Pharmacognostic Standardization of Ayurvedic Formulation of RajanyadiChoorna

Introduction

Herbs are the main source of drugs for various pharmaceutical products. Increasing demand of herbs, various methods of adulteration, non-availability of drugs have led to usage of spurious drugs.1 Though the standards for the raw drug is set by Ministry of AYUSH by publication of ‘The Ayurvedic Pharmacopoeia of India (API)’, quality testing of polyherbal formulations becomes essential to reassure the presence of genuine herbs being added in it. Therapeutic efficacy is mainly impacted by the quality of drugs being used in its preparation.2 The monograph in the Ayurvedic Formulary of India (AFI) provides the specific herb with its part to be utilized along with the method of formulation. In this study, Rajanyadi choorna (RC) standardization is considered, that has Rajani (Haridra), Daru (Devadaru), Sarala, Sreyasi (Gajapippali), Brhati, Kantakari, Prsniparni, Shatahva as ingredients. It was mentioned in Astanga Hrdaya Uttarasthana chapter 2 verse no 38. RC is one of the formulation that is prescribed for conditions like atisara (diarrhea), Grahani, Kamala (Jaundice), Pandu (anaemia), anaha (distension of abdomen), swasa (dyspnea), kasa (cough), dourbalya (weakness), bala roga (diseases of children), vaivarnya (discoloration of body parts). This study was taken up with the following objectives: Standardization of formulation RC- Physical, chemical analyses, microscopy and chromatographic (HPTLC) fingerprinting and development of monographs for RC.

Materials and methods

Collection of herbs of Rajanyadi Choorna

The raw materials for Rajanyadi Choorna, viz., Rajani (Haridra) (Curcuma longa L.), Daru (Devadaru) (Cedrus deodara (Roxb.) G. Don, Shatahva (Anethum sowa Kurz.) were procured from K Govindaraja setty son, D Devaraja Urs road, Mysuru, Karnataka; Sarala (Pinus roxburghii Sarg.) from Robin timber traders, Jammu; Sreyasi (Gajapippali) (Piper chaba L.) from Channabasappa & Co, Avenue road, Bangalore; Brhati (Solanum indicum Linn.), Kantakari (Solanum xanthocarpum Schrad. & H. Wendl.), Prsniparni (Uraria picta (Jacq.) DC) from Khajrekhar, raw drug distributor, Belgaum, Karnataka, India. Voucher specimens of the crude drugs (SDMDG/2018/06, SDMDG/2018/ 07, SDMDG/ 2018/08, SDMDG/2018/09, SDMDG/2018/ 10, SDMDG/2018/11, SDMDG/2018/12, SDMDG/ 2018/13 respectively) were deposited in the museum of Department of Dravyaguna, Sri Dharmasthala Manjunatheswara College of Ayurveda & Hospital, Hassan, Karnataka, India. These drugs were authenticated from Department of Pharmacognosy and Pharmachemistry, Sri Dharmathala Manjunatheshwara Centre for Research in Ayurveda and Allied Sciences, Kuthpady, Udupi and following were the specimen numbers.

Rajani: SDMRC/1067/18112301,

Daru: SDMRC/1067/18112302,

Sarala: SDMRC/1067/ 18112303,

Gajapippali: SDMRC/1067/18112304,

Brihati: SDMRC/1067/18112305,

Kantakari: SDMRC/1067/18112306,

Prishniparni: SDMRC/1067/18112308,

Shatahwa: SDMRC/1067/18112307.

Place of the study

Preparation of the formulation RC, macroscopic and organoleptic evaluation, powder microscopy, physicochemical, preliminary phytochemical analyses was conducted at Laboratory Department of Dravyaguna, Sri Dharmasthala Manjunatheswara College of Ayurveda & Hospital, Hassan, Karnataka.

High performance thin layer chromatography (HPTLC) was conducted at Department of Pharmacognosy and Pharmachemistry, Sri Dharmathala Manjunatheshwara Centre for Research in Ayurveda and Allied Sciences, Kuthpady, Udupi.

Preparation of Rajanyadi choorna

Rajanyadi Choorna was prepared using rhizomes of Rajani (Haridra) (Curcuma longa L.), heartwood of Daru (Devadaru) (Cedrus deodara (Roxb.) G. Don, and Sarala (Pinus roxburghii Sarg.), fruits of Sreyasi (Gajapippali) (Piper chaba L.), whole plants of Brhati (Solanum indicum Linn.) and Kantakari (Solanum xanthocarpum Schrad. & H. Wendl.), roots of Prsniparni (Uraria picta (Jacq.) DC), fruits of Shatahva (Anethum sowa Kurz.).3

The said ingredients were powdered and passed through sieve number 85 and then mixed together in specified proportions to get uniformly blended choorna. 3 Rajanyadi choorna was subjected to the following evaluation. The methodology was followed from the standard procedure mentioned in API.

