Standardization and Quality Control of Dadimastaka Curna –

An Ayurvedic Medicine

Neelesh Dwivedi*, Manoj Tripathi, Ashok Kumar Tiwari

Ayurveda Sadan, JRD Tata Foundation of Research in Ayurveda and Yoga Sciences,

Arogyadham, Deendayal Research Institute, Chitrakoot, Satna, M.P., India

*Corresponding Author E-mail: dwivedineelesh2013@gmail.com

ABSTRACT:

Standardization of the Ayurvedic medicine Dadimastaka Churn has been achieved by following modern scientific quality control procedures, both for the raw material and the finished product. Three samples from different batch were prepared in-house as per AFI and subjected to various physicochemical analysis and HPTLC fingerprinting. The obtained data of physical and chemical parameters for the finished product can be adopted to lay down new pharmacopoeial standards to be followed for classical preparation of Dadimastaka Churn with batch-to-batch consistency. The Phytochemical constituents found to be present in the raw material used for the preparation of churn facilitate the desirable therapeutic efficacy of the medicine.

KEYWORDS: Standardization, Ayurvedic Medicine, Physicochemical parameters, HPTLC Fingerprints.

INTRODUCTION:

Today, the world over, there is a great deal of interest in Ayurvedic System of Medicine. The therapeutic use of these medicines has gained considerable momentum during the past decade. The medical and research community is constantly searching for new natural agents. Natural products are now a multibillion dollar industry in the developed countries and these products are used by millions of people annually (K. Jayram Kumar, 2011). Currently, the herbal drug preparation itself is regarded as the active substance. Hence the reproducibility of the total configuration of herbal drug constituents is important, and the TLC/HPTLC/GC fingerprint profiles will serve as guiding line to the Phytochemical profile of the drug in ensuring the quality, while quantification of the marker compounds would serve as an additional parameter in assessing the quality of the sample.

Therefore, quality control standards of various medicinal plants used in Ayurvedic formulations are becoming more relevant today in view of commercialization of formulations. Standardization and quality control depends upon the nature of crude drug and compound drugs, on its source i.e. factors associated with raw materials which are beyond of human control like seasonal, geographical, age of the plant, time of collection, type of drying etc. Due to these natural conditions the percentage of chemical constituents of the drug does no remain uniform as our expectation (L V Asokar et al., 1972).

Dadimastaka Churn is a traditional compound formulation of Ayurvedic Formulary of India and therapeutically used in Grahani (Malabsorption syndrome) (Anonymous, 2000). The need of quality control for Ayurvedic drug is due to the fact that the preparation of drug according to the ancient method has been reduced due to the commercialization of ayurvedic pharmacy during past era. The World Health Organization has issued a detail protocol for the standardization of herbal drugs comprising of a single content, but very little literature is available for the standardization of these formulation. The absence of post-market surveillance and the paucity of test laboratory facilities also make the quality control of Aurvedic medicines exceedingly difficult at this time.

Table 1. Formulation Composition of Dadimastaka Churna

S.N.	Sanskrit Name	Botanical Name	Parts	Quantity
1	Tugãkṣiri (Vamsa)	Bambusa bambos	S.C.	12g.
2	Tvak	Cinnamomum zeylanicum Blume.	Stem bark	24g.
3	Patra (Tvakpatra)	Cinnamomum tamala Nees and Eberm	Leaf	24g.
4	Elã	Elettaria cardamomum Maton	Seed	24g.
5	Nãgakeśara	Mesua ferrea Linn.	Adr.	24g.
6	Yamãni (Yavãni)	Trachyspermu ammi Linn.	Fruit	48g.
7	Dhãnyaka	Corindrum sativum Linn.	Fruit	48g.
8	Ajãji (Śvetajiraka)	Cuminum cyminum Linn.	Fruit	48g.
9	Granthi(Pippalimũla)	Piper longum Linn.	Root	48g.
10	Suṇṭhi	Zingiber officinale Rose.	Rhizome	48g.
11	Marica	Piper nigrum Linn.	Fruit	48g.
12	Pippali	Piper longum Linn.	Fruit	48g.
13	Dãḍima	Punica granatum Linn.	Seed	48g.
14	Sitã (Sarkarã)	Sugar	-	38g.

Therefore, the work was undertaken to evaluate scientifically for its wider acceptance. In present study, the individual plant powders and three formulations, two in-house preparations and one sample from pharmacy were subjected to various standardization parameters viz. physicochemical analysis, Phytochemical analysis and HPTLC fingerprinting using authenticate ingredients as controls.

