INTRODUCTION:

Standardization is the need of the hour in Ayurvedic system of medicine. The traditional systems of medicine are really effective but the problem with them is they lack in quality assurance. This enables us to recognise the quality of the formulation. The Central Council of Research in Ayurveda and Siddha has prescribed the preliminary guidelines for testing the quality of these formulations. It is essential to derive a protocol or develop methods for evaluation of herbal formulation to maintain uniformity between batches during production.¹

Panchachurnam, an Ayurvedic polyherbomineral formulation consists of Cassia angustifolia Vahl. leaf, Terminalia chebula Retz. fruit, Zingiber officinale Rosc. rhizome, Foeniculum vulgare Mill. fruit and Saindhava lavana, traditionally used for managing constipation, haemorrhoids, pain in abdomen, flatulence, assimilatory disorder and rheumatic conditions, in managing all diseases of Kapha Dosha origin, improves digestion and ensures timely evacuation of faeces, improves functioning of liver, in hyperacidity, heart burn and acidic belching. It provides an antidote for pungent food and promotes the elimination of toxins, supports intestinal immunity and balances tridosha. ²

For present study we have prepared in-house Panchachurnam (IH) and selected three marketed Panchachurnam (SAN, VAN and CAP), we have pharmacognostically characterized the individual constituents of Panchachurnam with respect to anatomical studies, physical constant values (i.e. Extractive value, Total Ash value, Water soluble/ Water insoluble ash value, Acid soluble Ash value, Moisture content) and quantitative microscopy (i.e. Stomatal index, Palisade ratio, Vein islet number and Vein termination number) of Cassia angustifolia Vahl. leaf was performed for the authentication of the senna leaf. This paper reports on the standardisation of Panchachurnam based on organoleptic, microscopic, physical and physico-chemical characteristics to confirm test for identity, potency, purity, safety and efficacy. ³

MATERIALS AND METHODS:

Plant Materials:

The plant materials of Panchachurnam were procured from the local markets of Pithapuram, Samalkot and Peddapuram, East Godavari District, Andhra Pradesh, India. The specimens of the samples were authenticated by Mr. Raghava Rao, Department of Botany, M. R. College, Peddapuram, East Godavari District, Andhra Pradesh, India. Voucher specimens (SISP/LAB/PG-6/5) of the same have been deposited in the museum of Dept. of Pharmacognosy, Sri Sai Aditya Institute of Pharmaceutical Sciences and Research for future reference.

Preparation of Panchsakar Churna^4-6:

The churna was prepared as per the procedure given in Ayurvedic Formulary of India. All the ingredients were crushed to powder using grinder and were powdered separately and sieve no. #80. In-house Panchachurnam was prepared from these powders by mixing them in one part for each ingredient and passed through sieve no. #60. A physical mixture was made by mixing together in equal proportions to get uniformly blended churna.

Marketed Samples:

Marketed Panchachurnam supplied from three different companies were also procured and named as SAN, VAN and CAP. The in-house preparation (IH) along with the three marketed brands is standardized based on their organoleptic, microscopical, physical and physico-chemical characteristics.

Standardization Parameters^7,8:

The various standardization parameters studied were organoleptic properties, physico‐chemical investigations, determination of pH analysis, preliminary phytochemical analysis, determination of moisture content, determination of crude fibre, determination of total tannin content, determination of resin content and determination of physical characteristics of powder formulation.

Organoleptic Evaluation^9-12:

The colour, odour, taste, form and texture of the Panchachurnam and its marketed formulations were evaluated manually as per Pharmacopoeial Analytical Standards and have been tabulated in Table 1.

Determination of Foreign organic matter¹³:

250g or the quantity specified in the individual monograph, of the original sample was weighed accurately and spread out in a thin layer. The samples were inspected with the unaided eye or with the use of a magnifying lens (6X or 10X) and the foreign organic matter were separated manually as completely as possible and weighed. The percentage of foreign organic matter was weighed and determined with reference to the weight of the drug taken and has been tabulated in Table 2.

