Introduction
Kushta is a very potent and efficacious dosage form used in Unani. The most ancient manuscript written in the field of Kushtajat by Baracoles is 'Asagharba' which means collection of varieties. This terminology was used for drugs of mineral origin used in treatment of diseases, and their transformation into various dosage forms.[1]Kushtan in Persian means “to kill”. Kushtas are made to make the drug easy for use and purposeful, to remove the harmful and disadvantageous materials from it, to minimize the dose and increase the potency of the drug.[1] In this work Kushta of one drug of mineral origin viz Zahar Mohra (Serpentine/Bezoar stone) was evaluated [Figure 1].
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Figure 1: Zahar Mohra (Bezoar stone)
Kushta-e-Zahar Mohra (KZM) is one of the most popular and useful Unani formulations. Zahar Mohra (ZM) is also known as Fad-e-ZaharMa'dani (Antidote of mineral origin) and Hajr al-Sam (Stone for Poison) in Arabic. It is a type of stone having good smell and taste. It is a good-looking drug of mineral origin.[2]
It is imported from China and Khata (now part of China). The best quality is known as Zahar Mohra Khatai.[3] According to Arastatalis (Aristotleys) yellow coloured Zahar Mohra is best. According to Unani medicine the mijaz of Zahar Mohra is har yabis (hot & dry) in first degree. The actions of Zahar Mohra are mufarrih (exhilarant), muqawwī-i-d'dā' Ra' īsa (tonic for vital organs) and dafi'-i-samūm (antidote).[4] It acts as preservative of quwā(faculty) and rūh (pneuma). It gives protection from viral diseases and endemic conditions. It also protects from septic conditions in body fluids and acts as a muqawwī-i-a'sāb (nervine tonic) and muqawwī-i-bāh (aphrodisiac).[5] In the form of kushta preparation, Zahar Mohra acts as a mood elevator and cardio-tonic. It is also useful in palpitation, panic and nervousness. In this study formulation of KZM prepared in a Muffle furnace simulating classical Bhatti temperature was prepared and subjected to physico-chemical analysis. The formula contains Zahar Mohra (serpentine 100 g) and Arq-e-Gulab (rose water, Rosa damascene Mill. 375 ml). The formula was selected from Qarabadhin-e-Majidi compiled by Unani Tibbi conference, Hamdard, New Delhi.[6] Objectives of the study are preparation of KZM using contemporary methods and its physico-chemical and instrumental analysis.
Materials and Methods
Study was carried out in the Dept. of Ilmul Saidla (Unani pharmacy), NIUM, IISc (Indian Institute of Science) Bengaluru and other accredited labs. ZaharMohra (serpentine stone) was purchased from an apothecary shop in Bengaluru; Arq-e-Gulab (Rose water, Rosa damascene Mill.)[7] was purchased from Hamdard Waqf Laboratory, New Delhi. ZaharMohra (serpentine) was identified by an expert at NIUM, Bengaluru.
Method of Tadbeer(processing)
Zahar Mohra was taken and subjected to Tadbeer (detoxification/processing) by Arq-e-Gulab (rose water).[6]
150 g of ZaharMohra (serpentine) was powdered by Mortar and Pestle manually and filtered through muslin cloth; Arq-e-Gulab according to the need was mixed during grinding/triturating process and this process was carried out until 562 ml rose water was absorbed during trituration in an electric mortar and pestle in the pharmacy of NIUM, Bengaluru [Figure 2].
Figure 2: Kushta-e-Zahar Mohra preparation
Kushta-e-Zahar Mohra preparation
Pelletisation
Big size petridishes were taken and mudabbar (detoxified/processed) Zahar Mohra paste was made into pellets of 10 g each manually and was pressed to make the surface smooth. It was then kept for drying under shade. The dried pellets were separated easily as they do not stick to petridish after drying [Figure 2].
