Comparison and standardization of traditional siddha medicine pavala parpam

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Comparison and standardization of traditional siddha medicine pavala parpam

https://doi.org/10.21203/rs.3.rs-4316027/v1

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Bronchial asthma is the condition in which persons airways become inflamed, swell and narrow and produce extra mucus and it makes breathing difficult. There are number of allopathic and ayurvedic drugs available to treat bronchial asthma, but those drugs cause severe side effects to the patients. Hence majority of the people prefer Siddha drug to treat bronchial asthma due to less side effects. Siddha medicine is an ancient system of medicine that aims at spiritual and physical perfection. According to Siddha pharmaceutical principles, when pavalam (red coral) is combined with herbal juices and purified it obtains high range of medicinal value. Pavala parpam is a traditional herbo-mineral formulation. In present study two samples of pavala parpam, one containing Ziziphus mauritiana herbal juice and other containing Heliotropium indicum herbal juice were purchased. And the standardization of this drug samples was done based on organoleptic, physico-chemical, phytochemical analysis and morphological analysis. When comparing both drugs, the result showed only slight changes in organoleptic and physicochemical analysis and major changes showed in phytochemical analysis. The FE-SEM and XRD results showed that the drug have better absorption capacity due to presence of micro particles in both the samples.

Bronchial asthma

Heliotropium indicum

pavala parpam and ziziphus mauritiana

The knowledge of traditional medicine is based on the experiences and beliefs of different cultures (Fokunang et al. 2011). Siddha medicine is an ancient system of medicine of South India that aims at both spiritual and physical perfection of a person. The three humors like vadham, pitham and kapam must be at equilibrium with nature and when disturbed causes health problems. The sources of Siddha medicines are mooligai (herbs), thathu (minerals) and jeeva (animal products). Siddha medicine is cost effective and more reliable to poor and marginalized people than the conventional medicine. Standardization of a Siddha formulation is of significance to validate its potency, efficacy and enhance value in global market. In ancient times the Siddha formulation pavala parpam was found to be used for the treatment of bronchial asthma, stress ulcers, bleeding disorders, chronic respiratory diseases and life style diseases like cancer and diabetes (Rathinamala Rathinam et al. 2014). There are also reports that pavala parpam has anti-osteoporotic activity, anti-atherosclerotic, haemostatic activity and hepato-protective potential (Thanuja et al. 2021). The Ziziphus mauritiana herbal juice used in formulating siddha drug pavala parpam (PP1) due to presence of many medicinal properties like antibacterial, antioxidant and anti-inflammatory (Emad Mohamed Abdallah et al. 2016). The Heliotropium indicum herbal juice used in making siddha formulation pavala parpam (PP2) contains many valuable phyto components like alkaloids, flavonoids and saponins etc., it helps to enhance the pharmacological activity of pavala parpam (Anupam Roy 2015) (Jayachitra et al. 2016). A search of literature revealed that there was no report on standardization of the drug pavala parpam. The Siddha medicine pavala parpam has been reported for exhibiting “antimicrobial activity” & “In vitro evaluation of cytotoxicity and cell immunity as an ingredient in herbal capsule” (Thanigavelan et al. 2011; Radhakrishnan et al. 2021).

Two different brands of Siddha formulation pavala parpam were purchased from Siddha medicine shops in coimbatore district and the samples designated as PP1 and PP2. The samples were assessed for the organoleptic characteristics. Melting point of the samples were determined using electrical melting point apparatus. Temperature stability of the sample was analysed by thermo gravimetric analysis using TGA EXSTAR/6300 instrument. The elemental composition of the sample was analysed by EDX analyser. Crystalline analysis of the sample was done using X'Pert Pro Analytical XRD spectrometer. Surface topography, morphology (particle size and shape) of the sample was analysed using TESCAN -MIRA3 XMU FE-SEM analyser. The preliminary phytochemical analysis was carried out to find the presence of phyto constituents in the formulation. HPTLC was used for the qualitative analysis of alkaloid present in the sample. IR spectroscopic analysis of the samples was done using Shimatzu IR Spectrometer (Miracle 10).

