharmaceutics. The concept of improving cognitive functions is a newer concept to modern medicine. However, the nootropics are known, since 1972, to enhance cognitive functions. Though ageless, Ayurveda is dynamic enough to allow alteration of medicinal forms keeping the basic principles intact. Medicinal plants belonging to species Centellaasiatica Linn. (Mandukaparni) (Sharma, 2005), Glycyrrhiza glabra Linn. (Yastimadhu) (Yt, 1994), TinosporacordifoliaMiers (Guduchi), and Convolvulus pleuricaulisChois (Shankhapushpi) together constitute the MedhyaRasayan that is known for mental well-being. (Kulkarni, 2012)

Glycyrrhiza glabra, also known as Yashtimadhu, is one of the most used medicinal plants. It.is a hardy perennial shrub, which is consumed as a dried powder prepared from the roots and rhizome of this herbal plant. The active molecules of the plant include glabridin, glycyrrhizin, glycyrrhetenic acid, liquiritigenin, isoliquiritigenin, glabridin, and licochalcone A and E. (Pastorino, 2018) These different molecules contribute to different medicinal properties including anti-inflammatory, hepatoprotective, neuroprotective, sedative, oestrogenic, antiviral, anticarcinogenic, antimicrobial, and anti-oxidant activities and affects the skin in a good way. (Pastorino, 2018) The extracts of this herb are known to boost brain functioning, trigger the central nervous system (CNS) by increasing circulation, and act as a moderator for levels of blood sugar. (Rathee, 2008)

Despite the plethora of beneficial effects of the MedhyaRasayan, the potential of these MedhyaRasayan drugs have not yet been explored to its maximum capacity; there is an unmet need to further develop or refine the existing products such that they are easily taken up by the patients.

To work towards this unmet need, the present study was carried out with an aim to develop an adapted formulation of one of the nootropic agents, Glycyrrhiza glabra, such that the adapted formulation, i.e., YashtimadhuKalpa, has a longer shelf life and is more palatable. Yashtimadhuis a time-tested Ayurvedic medicine indicated for mental health that has shown promising results in attaining optimal intelligence in children. (Sheshagiri, 2015) It is available as YashtimadhuChurnaform that is less palatable and has shorter shelf-life. Here, we present the analyses of the properties of the novel YashtimadhuKalpaformulation.

Materials and Methods

Preparation of YashtimadhuKalpa

The roots of Glycyrrhizawere converted to a coarse powder using a pulveriser. Next, coarse Yashtimadhu powder (250g) was mixed in of water (4L), heated till volume reduced to 500 mL, forming the YashtimadhuKwatha (decoction). The decoction was filtered using a cotton cloth to which 1kg of Sita (candy sugar) was added and heated at a temperature of 100°C with continuous stirring till it became sticky. At this point, the heating was turned off and the mixture was stirred continuously to attain granules of YashtimadhuKalpa.

High-Performance Thin Layer Chromatography

HPTLC was outsourced to Anchrom Enterprises Pvt. Ltd., India and performed using Linomat 5 (CAMAG). The 0.2 mm pre-coated plates of silica gel 60 F₂₅₄ (Merck Millipore, US) were used with the solvent system consisting of ethyl acetate: formic acid: water: glacial acetic acid in the ratio 15:1:2:1 (v/v/v/v). To prepare the test solution, extract (1g) was mixed with 70% ethanol (10Ml) in Soxhlet apparatus consecutively three times. The mixture was then filtered and concentrated under vacuum to attain the powder that was resuspended in ethanol (10Ml). The standard solution was glycyrrhizin (10mg) dissolved in ethanol (10Ml).

The calibration curve was prepared where 2, 5, and 7 µl of the standard solution were spotted onto the TLC plate in duplicates. The plate was developed, dried for 5 minutes, and scanned for density at 254 and 366 nm using densitogram CAMAG TLC Scanner 4 (Camag, Muttenz, Switzerland) and image profiles using CAMAG TLC Visualizer 2 (Camag, Muttenz, Switzerland). Parameters used for high-performance thin-layer chromatography are shown in Table 1.

