Pharmacognostical and HPTLC Study of Unani Formulation Khamira Khas: A Cardiac Tonic

Medicinal plants have been known for millennia and are highly esteemed all over the world as a rich source of therapeutic agents for the prevention of diseases and ailments [1]. Over 60% of the world population relies on the use of traditional medicine, which is predominantly based on plant material [2]. In the health service sector, WHO also endorses and boosts up the exploration of traditional drugs by virtue of their facile availability, accessibility and affordability. However, paucity of pertinent authentication, rigorous quality control, documentation and standardization has created the main hurdle in the establishment of alternative drugs in the developed nations. The government of India has formal structures to regulate quality, safety, efficacy, practice and documentation of herbal medicine (National Policy on Indian system of Medicine and homeopathy-2002) [3]. Standardization of herbal drug is not an easy assignment as various components such as temperature, geographical localities, period and time of collection, age and part of the plant collected, method of collection and various other factors affect the bio efficacy and reproducible therapeutic effect [4]. In the manufacturing of poly-herbal formulations, adulteration, contamination and substitution or total absence or replacement of costly ingredients are dangerous practices that eventually affect the quality of the formulations. This has emphasized the necessity for the meticulous quality control and standardization of herbal preparations through proper scientific testing and evaluation methods [5]. 

The word Khamira represents the fermented confection in the Unani system of medicine, first established by the physicians of Mughal period. Khamira is a semi-solid sugar based preparation which is formulated by pouring a decoction of powdered herbal drugs to sugar or sugar & honey based qiwam (syrup) at a proper temperature. Khamiras usually act as a cardio-tonic and are also used in various other ailments like palpitations, weakness of heart, weakness of principal organs, cough, cold, catarrh, including respiratory and nervous disorders[6-8].Some of the findings have also disclosed the antioxidant and immunomodulator properties of Khamiras [9].

Preparation of the formulation 

All the ingredients were procured from the local market and identified botanically using pharmacognostical methods [10-11].The ingredients were further validated by comparing with the monographs available in API, Part I, Vol. III [12]. The formulation Khamira Khas was prepared as per the formulation composition given in NFUM, Part VI using the ingredients showed in table 1 [13]. 

Table 1: Formulation Composition of Khamira Khas. 

All the ingredients were taken of pharmacopoeial quality. The ingredient no.1 was cleaned and dried under shade to remove the moisture if any. It was ground to powder and sieved through mesh no.80. Ingredient no.2 & 3 were mixed together in a vessel and the content was boiled over a medium flame. At the boiling stage 2.5g of citric acid was added. The boiling of the content was continued until a qiwam (syrup) of 80% consistency was obtained. The vessel was removed from the flame and the ingredient no.1 was added immediately by thorough mixing. The mixture was vigorously beaten until it became homogenous and fluffy. Finally 2.5g sodium benzoate was added by thorough mixing. The content was allowed to cool at room temperature and the formulation so formed was stored in tightly closed glass container.

2g of the drug was taken and stirred gently with hot water in a beaker. The supernatant was discarded and the process was repeated each time rejecting the supernatant and keeping the sediment until a clear or transparent solution was obtained. A little sediment was stained with iodine solution and mounted with 50% glycerin. A small amount of residue was treated separately with chloral hydrate solution, washed with distilled water and mounted in 50% glycerin. The various characters were observed under the microscope [10,14].

The physico-chemical parameters such as moisture content, water and ethanol extractive values, ash values, pH values were analyzed by standard methods [15,16].

Preparation of extract for HPTLC 

After leaching out the sugar from the drug, 2g sample was extracted with 20ml of ethanol by refluxing for 20 minutes. The extract was filtered through Whatman No.1 filter paper. The filtrate was further concentrated up to 10 ml and used for HPTLC analysis. 

HPTLC fingerprinting was carried out by applying the ethanol extraction aluminum TLC plate pre-coated with silica gel 60 F₂₅₄ (E. Merck) using Desaga AS30 automatic sample applicator. The plate was developed up to a distance of 8cm in a twin trough glass chamber (10x10cm) using the solvent system of toluene: ethyl acetate (9: 1) as mobile phase. The plate was air dried at room temperature and observed underUV at 254nm & UV 366nm wavelengths. Later on the plate was derivatized by dipping it in 1% vanillin-sulphuric acid reagent followed by heating at 105°C till the colored bands appeared [17,18]. 

Estimation of Microbial Load 

The microbial load viz. total bacterial count (TBC), total fungal count (TFC), Enterobacteriaceae, Escherichia coli, Salmonella spp., Staphylococcus aureus and Pseudomonas aeruginosa were estimated as per standard methods [19].

Estimation of Heavy Metals 

The analysis of heavy metals like lead, cadmium, mercury and arsenic was carried out as per standardmethods [19-20].