Preparation of Kushta (classical method)
Pellets of Zahar Mohra were taken in clay crucible (buta) then the crucible was covered with clay lid and sealed by the process of Gille Hikmat,[1] this sealed crucible was subjected to put (fire) in five kg cow dung cakes as per classical reference. Temperature pattern was developed in the form of temperature graph by keenly observing and recording the heat pattern during the calcination process with the help of a thermocouple, sensor of which was placed near buta [Figures 2-4].[6]
Figure 3: Temperature pattern classical and Muffle Furnace method
Figure 4: Kushta-e-Zahar Mohra
Preparation by Muffle furnace method
Pellets of Zahar Mohra were again taken in buta. After sealing and drying in the shade the buta was then placed in Muffle Furnace. The temperature pattern recorded in the form of thermogram during classical method of preparation was followed in Muffle furnace method. It was developed, for the gradual increase and decrease of temperature during the classical method of preparation at 10 minutes time intervals with five kg cow dung cakes. A maximum temperature of 8040 C as obtained from classical method was set in Muffle furnace for the preparation of Kushta. Temperature was gradually increased from atmospheric temperature (i.e. 250 C) onward to the maximum and then it was again set to cooling upto the same level of atmospheric temperature [Figure 3]. Trituration with rose water and calcination in furnace was repeated number of times for different batches to prepare required quantity of KZM for analysis. Quantity of KZM obtained was observed and subjected to Physico-Chemical analysis [Figure 4].
Physico-chemical analysis
Organoleptic characters
Prepared KZM was evaluated for its Colour, Odour[18] Taste.[89][Figure 4]
Unani specification (test)
The Floating test,[89] Fineness test,[89] Loss of metallic luster,[89] Wall stick test,[8] Smokeless test[91011] [Table 1 and Figure 5] parameters are used for the analysis of proper calcinations process.
Table 1: Physicochemical parameters of Kushta-e-ZaharMohra
Figure 5: Finger thumb test → Floating test → Wall Stick Test of Kushta-e-Zahar Mohra
Powder characterizations
Bulk density, tapped density,[12] Hausner ratio and Compressibility index (Carr's index) were determined.[12] The angle of repose was determined by using fixed funnel method. The height of the tip of funnel was fixed 2cm above the horizontal surface. A graph paper was placed below the funnel on the table. The KZM was allowed to flow through the funnel freely on to the surface until the apex of the conical pile just touched the tip of the funnel. The diameter of the Kushta cone base was measured and the angle of repose was calculated by using this formula[13] Tan Ø =2h/D [h = height of powder (from graph paper to tip of funnel), D = mean diameter of the powder][13] Loss of weight on Drying (LoD) at 1050 C, pH, Total ash, Acid insoluble ash, Water soluble ash and Sulphated Ash[9] were determined as per method mentioned in Protocol for testing of Ayurvedic, Siddha, and Unani medicine.[9]
The Solubility in Petroleum Ether, alcohol and water[14] were determined as per Physicochemical Standards of Unani Formulations.[14]
Particle size analysis by mesh size
The KZM has been passed in different mesh from # No 80, 100, 170,200, 240, 300, 350 etc., Percentage (%) of material passed was recorded.
Qualitative estimation of constituents using chemical method
Organic
Qualitative tests for alkaloids (Dragendorff's test and Mayer's test) and Tannins was done as per method mentioned in Physicochemical standards of Unani formulations.[14] Tests for phenols, resin, steroids (Salkowski reaction), glycosides, proteins (Millon's test) and carbohydrate (Fehling's test) were done as per Physiochemical standardization of Unani formulation.[15] Tests for flavonoids, saponins and terpenoids were also carried out as per method adopted by Venkatesan et al.[16]
Inorganic compounds
The tests for Calcium, Aluminum, Arsenic, Mercury, Iron, Manganese, and Zinc compounds were carried out as per methods mentioned in Physicochemical Standards of Unani Formulations. Part IV.[14]
XRD Study of Kushta-e-ZaharMohra
XRD diffraction is a rapid and accurate method for identifying the crystal structure, study of phase equilibria and the measurement of particle sizeand so on. Samples can be easily prepared in this study. It does not help in case of Amorphous solids and trace element detection is often difficult.[1718] It works on the Principle of Bragg's law which may be written in the form: λ=2d sin θ. The distance between each set of atom planes (d) is determined with the help of wave length λ of x-ray beam and angle of diffraction θ by applying Bragg's Law.[1718] Powder method of diffraction has been adopted in this study. X-ray diffraction method for crystallographic study of sample was done at IISc Bangalore.