Table 2 Foreign organic matter of various ingredients of Panchachurnam and formulations

S. No.	Sample	Foreign organic matter (%w/w)
1.	C. angustifolia	0.383
2.	T. chebula	0.567
3.	Z. officinale	0.784
4.	F. vulgare	0.332
5.	IH	Nil
6.	SAN	Nil
7.	VAN	Nil
8.	CAP	Nil

Microscopic Evaluation:

Anatomical study of each ingredient of Panchachurnam^14-16:

For Anatomical study invariably slides were prepared. A transverse section of required part of ingredient was taken on a glass slide to which are added a few drops of saffranin for 1-2 minute then washed with water. Then it was mounted with glycerin. Care however is to be taken to avoid air bubbles and to see that there is sufficient glycerine under the cover slip. Excess of glycerine outside the cover slip is to be withdrawn using a blotting paper. Then the anatomy of the transverse section were observed and identified under Leica DME microscope (10x & 40x). Anatomical study for each ingredients of Panchachurnam i.e., transverse section of Cassia angustifolia leaf, Foeniculum vulgare fruit, Zingiber officinale rhizome and Terminalia chebula fruit were done and has been displayed in Figures 1,2,3,4.

Quantitative microscopy of Cassia angustifolia Vahl. Leaf^18,19:

Determination of Stomatal index of Cassia angustifolia Vahl. Leaf:

The leaf was teared in such a way that both the upper and lower epidermis was separated. A drop of chloral hydrate was added to the peeled or teared leaf part and then heated for few seconds. A drop of methanol was added after five minutes washed with water. A drop of safranin was added and washed immediately with water twice or thrice. The stained portion was kept in a slide and then mounted with glycerin. Then by using stage micrometer and camera lucida, one square mm was drawn in a black sheet. Then by the help of microscope (10x & 40x) and camera lucida the number of stomata and epidermal cells were counted within the square. Those cells were not counted more than half portions of which were outside the square. Ten observations were done to measure Stomatal index of both upper and lower epidermis.

Determination of Palisade ratio of Cassia angustifolia Vahl. Leaf:

Small pieces of leaves from the base, middle and apex position of lamina were taken. Leaf pieces were boiled in conc. Chloral hydrate solution by placing in test tubes. The test tubes were kept in water bath till the leaf pieces become chlorophyll free. Then by using camera lucida, four adjacent cells of upper epidermis were traced. Then focused on the palisade layer and traced off the palisade cells beneath the four epidermal cells which were already traced. The palisade cells which were outside more than half portion were not counted. 24 observations were.

Determination of Vein islet number and vein termination number of Cassia angustifolia Vahl. Leaf:

A few cut portions of the leaf from the central region of lamina of 4mm were boiled in conc. Chloral hydrate solution by placing in test tubes. The test tubes were kept in water bath till the leaf pieces become transparent. Then each portion was kept on a slide with lower portion placing upward so that vein were prominent on the lower surface and a small drop of glycerin was added. 5x eyepiece and low power objective 10x were used. Stage micrometer was focused and camera lucida was fixed. A drawing sheet was placed on the side of the microscope where camera lucida was fixed. Then using stage micrometer 1 mm sq. was drawn. Image of the leaf was made to super impose the square on the drawing sheet. Vein islet were traced and counted. 10 observations were done to measure the vein islet number and vein termination number.

Physico-Chemical Investigations²⁰:

Physico‐chemical investigations of formulations were carried out for the determination of ash values, extractive values, Moisture, crude fibre content and pH.

Determination of Ash values:

Determination of total Ash values²¹:

3g of air dried powder of ingredients of Panchachurnam and its marketed formulations were taken in a tarred silica dish and were incinerated at a temperature not exceeding 450⁰C until free from carbon, and it was then cooled and weighed. The percentage of ash was calculated with reference to air dried powder of ingredients of Panchachurnam and its marketed formulations and has been tabulated in Table 3.

Determination of water soluble and water insoluble ash values²²:

The total ash obtained from above section was boiled with 25ml water for five minutes and then filter through an ash less filter paper (whatmann#1). The filter paper was ignited in the silica crucible. Then cooled and the water insoluble ash was weighed. The water soluble ash was calculated by subtracting the water insoluble ash from the total ash. Then the percentage of water soluble ash was calculated with reference to the air dried drug and has been tabulated in Table 3.