Standardization of Siddha medicine is significant in establishing the chemical profile, consistent, biological action and quality assurance of a drug. The results of the standardization of two samples of pavala parpam are presented in the following sections,

Organoleptic characteristics

Organoleptic characteristics of the samples of pavala parpam are presented in Table 1

Table 1

Organoleptic characteristics of Pavala Parpam
Parameter	Observation
Parameter	PP1	PP2
Colour	Grey
Odour	Ash smell
Taste	Sour
State of drug	Powder
Consistency	Soft

Both samples of pavala parpam exhibited the same organoleptic characteristics.

Physico-Chemical Analysis

The solubility, pH value, behaviour of the samples with various reagents and temperature stability of the samples were analysed. Solubility is an important parameter for any drug, because it affects rate of dissolution and rate of absorption in the body. Hence solubility of the drugs in organic solvents and water. The drug was found to be insoluble in water and partially soluble in hot water. Melting point of both the samples was found to be above 196^oC.

The pH value of sample was found to be neutral in the range of 7.2–7.5. This pH confirms the expected requirements of drugs. The pH of any drug formulation plays an important role in biological system in order to support the absorption and distribution through systematic circulation.

Behaviour of Pavala Parpam with Reagents

The behaviour of the two samples of pavala parpam (PP1 & PP2) with various reagents was assessed. Both samples were completely soluble in concentrated acids. While PP1 was insoluble and slowly settles down on treating with 5% alkali, glacial acetic acid and iodine solution. PP2 was partly soluble on treating with 5% alkali, glacial acetic acid and iodine solution.

Phytochemical Analysis

For phytochemical screening, pavala parpam samples were extracted with ethanol. Phytochemical screening was done to identify the phytochemical constituents present in pavala parpam. The preliminary phytochemical analysis showed positive result for alkaloid in the sample PP2 and no phytochemicals was found to be present in PP1. Other phytochemicals were not present in both the samples.

HPTLC Fingerprinting

HPTLC analysis of PP2 indicated the presence of two unknown alkaloids. Since sample PP2 tested positive to alkaloids, an HPTLC analysis of this sample done and comparison made with standard alkaloids. The HPTLC chromatogram obtained under UV light (254nm and 366 nm) and visible light are given in Fig. 1. Figure 2 showed the HPTLC Chromatograms of PP2. Table 2 given the retardation factor of spots seen for sample PP2 in HPTLC.

Table 2

Observed R_f Values of PP2 in HPTLC
PP2
R_f value	Assigned Compound
0.56	Unknown
0.61	Standard (Colchicine)
0.71	Unknown

IR Spectroscopic Analysis

IR spectroscopic analysis of pavala parpam was done to found out the functional groups present in the sample.

FT –IR spectra of pavala parpam is showed in Figs. 3 and 4.

FT-IR Results of PP1

In the FT-IR spectra analysis of the pavala parpam sample (PP2) exhibits the peak value shows in Table 3 at the wave number of 1419.61, 1126.43, 709.80, 671.23 and 432.05 having C – O stretch, SO₄²⁻ symmetric stretch, Ca-O stretch, SO₄²⁻ out of plane bending and Mg-O stretch respectively. This indicates the presence of some metal oxides like CaO, CaCO₃, MgO and MgSO₄ (Rajamaheswari et al. 2016).