Fine powders of samples were prepared for investigation out of which 1g sample was separated for the study. X-ray powder diffraction was carried out using the X'pert Pro Pan-Analytical powder diffracto-meter equipped with Cu-target (Source; CU k α, α=1.5418 A°, operating voltage 40 KV/30 MA). Phases were identified using the automatic search program X'Pert High Score Plus. Other equipment used were Digital balance (Shimatzu corporation, Japan AY 220), standard Sieve # no. 200 (Test sieves IMPEX INDIA), X-Ray diffraction studies conducted on different sample of KZM are confirmed by comparing d-identified values with d-standard peak values. The 2-theta value and intensity of the peak (counts) are represented on X and Y-axis respectively, higher peak (count) value indicates higher crystallinity of the phase. Phase detection was done for the two samples prepared by the same method.[1718]
Heavy metal analysis
ICP-MS (Inductively Coupled Plasma–Mass spectrometry) and AAS (Atomic Absorption Spectrophotometry) test was also performed on the sample of KZM for heavy metal analysis.[1920]
Results
Observation regarding yield revealed that 150 g Raw drug ZM was subjectedto powdering and 147 g yield was collected as powdered form in one batch. This 147 g powdered Zahar Mohra was subjected for mudabbar process with 562 ml of Arq-e-Gulab (rose water) in one set of electric mortar and pestle [Figures 1-4]. After mudabbar process 223 g Zahar Mohra was collected in paste form. After pelletisation and drying in shade, the 145 g of sample was obtained and was subjected to calcination. The mean weight of Kushta was near about same as sample taken and obtained after pelletisation and drying. All the data are based on multiple observations. Value of heat pattern obtained by classical method was simulated in Muffle furnace. Thermogram of sample of KZM prepared by classical and Muffle Furnace method is depicted in [Figure 3].
Organoleptic properties
Prepared KZM was found to be slight yellowish white colour [Figure 4], odourless and tasteless. KZM completely passed in # No 350.
Unani specification test
Floating test, Loss of metallic lustre, Wall stick test, Smokeless test were positive and Fineness and Smoothness test results showed KZM to be very fine and smooth in both KZM- CM and KZM- MFM samples and were as per Unani specification [Figure 5].
Results for the physicochemical studies such as Ash Value, LoD at 1050 C, pH and solubility, are depicted in Table 1. Powder characterization is depicted in [Table 2].
Table 2: Powder characterization of Kushta-e-Zahar Mohra
Qualitative estimation of constituents/elements
Qualitative test of organic components such as Alkaloids, Phenols, Resins, Flavonoids, Saponins, Glycosides, Terpenoids, Tannins, Steroids, Proteins and Carbohydrates all were found to be negative in KZM. Inorganic components such as Arsenic, Mercury, Lead, Calcium, Iron, Manganese and Zinc compounds were found to be negative in KZM in this preliminary analysis. Aluminium compounds were found to be positive in KZM.
Heavy metal analysis
Elemental analysis of KZM done by ICP-MS and AASis depicted in [Table 3].
Table 3: Heavy metals test
XRD study
XRD findings reveal presence of Mg2 SiO4 (Magnesium Silicate/Forsterite) and Ca(ClO)2.3H2O (Calcium Chlorite Hydrate) phase, [Figure 6] Combined XRD of sample 1 and 2 of KZM revealed same phase and pattern [Figure 7].
