Comparative Analysis on Significance of Purification Process of Monosilai in Siddha medicine using Modern Instrumental Techniques: Standardization Approach

 

A. Sureka¹, R. Dhanalakshmi², C. Mary Sharmila³, R. Chithra Devi⁴

¹Emergency Medical Officer, National Institute of Siddha, Tambaram Sanatorium, Chennai 600047,

Tamil Nadu, India.

²Medical Officer, Naturalife Hospital, Thrichy, Tamil Nadu, India.

³ Resident Medical Officer, National Institute of Siddha, Tambaram Sanatorium, Chennai 600047,

Tamil Nadu, India.

⁴House Officer, National Institute of Siddha, Tambaram Sanatoruim, Chennai 600047, Tamil Nadu, India.

 

ABSTRACT:

Standardization is an integral part of the medicine system, which began since several centuries. The great Siddhars of the siddha system of medicine has Clearly documented both the preparation techniques and treatment methodology for each class of disease and their therapy. Siddha medicinal formulations has multiple components hence it’s involves greater challenge in standardization of the same. The standardization of herbal preparations can be achieved by systematic procedures. Manosilai is a metallo mineral drug composed of arsenic and sulphur as major components. According to siddha principle metals and minerals possess enormous healing properties as it can mediate some of the biologically significant metabolic reactions in the human body. Also it is utmost important to establish a detailed monograph of such preparation as a standard for future researchers who work on this novel formulation. The main aim of the present study is to comparatively analyze the siddha drug manosilai (MS) with special reference to its purification process by modern sophisticated analytical instrumentation techniques such as Fourier-transform infrared spectroscopy (FT-IR), scanning electron microscope (SEM) and X-ray powder diffraction (XRD). From the results of the present investigation it was observed that there was slight change in LOD value of the sample manosilai before purification (MSBP) and after purification (MSAP). In which the total ash value remains the same. There is no significant change were observed in the water soluble extractive value of both the sample. But there was marginal difference in alcohol soluble extractive value and pH of the samples MSBP and MSAP. FT-IR spectral data’s signifies the presence of increased functional group in the MSAP when compare to that of the MSBP. In which the sample MSBP depicts the presence of S=O and NH functional groups which were absent in MSAP. Whereas the sample MSAP has shown the presence of additional functional groups id N-N=O, C-Br and other benzyl groups were not observed in MSBP. There was no significant changes were observed with respect to the surface morphology and also average particle size of the both of these samples MSBP and MSAP. It was observed from the result analysis of the X-RD pattern of the sample MS before and after purification shows no significant change in the elemental composition since in both the preparations arsenic and sulphur remains the core components.

 

 

 

 

1. INTRODUCTION:

Siddha system of medicine is one of the holistic arts of healing prevailing in the southern region of India since ancient times. Siddha medicine has a unique way of identifying the diseases and the treatment modality for each kind varies independently. EnnVagaiThervu is a conventional diagnostic procedure followed in siddha medicine for diagnosis of diseases and includes 1. Nadi (Pulse), 2. Vizhi (Eyes), 3. Swara (Voice), 4. Sparisam (Touch), 5. Malam (Faeces) and 6. Moothiram (Urine) [1].

 

The Great Siddhars have formulated siddha medications with utmost care and uniqueness. The basic ingredients used in the siddha preparation are broadly classified in to three 1. Herbs 2. Metal and inorganic substances 3. Animal products. Further there exists the usage of combinational therapy which includes polyherbal, herbomineral, metallic preparation’s etc. The commonly adopted techniques commonly adopted for formulating the siddha drugs are extraction, decoction, incineration, distillation, pulverization and pasting. On application basic siddha drugs categorized for internal and external therapy.

 

Manosilai is one of such great metallic drug which was been prescribed to cure lot of disease, particularly of effective chronic disease, like, skin leprosy, fever with chills, asthma, eye diseases, urinary tract infection, kappa disease. Manosilai is also included as the ingredients in the preparations like Shivanaramirtham, Kasthoorikaruppu, Vishnu chakkaram, Bramananda Bairavam, Gandhaga Sudarthailam. Standardization of drugs not only reveals the identity and genunity of such preparations it also relies more on the safety aspect. Henceforth to be on the global platform its now a days become mandatory for the manufacturer to ensure the quality and safety of their drugs. As siddha system of medicine becomes the primary health care supportive therapy in majority of the people in the developing countries. It is of utmost responsibility falls in the formulators to ensure the safety and efficacy [2].