MATERIALS AND METHODS:

Plant Materials:

Dadimastaka Curna is a powder preparation made with the ingredients in the Formulation Composition. It consists of 14 ingredients (Table-1). All these plants parts were collected from forest and some plants were purchased by local market of Chitrakoot, and were authenticated with the help of Herbarium of Research laboratory, Deendayal Research Institute, Arogyadham, Chitrakoot, M.P., India.

Preparation of the Curna:

In-house formulation of Dadimastak Curna was prepared as per Ayurvedic Formulary of India. Took all the ingredients of pharmacopoeial quality. Cleaned, washed and dried ingredient numbered 2 to 13 in the powder separately and passed through 180 μm IS Sieve (sieve number 85). Cleaned and powdered ingredient numbered 14 and passes through 180 μm IS Sieve (sieve number 85). Weighed each ingredient separately and mixed together in specific quantity. Passed the cũrṇa through 355 μm IS Sieve (sieve number 44) to obtain a homogenous blend. Packed it in a container and make it air-tight to protect from light and moisture.

Standardization Parameters:

Organoleptic Evaluation:

The organoleptic characters of the samples were evaluated based on the method described by previous researcher (Siddiqui et al., 1995). Organoleptic evaluation refers to evaluation of the formulation by color, odor, taste and texture etc.

Physico-Chemical Investigations:

Physico-chemical investigations of formulations were carried out for the determination of extractive values and ash values (Mukerjee P K, 2002 and Anonymous 1996).

Total ash:

2g of powdered material of each batch and the individual ingredients of the powders were placed separately in a suitable tared crucible of silica previously ignited and weighed. The powdered drugs were spread into an even layer and weighed accurately. The materials were incinerated by gradually increasing the heat, not exceeding 450°C until free from carbon, cooled in desiccators, weighed and percentage ash was calculated by taking in account the difference of empty weight of crucible and that of crucible with total ash.

Acid Insoluble Ash:

The ash obtained as above was boiled for 5min with 25ml of dilute hydrochloric acid; the insoluble matter was collected on an ash less filter paper, washed with hot water and ignited to constant weight. The percentage of acid-insoluble ash with reference to the air-dried drug was calculated.

Alcohol Soluble Extractive:

2g of coarsely powdered air-dried drug was macerated with 100ml of alcohol in a closed flask for twenty-four hours, shaking frequently during six hours and allowed to stand for eighteen hours. It was then filtered rapidly; taking precautions against loss of solvent. 25ml of the filtrate was evaporated to dryness in a tared flat-bottomed shallow dish at 105°C to constant weight and weighed. The percentage of alcohol-soluble extractive was calculated with reference to the air dried drug and is represented as % value.

Water Soluble Extractive:

2g of coarsely powdered air-dried drug was macerated with 100ml of chloroform water in a closed flask for twenty-four hours, shaking frequently during six hours and allowed to stand for eighteen hours. It was then filtered rapidly, taking precautions against loss of solvent. 25ml of the filtrate was evaporated to dryness in a tared flat bottomed shallow dish at 105°C to constant weight and weighed. The percentage of water-soluble extractive was calculated with reference to the air-dried drug and is represented as % value.

Loss on Drying:

Placed 2 gm of powdered compound formulation (without primarily drying) after accurately weighing in a tarred evaporating dish. Dry the evaporating dish at 105⁰ for 5 hours, and weigh. Continue the drying and weighing at 30 minutes until difference between two successive weighing corresponds to not more than 0.25%. Amount of moisture present in the sample was calculated as reference to the air dried drug (Trease and Evans 1978).

Determination of pH:

1% solution of the churna was prepared in distilled water and _PH was determined using Digital pH meter. Operated the pH meter and electrode system according to the manufacturer’s instructions. Calibrated the meter and electrodes with 0.05M potassium hydrogen phthalate. At the end of a set of measurement, noted the reading of the aqueous solution.

Preliminary Phytochemical Analysis:

Preliminary qualitative phytochemical analysis of all the extracts was carried out by employing standard conventional protocols (Sazada et al., 2009 and Kokate et al., 2006).

HPTLC Finger Printing Profile:

The quality of the drug may be assessed on the basis of chromatographic fingerprint. HPTLC study of ethanol extracts of the formulations along with individual ingredients corresponding to the active ingredients was carried out to ensure the presence of active ingredients in all the formulations (Anonymous, 2003 and Anonymous, 2012). Test solution: Extracted 2g of the cũrna in ethanol (25 ml x 3) by refluxing on water bath for 30 minutes each. Filtered each of the extracts, combine and concentrate to 10 ml. Applied 9 μl of the extract as band at a height of 10 mm from the base of a 20x10 cm TLC plates and develop up to 8 cm from the base of plate using mobile phase Toluene: Ethyl Acetate (7: 3). Dried the plate in air and examined under UV 366 nm. Spread the plate with Anisaldehyde - sulphuric acid reagent and heat at 105⁰ till the color of the spots/bands appear without charring. TLC profile with test solution should match with the standard profile with respect to the major spots/bands.

