Preparation and Physico-chemical Characterization of Kushta-e-sadaf, A Traditional Unani Formulation

 

Nitin Dubey1*, Nidhi Dubey2, RS Mehta3 , AK Saluja3 and DK Jain1

 

1College of Pharmacy, IPS Academy, Indore (MP), India.,

2School of Pharmacy, D.A.V.V., Indore (MP), India

3A.R.College of Pharmacy, Vallab Vidyanagar (Gujarat)

* Corresponding Author E-mail: nitindubeympharm@yahoo.com

 

ABSTRACT

Kushta-e-sadaf is a traditional Unani medicinal preparation. It was prepared as per the method mentioned in Unani formulary. The changes occurring during process of kushta formation were characterized using Fourier transform infra- red spectroscopy (FTIR). The results were supported by X-ray powder diffraction (XRPD) studies and thermo gravimetric analysis (TGA).  The study shows that the raw material sadaf (pearl mother shell) is a mineral-organic matrix containing calcium carbonate in aragonite form and which during the process of kushta formation is converted to calcite form with decreased organic content and small percentage of calcium oxide. Physical evaluation reveals that, kushta-e-sadaf is a fine grayish white powder with average particle size of 20.5 µm, bulk density 0.918, tapped density 1.268 and angle of repose 35.47° .The value of Carr’s index is 46.10. Qualitative- quantitative determination indicates calcium content of the kushta, to be 39.67%. Trace element analysis of kushta by ICP revealed the presence of some other important metals like arsenic, lead, chromium, cadmium, mercury, tin under acceptable limits at prescribed dose. Microbial load for the formulation was found to be within limits.

 

KEY WORDS              Kushta; FTIR; calcination; Unani traditional medicine.

 

INTRODUCTION:

Unani is the tradition medicinal system that has been practiced in rural parts of India for centuries. Its popularity outside India is mainly in Arabians and Muslim world. The medicines have proven to have no noticeable side effect, within the recommended doses. Drugs of both herbal origin and metal preparation are used for oral administration.

 

Kushtas form an important class of Unani medicinal system and are inorganic in nature. Kushta is the finest powder form of medicinal preparation obtained by calcinations of metal, mineral and animal drugs.

 

Kushtas of iron, calcium, copper, tin silver, gold, lead and zinc are commonly used. Calcium containing kushtas are obtained using different sources of calcium. They differ from each other in their medicinal properties depending o n the so ur ce o f calciu m u sed d ur in g s ynt hes is.

 

Kushta e sadaf is calcium containing kushta. It was prepared using pearl mother shell as source of calcium. Kushta-e- sadaf    has mughalliz-e-mani (Impuissant to semen) , muqawwi-e-qalb (cardiac tonic), habis (retentive) activities and used for the treatment of sailan-ur-rahem (leucorrhoea), jiryan (spermatorrhoea), nazf-ud-dam (hemorrhage), Zof-e- qulb (cardiac arrhythmia) and quillat-e-kils (calcium deficiency).

 

The first step for synthesis of kushta involves purification [ghasl-e-adviyah], cleaning [tasfiyab] and detoxification [tadbir-e-adviyah] of raw drug material. Further raw drug material  is  finely  grounded  in  pestle  and  mortar  with specified juice of known drug for given time. Then the mixture is sealed in an earthen pot using the process of gil-e- hikmat, and calcinated in traditional furnaces, which are pits of different sizes having varying number of cowdung cakes with different intensity of heat. The process is repeated till the kushta is obtained 1. The metal is said to be completely converted into kushta when it complies with the tests and observations described in the Unani text (Table 1). These tests are highly subjective. With the advent of sophisticated instrumentation it  has  now become necessary to  develop tests that would not only through light on the changes taking place during formation of the kushta but would also help to establish quality control methods for standardization of the synthetic procedure of the kushta2,3 .The literature survey to the best of our knowledge   revealed that no  international work has been reported on standardization parameters for kushta. In present studies attempt has been made to understand the process of kushta formation.

 

In the present work the raw materials and final product along with the various intermediates obtained during the   synthesis   were   characterized   by   FTIR   and supported by XRD and TGA studies.

 

Table I: Traditional Tests for formation of Kushta

 

calcinations [QC K 204] is taken out from the apparatus; powdered, treated with sheer-e-madar and triturated for eight hours. The mixture was pressed in form of cake and dried. The   cakes   were   calcinated   as   above   to   obtain   the intermediate QC  K  208.The procedure was repeated two more times till the sample showed the entire traditional test for kushta positive, to obtain the final product QC K 212.

