Pharmacognostical identification of ingredients in Laghulai curna: An Ayurvedic compound formulation

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1 Indian Journal of Traditional Knowledge Vol. 14(4), October 2015, pp Pharmacognostical identification of ingredients in Laghulai curna: An Ayurvedic compound formulation Manoj Tripathi*, RLS Sikarwar, Ashok Tiwari & Neelesh Dwivedi Arogyadham (JRD Tata Foundation for Research in Ayurveda & Yoga Sciences), Deendayal Research Institute, Chitrakoot, Satna , MP Received 08 August 2014, revised 28 October 2014 Laghulai curna is a compound formulation prepared by 13 single ingredients, viz. Suta (Suddha parada), Gandha (Suddha gandhaka), Sunthi (Zingiber officinale Rose., rhizome), Marica (Piper nigrum L., Fruit), Pippali (Piper longum L., Fruit), Dipyaka (Yavani) (Trachyspermum ammi L., Fruit), Svetajiraka (Cuminum cyminum L., Fruit), Krisnajiraka (Carum carvi L., Fruit), Sauvarcala, Saindhava, Ramatha (hingu) (Ferula assafoetida Regel., Excude), Vida (Lavana), Sakrahvaya (Kutaja) (Holarrhena antidysenterica Wall., Seeds) and used in Grahani (mal absorption syndrome), Atisara (diarrhoea), Anaha (distension of abdomen due to obstruction to passage of urine and stools), Sūla (pain) in Indian System of medicine. The present paper provides microscopical diagnostic keys and HPTLC finger print profile that are needed for proper identification and authentication of an Ayurvedic compound formulation Laghulai curna. Keywords: Laghulai curna, Diagnostic keys, HPTLC finger print, Powder microscopy IPC Int. Cl. 8 : A61B 5/117, G06K 9/00, A61K, A01D 20/00 India is unique country with rich cultural heritage, diverse and hoary traditions and rich medicinal heritage. Plants are the main source of sustenance and medications to the human kind since ages. But rapid commercialization of traditional medicines led to adulteration and substitution of authentic drugs. The problem is compounded by the ready availability of raw plant material sometimes of questionable quality. Under section 33D in Chapter IV-A of the Drugs and Cosmetics Act, , and Rules there under, certain basic quality control measures have been introduced for Ayurvedic drugs. It now devolves upon the manufactures of these drugs, inter alia, to confirm that their raw materials are identified to be genuine and that the manufacture is carried out under sanitary and hygienic conditions. The Ayurvedic drug manufacturers are at a disadvantage with the former requirement, as uniformity in nomenclature as well as consensus over the correct identity of several Ayurvedic drugs in current use is lacking. Secondly, there is a dearth of scientific work of significance for use in regulatory analysis for Ayurvedic drugs. The present paper arises out of such needs and hopefully, may be beginning in overcoming the *Corresponding author deficiencies both for the controlling and controlled personnel. This paper establishes diagnostic powder characteristics and HPTLC finger print profile for an Ayurvedic compound formulation Laghulai curna and its ingredients which is formulated by 13 ingredients and used in Grahani (mal absorption syndrome); Atisara (diarrhoea); Anaha (distension of abdomen due to obstruction to passage of urine and stools); Sūla (pain) and its ingredients also widely used to cure various diseases and preparation of Ayurvedic compound formulations 2. The information generated by this particular study provides relevant microscopical diagnostic keys and HPTLC finger print profile data needed for proper identification and authentication of an Ayurvedic compound formulation Laghulai curna. Methodology Preparation of the curna All the ingredients of Laghulai curna were used in Pharmacopoeial quality 3, viz. Sũta (Parada Suddha), Gandha (Gandhaka Suddha), Śunthî (Zingiber officinale Rose., rhizome), Marica (Piper nigrum L., Fruits), Pippalî (Piper longum L., Fruits), Dîpyaka (Yavãnî) (Trachyspermum ammi L., Fruits), Śveta jîraka (Cuminum cyminum L., Fruits), Krsnajîraka

