Introduction

Yashtimadhu ghrita is a potent single herb ghee formulation mentioned in the classical Ayurveda texts like Ashtanga hrudaya, Sushruta Samhita, Bhaishajyaratnavali, Chakradutta, (Chakradutta & Sharma, 2007; Navre Shastri Krishna Rama Chandra DAMK, 2000; Susruta, 2007) in the context of management of sadya kshatavrana (wound of an acute origin as a result of injury, burns, contact with poisonous substances, strong and penetrating medications etc.) (Sushruta, 2003). It was also found to be effective in controlling the radiation and chemotherapy induced side effects in the head and neck malignancies (Das, Chandola, & Agarwal, 2011). Addition of liquorice not only improves therapeutic efficacy of ghee but also increases its shelf life by exerting a resistance towards the oxidative deterioration of the ghee (Bipinbhai & Aparnathi, 2017). The objective of this study is to create a finger print profile of yashtimadhu ghrita as a reference standard for future studies through its physico-chemical analysis. This was carried out systematically by procuring the herbal medicine, authentication of the specimen through organoleptic evaluation and cross-sectional microscopy, preparation of the formulation as per the classical texts of Ayurveda, physico-chemical analysis through TLC and HPTLC.

Materials and Methods

Yashtimadhu in its dried form was procured from cultivated resources at Hamirpur, Himachal Pradesh and Organic ghee from Ambadi hi-tech dairy farm, Tiruvamandoor, Kerala. The genuineness of the dry specimen of Yashtimadhu (Glycyrrhiza glabra Linn.) was confirmed through organoleptic evaluation and cross-sectional microscopy. The purity of the ghee was tested in the QC lab of the Department of Medicinal Chemistry and Pharmaceutics, Amrita School of Ayurveda. Furfural and Baudouin tests were performed to rule out the presence of vanaspati hydrogenated fat and refined vegetable oil. The purity of the ghee is ascertained and Yashtimadhu ghrita is prepared in the Department of Rasashastra and Bhaishajya Kalpana. The composition and ratio of ingredients of Yashtimadhu ghrita is mentioned in Table 1.

Method of Preparation of Yashtimadhu Ghrita

The medicine was prepared as per the sneha paka (preparation of unctuous formulations using ghee, oil etc.) procedure mentioned in Sarngadhara Samhita Madhyama khanda and the standard operative procedures specified in Ayurvedic pharmacopoeia of India (Dravyagunatvpm.files.wordpress.com, 2009; Murthy, 2010). Yashtimadhu decoction is made using eight kilogram of coarse powdered specimen in 64 Liters of water [Figure 1]. It is reduced to 32 Liters [Figure 2]. Into it eight liters of cow’s ghee and two kilograms of fine powdered yashtimadhu kalka are added and cooked in low flame until the entire moisture disappears and proper ghrita paka (consistency) is obtained. A final quantity of 6.5 liters of ghee is obtained [Figure 3]. Flame test confirmed the absence of moisture and the residue was perfectly rolled into a wick between fingers. The entire procedure is done using a traditional bronze vessel in manda agni (low flame). The finished product Yashtimadhu ghrita was prepared in four days and the finished product is semi solid in consistency, resembling colour of honey with a peculiar aroma and a mild sweet taste. Later the physico-chemical analysis of the finished product was carried out in the QC Laboratory of Arya Vaidyasala Kottakkal, Kerala.

Pharmacognostical evaluation of ingredients of Yashtimadhu ghrita

Organoleptic evaluation

The dry specimen powder is subjected to various sensory perceptions like color, taste, smell, and touch and recorded. Cross-sectional microscopy: A thin section of the specimen is observed under a compound microscope of 10X-15X resolution and studied. Physico-chemical evaluation: Yashtimadhu ghrita was analyzed using different definitive physico-chemical parameters like Acid value, saponification value, refractive index and Iodine value. HPTLC was carried out for the characterization of the plant material after making appropriate solvent system having methanolic extract of Yashtimadhu ghrita.