 

Physicochemical evaluation renders core data on the quality and adulteration in finished products. It is evident from several research outcomes that the physicochemical properties of the drug has an impact on solubility, absorption, transmission through barriers, binding affinity, metabolism and excretion. The greater dynamic phenomenon of developing standard for siddha formulation requires in-depth knowledge.

 

Modern analytical techniques joins hand in hand in the validation of multi component preparation’s. Spectroscopy, SEM and XRD is one among such techniques which provides the reliable data on basic finger printing of the drug above which in recent time used for identification of reference compound such as metals, minerals and other inorganic components. The main aim of the present study is to comparatively analyze the siddha drug manosilai (MS) with special reference to its purification process by modern sophisticated analytical instrumentation techniques such as Fourier-transform infrared spectroscopy (FT-IR), scanning electron microscope (SEM) and X-ray powder diffraction (XRD)

 

2. MATERIALS AND METHOD:

2.1. Method of Purification [3]:

Red orpiment of about 35gms were made in to small pieces and kept soaked in 175 gms of fermented butter milk in clay vessel using insulation and kindling frequently. sufficient time was allowed and the final product was collected and washed the same procedure was repeated thrice to get the purified form for further analysis.

 

2.2. Physicochemical analysis [4-6]:

Physicochemical standards of the prepared drug manosilai has been carried out in accordance with guideline prescribed for standard of Indian medicine.

 

2.3. Instrumental Analysis [7-10]:

Instrumental analysis of the manosialain has been carried out with FT-IR using Shimadzu –IR affinity 1S portal, XRD using advance X-ray diffractometer (Cu-Kα radiation; λ= 1.5405 Å) and SEM by S-4800 scanning electron microscope at the condition=Vacc=5kV, Mag=x5.00k WD=8.6mm

 

3. RESULTS:

3.1. Physicochemical Evaluation of Manosilai before and After Purification:

Result’s on organoleptic evaluation of MSBP and MSAP signifies the presence of characteristic orange coloured fine powder which reveals the quality and authenticity of the preparation. The results of the physicochemical evaluation reveals a loss on drying value of MSBP and MSAP was found to be 0.44 and 0.05%w/w. The results were tabulated in Table 01.

 

The total ash value of MSBP and MSAP was 3.79 and 0.12 %w/w. In which the acid insoluble ash value of MSBP (0.08 % w/w), MSAP (0.08% w/w). The results of water soluble extractive value of MSBP were 1.028% w/w and for MSAP it was 0.92%w/w. Similarly the alcohol soluble extractive value of MSBP and MSAP was found to be 0.27% and 2.3%w/w. pH plays a vital role in drug disintegration and absorption further the pH of MSBP and MSAP are 7.5 and 4 respectively. The results are tabulated in Table 01.

 

 

 

Table 1: Physicochemical Analysis of MSBP and MSAP

S. No	Parameters	Manosilai before Purification MSBP	Manosilai After Purification MSBP
1	Appearance	Orangecoloured fine powder	Yellowish Orangecoloured fine powder
2	pH	7.5	4
3	Loss on drying at 105oC	0.44 %w/w	0.05 %w/w
4	Total Ash	3.79 %w/w	0.12 %w/w
5	Acid Insoluble ash	0.08 % w/w	0.08 % w/w
6	Water Soluble Extractive (WSE)	1.02 % w/w	0.92%w/w
7	Alcohol Soluble Extractive (ASE)	0.27 % w/w	2.3%w/w

 

3.2. FT-IR analysis of Manosilai before and After Purification:

FT-IR spectrum of MSBP reveals the presence of IR absorption peak at 1127.75cm-1 may be due to the presence of =S. Wide predominant peak at 3431.75cm-1 due to free O-H vibration. Medium intensity peak at 1424.19 cm-1 and 876.29 due to presence of S=O functional group stretching. IR absorption peak at 1628.31 cm-1 due to NH2 scissoring. Absorption peak at 1127cm-1 due to presence of C-O stretching It is shown in spectrum figure 1.

 

Figure 1: FTIR Spectrum of the sample MSBP

 

Figure 2: FTIR Spectrum of the sample MSAP

FT-IR spectrum of MSAP reveals the presence of strong intense peak at 3432 cm-1 may be due to –CO-NH group. Medium peaks at 2924 cm-1 may be due to presence of hydrogen bond on –OH group. Sharp intense peak at 2860, 1138, 603 cm-1is due to the presence of free CHO group. Absorption Peak at 559 cm-1 due to presence of C-Br. Strong intense peak at 747 cm-1 due to presence of hydrogen at benzyl group and peak at 895cm-1 due to presence of alkenes. Peaks at 2285 cm-1 due to presence of amino group and peak at 1448 cm-1 due to N-N=O group it is shown in spectrum figure 2.

