Standardization of Rajanyādi cūrṇa: An ayurvedic preparation.

BACKGROUND: Rajanyādi cūrṇa (RC) is an ayurvedic classical preparation used in the treatment of digestive disorders, fever, jaundice, anemia, and asthma. We seek to standardize this drug to ensure its quality.
OBJECTIVE: The current investigation was aimed at the preparation of cūrṇa in three batches so as to standardize it.
MATERIALS AND METHODS: The cūrṇa was prepared in-house in three different batches according to directions given in The Ayurvedic Formulary of India. The cūrṇa was evaluated based on organoleptic characters, physical characteristics, and physico-chemical parameters. High performance thin layer chromatography was carried out for the quantification of curcumin.
RESULTS: The parameters were found to be comparable and sufficient for the evaluation of the cūrṇa.
CONCLUSION: Ayurvedic medicine, RC has been standardized using the various parameters and can be incorporated while developing the pharmacopoeial standards.

INTRODUCTION

Standardization of herbal medicines is the process of prescribing a set of standards or inherent characteristics, constant parameters, definitive qualitative and quantitative values that carry an assurance of quality, efficacy, safety, and reproducibility. It is the process of developing and agreeing upon technical standards. Specific standards are set to carry out the experimentation, which would lead to the development of a set of characteristics exhibited by the particular herbal medicine. Hence, standardization is a tool in the quality control process.[1]

Herbal medicines contain more than one active principles and the active compound is frequently unknown. For instance, the Chinese medicinal plant Huang-qin (Scutellaria baicalensis) has over 2000 compounds.[2] This makes the process of standardization challenging.

While assuring the quality of the drug, consistency of active principles and therapeutic efficacy, standardization of herbal formulations is an essential aspect. Rajanyādi cūrṇa (RC) is a polyherbal ayurvedic classical formulation used in agnimāndya (digestive impairment), atisāra (diarrhea), jvara (fever), kāmāla (jaundice), pāṇḍu (anemia) and śvāsa (asthma).[3] Literature survey did not reveal any standards for RC and hence the current study was undertaken to standardize the cūrṇa.

MATERIALS AND METHODS

Collection and authentication of plant materials

The crude drugs used in the in-house RC formulation were procured from the local market of Udupi, Karnataka, India, and authenticated by Dr. B. Satyanarayana, Principal and Chief medical officer, Muniyal Institute of Ayurveda Medical Sciences, Manipal, Karnataka, India. A voucher specimen of the crude drugs (PP 532A, PP 577A, PP 588 − PP 593) are deposited in the museum of Department of Pharmacognosy, Manipal College of Pharmaceutical Sciences, Manipal, Karnataka, India.

Preparation of cūrṇa

Rajanyādi cūrṇa was prepared in three different batches following the procedure as given in The Ayurvedic Formulary of India.[3] The ingredients viz Rajanī, Dāru, Saralā, Śreyasī, Bṛhatī, Kaṇṭakārī, Pṛṣṇiparṇī and Śatāhvā were powdered and passed through an number 80 sieve and then mixed together in specified proportions [Table 1 and Figure 1] to get uniformly blended cūrṇa. The three batches were labeled as RC-I, RC-II and RC-III respectively.

Table 1

Ingredients of Rajanyādi cūrṇa

Figure 1

Ingredients of Rajanyādi cūrṇa

Preparation of extract

Rajanyādi cūrṇa alcoholic extract (RCAL) was prepared by extracting 30 g of RC with ethanol using soxhlet extraction process. The ethanolic extract was evaporated till it was completely dry and the dried extract (yield 11.16%) was stored in the desiccator until its use.

Chemicals

All the solvents and chemicals used in the experiment were of analytical grade and were purchased from Changshu Yangyuan Chemical, China, Finar Chemicals Limited, Ahmedabad. The marker compound curcumin (purity 99%) was purchased from Loba Chemie, Mumbai, India.

Organoleptic evaluation

The cūrṇa was evaluated for organoleptic characters such as varṇa (color), gandha (odor), ruci (taste), and parimāṇa (size) were analyzed and recorded.

Microscopic evaluation

A small quantity of RC was taken on a slide. The clearing agent used was chloral hydrate (75%). The powder was heated with chloral hydrate over flame for a short while to clear the tissues and then stained with phloroglucinol (2 g phloroglucinol in 100 ml of 95% alcohol), along with concentrated hydrochloric acid (1:1) to stain lignified tissues to pinkish red. Few drops of glycerine was added and the slide was covered with a cover slip. The slide was viewed under a microscope connected with a computer for observing microscopic characters.[4]