Table 3

FT IR Results of Pavala Parpam Samples PP1 and PP2
PP1			PP2
Wave number (cm^− 1)	Vibration mode	Functional group	Wave number (cm^− 1)	Vibration mode	Functional group
432.05	Mg – O	MgO	424.34	Mg – O stretching	MgO
671.23	Out of plane bending of SO₄²⁻	CaSO₄	709.80	Ca-O	CaO
709.80	Ca – O stretching	CaO	871.82	C – O stretching	CaCO₃
871.82	C – O stretching	CaCO₃	1388.75	C-H bending	Alkane
1419.61	C – O stretching	CaCO₃	1643.35	C = C Stretching	Alkene
1126.43	Symmetric stretching of SO₄²⁻	MgSO₄	2360.87	C = O Stretching (strong)	Carbon dioxide
			2978.09	C-H Stretching	Alkanes
			3734.19	Free O-H Stretch	Alcohols

FT-IR Results of PP2

In the FT-IR spectra analysis of the pavala parpam sample (PP2) exhibits the peak value shows in Table 3 at the wave number of 3734.19, 2978.09, 2360.87, 1643.35, 1388.75, 871.82, 709.80, 424.34 having O-H Stretch, C-H Stretch, C = O Stretch, C = C Stretch, O-H in plane bending, =C-H bending, C – O stretch, Ca-O stretch and Mg-O stretch respectively (Raghavendra et al. 2017; Geetha et al. 2017; Rajamaheswari et al. 2016; Sahulhameed et al. 2015). This indicates the presence of some organic functional groups such as alkanes, esters and some metal oxides like CaO, CaCO₃ and MgO.

3.5 Elemental and Crystalline Analysis (EDAX)

Elemental analysis was done to determine the quantity and type of elements present in the herbal drug. The results of the elemental analysis of both samples indicated the presence of calcium and magnesium in their oxide form and the absence of toxic elements in pavala parpam. Elemental composition of pavala parpam samples PP1 and PP2 showed in Table 4. Figures 5 and 6 showed the EDAX spectrums of PP1 and PP2, respectively.

Table 4

Elemental Composition of Pavala Parpam Samples
PP1		PP2
Element	Weight %	Element	Weight %
O K	76.86	O K	61.06
Mg K	1.00	Mg K	3.75
Si K	0.33	Ca K	35.19
S K	8.76
Ca K	13.04

Magnesium is vital for healthy muscles and bones. It helps in easing muscle cramps and prevents osteoporosis. Magnesium is also essential for calcium absorption. Calcium is the most abundant mineral in the bone formation and other functions like muscle contraction. It also necessary to maintain the healthy communication between brain and other parts of the body. Calcium is used to cure bleeding disorders because Ca²⁺ much needed for blood clotting. Hence the presence of magnesium and calcium in both samples increases the medicinal value of pavala parpam.

Surface Morphological Analysis (SEM Analysis)

FE-SEM images of pavala parpam samples PP1 & PP2 are shown in Figs. 7 and 8. Particle size, surface area and shape will affect the homogeneity, efficiency and the stability of the drug. The particle size, shape and surface can be determined through SEM analysis. The particle size of sample of both the samples ranged from 0.5–5 µm and hence samples are micro sized.

The presence of nano and micro particles in the pavala parpam samples may improve the absorption and bio- availability of the herbal drug. As particle size is micro size, small amount of drug may be sufficient to cure diseases. The agglomeration of the particle in pavala parpam is observed. This may be due to the calcination process involved in preparation.

TGA/DTA Analysis

The thermal stability of the herbal drug was analyzed by thermo gravimetric analysis. In a controlled environment, the weight change of the drug was noted with change in temperature. Thermo gravimetric method given an important information about the herbal drug during standardization. The thermo characterization for plant based drug is to determine the amount of moisture content, volatile components and non-volatile compounds present in it. Figures 9 and 10 showed the TGA curves of PP1 and PP2 respectively. Decomposition temperatures and mass losses of samples PP1 and PP2 are given in Table 5.