Figure 6: XRD graph of Kushta-e-Zahar Mohra
Figure 7: Combined XRD of sample 1 and 2 of Kushta-e-Zahar Mohra
Discussion
Several formulations in Unani and traditional medicine contain mineral origin drugs. One such formulation is KZM. It is mentioned to act as mood elevator, cardio-tonic and useful in conditions of palpitation, panic and nervousness. Considering its vital pharmacological activities and beneficial use it becomes very essential to evaluate the constituents and margin of safety withrespect toheavy metal and also to establish data for its standardization. The mean temperature pattern of KZM for Muffle Furnace method was developed on the basis of classical method [Figure 3]. The sample was analysed for its organoleptic properties, preliminary tests and physicochemical studies [Tables 1-3].
Formulated Kushta passed the classical Unani specification test such as Finger thumb test, Floating test, Wall Stick Test and Smokeless Test [Figure 5]. Findings of the test revealed that Kamil (perfect) Kushta was prepared.[21] Powder characterisation data of KZM was set in Compressibility index (%), Hausnerx s ratio, Angle of Repose display poor flow property of KZM. Total Ash of 99% clearly indicates inorganic ingredients of KZM, and qualitative organic test displays no presence of organic ingredients despite presence of Rose water. It may be due to its degradation during calcination process. Chemical tests of inorganic ingredients displayed presence of Aluminium whereas XRD analysis clearly indicates presence of Mg2 SiO4 (Magnesium Silicate/Forsterite), Ca(ClO)2. 3 H2O (Calcium Chlorite Hydrate) phase [Figure 6]. Calcium chloride is commonly encountered as a hydrated solid; Calcium chloride injection may decrease cardiac toxicity as measured by electrocardiogram. It can also help to protect the myocardium from dangerously high levels of serum potassium in hyperkalemia. Intravenous calcium has been used in cardiac arrest.[22] Calcium is a very important element for heart. The importance of calcium-dependent signalling in the heart has been appreciated for decades; calcium is required for cardiac muscle contraction.[23]
XRD findings also revealed that sample 1 and 2 of KZM revealed same phase and pattern, [Figure 7] this finding can also be interpreted that process followed by method is reproducible. Heavy metal analysis indicates presence of lead, cadmium and arsenic under permissible limits [Table 3]. Slightly elevated level of Mercury above permissible limit serves as a caution and stresses the need regarding importance of screening the formulation for heavy metal owing to its mineral origin. These limit guidelines are for herbal origin traditional drug. These parameters of instrumental evaluation indicate safety of KZM. Mudabbar/Musaffa processes with Arq-e-Gulab on Zahar Mohra may have cause decrease in the particle size and the colour to become whitish. Kushta preparation is a nano-form of particle and can be used to modify or enhance the drug kinetics. The present study indicates further scope of evaluation of its safety by toxicological studies and indicates the immense potential of the formulation to validate its pharmacological action owing to its constituents.
Conclusion
Physicochemical data and process for standard of KZM was set in as a Quality specification for the same. KZM prepared by Muffle Furnace method could be a better option in respect of ease of preparation. The study revealed that KZM sample studied is kamil (perfect) according to Unani and contemporary instrumental analysis. KZM prepared accordingly can be a better option for utilisation as exhilarant, tonic for vital organs and antidote as mentioned in Unani texts.
Financial support and sponsorship
Self and Facilities provided by Director National Institute of Unani Medicine, Bengaluru India in terms of chemicals and other infrastructure to post graduate Department of Ilmul Saidla NIUM Bengaluru.
Conflicts of interest
There are no conflicts of interest.
Acknowledgement
The authors would like to express their thanks to, Director, National Institute of Unani Medicine (NIUM) Bangalore for his motivation to work, to all the Pharmacy staff of NIUM Pharmacy, Prof. Rajeev Ranjan, Department of Materials Engineering (In-charge CCD facility) and Mr. AN Krishnamurty (Technician XRD) Department of Material Engineering, IISc (Indian Institute of Science), Bangalore-560012, India, for help in XRD analysis and its interpretation.
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