High Performance Thin Layer Chromatography (HPTLC)

Both TLC and HPTLC were performed for obtaining the characterization of plant material. HPTLC is an instrumental version of TLC in order to get a reproducible result and is done using instruments where all parameters are fixed. 10 gm of the ghrita sample is taken and is extracted with 10 ml Methanol and spotted as 10 micro liters. Post chromatographic derivatization was done with Iodine vapor.

Chromatographic Conditions

• Stationary Phase: Merk, 1.05554.0007, TLC Silica gel 60 F_254, 10x10 cm Aluminium sheet.

• Mobile Phase: Toluene: Ethyl acetate: Formic acid: Methanol (7:5:1:0.5).

HPTLC Instrumentation

• Camag HPTLC instrument.

• Application mode: CAMAG Linomat 5

• Scanner: CAMAG TLC Scanner 3, CAMAG Reprostar 3.

• Development chamber: CAMAG 10 x 10 cm Twin trough chamber.

• Plates: Precoated silica gel 60 F_254, plates

• Chamber saturation: 30 min

• Development time: 30 min

• Development distance: 7 cm

• Detection: Deuterium lamp, Tungsten lamp

Thin Layer Chromatography (TLC)

TLC is exclusively used for herbal drug analysis in herbal chemistry. TLC depends on the principle of separation through adsorption, where it relies on the affinity of the compound towards a mobile phase and a stationary phase. The sample is run through a solvent system in a chamber. Phytochemicals will move (ascending technique as per the retention time of the chemical component). Spot is the representation of a chemical compound. These spots obtained is observed by exposing the developed plate in UV light of wave length 254nm, 366 nm, and white light.

Results

Macroscopic features of Yashtimadhu can be seen in Table 2. Various stages of preparation of Yashtimadhu ghrita shown in [Figure 3; Figure 2; Figure 1]. The results obtained by evaluating the physico-chemical parameters of the finished product is given in the [Table 3].

The HPTLC Results of Yashtimadhu ghrita is shown in the [Figure 4] and [Table 4] at 254 nm and in [Figure 5] and [Table 5] at 366nm respectively. The TLC documentation picture profiles of yashtimadhu ghrita sample at 254nm, 366 nm and in white light is shown in [Figure 6], [Figure 7] and [Figure 8] respectively. The derivatized TLC plate view of the ghrita sample is shown in [Figure 9], [Figure 10] and [Figure 11] respectively. The total number of peaks obtained is nine in a total area of 8300.4 (AU) at 254 nm and total number of peaks is five in a total area of 14041.6 (AU) at 366 nm.

Discussion

Yashtimadhu ghrita is a single herb ghee-based formulation having cold potency and wound healing property. On organoleptic evaluation and cross-sectional microscopy, the authenticity of the collected dry specimen of Yashtimadhu was confirmed.

The physicochemical parameters when applied for assessing the finished product revealed an acid value of 6.7 which indicates the quantity of fatty acid present in the ghrita formulation, a saponification value 213.4 is close to that of genuine cow’s ghee. The refractive index value 1.455 at room temperature is also close to that of genuine ghee. An Iodine value of 38.7 within the range of genuine ghee, implies the iodine molecules consumed by the free fatty acids. Loss on drying (LOD), 0.64 is in the admissible range.

During the HPTLC scanning a total of nine spots were distinguished at 254nm and five spots at 366 nm frequencies. Among the spots, the Rf values 0.33, 0.39, 0.45 and 0.57 are common in both 254 nm and 366nm.

Conclusion

The preliminary pharmacognostical study confirms the genuineness of the dry specimen of Yashtimadhu. The test result of the finished product shows Acid value 6.7, saponification value 213.4, and Refractive index value 1.455 at room temperature, Iodine value 38.7, LOD 0.64. The HPTLC scanning, distinguished nine peaks under 254nm and 5 peaks under 366nm. The test results of the present study can be considered as a reference standard and can be used for quality assessment of similar research undertakings in future.

Conflict of Interest

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

Source of Funding

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