Physicochemical evaluation

Physicochemical characteristics of RC samples were analyzed by quantitative analysis for total ash, water soluble ash, acid-insoluble ash, water soluble extractive, alcohol-soluble extractive, loss on drying, and pH (10% aqueous solution) as per the standard techniques.[5] Fluorescence analysis was carried out for the identification of cūrṇa.[67] RC samples were analyzed for presence of heavy metals like lead (Pb) and cadmium (Cd) by atomic absorption spectroscopy[8] (AA 240, Varian, The Netherlands). Level of arsenic was determined by following the WHO protocol.[9] The cūrṇa was also subjected to the determination of microbial contamination such as Escherichia coli as per the WHO procedure.[9]

Powder characteristics

Micromeritic characteristics like bulk density, tap density, angle of repose, Hausner ratio and Carr's index were determined for RC samples.[1011]

High performance thin layer chromatography determination of curcumin

High performance thin layer chromatography (HPTLC) was carried out for RCAL using curcumin as the reference standard. The standard and test extract were spotted in triplicate on silica gel 60F254 HPTLC plates and developed with the solvent system Chloroform-Methanol (9.6:0.4) as the mobile phase.[12] After development, the plates were dried and then scanned at 366 nm with a Camag TLC Scanner-3, CAMAG, Switzerland.

RESULTS AND DISCUSSION

As a part of standardization, all the three batches of finished product of RC were tested for the relevant physical and physico-chemical parameters. The samples of cūrṇa were found to be yellowish green colored, moderately fine powder, characteristic turmeric odor with slightly bitter in taste. Microscopic evaluation revealed the presence of cortical parenchyma cells which are mostly thin walled, rounded and filled with gelatinized starch grains and permitted with a bright yellow coloring matter (a-Rajani),[13] narrow, five or six sided tracheid's, having very thick wall with pits and narrow lumen (b-Devadāru), medullary rays and resin debris (c and c1-Saralā), isodiametric parenchymatous cells filled with brown content (d-Śreyasī), fibers surrounded by crystal sheath (e-Bṛahtī), lignified stone cells of primary cortex (f-Kaṇṭakāri), simple pitted vessels and fibers (g and g1-Pṛṣṇiparṇī) and Vittae and endosperm parenchyma cells containing fixed oil and aleurone grains (h-Śatāhvā)[14] as shown in the Figure 2a-h. Physicochemical parameters such as ash values, extractive values, loss on drying including the pH were determined and the values are presented in Table 2. Physical characteristics such as bulk density, tapped density, angle of repose, Carr's compressibility index and Hausner ratio were determined for all the three batches. Flow property[11] was found to be very poor for angle of repose and poor for Carr's compressibility index and Hausner ratio and this could be attributed due to the oleo-resinous matter of Turmeric[15] and Saralā[16] of the formulation in all the batches. The values are depicted in Table 2. Fluorescence analysis was carried out to check the fluorescence behavior of the cūrṇa formulations with different reagents as identification characters and the observations are given in Table 3. Heavy metals cause gastrointestinal disturbances, liver damage and bone and nervous system defects.[171819] WHO prescribes limits for the presence of such heavy metal contamination. Therefore, heavy metals were determined in RC samples as per WHO guidelines. Lead and Cd content were 17 ppm and 90 ppm respectively and found to be higher than the prescribed limits. This could be attributed to reasons such as, plants imbibing toxic metals in various ways including the environment they grow in, contaminated water, agricultural expedients, storage environment, and manufacturing processes.[20] The cūrṇa showed absence of E. coli.

Figure 2

Powder microscopy of Rajanyādi cūrṇa

Table 2

Physico-chemical analysis of Rajanyādi cūrṇa

Table 3

Fluorescence analysis of Rajanyādi cūrṇa

In the present study, curcumin has been determined by HPTLC method and it was used as marker compound.[15] Curcumin, a yellow pigment present in Curcuma longa has been linked with various biological activities such as suppression of inflammation; angiogenesis; tumorigenesis; diabetes; diseases of the cardiovascular, pulmonary, and neurological systems, of skin, and of liver; loss of bone and muscle; depression; chronic fatigue; and neuropathic pain.[21] Due to the shortage and lack of availability of markers including the purity, HPTLC determination of all the ingredients was not possible except for curcumin. The chromatographic system afforded a satisfactory Rf value of 0.69 for curcumin of test extract which was comparable with the standard. The curcumin content of the RCAL was found to be 0.335% w/w. The video densitometry of the developed plate is shown in Figure 3. Curcumin[22] (may be actually mixture of three curcuminoids) has therefore shown three spots. The chromatogram of standard curcumin and RCAL is shown in the Figures 4 and 5 respectively. From the above results, Analytical specifications for RC have been represented in Table 4.

Figure 3

Video densitometry of standard curcumin and Rajanyādi cūrṇa alcoholic

Figure 4

Chromatogram of standard curcumin

Figure 5

Chromatogram of Rajanyādi cūrṇa alcoholic

Table 4

Analytical specifications of Rajanyādi cūrṇa

CONCLUSION

Ayurvedic classical preparation, RC has been standardized using the various modern scientific quality parameters. The results obtained can be used as reference while setting the pharmacopoeial standards for RC to ensure the quality of the medicine.