Modulation and Validation of YashtimadhuKalpa (formulated from Glycyrrhiza glabra) asMedhyaRasayana: A Neuro-nutrient

Mrudul Y Chitrakar1, Subhash S Kudale2, Manish P Deshmukh3, Ashish B Budhrani*4 1Department of Swasthavritta and Yoga, School of Ayurveda, D. Y. Patil University, Mumbai-400706, Maharashtra, India 2Department of Biotechnology and Bioinformatics, D. Y. Patil University, Mumbai-400706, Maharashtra, India 3Department of Pharmacology, Datta Meghe College of Pharmacy, Datta Meghe Institute of Medical Sciences (DU), Wardha-442001, Maharashtra, India 4Department of Pharmaceutics, Datta Meghe College of Pharmacy, Datta Meghe Institute of Medical Sciences (DU), Wardha-442001, Maharashtra, India


A
The present study was aimed to develop an adapted formulation of the Med-hyaRasayan, i.e. YashtimadhuKalpa to make Yashtimadhu (Glycyrrhiza glabra) more palatable and well-preserved. The classical reference from Charak-Samhita (ChikitsaSthana 1-3/30) elaborates the use of YashtimadhuChurna (coarse powder) along with milk as a MedhyaRasayana. The novel modi ied version, i.e. YashtimadhuKalpa (formulation), was prepared from Yashtimad-huChurna. In HPTLC inger printing clear evident bands with medium intensity were observed at R f values 0.19 (pink), 0.24 (light brown), and 0.49 (yellow) in YashtimadhuKalpa as well as YashtimadhuChurna. The content of glycyrrhizin was quanti ied from YashtimadhuChurna and YashtimadhuKalpa by HPLC and HPTLC by comparing the peak area of the standard. It was conirmed that the same active component was present in the YashtimadhuKalpa and YashtimadhuChurna. The content of glycyrrhizin was identi ied and quanti ied and showed the same comparable amounts in YashtimadhuKalpa and YashtimadhuChurna. In this study, we presented the classic Ayurvedic Medhya Rasayan in a novel formulation -YashtimadhuKalpa-in a simpli ied manner. The authenticity of the formulation was corroborated by HPTLC and HPLC. This study enforces the fact that the modi ication of classic Ayurvedic formulations is possible such that simpli ied and adapted formulations can be generated.

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 et al., 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 et al., 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 et al., 2018) These different molecules contribute to different medicinal properties including anti-in lammatory, hepatoprotective, neuroprotective, sedative, oestrogenic, antiviral, anticarcinogenic, antimicrobial, and antioxidant activities and affects the skin in a good way. (Pastorino et al., 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 et al., 2008) Despite the plethora of bene icial effects of the Med-hyaRasayan, the potential of these MedhyaRasayan drugs have not yet been explored to its maximum capacity; there is an unmet need to further develop or re ine 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 et al., 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.

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 iltered 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 F 254 (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: To prepare the test solution, extract (1g) was mixed with 70% ethanol (10Ml) in Soxhlet apparatus consecutively three times. The mixture was then iltered 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 pro iles using CAMAG TLC Visualizer 2 (Camag, Muttenz, Switzerland). Parameters used for highperformance 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 irst 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 pro iles 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 ingerprints 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 Yashti-madhuChurna, YashimadhuKwatha, and Yashtimad-huKalpa, HPLC was done. The extracts from Yashti Plates were derivatised by dipping (speed: 5, time: 0) in Natural product A reagent and then heated at 100 • C for 3 min Derivatization 2 Plates were dipped (speed: 5, time: 0) in AnisaldehydeSulphuric acid reagent and heated at 100 • C for 3 min Documentation after derivatization 1 or 2 UV 366 nm, UV 256 nm and white light

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

Synthesis of YashtimadhuKalpa
In pilot preparation, in 1 hour 30 minutes Yashimad-huKwatha was obtained and inally, 1.1 kg of Yashti-madhuKalpa 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  In YashtimadhuChurna, YashimadhuKwatha, and YashtimadhuKalpa, the content of glycyrrhizin was quanti ied 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 Yashtimad-huKalpa. The amount of glycyrrhizin present in YashtimadhuKalpa was comparable to that in the YashtimadhuChurna.

High-Performance Liquid Chromatography
HPLC resulted in similar indings as compared to the HPTLC. In HPLC, the retension time for glycyrrhizin ranged from 1.4 to 1.5 minutes. HPLC con irmed the presence of homogenous glycyrrhizin in the same proportion and purity in the Yashti-madhuChurna, YashimadhuKwatha, and Yashtimad-huKalpa, con irming the composition of Yashtimad-huKalpa to be similar to that of YashtimadhuChurna. These results con irmed 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 inal 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 et al., 2018). Yashtimadhu has glycyrrhizin as one of its major constituents and shown to have many health bene its. (Karahan et al., 2016;Dhama et al., 2018) 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 et al., 2002), Hepatitis C virus (Rossum et al., 1999), and H5N1 in luenza virus (Michaelis et al., 2011) by inhibiting the processes like ROS, protection against lipid peroxidation of liposomal membrane, oxidative DNA damage and induction of factors involved in apoptosis, in lammatory lipopolysacchride mediators (Račková et al., 2007) like COX-2 PG2, TNFalpha, interleukins. (Grippaudo and Russo, 2016;Yin et al., 2017) Glycyrrhizin has also been used for the treatment of ulcers as it stimulates mucus secretion in the stomach and the prostaglandins. (Jafarian et al., 2007) Yashtimadhu, traditionally, has been used as the powdered YashtimadhuChurna, ingested with milk. This causes low palatability, shorter shelf life and dif icult 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 bene its. 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, in lammation of certain parts of the brain and eventually suffers from dementia as one of the symptoms. (Chakravarthi and Avadhani, 2013;Dhingra and Sharma, 2006) This study showed that YashtimadhuChurnaand YashtimadhuKalpa have the same proportion of glycyrrhizin, indicating that the novel Yashti-madhuKalpaformulation might also have similar effects as conferred by the conventional Yashtimad-huChurna. 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 Yashtimad-huKalparequires 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 simpli ied manner. The authenticity of the formulation was corroborated by HPTLC and HPLC. This study enforces the fact that the modiication of classic Ayurvedic formulations is possible such that simpli ied and adapted formulations can be generated.