Later, the plate was subjected to derivatisation for detection of analytes which cannot be detected using visible or UV spectrum. The first derivatisation was done using a solution of 2-aminoethyl diphenylborinate in ethyl acetate and the second derivatisation was done using a solution of anisaldehyde sulphuric acid reagent. After each derivatisation, images were captured using CAMAG TLC Scanner 4 and image profiles using CAMAG TLC Visualizer 2. Peak areas were recorded using the densitogram for each spot to prepare the standard curve by plotting area under the peak versus the concentration of glycyrrhizin at each spot. HPTLC fingerprints before and after derivatization are shown in Figure 1.

To estimate the glycyrrhizin present in the test solution (YashtimadhuChurna, YashtimadhuKwatha, and YashtimadhuKalpa), 2, 5, and 7 µl of the test solution was applied to the TLC plate in triplicate and the plate was developed and dried. The peak area and R_f were then plotted on the calibration curve to estimate the amount of glycyrrhizin present in the test solution. HPTLC densitiograms showing the mean area are shown in Figure 2.

High-Performance Liquid Chromatography

To assess the presence of glycyrrhizin in YashtimadhuChurna, YashimadhuKwatha, and YashtimadhuKalpa, HPLC was done.

The extracts from Yashti madhu Churna, Yashimadhu Kwatha, and Yashtimadhu Kalpa were examined on Kromasil C₁₈ column using phosphate buffer and acetonitrile as the mobile phase (65:35; v/v). Detection was done according to the method previously described by (De, 2012) Commercially available glycyrrhyzic acid was used as the standard control, for identification of glycyrrhyzin in Yashtimadhu Churna, Yashimadhu Kwatha, and Yashtimadhu Kalpa. Shown in Figure 3.

Microbial Analysis

The novel formulation, Yashtimadhu Kalpa, was further analyzed for the total bacterial count and total fungal count. It was specifically tested for pathogens such as Escherichia coli, Salmonella, Pseudomonas aeruginosa, and Staphylococcus aureus and microbial analysis of novel Glycyrrhiza glabra formulation – Yashtimadhu Kalpa has shown in Table 2. Additionally, the formulation was also examined for organoleptic parameters of novel Glycyrrhiza glabra formulation- Yashtimadhu Kalpa has shown in Table 3.

Results

Synthesis of YashtimadhuKalpa

In pilot preparation, in 1 hour 30 minutes Yashimadhu Kwatha was obtained and finally, 1.1 kg of Yashtimadhu Kalpa was obtained in 3 hours 45 minutes. It was observed that continuous stirring was required due to the sticky nature of the mixture.

High-Performance Thin Layer Chromatography

The Yashtimadhu Churna, Yashimadhu Kwatha, and Yashtimadhu Kalpa were individually investigated for the presence of glycyrrhizin using the HPTLC method. The fingerprints thus generated were analysed to calculate the R_f values. After derivatisation clear evident bands with medium intensity were obtained with R_f values 0.19 (pink), 0.24 (light brown) and 0.49 (yellow) for YashtimadhuKalpa as well as YashtimadhuChurna. The light brown band having R_f around 0.25 was observed in all the samples confirming the presence of glycyrrhizin. The fingerprint of Yashtimadhu Kalpa was comparatively intense for the 0.24 band than the Yashimadhu Kwatha and the Yashtimadhu Churna.

In YashtimadhuChurna, YashimadhuKwatha, and YashtimadhuKalpa, the content of glycyrrhizin was quantified by the HPTLC method using a known standard solution containing glycyrrhizin at 0.1 mg/mL. The percentage of glycyrrhizin was calculated in the above-mentioned samples using the area values under the curve obtained after densitometric scans of the TLC sheet. YashtimadhuChurnawas found to contain 8.7% of glycyrrhizin, YashimadhuKwatha had 3.8% of glycyrrhizin and 7.4% of glycyrrhizin was found to be present in YashtimadhuKalpa. The amount of glycyrrhizin present in YashtimadhuKalpa was comparable to that in the YashtimadhuChurna.

High-Performance Liquid Chromatography

HPLC resulted in similar findings as compared to the HPTLC. In HPLC, the retension time for glycyrrhizin ranged from 1.4 to 1.5 minutes. HPLC confirmed the presence of homogenous glycyrrhizin in the same proportion and purity in the YashtimadhuChurna, YashimadhuKwatha, and YashtimadhuKalpa, confirming the composition of YashtimadhuKalpa to be similar to that of YashtimadhuChurna. These results confirmed and lead us to an agreement that YashtimadhuKalpa contained the active molecule glycyrrhizin in considerable amounts as compared to YashtimadhuChurna.

