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the analysis of bulk samples of ketoprofen and salicylic acid. Asian J 2006;43:516-8. Chem 2006;18:393-6. 10. Maheshwari RK, Chaturvedi SC, Jain NK. Application of hydrotropy 2. Maheshwari RK. New application of hydrotropic solubilization in the in spectrophotometric analysis of pharmaceutical dosage form. Indian spectrophotometric estimation of ketoprofen in tablet dosage form. Drugs 2005;42:760-3. Pharm Rev 2005;3:123-5. 11. Jain NK, Agrawal RK, Singhai AK. Hydrotropic solubilization of 3. Maheshwari RK. Application of hydrotropic solubilization in the nifedipine. Pharmazie 1990;45:221-5. analysis of aceclofenac. Asian J Chem 2006;18:1572-4. 12. Da Silva RC, Spitzer M, Da Silva LHM, Loh W. Investigations on the 4. Maheshwari RK. Analysis of frusemide by application of hydrotropic mechanism of aqueous solubility increase caused by some hydrotropes. solubilization phenomenon. Indian Pharma 2005;4:55-8. Thermochim Acta 1999;328:161-7. 5. Maheshwari RK. Spectrophotometric determination of cefixime in 13. Etman MA, Hada AH. Hydrotropic and cosolvent solubilization of tablets by hydrotropic solubilization phenomenon. Indian Pharma indomethacin. Acta Pharm 1999;49:291-8. 2005;4:63-8. 14. Poochikian GD, Cradock JC. Enhanced chartreusin solubility by 6. Maheshwari RK. Novel application of hydrotropic solubilization in hydroxyl benzoate hydrotropy. J Pharm Sci 1979;68:728-32. the spectrophotometric analysis of tinidazole in dosage form. Asian J 15. Shukla RS, Patel A, Soni ML, Modi V, Jaliwala YA. Quantitative Chem 2006;18:640-4. spectrophotometric estimation of cefadroxil using hydrotropic 7. Maheshwari RK. Spectrophotometric analysis of amoxycillin in tablets solubilization technique. Asian J Pharm 2008;2:146-7. using hydrotropic solubilization technique. Asian J Chem 2006;18: 3194-6. 8. Maheshwari RK, Chaturvedi SC, Jain NK. Application of hydrotropic Accepted 21 March 2010 solubilization phenomenon in spectrophotometric analysis of Revised 16 November 2009 hydrochlorothiazide tablets. Indian drugs 2005;42:541-4. Received 30 June 2008 9. Maheshwari RK, Chaturvedi SC, Jain NK. Analysis of aceclofenac in tablets using hydrotropic solubilization technique. Indian Drugs Indian J. Pharm. Sci., 2010, 72 (2): 258-261

Pharmacognostic and Antifungal Investigations of ganitrus (Rudrakasha)

B. SINGH*, A. CHOPRA, M. P. S. ISHAR, A. SHARMA1 AND T. RAJ Pharmacognosy and Phytochemistry Laboratory, Department of Pharmaceutical Sciences, Guru Nanak Dev University, Amritsar-143 005, India, 1University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh-160 014, India

Singh, et al.: Pharmacognostic and Antifungal Activity of Rudrakasha

Rudrakasha is the dried bead obtained from the ripe fruit of Elaeocarpus ganitrus Roxb. (Family: ). Microscopic studies revealed the presence of a hard endocarp with lignifi ed isodiametric sclereids, with membranous coat, which enclosed a dense cellular endosperm comprising of calcium oxalate druses. Physicochemical parameters showed that total ash was 1.36 times and 1.56 times more than the acid insoluble ash and water-soluble ash, respectively. Further, ethanol had a maximum extractable value of 2.4% and moisture content was found to be 9.7%. Different extracts, petroleum ether, chloroform, ethanol and water were prepared. Chemically the extracts showed the presence of , fats, alkaloids, fl avonoids, carbohydrates, proteins and tannins. The extracts were evaluated for antifungal activity on different fungal strains. Chlorofom and ethanol extracts have high antifungal activity against Candida albicans. Whereas, chloroform, ethanol and water extracts showed moderate inhibition against Aspergillus niger.