2 532 INDIAN J TRADIT KNOWLE, VOL. 14, No. 4, OCTOBER 2015 (Carum carvi L., Fruits), Sauvarcala (Black salt), Saindhava (Rock salt), Rãmatha (Hingu) (Ferula assafoetida Regel., Exude), Vida Lavana (Black salt) and Śakrãhvaya (Indrayava) (Holarrhena antidysenterica Wall., Seeds). Treated Sũta and Gandha to prepared Suddha parada and Suddha Gandhaka respectively, and prepared the Kajjali 4. Ground to make powder and passed through 180 um IS Sieve (old sieve number 85). Cleaned, washed, dried and powdered the Śunthî, Marica, Pippalî, Dîpyaka (Yavãnî), Śvetajîraka, Krsnajîraka and Śakrãhvaya (Indrayava) for the formulation composition separately and passed through 180 µm IS Sieve (old sieve number 85). Cleaned and roasted fine powder Sauvarcala, Saindhava and Vida Lavana in a stainless steel pan separately till it become free from moisture and powder enough to pass through 180 µm IS Sieve (old sieve number 85). Treated Rãmatha (Hingu) to prepared Suddha-Hingu 5 and powder enough to passed through 180 µm IS Sieve (old sieve number 85).Weighed each ingredients separately and mixed together in specific quantity and enough to passed through 355 µm IS Sieve (old sieve number 44) to obtained a homogenous blend. It was stored in air-tight container to protect from light and moisture. Powder microscopy For powder microscopy analysis 6,7 about 50 gm of compound formulation curna along with the genuine samples, i.e., Suta (Suddha parada), Gandha (Suddha gandhaka), Sunthi (Zingiber officinale Rose., Rhizome), Marica (Piper nigrum L., Fruit), Pippali (Piper longum L., Fruit), Dipyaka (yavani) (Trachyspermum ammi L., Fruit), Svetajiraka (Cuminum cyminum L., Fruit), Krsnajiraka (Carum carvi L., Fruit), Sauvarcala, Saindhava, Ramatha (hingu) (Ferula foetida Regel., Excude), Vida (lavana), Sakrahvaya (Kutaja) (Holarrhena antidysenterica Wall., Seeds) on a 106 µm IS Sieve; was used lukewarm water from a wash bottle sprayed a jet on to the material; stirred the sample from side to side simultaneously used a scalpel so that the jet of water removed minerals or bhasma particles or dissolved salts through the sieve; it was repeated the process; collected the material left on the sieve, without loss of material; mounted a small portion in glycerin; warmed a few mg with chloral hydrate solution, washed in water and mounted in glycerin; treated a few mg with iodine in potassium iodide solution and mounted in glycerin; heated a few mg in 2 % aqueous potassium hydroxide, washed in water and mounted in glycerin and Observed the characteristics under microscope at 40 X 10X magnification of the trinocular research microscope (Fig. 1). HPTLC (High performance thin layer chromatography) profile For HPTLC, 2 gm of each sample was extracted with 25 ml of methanol on boiling water bath for 20 min. consecutively of 3 times used fresh portion of 25 ml methanol, filtered and concentrated. Similarly, methanolic extracts were prepared for all 13 ingredients 8 (Figs. 2-5). TLC of methanolic extracts of all the samples and the reference ingredients was carried out on silica gel 60 F 254 percolated plates (0.2 mm thickness; from Merck India Limited Mumbai). An applicator from Camag Linomat-5 Camag Switzerland: ) was used for band application and photo documentation unit (Camag Reprostar-3: ) was used for documentation of chromatographic fingerprints. The mobile phase used Toluene: Ethyl acetate (7:3). The plate was developed over a distance of 9 cm in a saturated development chamber (Twin through chamber 20X10 cm with SS lid and visualized under 254nm, 366nm and visible light. After spraying with 5% methanolic sulphuric acid followed by heating at C for 5-10 min Results and discussion The polyherbal formulation has been described in various Ayurvedic texts and used for the treatment of various chronic ailments and diseases since ancient times. But present times it is mandatory to testify the polyherbal formulations on modern research parameters. On the basis of modern research parameters, the study on Unani polyherbal formulation Suffoof-e-Mohazzil 12 and ayurvedic polyherbal formulations Katphaladi curna 13, Yogaraja guggulu 14, Palashbijadi curna 15 and Triphala curna 16 have already been carried out by various workers. Our study was subjected to identification of ingredients in Laghulai curna through various analytical techniques. Botanical parameters revealed that the formulations powder colour is black, odour characteristic and taste is salty. Powder microscopic examination was carried out for individual ingredients present in the formulation. The following diagnostic characters are observed in the various mounts. Presence of parenchymatous cells containing oleo-resin, oval to round starch grains not less than 15-30µ and several up to 70µ with hilum ecentric, lamellae distinct; pitted, septate fibres with

