Quality Control and Shelf-Life Study of Dasmula Taila- A Classical Ayurvedic Formulation for Nasya

Ayurveda is a science of life and it is the ancient and comprehensive medical system, focuses on holistic healing using natural remedies, including herbal formulations and purification procedures [1]. In Ayurveda, Dash stands for ten (10) and Moola stands for Root. Dasmula, a combination of Brihat Panchamoola and Laghu Panchamoola, contains ten roots of ten different plants in equal ratio. Out of the ten roots five roots are of trees, known as Brihat Panchmoola and five roots are of shrubs, known as Laghu panchmoola. Brihat Panchmoola contains Bilva (Aegle marmelos (L.) Corrêa), Shyonaka (Oroxylum indicum (L.) Kurz), Gambhari (Gmelina arborea Roxb), Patala (Stereospermum suaveolens (Roxb.) DC) and Agnimantha (Clerodendrum phlomidis Linn) whereas Laghu Panchmoola contains Shalaparni (Desmodium gangeticum (L.) DC), Prishniparni (Uraria picta (Jacq.) Desv), Kantakari (Solanum xanthocarpum Schrad. & H. Wend), Brihati (Solanum indicum Linn) and Goksura (Tribulus terrestris L.) [2].  Ayurveda makes extensive use of a mixture of these ten (10) roots, which work on Vata and Dosha to lessen their aggravation. There are a plethora of health benefits associated with Dasmula, gout, Parkinson's disease, arthritis, asthma, headaches, puerperal disorders, muscle spasms, lower back aches are the some of those. It has strong analgesic, antioxidant, and anti-inflammatory effects [3-4]. In the Ayurvedic system, health is defined by the equilibrium of Dosha, Dhatu, and Malas, which are the body's regulatory, structural, and waste-removal components [4]. The polyherbal combination is one of the most often used ingredients in Ayurveda medicine, where it is utilized to make various kinds of medicine that are used to treat different disorders. Dasmula Taila is a herbal oil blend prescribed for Nasya treatment in cases of Shiro-roga, a category of head-related ailments [2-3]. Sharma et al. recently conducted a clinical study on the Dasmula Taila Nasya and Shirodhara in Vatika Shiroroga (Tension Headache) [5] which showed that both therapies were approximately equally beneficial, but patients who were administered  Shirodhara with Dashmoola Taila showed better results in comaparison to those administered Nasya Karma with Dasmula Taila. This preparation is also linked to treating Ardhavbhedaka, which is comparable to migraines. The Taila formulation is crafted to harmonize the body’s elements, specifically to soothe Vatadosha without worsening Kapha [6]. During its preparation, known as Snehpaka, Taila uniquely integrates the qualities of the herbs infused into it while retaining its inherent characteristics [7]. This study chooses Dasmula Taila for managing Ardhavbhedaka due to its predominantly Tridoshamaka contents.

MATERIALS AND METHODS

Plant Materials

In the present study, roots of the ten (10) selected plants  roots viz. Bilva (Aegle marmelos (L.) Corrêa), Shyonaka (Oroxylum indicum (L.) Kurz), Gambhari (Gmelina arborea Roxb), Patala (Stereospermum suaveolens (Roxb.) DC), Agnimantha (Clerodendrum phlomidis Linn), Shalaparni (Desmodium gangeticum (L.) DC), Prishniparni (Uraria picta (Jacq.) Desv), Kantakari (Solanum xanthocarpum Schrad. & H. Wend), Brihati (Solanum indicum Linn), Goksura (Tribulus terrestris L.) was collected [2]. The selected plant roots were authenticated by the Pharmacognosy department of CARI, Kolkata, along with macroscopical and powder microscopical characterization as per API. [8-9]