RESULTS AND DISCUSSION:

As part of standardization procedure, the formulations were tested for relevant physical and chemical parameters along with samples from three different batches, 01, 02 and 03. The formulation was subjected to various analytical techniques. Organoleptic parameters revealed that all the samples are creamish-brown smooth powder with a spicy odour and sweetish taste; the powder completely passes through 355 μm IS Sieve (sieve number 44) and not less than 50 percent through 180 μm IS Sieve. Quality tests for different batches of Dadimastaka curna and its individual ingredients were performed for moisture content, ash content, acid insoluble ash, water soluble extractive, alcohol soluble extractive, and were found to be within standard ranges (Anonymous, 2000). The Results of physicochemical analysis are given in (Table -2, 3).

The results are expressed as mean (n=3) ± Standard deviation (SD). The total ash value is an indicative of total amount of inorganic material after complete incineration and the acid insoluble ash value is an indicative of silicate impurities, which might have arisen due to improper washing of drug. Ash value is useful in determining authenticity and purity of drug and also these values are important quantitative standards (Dwivedi et al. 2014). The extractive values names water soluble and alcohol soluble indicates the amount of active constituents in given amount of plant material when extracted with respective solvent. The loss on drying value obtained is an indicative of amount of moisture content present in the drug. The less value of moisture content could prevent bacterial, fungal or yeast growth. The results of preliminary phytochemical screening are given in (Table-4).

Table 2. Physico-chemical parameters of different batches in Dãḍimãṣṭaka cũrṇa

Parameters	Dãḍimãṣṭaka cũrṇa			Average	Limits
	Batch 01	Batch 02	Batch 03	(w/v) %
LOD at 105° C	3.74	3.72	3.61	3.69	NMT 4
Total Ash	4.11	4.18	4.22	4.17	NMT 5
Acid-insoluble ash	1.4	1.5	1.4	1.43	NMT 2
Water-soluble extract	44.52	45.40	44.37	44.76	NLT 44
Alcohol-soluble extract	13.17	13.18	13.11	13.15	NLT 13
Volatile oil	2.0	1.9	2.1	2.0	NLT 1.5
pH (10% aqua solution)	4.8	4.9	4.9	4.87	4 - 5

Table 3: Physico-chemical parameters of single ingredients of Dãḍimãṣṭaka cũrṇa

Name of Ingredients	LOD (%w/w)	Total ash (% w/w)	AI ash (%w/w)	ASE (%w/w)	WSE (% w/w)	Volatile oil (% v/w)	Compliance with API
Tvak (St. bk.)	8.15	NMT3 2.9	NMT2 1.8	NLT2 3.96	NLT3 4.73	NLT1 1.3	Complies
Patra (Tvakpatra-Lf.)	6.95	NMT5 4.60	NMT1 0.97	NLT6 6.14	NLT9 15.45	NLT1 1.4	Complies
Elã-(Sd.)	6.35	NMT6 4.1	NMT2 3.61	NLT10 10.37	NLT4 15.11	NLT4 7.11	Complies
Nãgakeśara (Stmn)	6.96	NMT6 5.33	NMT3 2.56	NLT15 17.63	NLT12 15.19	-	Complies
Yãmanî (Yavãni) (Fr.)	8.49	NMT9 5.56	NMT 02 0.14	NLT2 4.22	NLT13 18.78	NLT2.5 3.3	Complies
Dhãnyaka	5.79	NMT6 4.69	NMT1.5 0.94	NLT10 23.55	NLT19 22.22	NLT0.3 0.5	Complies
Ajãjî (Fr.) (śvetaji raka)	8.98	NMT8 6.26	NMT1 0.65	NLT7 17.49	NLT15 31.79	3.0	Complies
Granthi (St.) (Pippali mũla)	12.91	NMT5.5 4.87	NMT 0.2 0.13	NLT4 5.3	NLT12 17.92	2.20	Complies
Suṇṭhi	8.68	NMT6 5.84	NMT 1.5 0. 97	NL3 4.14	NLT10 15.45	1.7	Complies
Marica (Fr.)	6.95	NMT5 4.28	NMT 0.5 0.43	NLT6 7 .14	NLT6 11.45	2.7	Complies
Pippali (Fr)	11.25	NMT7 6.61	NMT 0.5 0.36	NLT5 7 .85	NLT7 19.5	1.5	Complies
Dãḍima	7.01	NMT4 3.32	NMT 0.4 0.31	NLT9 11.17	NLT20 22.44	-	Complies