Similarly, hypodermal parenchyma interspersed with stone cells, thick walled polygonal stone cells from testa indicated the presence

3 TRIPATHI et al: PHARMACOGNOSTICAL IDENTIFICATION OF INGREDIENTS IN LAGHULAI CURHA 533 indentations on its walls confirm the presence of Śunthî. Similarly, hypodermal parenchyma interspersed with stone cells, thick walled polygonal stone cells from testa indicated the presence of Marica. Fig. 1 Microscopical characters of Laghulai curna Likewise, lignified spindle shaped stone cells with wide lumen associated with vascular elements confirm the presence of Pippalî. Similarly, epidermis with papillose and glandular out growths with

534 INDIAN J TRADIT KNOWLE, VOL. 14, No. 4, OCTOBER 2015 Fig.

Gandha; 3: Sunthi; 4: Marica; 5: Pippali; 6: Dîpyaka; 7: Śvetajîraka; 8: Krsnajîraka; 9: Sauvarcala; 10: Saindhava; 11: Rãmatha; 12: Vida Lavana; 13: Śakrãhvaya Fig.

curna; 1: Suta; 2: 3 HPTLC finger prints of test solution of Laghulai curna at 366nm Test solution of Laghulai curna for A: Batch A-Laghulai curna; B: Batch B-Laghulai curna; C: Batch C- Laghulai

striations radiating from base confirm the presence of Dîpyaka.

4 534 INDIAN J TRADIT KNOWLE, VOL. 14, No. 4, OCTOBER 2015 Fig. 2 HPTLC finger prints of test solution of Laghulai curna at 254nm Test solution of Laghulai Curna for A: Batch A-Laghulai curna; B: Batch B-Laghulai curna; C: Batch C -Laghulai curna; 1: Suta; 2: Gandha; 3: Sunthi; 4: Marica; 5: Pippali; 6: Dîpyaka; 7: Śvetajîraka; 8: Krsnajîraka; 9: Sauvarcala; 10: Saindhava; 11: Rãmatha; 12: Vida Lavana; 13: Śakrãhvaya Fig. 4 HPTLC finger prints of test solution of Laghulai curna at 366nm after derivatization Test solution of Laghulai curna for A: Batch A-Laghulai curna; B: Batch B-Laghulai curna; C: Batch C- Laghulai curna; 1: Suta; 2: Gandha; 3: Sunthi; 4: Marica; 5: Pippali; 6: Dîpyaka; 7: Śvetajîraka; 8: Krsnajîraka; 9: Sauvarcala; 10: Saindhava; 11: Rãmatha; 12: Vida Lavana; 13: Śakrãhvaya Fig. 3 HPTLC finger prints of test solution of Laghulai curna at 366nm Test solution of Laghulai curna for A: Batch A-Laghulai curna; B: Batch B-Laghulai curna; C: Batch C- Laghulai curna; 1: Suta; 2: Gandha; 3: Sunthi; 4: Marica; 5: Pippali; 6: Dîpyaka; 7: Śvetajîraka; 8: Krsnajîraka; 9: Sauvarcala; 10: Saindhava; 11: Rãmatha; 12: Vida Lavana; 13: Śakrãhvaya cuticular striations radiating from base confirm the presence of Dîpyaka. Likewise, epidermis with multicellular, multiseriate covering trichomes, fragments of epicarp showing stomata and trichomes in surface view, sclereids from mesocarp not much longer than broad indicated the presence of Śveta jîraka. In case of Krsnajîraka, sclereids from mesocarp much longer than broad, surface view of epidermis with stomata and striated cuticle but without trichomes were observed. Similarly the presence of Śakrãhvaya, can be detected by the presence of seed coat of testa in surface view, thin walled parenchymatous cells containing aleurone grains, comose trichomes with annular rings, endosperm along with oil globules, pappilose epidermal cells, oval to rectangular parenchymatous cells containing prismatic crystals of calcium oxalate (Fig. 1). Fig. 5 HPTLC finger prints of test solution of Laghulai curna visible light after derivatization Test solution of Laghulai curna for A: Batch A-Laghulai curna; B: Batch B-Laghulai curna; C: Batch C- Laghulai curna; 1:Suta; 2: Gandha; 3:Sunthi; 4: Marica; 5: Pippali; 6: Dîpyaka; 7: Śvetajîraka; 8: Krsnajîraka; 9: Sauvarcala; 10: Saindhava; 11: Rãmatha; 12: Vida Lavana; 13: Śakrãhvaya HPTLC fingerprint profile of the formulations are depicted in (Figs. 2-5) indicates the presence of all the ingredients in proportional quantity in the formulations. This confirms the batch-to-batch consistency of the finished products. Development fingerprint profile would serve as a reference standard of the formulation. The TLC plate was examined under 254nm, 366nm, after derivatization 366nm and visible light. The R f values and colours of the bands obtained were recorded. It shows major spots at 254nm R f 0.15, 0.18, 0.35, 0.40, 0.45, 0.51, 0.59 and 0.85(all black). At 366nm R f 0.15, 0.20 (both blue), 0.27(fluorescent), 0.31, 0.36, 0.44, 0.50, 0.58, 0.62 (all blue), 0.71(fluorescent), 0.79(blue). After derivatization the plate shows major spots at 366nm and visible light given (Tables 1 & 2).