Key words: Rudrakasha, Elaeocarpus ganitrus, antifungal activity, Candida albicans, Aspergillus niger

Elaeocarpus ganitrus Roxb. (Syn. E. sphaericus also called blueberry beads[1]. It finds a prominent Gaertn; family Elaeocarpaceae), is popular for its place in Hindu religion and Ayurveda, the ancient attractive fruit stones. Its beads are covered by an Indian system of medicine. In Hindi it is known as outer shell of blue color on fully ripening, hence [2]. Rudrakasha fruits are thermogenic, sedative and are useful in cough, bronchitis, neuralgia, *Address for correspondence cephalagia, anorexia, migraine, manic conditions E-mail: [email protected] and other brain disorders[3]. The flesh or pulp

March - April 2010 Indian Journal of Pharmaceutical Sciences 261 www.ijpsonline.com of drupe is given in epilepsy, diseases of head blue as per well documented method[24]. Wherever and in mental illness[4]. Besides it is reported to necessary sections were also stained with safranin, exhibit multifarious pharmacological activities that fast-green and iodine solution. Powdered material was include antiinflammatory[5], analgesic[6], sedative[6], cleared with chloral hydrate (S. D. Fine Chemicals antidepressant[7], antiasthmatic[8], hypoglycemic[4], Ltd., Mumbai, India) and mounted in glycerine antihypertensive[9-11], smooth muscle relaxant[12], (Glaxosmithkline Pharmaceuticals Ltd. Mumbai, hydrocholeretic[12], antiulcerogenic[7] and India) and stained where ever necessary. The fruit anticonvulsant[13]. A detailed study on pharmacognostic had a hard, stony endocarp or sclerotesta. The surface standards of Rudraksha is lacking. In ancient Indian of the endocarp was black, deeply folded, and hard medicine, the fruits were employed to ward off evil with ridges and furrows. Mesocarp was not seen spirits and omens[14] which can be considered to be in the fruit. The fruit included five or six carpels, at least partly manifestations of microbial infection. each carpel having a single large seed (fig. 1). In In addition, the Rudrakasha extracts also known to sectional view, the seed was elliptical and consisted exhibit very high antimicrobial activity[15]. Therefore, of a membranous seed coat. The seed coat enclosed it was decided to evaluate the drug (beads) for a dense cellular endosperm (fig. 2). The seed coat, various pharmacognostic parameters and beads though membranous, is differentiated into outer darkly extracts for antifungal activity against different fungal staining cell layer and inner vertically oblong compact strains. dark cell layer. They have dense accumulation of tannins. The middle seed coat has two or three layers Dried fruits (beads) of Elaeocarpus ganitrus were of circular brachysclereids or stone cells. Their walls procured from a cultivated source, Hind Herb Store, are highly lignified. The sclereids have dark cell Saharanpur, Uttar Pradesh, India, in the month of contents. The endosperm cells are in parallel compact September 2007. Identity of dried fruit was confi rmed rows. They extend from periphery to the centre; through Anatomy Research Centre, Medicinal the cells are squarish and thin walled. The cells Plant Research Unit, Chennai. Voucher specimen no. towards the periphery are smaller and they become PARC/2008/164 has been deposited in the herbarium gradually larger towards the centre of the seed. The of the same institute. Coarsely powdered dried fruits endosperm cells have large calcium oxalate druses or were successively extracted with petroleum ether, sphaerocrystals. The crystals are either one or two chloroform and ethanol for 48 h each using Soxhlet per cell. They are random in distribution (fi g. 3). The apparatus and finally boiled with distilled water druses are 10 μm in diameter. The stony endocarp or for 6 h. The extracts were filtered, concentrated in sclerotesta consists of only sclereids. No other cell vacuo and dried in an oven at 40-50º. After removal types or cell inclusions are evident in the powder of solvents under vacuo from various extracts, the (fi g. 4). The sclereids may be short or elongated. The percentage of various extractives obtained was: short sclereids are isodiametric. The sclereid walls petroleum ether extract (PE) 1.655, chloroform are highly lignifi ed; the cells have wide lumen with extract (CE) 0.458, ethanol extract (EE) l.538 and brownish content. water extract (WE) 1.013. Other physicochemical parameters[16,17] showed total ash of 1.5%, acid In vitro antifungal activity of all the extracts was insoluble ash 1.1%, water-soluble ash 0.96%, pet carried out using the disk-diffusion assay[25] and ether soluble extractive 1.3%, chloroform soluble broth dilution test[26,27]. The disk-diffusion assay was extractive 0.5%, ethanol soluble extractive 2.4%, applied to determine the growth inhibition of fungi water soluble extractive 1.8% and loss on drying by extracts to be tested. Overnight fungal cultures 9.7%. Phytochemical screening[18-22] gave positive (100 μl) were spread onto SDA. The extracts were test for phytosterols, fats, alkaloids, , applied to 8 mm disks (Whatman paper No.1). After carbohydrates, proteins and tannins. 48 h of incubation at 25o, the diameter of growth inhibition zones was measured. MIC of all extractives For microscopic studies the paraffin embedded was determined by broth dilution test which was specimens were sectioned with the help of rotary performed in test tubes. The conidial suspension, microtome. Thickness of the sections was 10-12 μm. which gave the fi nal concentration of 1×105 CFU/ml, De-waxing of the sections was done by customary was prepared. A growth control tube and a sterility procedure[23]. The sections were stained with toluidine control tube were used in each test. After 24-72 h