Pharmacognostic evaluation of Rajanyadi Choorna included:

a. Macroscopic and organoleptic evaluation

b. Microscopic evaluation and powder microscopy

c. Physicochemical evaluation4

d. Preliminary phytochemical evaluation5

e. High performance thin layer chromatography (HPTLC):

one gram of powdered samples of Curcuma longa L. (Haridra), Cedrus deodara (Roxb.) G. Don (Devadaru), Pinus roxburghii Sarg. (Sarala), Piper chaba L. (Gaja pippali), Solanum indicum Linn. (Brihati), Solanum xanthocarpum Schrad. &H. Wendl. (Kantakari), Anethum sowa Kurz (Shatapushpa), Uraria picta (Jacq.) DC (Prishniparni), Rajanyadi choorna were suspended in 10 mL ethanol (99.9%) and kept for cold percolation for 24h and filtered. 5 µL of the above samples were applied on a pre-coated silica gel F254 on aluminum plates to a band width of 7 mm using Linomat 5 TLC applicator. The plate was developed in Toluene: Ethyl acetate (9.0: 1.0). The developed plates were visualized in short UV, long UV and then derivatised with vanillin sulphuric acid reagent and scanned under UV 254 nm, 366 nm and 620 nm following derivatization. Rf , colour of the spots and densitometric scan were recorded.

Observation and Results

The observations and results of physical, chemical analyses, microscopy and chromatographic finger printing of the formulations were as follows:

Macroscopic and Organoleptic evaluation

Rajanyadi choorna was a brownish powder. It had bitter and astringent taste.

Microscopic evaluation

Powder microscopy: Rajanyadi choorna showed the presence of sclereids, trichomes, crystal of calcium oxalate, bunches of starch grains like grapes, mesocarp containing groups of stone cells and pitted tracheids. (Figure 1)

Physicochemical evaluation of Rajanyadi choorna

The results of the physicochemical evaluation were as follows (Table 2). 

Results of preliminary phytochemical evaluation of Rajanyadi choorna are interpreted in Table 3.

High performance thin layer chromatography (HPTLC)

Results of the chromatography of alcoholic extract of Rajanyadi choorna showed presence of various compounds as represented at various wavelengths. At 254 nm wavelength, 5 spots with Rf values 0.12, 0.23, 0.29 (Dark green), 0.35, 0.84 (Light green) were observed. At 366 nm wavelength, 5 spots at Rf values 0.12, 0.35, 0.42, 0.51, 0.70 (F: blue) were observed. After derivatization, 2 spots with Rf values 0.32 0.84 (both purple) were observed. (Table 4, Table 5, Table 6) (Figure 2).

Discussion

Rajanyadi choorna is a brown powder. It has bitter and astringent taste. The combination of herbs has produced a brown colour and as most of the ingredients are bitter and astringent, the same was evident in the formulation.

Rajanyadi choorna showed the presence of sclerids of Shatapushpa; 6 trichomes of Kantakari, 7 Brhati; 8 crystal of calcium oxalate of Shatapushpa, 6 Prishniparni, 9 Brhati; 8 bunches of starch grains like grapes of Gajapippali,10 Kantakari, 7 Brhati, 8 Haridra; 11 mesocarp containing groups of stone cells of Gajapippali and pitted tracheids of Prishniparni, 9 Sarala,12 Devadaru. 13 The microscopic features of the formulation relate to the individual ingredients constituting the formulation and may be used as an identifying microscopic feature for the formulation. The physicochemical standards obtained from the present study could be used as a reference for setting a pharmacopeial standard for this formulation.

The preliminary phytochemical evaluation of the formulation revealed the presence of tannins, flavonoids, glycosides, proteins. These constituents could be attributed to pharmacological action of the formulation. The chromatographic profile of the formulation revealed constituents which relate to the individual ingredients. At 254 nm wavelength, spots with Rf value 0.23 and 0.29 (D. green), 0.35, 0.84 (L. green) were observed. Among these, 0.23 and 0.29 spots correlate with the Rf value of Gajapippali, 0.35 coincides with Rf value of Sarala and Gajapippali. Haridra, Gajapippali and Shatapushpa has spots at Rf values 0.84. At wavelength 366 nm, 0.12, 0.42, 0.70 (F. blue), 0.51 (F. red), 0.32, 0.85 (purple) Rf values were observed. 0.42 (F. blue) coincided with the Rf value of Devadaru, Sarala, Gajapippali, Brihati, Kantakari, Prishniparni, Shatapushpa. Devadaru and Gajapippali had 0.12 (F. blue). 0.51 (F. red) Rf value correlated with the Rf values of Kantakari, Shatapushpa, Prishniparni. Gajapippali has a spot at Rf value 0.70 (F. blue). After derivatization, two spots at Rf value 0.32 and 0.85 (purple) were observed. Haridra and Gajapippali showed spots at these. Shatapushpa also has spot at 0.85 Rf value.