Table 4. Preliminary Phytochemical Screening

S.N	Active constituents	Name of the test	01	02	03
1	Alkaloid	Wagner’s test	+	+	+
2	Flavanoids	HCl+ Mg	+	+	+
3	Tannin	FeCl3	+	+	+
4	Saponin	NaHCO3	+	+	+
5	Resin	Acetone	+	+	+
6	Carbohydrate	Fehling test	+	+	+
7	Protein	NaOH+CuSO4	+	+	+
8	Terpenoid	CHCl3 + conc. H2SO4	+	+	+
9	Steroid	Acetic anhydride + H2SO4	+	+	+

(+) Presence of phyto-constituents and (-) Absence of phyto-constituents

Various tests have been conducted qualitatively to find out the presence or absence of bioactive compounds (Kokate et al., 2006). Various chemical compounds such as carbohydrate, protein, amino acids, tannin, flavanoids, alkaloids, glycosides are detected in the formulation which could make the drug useful for treating different ailment as having a potential of providing useful drugs for human use. HPTLC chromatograms of three batches of Dadimastak Churna samples were are depicted in (Plate-1). The Rf values and color of the resolved bands are tabulated in Table-5.

These bands were chosen to identify the presence or absence of the individual ingredients in each of the studied samples. Developed chromatograms indicate the presence of all the raw ingredients in proportional quantity in the formulations. This confirms the batch-to-batch consistency of the finished products and can serve as Quality standard for manufacturer of same drug in future.

Table 5: R_f values in test solutions of Dãḍimãṣṭaka cũrṇa at 254 nm

Rf values	254nm				366nm				366nm After derivatization
	T1	T2	T3	color	T1	T2	T3	color	T1	T2	T3	color
Rf 1	0.14	0.14	0.14	All Black	0.14	0.14	0.14	Blue	0.11	0.11	0.11	Light yellow
Rf 2	0.37	0.37	0.37		0.18	0.18	0.18	Blue	0.32	0.32	0.32	White
Rf 3	0.43	0.43	0.43		0.30	0.30	0.30	Blue	0.35	0.35	0.35	White
Rf 4	0.48	0.48	0.48		0.39	0.39	0.39	Grey	0.51	0.51	0.51	Light brown
Rf 5	0.49	0.49	0.49		0.46	0.46	0.46	Brown	0.58	0.58	0.58	Blue
Rf 6	0.62	0.62	0.62		0.61	0.61	0.61	Red	0.67	0.67	0.67	Sky blue
Rf 7	0.79	0.79	0.79		0.64	0.64	0.64	Blue	0.74	0.74	0.74	Blue
Rf 8	0.88	0.88	0.88		0.71	0.71	0.71	Blue	0.84	0.84	0.84	Sky blue
Rf9					0.81	0.81	0.81	Blue	0.92	0.92	0.92	Blue

T₁ T₂ T₃ T₁ T₂ T₃ T₁ T₂ T₃

Plate 1 : TLC Finger prints in test solution of Dãḍimãṣṭaka cũrṇa

Fig 1. TLC profile of Dãḍimãṣṭaka cũrṇa RS observed under (I) 254 nm; (II) 366 nm; and (III) 366nm after spraying with Anisaldehyde - sulphuric acid reagent

Track 1: Batch 01, Track 2: Batch 02, Track 3: Batch

CONCLUSION:

From the present studies, it can be calculated that the distinguishing band in the HPTLC profiles are very important for monitoring the quality of the Cûrna formulation as well as for establishing whether all the required ingredients are present in them. Also, standardization and development for reliable quality protocols for Ayurvedic formulations are important for keeping a check on batch to batch variations. Hence, the physiochemical parameters, quantitative analysis and HPTLC fingerprinting profiles together may be used for quality evaluation and the standardization of compound formulations and maintaining their quality, purity and efficacy.

ACKNOWLEDGEMENTS:

Authors gratefully acknowledge the research facilities provided by Dr. Bharat Pathak, General Secretary Deendayal Research Institute, Chitrakoot. Authors are also thankful to Department of AYUSH, Ministry of Health and Family Welfare, Government of India for providing financial assistance.

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Received on 16.03.2015 Modified on 29.03.2015

Accepted on 11.04.2015 ©A&V Publications All right reserved

Res. J. Pharmacognosy and Phytochem. 7(2): April-June 2015; Page 111-115

DOI: 10.5958/0975-4385.2015.00019.9