 

3.3. Results of SEM image Analysis of MSBP and MSAP:

SEM analysis reveals that MSBP possesses micro particles with varying shapes and the surface morphology depicts the nature of metals and minerals. Further it is observed that the average particle size of the sample ranges from 13.3 µm to 36.42 µm. As shown in figure 3.

 

SEM analysis of MSAP showing the presence of wide range of micro particles with varying shapes and surface morphology depicts the nature of metals and minerals. Further it is observed that the average particle size of the sample ranges from 16.06 µm to 30.70 µm. As shown in figure 4.

 

 

A.       Clustered View

B.  Categorized View

Figure 3: SEM analysis of the sample MSBP

 

 

A-Clustered View

B- Categorized View

Figure 4: SEM analysis of the sample MSAP

 

3.4. XRD analysis of Manosilai before and After Purification:

The X-ray diffraction pattern of the prepared sample MS before purification reveals the presence of major peak with 2- Theta value of 28.12 which exactly matches to the ICDD (International Centre for Diffraction Data) 71- 2434. ICDD 71- 2434 corresponds to the crystalline pattern of Arsenic Sulfide (AsS). The XRD pattern of the test sample AsS (Before) exactly matches with the reference material AsS, which justifies the presence of stable and purified AsS in the formulation, as shown in table 2 and figure 5.

 

Table 2: XRD peak analysis table of sample MSBP

Sample Name

Left Angle

Right Angle

Left Int.

Right Int.

Obs. Max

d (Obs. Max)

Max Int.

Net Height

FWHM

Chord Mid.

I. Breadth

Gravity C.

d (Gravity C.)

Raw Area

Net Area

2-Theta °

2-Theta °

Cps

Cps

2-Theta °

Angstrom

Cps

Cps

2-Theta °

2-Theta °

2-Theta °

2-Theta °

Angstrom

Cps x 2-Theta °

Cps x 2-Theta °

As2S2 before

28.020

28.220

993

993

28.125

3.17026

1371

378

0.134

28.123

0.129

28.123

3.17048

247.4

48.74

 

Figure 5: XRD Spectrum of the Sample MSBP

 

The X-ray diffraction pattern of the prepared sample MS after purification reveals the presence of major peak with 2- Theta value of 35.57 with the relative intensity of 29.9% corresponds to Arsenic and their corresponding intensities 34.4% matching with the material sulphur. As shown in table 3 and figure 6

.

Table 3: XRD peak analysis table of sample MSAP

Index	Name	Angle	D Value	Net Intensity	Gross Intensity	Rel. Intensity
1	Peak # 1	13.124	6.74054	20.7	35.1	24.0%
2	Peak # 2	15.419	5.74223	86.1	98.5	100.0%
3	Peak # 3	17.827	4.97156	43.1	56.7	50.0%
4	Peak # 4	18.273	4.85110	16.1	30.1	18.7%
5	Peak # 5	21.590	4.11266	18.7	30.8	21.8%
6	Peak # 6	22.695	3.91488	31.6	44.3	36.7%
7	Peak # 7	28.086	3.17458	33.0	46.9	38.4%
8	Peak # 8	29.206	3.05532	29.6	45.7	34.4%
9	Peak # 9	29.853	2.99052	67.8	85.0	78.8%
10	Peak # 10	31.141	2.86969	74.4	90.9	86.4%
11	Peak # 11	31.838	2.80850	20.2	35.5	23.5%
12	Peak # 12	32.975	2.71422	11.9	23.0	13.8%
13	Peak # 13	35.573	2.52170	25.7	37.7	29.9%
14	Peak # 14	37.907	2.37159	17.4	27.6	20.2%
15	Peak # 15	40.104	2.24658	12.5	24.4	14.5%
16	Peak # 16	49.648	1.83477	18.9	32.9	22.0%

 

Figure 6: XRD Spectrum of the Sample MSAP

 

4. DISCUSSION:

Since olden days siddhars the pioneer inventors of he siddha system of medicine have clearly detailed the 5 fundamental elemental composition of the human body which are The Earth (Mann), Water (Neer), Fire (Thee), Air (Vayu) and Ether (Akayam).Basic physiology of the biological system digestion, metabolism, circulation, conduction, transmission, secretion, exchange, production, regeneration mainly depends on Tridoshas which are Vatham, Pitham and Kabam, imbalance or change in the proportion of the three components ultimately results in fluctuations of the fundamental physiology which paves a way to several diseases in humans. Hence treatment in siddha system of medicine targets to correc the proporation of he disturbed doshas, whereby the pathology can be reversed.