Preparation of Dasmula Taila

 The oil 'Dasmula Taila' was prepared by the roots of ten (10) plants along with Tila taila (sesame oil), which was chemically standardized by testing of the parameters, i.e. Acid value, Saponification value, Unsaponifiable matter, Iodine value, Peroxide value etc. The physicochemical parameters (Loss on drying, Total ash, Acid insoluble ash, water soluble extractive, alcohol soluble extractive, pH, HPTLC) and safety parameters (Heavy metals, microbial load, specific pathogens, pesticide residue testing) analysis were carried out for all the mentioned raw drugs as per API (Table 1). The murchit tila taila was obtained by following the murchana process of Tila taila using the raw drugs viz. Manjistha (Root), Musta (Rz.), Tvak/Nalika (St. Bk.), Haritaki (Fr.), Haridra (Rz), Ketaki (Root), Bibhitaka (Fr.), Amalaki (Fr.), Hrivera (Root), Vatankura (St. Bk.) and Lodhra (St. Bk.) which were macroscopically studied and chemically standardized as per API [10, 11]. At first, all the plant roots were cleaned, washed, dried and powdered separately. After that, all powdered raw drugs were passed individually through a sieve size 80. After that, all these raw drugs, 320 gm each, were taken, and 16 litres of water was added to make a homogeneous mixture. The process began by adding 1 litre Murchit Tila Taila to the mixture and heating it at a temperature range of 50-60 °C for three hours. The heat was turned off once the three hours had elapsed, and the boiler was left to rest overnight. The next day, heating resumed, and froth development was monitored while regularly checking the kalka for varti formation until the froth eventually settled. Subsequently, the varti was strained while still hot (around 80 °C) through a muslin cloth. After cooling, the clear liquid collected was the final Dasmula Taila product.

Physicochemical ananlysis

The powder form of each plant part were used for the physicochemical analysis as per the WHO/API guidelines. For determining the water and alcohol extractive values, the crude powder (4 g) was soaked in the respective solvents (100 mL) with occasional shaking for up to 6 h, followed by keeping the mixture overnight (18 h) at room temperature. The extracts were subsequently concentrated in a hot water bath to obtain constant weights, and the extractive values were calculated. In order to determine Loss on drying, the crude powder (4 g) was kept in hot air oven at 105 °C for 6 h, followed by cooling weight is noted and LOD at 105 °C was calculated. For total ash determination, 2 g of each raw drugs (powder form) were taken in a silica crucibe and incinerated at 450 °C for 6 h. On the next day the weight of the crucible is noted and result is calculated. Now the ash is ready for determination of acid insoluble ash. The ash obtained on the previous day was treated with 25 mL 2(N) HCl and warmed in a water bath, filtered through a ashless filter paper. Once all the liquid were drained, the ashless filter paper was transferred in the same silica crucible and incinerated at 450 °C for 6 h. On the next day the weight of the crucible is noted and result is calculated. In order to check the pH, 10 % aqueous suspension was prepared, kept for 24 h and pH was noted. The formulation Damula Taila is liquid and three (03) batches of formulations have been prepared. Physicochemical investigations viz. determination of Refractive index at 40 °C,  Viscosity, Specific optical rotation, Acid value, Iodine value, Saponification value, Unsaponifiable matter, Peroxide value, Specific gravity and Wt per ml (at 25 °C) of the three batches of Dasmula Taila were carried out following API methods. [12-13]

High Performance Thin Layer Chromatography (HPTLC) study [14-15]

Equipment

A CAMAG HPTLC system (Switzerland), comprising of a CAMAG ATS 4, CAMAG TLC scanner 3, CAMAG Wincats Software, Version 1.44, a CAMAG TLC plate heater, and a CAMAG Visualizer, was used for the study.

Preparation of samples

1. Murchit Tila Taila (oil) along with raw drugs used for Murchana

For each sample, 1 gram was refluxed with 20 ml of Methanol for one hour. The resulting extracts were then passed through filter paper. The filtrates obtained were concentrated for Thin Layer Chromatography (TLC) analysis.

2. Dasmula Taila along with its ten (10) raw ingredients and markers

The High-Performance Thin Layer Chromatography (HPTLC) analysis of the Dasmula Taila formulation and the raw materials and reference markers was conducted across three separate batches. Each sample, weighing 1 gram (or 2 mL for Taila), underwent reflux with 20 mL of Methanol for one hour; post-reflux, the solution was filtered through filter paper. The resulting filtrate was then reduced in volume and prepared for TLC analysis. Reference standards were created by dissolving 1 mg each of marmelosin, beta-sitosterol, and lupeol acetate in 10 mL of Methanol.

Stationary phase

Pre-activated 20 × 10 cm aluminum-supported precoated silica gel 60 F₂₅₄ plates (Merck, India, Batch No. 1.05554.0007) were used.

Application of samples

A 5 µL sample was applied to the plate as an 8 mm band, positioned 15 mm above the plate's base using CAMAG ATS4 applicator.

Mobile phase

3. Murchit Tila Taila (oil) along with raw drugs used for Murchana

The solvent system used was a mixture of Toluene, Ethyl acetate, and Formic acid in the ratio of 7:3:0.1(v/v).

4. Dasmula Taila along with its ten (10) raw ingredients and markers

The solvent system comprised a blend of toluene, ethyl acetate, and formic acid in a 7.5:2.5:0.4 (v/v) ratio.