 

Table III: Calcium carbonate and calcium content of Sadaf and Kushta e sadaf

Material	Calcium carbonate content by acid-base titration	Calcium carbonate content by complexome tric titration	Calcium content by acid-base titration	Calcium content by complexome tric titration
Sadaf	94.55%	95.32%	34.69 %	34.98%
Kushta e sadaf	98.81%	99.55%	36.26%	36.53%

 

Presences of mineral components play a crucial role in the physiological action attributed to the dosage form, but trace mineral elements in higher concentration can exert toxic effect, therefore stringent control on sources of contamination of trace elements in the final product is required4-6. ICP analysis of kushta was carried out to determine trace element contamination.

 

It is important to study other significant pharmaceutical parameters to standardize the quality of product, thus an attempt has been made here to evaluate kushta for some physical, chemical and microbial parameters.

 

MATERIALS AND METHODS:

Materials:

Materials were procured and authenticated by Tayabee Unani Hospital. Intact pearl mother shells (Sadaf) of Pinctada Margaritifera (Linn.) genus Pinctada Roding (pteriidae) were selected for synthesis of kushta. Sheer-e-madar is alkaloidal resin obtained from arial parts of Calotropis Gigantea.

 

Preparation of kushta-e-sadaf 1:

Small pieces of sadaf [QC K 201] were first cleaned

with hot water. Cleaned sadaf were grounded for eight hours  with  equal  proportion  of  sheer-e-madar,  this were than dried and pressed to form small cakes. Drug is  kept and  sealed in  earthen disk by gil-e- hikmat process, where two earthen disks were kept opposite, drug  is  placed  between  them  and  sealed  using  soil paste. This apparatus was then dried and kept in pits. Pit is filled half with 15 kg of cowdung cake, ignited and  then  cooled.  Drug  cakes  obtained  after  first

 

Physical evaluations:

Loss on drying, loss on ignition, bulk density and tapped density were determined using standard pharmacopoeial methods7. Values of bulk density and tapped density were used   to   indirectly   calculate   flowability   by   deriving percentage compressibility (Carr’s index). Static angle  of repose was determined by funnel method8.   Particle size distribution studies were carried out using particle size analyzer (Sympatec, Germany). The kushta was added to distilled water and sonicated for 30 seconds and analyzed.

 

Chemical evaluations:

Fourier transform infra-red spectroscopic studies:

The   raw   material,   intermediates   obtained   after   each calcinations process and final product were scanned using Thermonicolet IR-200 spectrophotometer with DTGS detector in the region of 650 to 3600cm-1. Each spectra is an average of 24 scans of 2 cm-1 resolution. Sampling was done using attenuated total reflectance (ATR) assembly with a sample holder of Zn-Se crystal.

 

Figure 1 [a] FTIR spectrum of Sadaf [QC K 201] and

Figure 1 [b] FTIR spectrum of Kushta-e-Sadaf [QC K 212]

 

X-ray powder diffraction analysis:

All the samples were scanned on Phillips make X-pert powder diffractometer and 2θ scan was from 10 º to 100 º using Ni filter Cu K alpha radiation and NaI scintillator.

 

Thermo gravimetric Studies:

The thermo gravimetric analysis was done on Perkin Elmer series TG analyzer. The thermogram were recorded from 40º to 1000º at the heating rate of 10ºC per min. in air atmosphere.

 

Figure 2[a]  XRPD graphs of Sadaf [QC K`201] and

Fig 2[b] XRPD graphs of Kushta-e-Sadaf [QC K 212].

 

Quantitative determination of calcium carbonate and calcium content:

Both raw material and final product were analyzed for calcium carbonate content. Calcium carbonate content was determined using acid base titration and complexometric titration. Assay results for calcium carbonate were used for indirect determination of calcium content of kushta.

 

I)    Acid base titration: Sample (2gm) was suspended in 50ml of water, 50ml of 2N HCl was added and the mixture was boiled and cooled .Excess HCl was back titrated with 1N NaOH using bromophenol blue as an indicator.

 

II)   Complexometric  titration:  The  sample  (0.1gm) was dissolved in 3 ml of dilute HCl and further diluted with 10ml of distilled water. The mixture was boiled for 10minutes, cooled and diluted to 50ml with distilled water. The mixture was then back titrated with 0.05M disodium edetate using eriochrome black as indicator.