Determination Acid insoluble ash values²³:

About 3g of the powdered drug was accurately weighed in a tarred silica crucible. Incinerate the powdered drug by gradually increasing the heat until free from carbon and cooled before weighing. The ignition was repeated until constant weight was observed. The total ash obtained was boiled for five minutes with 25ml of 6N HCl and then filtered through ash less filter paper (whatmann#1). The filter paper was ignited in the silica crucible, cooled and acid soluble ash was calculated by weighing and has been tabulated in Table 3.

Table 3 Ash values of various ingredients of Panchachurnam and formulations

S. No.	Sample	Total Ash Value (%w/w)	Water Soluble Ash Value (%w/w)	Water Insoluble Ash Value (%w/w)	Acid Insoluble Ash Value (%w/w)
1.	C. angustifolia	9.295	1.970	7.410	1.080
2.	T. chebula	3.125	2.776	0.793	2.423
3.	Z. officinale	5.780	1.880	3.780	1.553
4.	F. vulgare	6.005	2.413	3.090	1.477
5.	IH	20.85	16.72	4.343	1.427
6.	SAN	23.77	18.70	4.990	1.710
7.	VAN	22.93	18.04	4.767	1.450
8.	CAP	24.09	19.14	5.003	1.790

Determination of Extractive Values:

Determination of Water soluble Extractives:

5g of air dried powder of ingredients of Panchachurnam and its marketed formulations were taken and macerated with 100ml of chloroform water (0.25ml of chloroform with volume made up to 100ml with distilled water). In a closed flask for 24 hours, shaking frequently for the first 6 hrs and then was allowed to stand for 18 hrs. It was then filtered taking precautions against loss of chloroform water. 25ml of the filtrate was evaporated to dryness in a tarred flat bottomed shallow dish and was then dried at 105⁰C and weighed. The percentage of the water soluble extractives was calculated with reference to air dried powder of ingredients of Panchachurnam and its marketed formulations and has been tabulated in Table 4.

Determination of Alcohol soluble Extractives²⁴:

5g of air dried powder of ingredients of Panchachurnam and its marketed formulations were taken and macerated with 100ml of methanol of the specified strength (95%) in a closed flask for 24 hours, shaking frequently for the first 6 hrs and then was allowed to stand for 18 hrs. It was then filtered taking precautions against loss of methanol. 25ml of the filtrate was evaporated to dryness in a tarred flat bottomed shallow dish and was then dried at 105⁰C and weighed. The percentage of the methanol soluble extractives was calculated with reference to air dried powder of ingredients of Panchachurnam and its marketed formulations and has been tabulated in Table 4.

Table 4 Extractive values of various ingredients of Panchachurnam and formulations

S. No.	Sample	Alcohol Soluble Extractive Value (%w/w)	Water Soluble Extractive Value (%w/w)
1.	C. angustifolia	17.6	30.0
2.	T. chebula	62.4	62.8
3.	Z. officinale	7.60	14.8
4.	F. vulgare	8.80	6.80
5.	IH	34.8	40.0
6.	SAN	30.0	35.6
7.	VAN	31.2	38.4
8.	CAP	27.2	34.8

Determination of Moisture Content²⁵:

Moisture content was determined by loss on drying (LOD) method. 5gm of the weighted quantity of the drug was taken and kept in oven at 105⁰C till a constant weight was obtained. Amount of moisture present in the sample was calculated as reference to the air dried drug and has been tabulated in Table 5.

Table 5 Moisture content of various ingredients of Panchachurnam and formulations

S. No.	Sample	Moisture content (%w/w)
1.	C. angustifolia	3.8
2.	T. chebula	2.6
3.	Z. officinale	4.0
4.	F. vulgare	2.8
5.	IH	2.6
6.	SAN	2.8
7.	VAN	2.6
8.	CAP	3.2

Determination of physical characteristics of powder formulation^26,27,28:

Physical characteristics like bulk density, tap density, angle of repose, Hausner ratio and Carr's index were determined for different formulations and has been tabulated in Table 6. The term bulk density refers to method used to indicate a packing of particles or granules. The equation for determining bulk density (BD) is BD =M/V_b where M is the mass of particles and V_b is the total volume of packing. The volume of packing can be determined in an apparatus consisting of graduated cylinder mounted on mechanical tapping device (Jolting Volumeter) that has a specially cut rotating can. Hundred gm of weighed formulation powder was taken and carefully added to cylinder with the aid of a funnel. The initial volume was noted and sample was then tapped until no further reduction in volume was noted. The initial volume gave the bulk density value and after tapping the volume reduced, giving the value of tapped density. Angle of repose has been used as an indirect method quantifying powder flowability, because of its relationship with interparticle cohesion. The fixed funnel and the free standing cone method employs a method that is secured with its tip at a given height (H), above the glass paper that is placed on a flat horizontal surface. Powder or granules were carefully poured through the funnel until the apex of the conical pile just touched the tip of funnel. Thus, with R being the radius of the conical pile. a = tan-1 H/R, where a is the angle of repose. Hausner ratio is related to interparticle friction and as such can be used to predict the powder flow properties. The equation for measuring the Hausner ratio is D_f /D_o Where, D_f = Tapped density and D_o = Bulk density. Carr's index is another indirect method of measuring the powder flow from bulk density. The equation for measuring Carr's index is 1- D_o/ D_f×100 Where D_f = tapped density, D_o = Bulk density.

Table 8 Phytochemical screening of different Panchachurnam formulations

S. No.	Phytoconstituents	IH	SAN	VAN	CAP
1.	Alkaloids	-ve	-ve	-ve	-ve
2.	Glycosides	+ve	+ve	+ve	+ve
3.	Anthraquinone Glycosides	+ve	+ve	+ve	+ve
4.	Carbohydrates	+ve	+ve	+ve	+ve
5.	Steroids	-ve	-ve	-ve	-ve
6.	Terpenoids	+ve	+ve	+ve	+ve
7.	Tannins	+ve	+ve	+ve	+ve
8.	Flavonoids	+ve	+ve	+ve	+ve
9.	Volatile Oils	+ve	+ve	+ve	+ve
10.	Proteins	+ve	+ve	+ve	+ve
11.	Fixed Oils	+ve	+ve	+ve	+ve
12.	Resins	+ve	+ve	+ve	+ve

Table 9 Screening of different Panchachurnam formulations for inorganic constituents

S. No.	Inorganic constituents	IH	SAN	VAN	CAP
1.	Sodium	+ve	+ve	+ve	+ve
2.	Chloride	+ve	+ve	+ve	+ve

Estimation of phytoconstituents:

Determination of Crude Fibre Content³¹:

2g of the churna wad added with 50ml of 10% nitric acid. This was boiled and filtered. The retains was washed with hot water and added with 50ml of 2.5% v/v sodium hydroxide solution. This was again filtered, washed with hot water and the residue was transferred into a crucible. The weight of the residue was taken for determining the crude fibre present in the churna and has been tabulated in Table 10.

Quantitative Estimation of Tannins³²:

1 gm of powdered drug was refluxed in 100 ml of 70% aqueous acetone for 2 hours followed by filtration. The filtrate was concentrated and partitioned with solvent ether (3 times) and then with n- butanol previously saturated with water. The n- butanol soluble portion was dried over a water bath until constant weight and has been tabulated in Table 10.

Total tannin content was calculated by formula:

% w/w Total tannin content=

weight of n-butanol fraction in gm

---------------------------------------------X 100

weight of sample in gm

Determination of resin content³¹:

The accurately weighed drug sample (5gm) was rapidly refluxed with acetone (3 X 200ml) for 6 hours to exhaust the drug for the resin content. The excess solvent was removed by distillation on a water bath. The residue so obtained was suspended in water and transferred to a separating funnel, repeatedly extracted the suspension with solvent ether (2 X 200ml) to extract all the resin contents. The ether extracts were cooled out dried over anhydrous sodium sulphate and excess ether removed over a water bath. It was transferred to a weighed beaker and the final weight is noted and has been tabulated in Table 10.

Table 10 Estimation of phytoconstituents in different Panchachurnam formulations

S. No.	Phytoconstituents	FORMULATIONS (% W/W)
S. No.	Phytoconstituents	IH	SAN	VAN	CAP
1.	Crude Fibre Content	13.89	14.93	14.37	15.53
2.	Total Tannin Content	5.18	5.74	6.3	5.91
3.	Resin Content	7.78	7.67	7.75	6.87

RESULTS AND DISCUSSION:

In house formulation was prepared in accordance with the Ayurvedic Formulary of India. As part of standardization procedure, the finished product Panchachurnam was tested along with samples from three different manufacturers, SAN, VAN and CAP for a comparative study. The organoleptic properties of the marketed formulations and the in-house formulations were compared and all the formulations were light green to greenish brown in colour with characteristic odour and salty taste. The powders are smooth with fine texture. The In-house formulation has an aromatic odour when compared with marketed formulations. The foreign organic matter of powdered individual ingredients of Panchachurnam was found to be within the limits. No foreign organic matter was present in all the formulations.