Plate development and detection of spots

The plates were developed to a distance of 90 mm in a CAMAG Twin trough chamber, with a pre-conditioning period of 20 minutes at 25°C and 42% average relative humidity. 20% aqueous sulphuric acid solution was used as the derivatising agent. The TLC plate was developed up to 90 mm in a pre-saturated CAMAG twin trough chamber after preconditioning the plate at 25 °C and relative average humidity of 42%. Images of the developed plate were captured at 254, 366 nm and in white light.

Shelf life study [16-17]

The stability of the formulation was evaluated in a stability chamber (Model: REMI 12C) under regulated conditions (temperature at 38 °C and relative humidity at 75%) over a period of six months, with assessments at intervals of one month (01), three months (03), and six months (06). The stability profile, composed of specific parameters, is essential for determining appropriate storage conditions and the expiration period for pharmaceuticals. The construction of the stability study should rely on an understanding of the drug's characteristics and the physical form in which it is administered. The purpose of the present study was to establish stability data by taking three batches of drug formulation at specified intervals of 0 days, 1st month, 3^rd month, and 6^th month. Various physical parameters, like the refractive index, viscosity, and specific gravity, as well as chemical parameters, like saponification, acid, and iodine values, were studied.

RESULT AND DISCUSSION

Organoleptic and quality control studies carried out on the ingredients of Dasmula Taila

In Dasmula Taila the ingredients present are the roots of the following ten (10)  plants viz. Bilva (Aegle marmelos (L.) Corrêa), Shyonaka (Oroxylum indicum (L.) Kurz), Gambhari (Gmelina arborea Roxb), Patala (Stereospermum suaveolens (Roxb.) DC), Agnimantha (Clerodendrum phlomidis Linn), Shalaparni (Desmodium gangeticum (L.) DC), Prishniparni (Uraria picta (Jacq.) Desv), Kantakari (Solanum xanthocarpum Schrad. & H. Wend), Brihati (Solanum indicum Linn), Goksura (Tribulus terrestris L.) and Murchit Tila Taila. In The following table (Table 1) photographs, organoleptic findings, physicochemical parametrs and safety parameters of the ingredients of Dasmula Taila are documented.

Abbreviations: LOD- Loss on drying; TAV: Total ash value; AIA: Acid insoluble ash; WSE- Water soluble extractive; ASE- Alcohol soluble extractive

HPTLC study of Murchit Tila Taila (oil) along with raw drugs used for Murchana

The murchit tila taila was obtained by following the murchana process of Tila taila using the raw drugs viz. Manjistha (Root), Musta (Rz.), Tvak/Nalika (St. Bk.), Haritaki (Fr.), Haridra (Rz), Ketaki (Root), Bibhitaka (Fr.), Amalaki (Fr.), Hrivera (Root), Vatankura (St. Bk.) and Lodhra (St. Bk.). For each sample, 1 gram was refluxed with 20 ml of Methanol for one hour. The resulting extracts were then passed through filter paper. The filtrates obtained were concentrated for Thin Layer Chromatography (TLC) analysis. The TLC was performed on pre-coated Silica Gel Plates (60 F254) supported on aluminium sheets from Merck. The solvent system used was a mixture of Toluene, Ethyl acetate, and Formic acid in the ratio of 7:3:0.1(v/v). A 5 µL sample was applied to the plate as an 8 mm band, positioned 15 mm above the plate's base. The plates, measuring 20x10cm, were developed to a length of 90 mm in a CAMAG Twin trough chamber, with preconditioning at 25 °C and 42% relative humidity. Figure 1 documents the TLC plates scanned at 254 nm and 366 nm while Table 2 lists the R_f valus.

Figure 1. Photography of Developed HPTLC Plate in (a) 254 nm and (b) 366 nm

Organoleptic studies of Dasmula Taila (oil)

The Dasmula Taila formulation was prepared by the Pharmacy department of CARI, Kolkata, in three batches (Batch-I, II & III). Organoleptic characteristics, macroscopical analysis and photographs of three batches of Dasmula Taila are listed in Table 3.

Table 3: Organoleptic characteristics and photographs of three batches of Dasmula Taila

Chemical Standardization of Dasmula Taila

The chemical standardization of Dasmula Taila was performed by analysing the following parameters, i.e. determination of Refractive index at 40 °C,  Viscosity, Specific optical rotation, Acid value, Iodine value, Saponification value, Unsaponifiable matter, Peroxide value, Specific gravity and Wt per ml (at 25 °C). Safety parameters i.e. testing of heavy metals, aflatoxins, microbial load, test for specific pathogens and pestiside residue were also carried out. Comparative marker-based HPTLC fingerprinting profile of all ten (10) raw drugs &  three (03) batches of the formulation were also carried out. All these results are documents in Table 4-9.