Conclusion

Standardization of Rajanyadi choorna will enable to test the quality of prepared formulations of various pharmaceutical companies and thereby ensures safety and efficacy of the product. Quality assurance of the formulation will have direct impact on therapeutic efficacy.

Monograph of Rajanyadi Choorna

Rajanyadi choorna is a powder preparation made with the ingredients in the formulation composition given in Table 1.

Method of preparation

Take all the ingredients of pharmacopoeial quality. Clean, dry and powder the ingredients 1 to 8 individually and pass through sieve number 85. Weigh each ingredient separately, mix together in specified proportion and pass through sieve number 44 to obtain a homogeneous blend. Pack it in tightly closed containers to protect from light and moisture.

Description

Rajanyadi choorna is a brown coloured smooth powder with bitter and astringent taste.

Identification

Microscopy:

Take a few mg of Choorna and warm with chloral hydrate, wash and mount in glycerine; wash a few mg of Choorna in water and mount in glycerine; treat a few mg of Choorna with iodine solution and mount in glycerine; observe the following characters in the different mounts.

Parenchyma with fibres and starch, vessels, crystals of calcuim oxalate, starch grains in bunches, fibre with thin lumen (Haridra), tracheids with underlying parenchyma, fragments of pitted tracheids, parenchyma with crystals, bunch of starch grains (Devadaru), xylem fibres crossing medullary rays, tracheids in groups with pits, vessels, parenchyma with cell content (Sarala), fragments of thin walled cells, bunch of starch grain like grapes, parenchyma with starch content, epidermal layer in surface view, epidermis of seed coat layer in surface view (Gajapippali), starch grains, vessels with reticulate thinkings, bundle of fibers, testa in surface view, fragments of stellate trichomes, crystals in clusters (Brhati), vessels with reticulate thickening, spiral vessels, fragments of vessels, fragments of fibre, trichomes, bundle of fibres and starch grains (Kantakari), vessels with thickenings, crystals of calcium oxalate, xylem vessels, tracheids, thick walled parenchymatous cells of wood (Prishniparni), fragments of parenchyma with content, mesocarp with thick walls, mesocarp and endocarp, sclereids of the wing, bundle of fibres with crystals (Shatahva).

High performance thin layer chromatography (HPTLC):

Extract 1 gram of sample of Rajanyadi choorna in 10 mL ethanol and keep for cold percolation for 24h, and filter. Apply 5 µL of the extract on a pre-coated silica gel F254 on aluminum plates to a band width of 7 mm using Linomat 5 TLC applicator. Develop the plate using toluene: ethyl acetate (9.0: 1.0) as mobile phase. After development, allow the plate to dry in air, spray the plate with vanillin sulphuric acid reagent and observe under various wavelengths. Under 254 nm, 5 spots with Rf values 0.12, 0.23, 0.29 (dark green), 0.35, 0.84 (light green) were seen. Under 366 nm, 5 spots with Rf values 0.12, 0.35, 0.42, 0.51, 0.70 (all F. blue) were seen. After derivatization, under 620 nm, 2 spots were detected with Rf value 0.32, 0.85 (all purple).

Physico-chemical parameters

Loss on drying at 105o C: Not more than 5.90 per cent

Total ash : Not more than 5.33 per cent

Acid insoluble ash : Not more than 0.67 per cent

Water- soluble extractive : Not less than 8.33 per cent

Alcohol- soluble extractive: Not less than 5.33 per cent

Storage

Store in a cool place in tightly closed containers, protected from light and moisture.

Therapeutic uses

Atisara, Grahani, Kamala, Pandu, Jwara, Agnimandhya, Anaha, Shwasa, Kasa, Balaroga, Daurbalya, Vaivarnya

Dose: ½ to 2 g

Anupana: Ghee, Honey

Acknowledgements

This work was funded by Advance Research Wing, Rajiv Gandhi University of Health Sciences, Bengaluru. ( R G U : R G U / A D V. R E S / G R A N T S / 0 5 9 / 2 0 1 6 - 17DATED: 30.01.2017). Authors are also thankful to Principal, Sri Dharmasthala Manjunatheshwara College of Ayurveda and Hospital, Hassan for providing the essential laboratory facility for conducting the experiments and Dr. Prakash L Hegde Head, Department of Dravyaguna Sri Dharmasthala Manjunatheshwara College of Ayurveda and Hospital, Hassan for the support by providing the laboratory facilities required for conduction of experiments.

Conflicts of Interest

Declared.