Microbial Analysis

The newly formulated YashtimadhuKalpa was further examined for organoleptic parameters, physicochemical and microbial contaminants viz. Escherichia coli, Salmonella, Pseudomonas aeruginosa, and Staphylococcus aureus. The organoleptic evaluation showed that the YasthimadhuKalpa was light brown in colour, having its characteristic odour. The final formulation was solid crystalline granules having a sweet, yet slightly bitter, taste. The microbial growth test determined that the total plate count was 20 colony-forming unit (CFU)/g. Total yeast and mold count was less than 10 CFU/g, and Escherichia coli was absent with the most probable unit being less than 1 CFU/g. Additionally, Salmonella, Pseudomonas aeruginosa, and Staphylococcus aureus were absent in the novel Glycyrrhiza glabra formulation. All these analyses validated the safety of YashtimadhuKalpa for internal use.

Discussion

The use of MedhyaRasayan has been known since ages; despite this fact, Glycyrrhiza has gained importance in prophylactics and as a dietary supplement in the recent decade (Karkanis, 2018). Yashtimadhu has glycyrrhizin as one of its major constituents and shown to have many health benefits. (Dhama, 2018; Karahan, 2016) The in-vitro studies done with cell-lines demonstrated that glycyrrhizin inhibits EV71 (Kuo, 2009), coronavirus (Cinatl, 2003), nonsyncytium-inducing HIV variant, HIV-1 (Sasaki, 2002), Hepatitis C virus (Rossum, 1999), and H5N1 influenza virus (Michaelis, 2011) by inhibiting the processes like ROS, protection against lipid peroxidation of liposomal membrane, oxidative DNA damage and induction of factors involved in apoptosis, inflammatory lipopolysacchride mediators (Račková, 2007) like COX-2 PG2, TNF-alpha, interleukins. (Grippaudo & Russo, 2016; Yin, 2017) Glycyrrhizin has also been used for the treatment of ulcers as it stimulates mucus secretion in the stomach and the prostaglandins. (Jafarian, 2007)

Yashtimadhu, traditionally, has been used as the powdered YashtimadhuChurna, ingested with milk. This causes low palatability, shorter shelf life and difficult patient compliance. As it is a well-stated fact that our cognitive system starts developing from the gestation period, and therefore, it is necessary to have supplements like Yashtimadhu inculcated in our food habits to aid the process of cognitive development, apart from other health benefits. Studies on mice model have speculated Yashtimadhu to be neuroprotective and to enhance the functioning of the brain; therefore, it might be used to develop the treatment of diseases like Alzheimer where the patient loses the memory, inflammation of certain parts of the brain and eventually suffers from dementia as one of the symptoms. (Chakravarthi & Avadhani, 2013; Dhingra & Sharma, 2006)

This study showed that YashtimadhuChurnaand YashtimadhuKalpa have the same proportion of glycyrrhizin, indicating that the novel YashtimadhuKalpaformulation might also have similar effects as conferred by the conventional YashtimadhuChurna. In the study, YashtimadhuChurnawas transformed into YashtimadhuKalpa, which rendered it increased shelf-life and more palatability. The HPLC and HPTLC data, lead us to conclude that the active component of the Glycyrrhiza, i.e. glycyrrhizin is intact in YashtimadhuKalpa and is safe for consumption. The essentiality of this study was due to the fact that the synthesis of YashtimadhuKalparequires extensive boiling that may lead to de-activation of the active component.

The effects of YashtimadhuKalpa on cognitive health need to be further studied and validated through pre-clinical and clinical studies. The longer shelf-life and enhanced palatability of the YashtimadhuKalpa is promising for its use as an alternative to currently used YashtimadhuChurna. The outcomes of pre-clinical and clinical studies will demonstrate the activity and role of YashtimadhuKalpa.

Conclusions

In this study, we presented the classic Ayurvedic Medhya Rasayan in a novel formulation —Yashtimadhu Kalpa— in a simplified manner. The authenticity of the formulation was corroborated by HPTLC and HPLC. This study enforces the fact that the modification of classic Ayurvedic formulations is possible such that simplified and adapted formulations can be generated.

Conflict of Interest

The authors declare that they have no conflict of interest for this study.

Funding Support

The authors declare that they have no funding support for this study.