Details of the Instrument and operating parameters 

Atomic Absorption Spectrophotometer (AAS) model LABINDIA AA7000 was used for heavy metals analysis. The operating parameters were as follows: 

Lead and Cadmium: Instrument technique  - Flame atomization; wavelength (Lead) - 217 nm; wavelength (Cadmium) - 228.8 nm; slit width - 0. 5 mm; lamp current (Pb) - 4.0 mA; lamp current (Cd) - 3.0 mA; carrier gas and flow rate - air and acetylene, 1.1 L/min; sample flow rate - 2 ml/min. Mercury: Instrument technique - Cold vapour technique; wavelength - 253.7 nm; slit width - 0. 5 mm; lamp current - 3.0 mA; carrier gas and flow rate - argon, 1.1 L/min; sample flow rate - 5ml/min. Arsenic: Instrument technique - Cold vapour technique; wavelength - 193.7 nm; slit width - 0. 5 mm; lamp current - 6.0 mA; carrier gas and flow rate - acetylene, argon, 1.1 L/min; sample flow rate - 5ml/min. The hallow cathode lamps for Pb, Cd, Hg and As analysis were used as light source to provide specific wavelength for the elements to be detected.

Analysis of Aflatoxins 

Aflatoxins B₁, B₂, G₁ and G₂ were analyzed as per Official Analytical Methods of the American Spice Trade Association (ASTA), 1997 [21].

Details of Instrument and operating parameters 

High Performance Liquid Chromatography (Thermo Fisher) was used for the analysis of aflatoxins. Column - Ultra C18, 250 X 4.6 mm, 5 µm particles; Mobile phase: Water: Acetonitrile: Methanol (65: 22.5: 22.5); Flow rate: 1 ml/min; Temperature: 35?; Detector: Fluorescence detector at 360 nm; Injection: 20 µl (Aflatoxins mixture and sample)

Analysis of pesticide residue 

The analysis of pesticide residues was carried out as per the method described in AOAC, 2005 [20]. Pesticide residues were analyzed by employing Gas Chromatography-Mass Spectrometry (GC-MS) (Instrument-Agilent, detector-mass selective detector, column specification-DB5MS, carrier gas - helium, flow rate - 1ml/min, column length - 30 m, internal diameter - 0.25 mm, column thickness - 0.25 μm).

Khamira Khas is a light brown semi-solid drug with pleasant aromatic smell and sweet taste.

Identification 

Microscopy: Microscopy of khamira khas showed anatomical markers such as thin walled parenchyma cells (non lignified) and hexagonal parenchyma cells, tuft of fibre cells and fibre cells with broad lumen, pitted and reticulate vessel, large rounded parenchyma cell, large and thick walled sclerenchyma, sieve tube element, elongated cells with yellowish oil globule, cell filled with oil globules, druse crystals and starch granules, sclerenchyma sclereids (thick walled) with group of sclereids cells (Khas Hindi) (Figure 1). 

Figure I: Microscopy of Khamira Khas.

Physico-chemical parameters 

The results of physico-chemical parameters of Khamira Khas (five samples) are tabulated in table 2. Quantitatively evaluated data revealed that the moisture content of the drug ranged between 9.02 - 9.76 % which is apt in case of Khamiras. Total ash content was not more than 0.70 % and acid insoluble ash remained in traces which indicated that siliceous matter was present in the drug samples in negligible amount. The water extractive valuesturned out to be on higher end, ranged between 82.15 - 82.88 % which indicated the presence of inorganic and more polar organic content and the ethanol soluble extractive values were also high and ranged between 71.20 - 72.35 % which indicated the extraction of polar constituents. 

Table 2: Physico- chemical Parameters.

HPTLC studies of ethanol extract: The HPTLC data of ethanol extract is tabulated in table 3. The drug sample showed 02 bands under UV254nm and 05 major fluorescent band sunder UV 366nm. After derivatization of the plate with 1% vanillin - sulphuric acid reagent, 06 major colored bands were observed under visible light. (Figure 2).

Figure 2: HPTLC of Khamira Khas. 

Table 3: R_f values of ethanol extract.

Evaluation of quality (WHO) parameters

In order to assess the quality of drug samples, the microbial loads viz. Total Bacterial Count (TBC), Total Fungal Count (TFC), Enterobacteriaceae, Escherichia coli, Salmonella spp., Staphylococcus aureus and Pseudomonas aeruginosa were analyzed and found to be in permissible limit (Table 4). The heavy metals lead, cadmium, mercury and arsenic were found to be below detection Limit (LOD) in the drug samples (Table 5). Moreover, aflatoxins B₁, B₂, G₁ and G₂ were below the Limit of Quantification (LOQ). Similarly pesticide residues such as organo chlorine group, organo phosphorus group, alachlor, aldrin, chlordane, DDT, endosulfan, heptachlor, lindanewere also below the Limit of Quantification (LOQ). The study indicates that the drug samples were free from heavy metals, aflatoxin and pesticide contaminations (Table 6).

Table 4: Microbial Load. 

 Table 5: Analysis of Heavy Metals. 

Table 6: Pesticide residues.

Standardization of the traditional medicines is essential to maintain their purity and efficacy. Thus, in modern era more attention and stress have been given on the standardization of Unani formulations. The HPTLC fingerprinting and evaluated pharmacognostical&physico-chemical parameters of Khamira Khas will be productive for fixing pharmacopoeial standards of the drug. All the quality parameters were found to be in permissible limits which indicate that Khamira Khasis free from toxic materials and is safe for human consumption. Hence, the present study holds high significance to ensure authenticity and quality of the drug.

The authors are extremely thankful to the Director-General, CCRUM, New Delhi for their valuable guidance, encouragement and providing necessary research facilities to carry out the studies.