5 TRIPATHI et al: PHARMACOGNOSTICAL IDENTIFICATION OF INGREDIENTS IN LAGHULAI CURHA 535 Table 1 R f values in test solution of Laghulãi cũrna at 366 nm (after derivatization) R f values Laghulãi cũrna Single ingredients A B C R f1(blue) R f2(blue) R f 3(sky blue) R f4(brown) (fluorescent) R f5(brown) (fluorescent) R f 6(blue) (fluorescent) R f 7(blue) (yellow 0.65 (yellow) R f8(brown) R f 9 (blue) R f 10(brown) R f 11(brown) : Not Appeared Table 2 R f values in test solution of Laghulãi cũrna at visible light (after derivatization) R f values Laghulãi cũrna Single ingredients A B C R f1(brown) R f2(brown) 0` R f3(brown) R f4(brown) R f5(brown) R f6(brown) R f7(brown) R f8(red) The powder microscopic diagnostic features have been established to identify ingredients of Laghulai curna. These diagnostic characters can be used for checking the adulteration and purity of this compound formulation. HPTLC finger print profile and obtained R f values helped in identification of various ingredients present in the Laghulai curna for substantiating and authenticating of crude drug. Conclusion Laghulai curna and its ingredients have numerous uses in Ayurvedic medicine and traditional medicine to treat several ailments like used in Grahani (mal absorption syndrome); Atisara (diarrhoea); Anaha (distension of abdomen due to obstruction to passage of urine and stools); Sūla (pain). Thus, the present study revealed that the characteristics microscopical features and the distinguished finger prints in the HPTLC profiles may be utilized as marker parameters for monitoring the quality of the formulation. Both parameters may be also used for standardization and quality evaluation of compound (brown) formulations. Spiking of the formulations with the different genuine ingredients further confirms the presence of individual components in them. Acknowledgement Authors are grateful to Dr Bharat Pathak General Secretary, Deendayal Research Institute, Chitrakioot for providing the research facilities. Authors are also thankful to Department of AYUSH, Ministry of Health and Family Welfare, Government of India, for financial support under the scheme Centre of Excellence. References 1 Anonymous, The drugs and cosmetics act 1940 and Rules 1945, (Stationary office, London), 1970, 395,661 and 8/9, Anonymous, The Ayurvedic Pharmacopoeia of India, Part-1, Vol-1, (Govt. of India, Ministry of Health & Family Welfare, Deptt. of Indian systems of Medicine and Homoeopathy, New Delhi) 1990, 49,73,103, 106, Anonymous, The Ayurvedic formulary of India, 1 st edn, Part II, (Government of India, Ministry of Health & Family Welfare Department of Indian Systems of Medicine and Homoeopathy, New Delhi), 2000, 7.15, 126.

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Available online at www.ijpab.com ISSN: 2320 7051 Int. J. Pure App. Biosci. 2 (5): 88-93 (2014) INTERNATIONAL JOURNAL OF PURE & APPLIED BIOSCIENCE Research Article Scientific Evaluation of Ayurvedic Compound