 

Inductive couple plasma analysis of kushta:

A Perkin Elmer ELAN 6000 ICP equipped with an As-91 auto sampler was used. Instrument was calibrated using reference standards of  1  ppm  and  10  ppm.  0.1  gram of sample was accurately weighed into a metal free container and dissolved in 1ml of aquaregia and  heated  on a hot plate to extract the metal .Then solution   was filtered in a volumetric flask and washing of  deionized water was added to it and volume made up to 10 ml. The solution was used for analysis.

 

Microbial evaluations:

Microbial evaluation of kushta was carried out according to pharmacopoeial method7. The kushta was tested for presence of contaminating fungus (yeast and moulds), Staphylococcus aureus, Salmonella sp., Escherichia coli and total Aerobic microbial count.

 

Figure 3 [a] TGA curves of Sadaf [QC K 201] and

Figure 3[b] TGA curves of Kushta-e-Sadaf [QC K 212].

 

RESULTS AND DISCUSSION:

Kushta-e-sadaf was prepared by traditional method as given in Unani formulary. FTIR, XRD, TGA graph of raw material and final products are as shown in Fig 1(a) and (b), 2(a)and (b), and 3(a)and (b) respectively.

 

The FTIR studies of sadaf as active ingredient shows numerous bands in the region of 4000cm-1  to 600cm-1.The carbonate ions of the mineral was shown by the internal vibration modes of CO32- - ν4 mode at 714 cm-1 and 704 cm-1, ν2 mode at 865cm-1and 845 cm-1  and ν1  mode at 1097 cm-1 and 1496cm-1 respectively. The strong IR band at 1711cm-1

 

Table III.  Evaluation of kushta-e-sadaf

Test Parameter	Test	Methods	Inference
Identity	Macroscopic		Non lustrous, grayish white ,fine powder .
	Physical properties	Pharmacopoeial	Bulk density 0.918, Tapped density 1.268 Angle of repose 35.47° . Carr’s index is 46.10. Loss on drying at 110°C : < 0.5 %w/w loss on ignition:< 0.05% w/w.
	XRPD		Characteristic d-spacing value at 3.04 Å, 2.28 Å, 1.91 Å, 1.87 Å, 1.42 Å. (calcite form of calcium carbonate)
	TGA		42 % weight loss at 850°C.
	FTIR-ATR		882cm-1, 714 cm-1 (calcite form of calcium carbonate)
Purity	Contaminating fungus (Yeast and mould)	WHO	<1 X 104 CFU/g
	Total Aerobic Count	WHO	<1 X 105 CFU/g
	Escherichia Coli	WHO	Absent
	Salmonella spp.	WHO	Absent
	Staphylococcus aureus	WHO	<1 X 102 CFU/g
	Arsenic	Pharmacopoeial	<0.14 µg/k.g. b.w./day
	Cadmium	Pharmacopoeial	<0.09 µg/k.g. b.w./day
	Lead	Pharmacopoeial	<0.29 µg/k.g. b.w./day
	Total mercury	Pharmacopoeial	<0.29 µg/k.g. b.w./day
Quantity	Quantitative Test	Titrimetric methods	Calcium content :39.67%

 

can be attributed to C=0 groups of the carbonate ions. The splitting of ν4 is a characteristic of aragonite structure of calcium carbonate. The bands for OH and NH stretching modes of the organic matrix were found in the region of 3000-3450cm-1 and C-H stretching in region of 2800-2980cm-1. IR bands at 1664cm-1  and 1638cm-1   were  attributed  to  amide  C-O  and  C-N groups of  matrix. The  band  in  the  region of  2520- 2650cm-1 is due to OH group of organic matrix. The d- spacing values obtained by XRPD investigation [3.03 Å,2.70 Å,2.49 Å and 2.37Å] and 42% loss in weight above  800°C-900°C observed  in  TGA  studies confirms that   sadaf is composed of a organic matrix and aragonite crystal of calcium carbonate as mineral phase.

 

FTIR data of final product shows loss of ν1 vibration mode of CO32-, shift of ν2 mode from 865 to 882cm-1 and degeneration of ν4 mode and single band at 714 cm-1,   which   indicates   calcite   structure   of   calcium carbonate. Thus,  during  the  synthesis of  kushta  the lines  of  aragonite  disappeared and  product  contains purely  calcite  form  of  calcium  carbonate.  A  small percentage of calcium carbonate   is   transformed to calcium oxide .The decreased carbonate content was indicated by the lowering of    intensity of the characteristic bands and peak for calcium oxide is  at 452cm-1.The bands for organic matrix were modified at different stages of process  .The partial destruction of organic matrix was indicated by   weight loss during synthesis and decreased   band intensities .The FTIR data is further supported by XRD of intermediates and final product having d-spacing values [3.04 Å, 2.28 Å, 1.91

Å, 1.87 Å, 1.42 Å] and TGA showing 42 %  weight loss at 850°,which  indicates  presence  of  calcium  carbonate  in calcite form. The final product, kushta-e-sadaf is calcium carbonate in calcite form with decreased organic content and small percentage of calcium oxide. Above result for FTIR, XRD, TGA studies are in coordination with studies reported for pearl mother shell and different crystal forms of calcium carbonate9,10.