Table 5

Decomposition Temperatures and Mass Losses of Samples PP1 and PP2
De- composition stages	PP1		PP2
De- composition stages	Temperature (⁰C)	Mass loss (%)	Temperature (⁰C)	Mass loss (%)
I	31⁰C – 130⁰C	1.6	31⁰C – 139⁰C	0.8
II	130⁰C − 682⁰C	8.4	139⁰C − 342⁰C	1.1
III	682⁰C − 944⁰C	14.1	342⁰C − 696⁰C	11.4
IV	944⁰C − 972⁰C	24.1	696⁰C − 867⁰C	29.6

Thermo Gravimetric Analysis (TGA) of PP1

The minor decomposition stage occurred in the range of 31⁰C − 130 ⁰C corresponding to the

mass loss nearly 1%, which was due to the elimination of water. The first major decomposition was noted in the range of 130⁰C – 682⁰C with the mass loss of 8.4% which corresponds to the conversion of metal into metal oxides. Second decomposition stage was found in the range of 696⁰C – 867⁰C with the mass loss of 24.1% correlates to the non- degradable residues like mineral residues. After 972 ⁰C the parpam disintegrated completely.

Thermo Gravimetric Analysis (TGA) of PP2

The curves showed some minor changes before 400⁰C and two major decomposition changes after 400⁰C. The minor decomposition stage occurred in the range of 31⁰C − 130 ⁰C which corresponds to the mass loss of 1%, which was due to the elimination of water. The slight decomposition changes was observed at 130⁰C − 342⁰C corresponding to the mass loss of 1% (degradation of micro and macro compounds). The first major decomposition was noted in the range of 342⁰C – 696⁰C with the mass loss of 11.4% which corresponds to the conversion of metal into metal oxides. Second decomposition stage was found in the range of 696⁰C – 867⁰C with the mass loss of 29.6% correlates to the non- degradable residues like mineral residues. After 900⁰C the parpam disintegrated completely.

Differential Thermal Analysis (DTA)

The difference in the peak heights between the samples at the selected temperature were noted from the DTA curves of pavala parpam.

DTA curve of both PP1 and PP2 showed three endothermic and two exothermic peaks. The endothermic peak may be attributed to the elimination of physically absorbed humidity. The exothermic peak indicated the breakdown of metallic mixtures in the pavala parpam. DTA results of Samples PP1 and PP2 are shown in Table 6. Figures 11 and 12 indicated the DTA curves of PP1 and PP2, respectively. DTG curves of PP1 and PP2 shown in Figs. 13 and 14, respectively.

Table 6

DTA Results of Samples PP1 and PP2
Decomposition stages	PP1		PP2
Decomposition stages	Peak Height (Mw/mg)	Peak Temperature (⁰C)	Peak Height (Mw/mg)	Peak Temperature (⁰C)
I	-5.9601	168.02	-5.5669	126.21
II	7.4135	342.28	-4.4236	330.87
III	-0.2882	375.00	-0.2842	368.24
IV	-36.9366	738.02	-42.3592	762.36
V	-31.0602	763.51	-38.7812	795.30

XRD Analysis

Phase identification and crystalline size of the sample was analysed using XRD powder diffraction method. Due to random orientation of the powdered sample, when it is scanned through the range of 2θ angles all possible diffraction directions of lattice will be obtained. Identification of minerals is done by converting diffraction peaks to d- spacing because each mineral has unique set of d- spacing. Table 7 contains d- Spacing and 2 – θ values of identified compositions in PP1 and PP2 determined from XRD analysis. Calculated crystalline size of samples PP1 and PP2 shown in Table 8. XRD Patterns of PP1 and PP2 are given in Fig. 15 and Fig. 16, respectively.

Table 7

d- Spacing and 2 – θ Values of Identified Compositions in PP1 and PP2
PP1				PP2
Pos. [°2Th.]	d-spacing [Å]	Height [cts]	Identified composition	Pos. [°2Th.]	d-spacing [Å]	Height [cts]	Identified composition
20.7311	4.28470	595.80	CaSO₄	29.4269	3.03536	3562.89	CaO
25.4757	3.49647	1242.74	SiO₂	39.4753	2.28281	574.76	CaO
29.8148	2.99675	875.20	CaCO₃	43.1993	2.09426	555.33	CaO
39.9403	2.25730	136.24	CaO	48.5044	1.87688	559.76	MgO
43.5325	2.07900	110.21	CaO	57.4254	1.60472	1.60472	MgO
49.2296	1.85092	133.58	MgO	57.4254	1.60472	1.60472	MgO

