The plant materials of formulation were purchased from a reliable herbal drug supplier at Bengaluru, Karnataka, India during the month of June-July 2016. Plant materials were identified and authenticated by S. Noorunnisa Begum, Assistant Professor, Dept. of Pharmacognosy, Centre for Repository of Medical Resources (C-RMR), Trans-Disciplinary University (TDU), FRLHT Bengaluru (accession number 3801-17). Specimens of all the ingredients used were deposited in the Drug Museum, National Institute of Unani Medicine, Bengaluru (voucher specimen no. 40/IS/Res/2016) for future reference.

All the ingredients were manually cleaned and excluding zafran and mastagi, washed with running tap water to remove dirt. They were spread in the shade for a few hours to remove water. Further, the ingredients were placed in the hot air oven at 60ºC for 4-6 hours to dry them completely. All the drugs except zafran and mastagi were powdered separately in an electric pulveriser and passed through the sieve size number 80. Zafran was finely ground manually with rose water (arq gulab) in mortar and pestle (kharal). Mastagi was grounded gently in porcelain mortar and pestle to avoid melting. For the preparation of honey syrup (qiwam), honey was placed on the low flame till it begins to boil. Its impurities were removed and cooled to room temperature. The powder of all the ingredients was mixed uniformly and entered into honey syrup with continuous stirring. Then, zafran paste was incorporated into honey syrup and mixed well. At the last, powdered mastagi was dissolved in 100 ml of slightly warmed cow ghee (roghan gao zard) and sprinkled on the mass and rigorously mixed and JJ was prepared. Prepared JJ was allowed to cool at room temperature (NFUM Part-I, 2006). No preservatives were added in the preparation.

JJ was filled/packed in transparent, airtight, polyethylene terephthalate (PET) containers of 250ml capacity, purchased from the local market of Bengaluru. In each container, 200gm of JJ was filled. Maximum possible attention was paid to avoid any contamination during preparation and packaging.

JJ packed in PET containers were subjected to the light challenge with overall illumination of 1.2 million lux hours and integrated near ultraviolet energy of 200watt hours per square meter by using option 2 as per the ICH guideline Q1B. A cool white fluorescent lamp emitting light energy comparable to that stated in ISO 10977(1993) and near UV fluorescent lamp producing a spectral distribution, 320-400 nm with an extreme energy discharge between 350nm and 370nm and a significant quantity of UV light in the band of 320 to 360nm and 360 to 400 nm were utilized (ICH Q1B, 1996).

Exposure time for white fluorescent and UV light was calculated for photostability chamber model-Osworld photostability chamber OPSH G-4 1258 as follows.

To achieve the overall illumination of 1.2 million lux hours and integrate near ultraviolet energy of 200-watt hours per square meter, JJ was placed in a photostability chamber. At the commencement, both UV lamp and white fluorescent lamp were concurrently switched on. After completion of 22 hours and 13 minutes, the UV lamp was switched off and the fluorescent lamp was kept on to complete a total duration of 4 days and 12 hours. During the light challenge temperature and humidity in the stability, the chamber was regulated at 30±2°C/70±5%RH (ICH Q1B, 1996).

Before and after the light challenge JJ was evaluated for organoleptic characters (appearance, colour, odour and taste) and various physicochemical parameters viz. loss of weight on drying, alcohol and water-soluble matter, successive extractives value, pH, viscosity, ash value, total sugar (Total Carbohydrate protocol), reducing sugar (Baskan et al., 2016), total alkaloids, HPTLC and total and specific microbial contamination.

Total alkaloid estimation was carried out by HPLC method using atropine as standard. To prepare a standard sample 10mg of atropine was dissolved in 25ml of mobile phase i.e., methanol: water (4:6). To prepare the test sample, a specific quantity of test drug was dissolved in 20 ml of mobile phase, subjected to centrifuge and supernatant was separated and filtered out. The filtrate was used for HPLC analysis. Ultra-fast liquid chromatography system (Waters 510) isocratic with stationary phase column C18-5μn, (length 250mm, diameter 4.6mm) and Rheodyne manual injector were used for analysis. Injection volume was 20 μl, flow rate 1 ml/minute using mobile phase methanol: water (4:6 ratio), the pressure of 1300 PSI with a run time of 10 minutes were adopted to run the HPLC. Analysis was carried out at wavelength 274nm by UV detector using IRIS-HPLC SPECTRAL PROCESSING SOFTWARE 32. Qualitative analysis of alkaloid in the test sample was done by equating retention time of atropine (2.46 minutes) and quantitative estimation was carried out by comparison of area under the peak in densitogram of atropine (taken as 100%) and test sample.