 

Physical, chemical and microbial properties of kushta-e- sadaf are given in table II. Physical evaluation of the product revealed that kushta-e-sadaf is a fine grayish white powder with poor flow property.

 

The  calcium carbonate and  calcium content of  raw drug material  sadaf  and  kushta-  e-sadaf as  given  in  table  III, indicates that final product has high proportion of calcium carbonate and least organic matter, this may be due to the elimination of organic species at high temperature. The acid base titration determines carbonate ion and complexometric titration determines the calcium ion.  The results obtained by both acid-base titration and complexometric titration are in good coordination.

 

The ICP analysis reveals that trace elements were present in reasonable amounts in starting material; but the final product (kushta) contains a very minute amount of them. It may be attributed to volatilization of elements at high temperature and absorption in earthen pot. Reaction of metal oxides with active carbon of organic component used in synthesis may result into metallic forms of element which are least toxic.

 

The results of ICP analysis revealed that metals like arsenic, lead,  chromium, cadmium,  mercury, tin  are under acceptable limit at prescribed dose of 300-400mg daily as per Canadian guideline 200311.

 

Microbial load of the preparation was found negative for   the   presence  of   Escherichia  coli,   Salmonella species and Staphylococcus aureus. Total aerobic count was under acceptance limit11.

 

CONCLUSION:

The  characterization    techniques  like  FTIR,  XRD, TGA, PSA which have been used in the present studies can be used as quality control methods for characterization  of  samples  in  industry  not  only  to check uniformity of the samples marketed by manufacturer but also to ensure that each step is been followed and product marketed are kushta and not the intermediate.   A   routine   use   of   such   scientific techniques will lead to standardization of the product to a certain extent and would definitely help in building confidence in use of such products for medication.

 

ACKNOWLEDGEMENT:

Authors are thankful to The Tayyebi Dawakhana Unani (Indore) Pvt. Ltd and for providing facilities for synthesis by traditional method and SICART, Vallbh Vidya Nagar for analysis of kushta.

 

REFERENCES:

1.       Anonymous, The National formulary of Unani India, Govt. of India,

2.       Ministry of health and family planning, part I, 1981.

3.       Lalla  JK,  Hamrapurkar  PD,  Patil  PG  and  Mamania  HM. Preparation,   characterization   and   analysis   of   shankha bhasma. Indian drug.2002; 39(3): 152 -156.

4.       Mishra  LC.  Scientific  bases  for  ayurvedic  therapy,  CRC press, Washington DC. 2004; 83- 100.

5.       Kumar A, Nair AGC, Reddy AVR and Garg AN. Bhasmas Unique Ayurvedic Metallic-Herbal preparations, chemical characterization.  Biol. Trace Elem. Res. 2006; 109: 231-254.

6.       Ernst E. Heavy metals in traditional Indian remedies. Eur J Clin Pharmacol 2002; 57 : 891-896.

7.       Saper RB, Kales SN, Paquin, J, Burns MJ, Eisenberg DM, Davis   RB   and   Phillips   RS.   Heavy   Metal   Content   of Ayurvedic  Herbal  Medicine  Products.  JAMA.  2004;  292: 2868-2873.

8.       Anonymous Pharmacopoeia of India vol. II. Government of India: 1996, A-34-35, A72-77, 89,100-127.

9.       Martin AH, Swarbrick J, Cammarata A. Physical Pharmacy. Varghesse Publication: 1991, 492-519.

10.    Balmain  J,  Hannoyer  B  and  Lopez  E.  FTIR  and  XRD analyses of mineral and organic marix during heating of mother of pearl from the shell of the mollusk Pinctada maxima. J. Biomed. Mater. Res. 1999; 48(5):749-754.

11.    Dickinson SR, and McGrath, Quantitative determination of binary and tertiary calcium carbonate mixtures using powder X-ray diffraction. Analyst. 2001; 126: 1118-1121.Canadian guideline                   http://www.                   hc-sc.gc.ca/dhp- ps/prodnatur/legislation/docs/eqpaq_e. 12.  Natural health product directorate, Canada, 2003.

 

Accepted on 20.07.2008   © RJPT All right reserved