Table 8

Calculation of Crystalline Size of Samples PP1 and PP2
PP1					PP2
K	λ (Å)	Peak position 2θ (°)	FWHM (°)	D (nm)	K	λ (Å)	Peak position 2θ (°)	FWHM (°)	D (nm)
0.9	1.5406	20.7311	0.0836	100.9	0.9	1.5406	29.4269	0.1840	44.639
0.9	1.5406	25.4757	0.1338	63.578	0.9	1.5406	39.4753	0.2007	42.054
0.9	1.5406	29.8148	0.2342	36.663	0.9	1.5406	43.1993	0.1004	85.102
0.9	1.5406	39.9403	0.2676	32.99	0.9	1.5406	48.5044	0.1338	65.122
0.9	1.5406	43.5325	0.6691	13.353	0.9	1.5406	57.4254	0.1338	67.699
0.9	1.5406	49.2296	0.2676	34.106	0.9	1.5406	57.4254	0.1338	67.699

A sharp line in the XRD patterns of the sample indicated the crystalline nature of pavala parpam. All the preferred orientations in the Figs. 15 and 16 confirmed the pure cubic phase of CaO. The mean crystallite size was determined using Debye Scherrer’s equation,

D = 0.9λ / β cosθ

Where, D = Crystalline size (nm), λ = X-ray wavelength (Å), β = FWHM of diffraction peak, θ = Bragg angle. The mean crystalline size of PP1 is 46.9316 nm and PP2 is 60.9232 nm.

The present work undertaken to standardize two samples of pavala parpam which contain red coral fused with juices of plant extracts Ziziphus mauritiana and Heliotropium indicum respectively. Samples were characterised based on organoleptic, physico-chemical, phytochemical analysis and morphological study and the results revealed that both the samples have acceptable organoleptic, physico-chemical, phytochemical analysis and morphological characteristics for use as siddha medicines. This study clearly indicates that both samples of pavala parpam have same organoleptic characteristics, however physico-chemical characteristics and thermo gravimetric parameters slightly differed. Sample PP2 tested positive to alkaloid however sample PP1 tested negative, probably because of the difference in the plant juices used to prepare the parpam samples. HPTLC fingerprinting also confirmed the presence of an unknown alkaloid in PP2. FT-IR results clearly indicates the presence of metal oxides of Magnesium and Calcium in the both the samples. Elemental analysis also revealed the predominant presence of calcium in both the samples in addition to magnesium also thus validating the significance of using pavala parpam for treating osteoporosis. Morphological studies and XRD analysis indicated that both the samples are micro sized and crystalline. The micro sized nature of the samples may contribute to improved bio- availability and drug absorption. The above study focused on standardization of two samples of pavala parpam to aid in authentication of commercial samples.

ACKNOWLEDGEMENT

The authors thank staff members of department of chemistry, Avinashilingam Institute for their continuous support and providing facilities throughout the research work.

Ethics approval

No human and animal studies were used in this study, so we didn’t get any ethical clearance.

Competing interests

The authors have no competing interests to declare that are relevant to the content of this article.

Author’s contributions

All authors contributed to the study and designing of this work. Material preparation, data collection and analysis were performed by [Keerthana.L] and [Pavithra.P]. The first draft of the manuscript was written by [Keerthana.L] and all authors commend on previous version of the manuscript. All authors read and approved the final manuscript.

Funding .

The authors did not receive support from any organization for the submitted work.

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Comparison and standardization of traditional siddha medicine pavala parpam

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Abstract

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1. INTRODUCTION

2. MATERIALS AND METHODS

3. RESULTS AND DISCUSSION

3.5 Elemental and Crystalline Analysis (EDAX)

4. CONCLUSION

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References

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