 

Standardization techniques identity adulteratants in drugs which may be intentional or unintentional. Addition of spurious drugs to raise the volume leads to deviation in basic physiochemical properties of the drugs. Physicochemical analysis of Manosilai in the present research work reveals that there is a slight change in LOD value of the sample MS before and after purification. In which the total ash value remains the same. There is no significant change observed in the watersoluble extractive value of both the samples. But there is significant difference in alcohol soluble extractive value and pH values of the sample MSBP and MSAP.

 

In analytical method FT-IR techniques grabs attention because of its simplicity and reliability in results. Each active ingredient irrespective of the combination has a functional group on its core. These functional groups attains different posture under vibrational changes in the presence of infrared radiation. These vibrational changes are captured in the form of peaks some are weak and some are strong. Based on the region of vibrations each functional group present in the formulation can be located. Identifying the functional group is be highly helpful in predicting the mechanism of action of such drugs. Spectral analysis results are highly essential for development of basic monograph. Documentation of spectrum of each formulation is mandatory for the future researcher to work on the change of functional group, seasonal variation, batch variations, adulteration’s etc. In the present study FT-IR spectral data’s signifies the presence of increased functional group in MSAP when compared to MSBP. In which the S=O and NH functional groups presen in MSBP were absent in MSAP. Whereas there is presence of additional functional groups id N-N=O, C-Br and other benzyl groups in MSAP which is absent in MSBP.

 

SEM is an advanced microscopic techniques applied in validating the surface morphology of the individual particles present in medicinal formulations. Surface characterization and particle size identification are the most important factors which determine the permeability of the drug in crossing biologically significant membranes. A significant problem in interpreting the SEM images of siddha drugs is aggregation. To avoid such an issue proper dilution of sample in solvent of different solubility provides high resolution image of the individual particles. Information ON cracking, shrinkages, fractures, can be elucidated from particle size and morphology with this the nature of formulation and the stability of each raw ingredients used can be ascertained. [11-13]. Hence SEM made easy prediction on quality and stability of the formulation particle size, morphology and distribution volume advise the formulators to correct the formulation and other processing errors involved if any. There is no significant change observed in surface morphology and average particle size of the both MSBP and MSAP samples.

 

In regard to Micro and nano particle characterization SEM analysis assists the researcher due to versatile implicaion of the technique in proper detection and distribution of nano size particle and their surface topography. Presence of micro particle aids in greater drug absorption as it is proven that decrease in size of the particle increases the surface area. Greater the surface area higher the rate of absorption. SEM analysis works behind the principle of electron bombardment as the sample under investigation is exposed to set of primary electrons. According to the surface and morphological nature of the sample the bombarded secondary electron sensed by the detectors is be captured in form of high resolution images. This images is useful in surface imaging analysis [14].

 

In recent times XRD analytical techniques have been widely utilized for evaluating the quality and stability of formulation in the process of drug development. [15]. Presence of minerals and inorganic components are easily identified by XRD analysis. Based on the X-RD diffraction pattern of the sample MS before and after purification shows no significant change in the elemental composition since in both the preparations arsenic and Sulphur as the core components. Hence it can be concluded that the purification process doesn’t alter the nature of the drug MS. Presences of inorganic minerals is quite an often in siddha formulation as the raw materials may have traces of these minerals and other inorganic components. As per siddha literatures certain minerals and inorganic components have potential pharmacological action as it mediates some of the key enzymatic reactions in our biological system.

 

5. CONCLUSION:

Standardization and analytical validation of drugs are important to ascertain the quality and stability of the preparations. In the present investigation manosilai is purified with butter milk as per the standard in Siddha Medicinal literature and is subjected to SEM, XRD and FT-IR analysis to confirm its nature and purity. Physiochemical analysis of the formulations MSBP and MSAP confirms the quality of the preparation and FT-IR provides the information of functional moieties present in manosilai before and after purificaion. Further the XRD data justifies the nature of chemical composition and the presence of Arsenic and sulphur in both these preprations. SEM analysis assured the presence of micro particles and its surface morphology in both MSBP and MSAP. Data of these studies may render some useful information with respect to the elemental composition and nature of the drug manosilai before and after purification with the specified purification process which may be essential to correlate the actual biological activity of the drug upon clinical usage.

 

6. ACKNOWLEDGEMENT:

I wish to acknowledge my sincere thanks to Sastra University, Thanjavur 613401, Tamil Nadu, India and The Noble research solutions, Chennai, Tamil Nadu, India for their for analytical and technical support in this research work.

 

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Received on 27.04.2019                 Accepted on 06.05.2019

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Asian J. Pharm. Ana. 2019; 9(2):99-104.