JJ was extracted in HPLC grade absolute alcohol using Soxhlet apparatus. The dry extract was dissolved in the mobile phase [Toluene: Ethyl acetate (9:1)] and filtered for application. HPTLC analysis was performed on a plate size of 20×10 cm silica gel 60 F₂₅₄ plate. The sample solution was applied on the plate using TLC sample applicator Linomat 5 (CAMAG Switzerland) automated spray-on band. Applicator was equipped with 100μl Hamilton syringe and operated with the settings of band length 10mm, application position Y 12 mm, number of track 4.

Ethyl acetate (9:1) was used as a solvent system for plate development (Meena et al., 2013). The twin trough chamber was saturated with the stated solvent system for about 20 minutes and the plate was placed within for development. Densitometric scanning of dried developed plates was carried out at UV 254nm, 366nm, and 550nm. The plate was derivatised with anisaldehyde-sulphuric acid, heated at 110ºC and evaluated under visible light using CAMAG TLC VISUALISER and screened using CAMAG TLC SCANNER 3 operated by win CATS software (V 1.4.2, Camag) (Sethi, 1996).

Microbial contamination in the test samples was analysed for the total bacterial count, total fungal count using the pour plate method. The presence of specific pathogenic bacteria, viz E. coli, Salmonella, Staphylococcus aureus, Pseudomonas aeruginosa were carried out by conventional culturing and colony counting method (Quality control methods for herbal materials, 2011).

Organoleptic characters did not show any significant changes in the appearance, colour, odour, and taste in the baseline and photostability sample of JJ. (Table 1)

Physicochemical parameters viz., loss of weight on drying at 105°C, ash value (total ash, water soluble ash, acid insoluble ash), alcohol soluble matter, water soluble matter, successive extractives (in petroleum ether, chloroform and ethanol) pH at 1% and 10% aqueous solution, and total and reducing sugar exhibited less than 5% change at post light challenge sample values in comparison to baseline challenge. However, the total alkaloid showed a 10% reduction after photostability testing (Table 2). In this view, analysis and comparison of baseline and post light challenge data showed that changes in physicochemical parameters and chromatographic fingerprinting were less than 25 per cent and changes in total alkaloidal content were only 10%. Therefore, the authors infer those changes in JJ due to light challenges is not noteworthy and confirmed that JJ is photostable as per the ICH guideline. ICH guideline describes a limit of 5% change in assay from its initial value is acceptable and any surpass in it is taken as failing to meet the acceptance criteria. Appearance and physical attributes are subjectively evaluated and should not exhibit significant change for acceptance. However, O’Donnell and Bokser (2006) mentioned that 90% of labelled potency is considered as the minimum acceptable potency level for any drug. Further, as per the Ayurvedic Pharmacopoeia of India, physicochemical parameters shall not vary beyond 25 per cent of the initial value and ±15 per cent change from the initial assay value if the drug is analyzed for its active compound is acceptable to confirm the stability of the ASU drug.

HPTLC fingerprinting shows justified variation summarized in table 3 and Fig. 1 and 2.

Microbial analysis: The total bacterial and fungal count were within the WHO prescribed limit. The specific pathogenic bacteria, viz E. coli, Salmonella, Staphylococcus aureus, Pseudomonas aeruginosa were absent (Table 2).

Table 4 summarises the retention time and area under the peak in HPLC for total alkaloid in JJ at baseline and post light challenge.

Stability study of drug substances and drug products is carried out using heat, humidity and light challenges. Many stability studies of herbs and their finished products were also carried out and published in reputed journals, using thermal and humidity challenges. Although the effect of sunlight on various materials including herbal drugs was observed in day to day life and the ICH guideline on stability testing also mentioned that photostability should be an integral part of stress testing, however, to date no photostability study is carried out on herbs and their finished products. Likewise, no specific method for the application of light challenge and parameters for photostability evaluation were discussed in any stability study guidelines for traditional medicinal products.

To the best of the author’s knowledge, no published data on photostability study of herbs and their finished product are available to compare and interpret them with the present study. This is the first kind of its study where a Unani compound formulation was subjected to the light challenge as per the photostability guideline provided for conventional drug products. Furthermore, quality control parameters for herbal were used to assess any significant change.