Anatomical studies of the each ingredient are showing same anatomical characteristics of the selected ingredients. Powder microscopy of the formulations shows the presence of each ingredient in the all formulations. Stomatal index of upper epidermis, Stomatal index of lower epidermis, Palisade ratio, Vein islet number and Vein termination number of Cassia angustifolia Vahl leaf were found to be 17.24-20.00; 16.98-19.29; 4.00-7.25; 19-22; 24-33 respectively, which are between the reference ranges.

Quality tests for different Panchachurnam and its individual ingredients were performed for moisture content, ash content, water soluble extractive, methanol soluble extractive, acid insoluble ash and water insoluble ash, and were found to be within standard ranges. Water soluble ash value was increased due to presence of Saindhava lavanam in the Panchachurnam. Ash values and Moisture content of the ingredients and formulations passed the Indian pharmacopoeial standards. P^H of the Panchachurnam of different brands and in house formulation was found to be ranging from 6.8 to 7.5, almost neutral.

The physical characteristics of the market formulations and in house formulation were found to be comparable. Among all the formulations, In-house formulation has shown excellent flow property.

Phytochemical Screening revealed the presence of Glycosides, Anthraquinone Glycosides, Carbohydrates, Triterpenes, Tannins, Flavonoids, Volatile Oils, Proteins, Fixed Oils and Resins. Screening of formulations was also done for the detection of inorganic constituents by performing Tests for Sodium and Tests for Chloride. The churnas showed the presence of sodium and chloride due to Saindhava Lavana (Rock Salt).

The quantitative analysis of phytoconstituents was carried out and the values of crude fibre, Tannin and resin content of the churnas have very insignificant difference.

CONCLUSION:

The present study revealed that the set parameters for investigation can be used for correct identification of the ingredients of Panchachurnam. All the ingredients were found to be genuine and passed the pharmacognostical, physicochemical and phytochemical studies. Ash values, Extractive values had passed the Ayurvedic Pharmacopoeial standards and moisture content values had passed Indian Pharmacopoeial standards for the ingredients, while all these standards were established for the formulations of Panchachurnam and can be used for routine standardization purpose. The results of the present study will also serve as a reference monograph in the preparation of Panchachurnam. Hence, these quality-control parameters may be considered as a tool for assistance for scientific organizations and manufacturers in developing standards for polyherbal formulations.

ACKNOWLEDGEMENT:

We are grateful to our principal Dr. K. Ravi Shankar and management of Sri Sai Aditya Institute of Pharmaceutical Sciences and Research, Surampalem for providing us necessary facilities to carry out the research project.

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Received on 10.05.2016 Modified on 25.05.2016

Res. J. Pharmacognosy and Phytochem. 2016; 8(3): 125-132.

DOI: 10.5958/0975-4385.2016.00023.6

S. No.	Name of formulation	Colour	Odour	Taste	Form	Texture
1.	IH	Greenish Brown	Aromatic, Characteristic	Salty	Powder	Fine
2.	SAN	Greenish Brown	Characteristic	Salty	Powder	Fine
3.	VAN	Green	Characteristic	Salty	Powder	Fine
4.	CAP	Light Green	Characteristic	Salty	Powder	Fine

S. No.	Parameters	Formulation Code
S. No.	Parameters	IH	SAN	VAN	CAP
1.	Angle of repose Ø=tan^-1(h/r)	28.39	30.94	30.75	36.27
2.	Bulk Density (gm/cc)	0.352	0.384	0.361	0.416
3.	True density (gm/cc)	0.483	0.517	0.5	0.566
4.	Carr’s Index	10.2	12.3	10.8	17.2
5.	Hausner’s ratio	1.11	1.14	1.12	1.20

S. No.	Name of formulation	P^H
S. No.	Name of formulation	1% w/v solution	10% w/v solution
1.	IH	6.85	7.0
2.	SAN	7.1	7.3
3.	VAN	6.98	7.2
4.	CAP	7.4	7.5