Table 4:  Physicochemical parameters

Table 5: Heavy metals testing:

^*1,2,3 Limit of Detection = < 0>

Table 6: Aflatoxins:

Table 7: Microbial Load:

Table 8: Pathogens testing:

*Abs- Absent

Dasmula Taila along with its ten (10) raw ingredients and markers

The High-Performance Thin Layer Chromatography (HPTLC) analysis of three batches of Dasmula Taila formulation and the raw materials and reference markers was conducted across three separate batches. Each sample, weighing 1 gram (or 2 mL for Taila), underwent reflux with 20 mL of Methanol for one hour; post-reflux, the solution was filtered through filter paper. The resulting filtrate was then reduced in volume and prepared for TLC analysis. Reference standards were created by dissolving 1 mg each of marmelosin, beta-sitosterol, and lupeol acetate in 10 mL of Methanol. For the chromatography, precoated Silica Gel Plates (60 F254) on aluminium sheets supplied by Merck were utilized. The solvent system comprised a blend of toluene, ethyl acetate, and formic acid in a 7.5:2.5:0.4 ratio. The plant extracts, 5 µL each, were applied to the plate as 8 mm bands, placed 15 mm from the bottom edge. The plates, sized 5x10 cm, were developed to a distance of 90 mm in a CAMAG Twin trough chamber, with a preconditioning period of 20 minutes at 25°C and 42% average relative humidity. The chemical development was completed using a 20% aqueous sulphuric acid solution as the derivatising agent.

Shelf life Study of Dasmula Taila

Three batches of Dasmula taila formulation were prepared at Pharmacy department of CARI Kolkata and were kept in stability chamber at the Department of Chemistry for stability assessment and accelerated stability study. Understanding shelf life ensures that the prepared Dasmula Taila maintain acceptable quality and safety standards. Table 11 documents the comparative test result of Dasmula Taila kept in controlled environmental condition at temperature 38 °C and relative (RH) humidity 75% over a period of six months, with assessments at intervals of one month (01), three months (03), and six months (06).

Table 11: Comparative test result of Dasmula Taila kept in controlled environmental condition Temperature 38 °C and Relative (RH) Humidity 75% (Test reports after sample standing in Stability chamber)

The refractive index (RI) serves as an indicator of a sample's purity. It is commonly measured using refractometers in the pharmaceutical industry to ensure the quality of raw, intermediate, and final products. Typically, this measurement is conducted at the sodium D-line (NaD) wavelength of approximately 589 nm589 nm. The recorded values for batches I, II, and III are 1.4670, 1.4665, and 1.4665 respectively, suggesting consistency across the batches with no significant variation. Viscosity is a fluid's resistance to flow or change shape at a specified rate. The values obtained in (Pa-sec) are 86.91, 82.73, 83.51 of batches I, II and III, respectively at 0 day and 101.5, 95.96, 97.82 (Pa-sec) of batches I, II and III at 6 months [Figure 3(a)] thus indicating that a slight increase viscosity of Dasmula Taila upon storage.

       
           
       
Figure 3. (a): Graph of Viscosity vs No. of days; (b): Graph of Acid value vs No. of days

High-Performance Thin Layer Chromatography Profile of Dasmula Taila (B-I, II, III)

For the analysis, 2 mL of each Dasmula Taila batch (B-I, II, III) was refluxed with 20 mL of Methanol for one hour. The mixture was filtered through filter paper, and the filtrate was reduced in volume for Thin Layer Chromatography (TLC). A TLC Silica Gel 60F₂₅₄ plate, supported on aluminium sheets, served as the stationary phase. The solvent mixture used was Toluene, Ethyl acetate, and Formic acid in a 7.5:2.5:0.4 (v/v) ratio. A 5 µL sample was applied as an 8 mm band, 15 mm from the plate's bottom. The plate, measuring 5x10 cm, was developed to 90 mm in a CAMAG Twin trough chamber under conditions of 25 °C and 42% humidity. Finally, 20% aqueous sulphuric acid was employed as the derivatising agent. Table 12 documents the comparative test result of HPTLC of Dasmula Taila kept in controlled environmental condition at temperature 38 °C and relative (RH) humidity 75% over a period of six months, with assessments at intervals of one month (01), three months (03), and six months (06).