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Fig. 1: TS of stony endocarp of E. ganitrus fruit Fig. 2: TS of E. ganitrus seed Transverse section of the stony endocarp of E. ganitrus fruit showing Transverse section of E. ganitrus seed showing En- endosperm, Fu- S seeds and SE stony endocarp funicle, Isc- inner seed coat and Osc- outer seed coat

Fig. 3: TS of E. ganitrus seed showing calcium oxalate druses Fig. 4: TS of E. ganitrus seed showing thick masses of sclereids Transverse section of E. ganitrus seed with calcium oxalate druses (Dr) in the endosperm incu bation at 25o, the MIC was determined visually here that various plant extracts showed no sign of as the lowest concentration that inhibits growth, inhibition on C. glabrata and G. candidum even at evidenced by the absence of turbidity on the fungal higher concentration. strains, Asperagillus niger (MTCC-281), Candidum geotrichum (MTCC-3993), Candida albicans (MTCC- In conclusion, microscopic studies revealed the 227), C. glabrata (MTCC-1637) and C. tropicalis presence of a hard stony endocarp and elliptical seed (MTCC-230) using ketoconazole as the positive with a membranous seed coat, which enclosed a dense control. Minimum inhibitory concentration (MIC) cellular endosperm and had a dense layer of circular is the concentration required to inhibit fungal cell brachysclereids. Endosperm had a large calcium proliferation by 50% after exposure of cells to test oxalate druses or spaheorocrystal. Powder microscopy compounds. Inhibitory concentration in terms of MIC revealed that stony endocarp had only isodiametric (mg/ml) was determined using turbidimetry method sclereids, with lignified walls. Physicochemical (Table 1). Maximum inhibition was observed for CE parameters viz total ash, acid insoluble ash; water (MIC 1.5 mg/ml), followed by EE (MIC 4.0 mg/ml) soluble ash, extractive values and loss on drying on C. albicans. In the case of C. tropicallis maximum were performed. Total ash was about 1.36 times inhibition of MIC 5.0 mg/ml was observed for CE, more than the acid insoluble ash indicating the whereas, no inhibition was observed for EE and WE. presence of good acid soluble inorganic matter Maximum inhibition of MIC 3.0 mg/ml on A. niger in E. ganitrus. The water soluble ash was 1.56 was observed for CE and EE, which is followed by times less than the total ash. Out of all the solvents WE (MIC 5.0 mg/ml). It is also pertinent to mention used, ethanol had a maximum extractable value of

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TABLE 1: IN VITRO ACTIVITY OF E. GANITRUS ON VARIOUS FUNGAL STRAINS Strains Extractives Dilutions (mg/ml) 0.125 0.5 1.0 1.5 2.0 3.0 4.0 5.0 Ketocon-azole C. albicans CE +++ ++ + ------MTCC 3017 EE +++ +++ +++ +++ ++ + - - - WE +++ +++ +++ +++ +++ +++ +++ +++ - C. tropicallis CE +++ +++ +++ +++ +++ ++ + - - MTCC 230 EE +++ +++ +++ +++ +++ +++ +++ +++ - WE +++ +++ +++ +++ +++ +++ +++ +++ - C. glabrata CE +++ +++ +++ +++ +++ +++ +++ +++ - MTCC 1637 EE +++ +++ +++ +++ +++ +++ +++ +++ - WE +++ +++ +++ +++ +++ +++ +++ +++ - C. geotricum CE +++ +++ +++ +++ +++ +++ +++ +++ - MTCC 3993 EE +++ +++ +++ +++ +++ +++ +++ +++ - WE +++ +++ +++ +++ +++ +++ +++ +++ - A. niger CE +++ +++ ++ ++ + - - - - MTCC 1344 EE +++ +++ ++ ++ + - - - - WE +++ +++ +++ ++ ++ + + - - *+++ Highly turbid, ++ moderately turbid, + weakly turbid and - No turbidity. The microorganisms tested were, Candida albicans, C. tropicalis, Candida glabrata, Candidum geotrichum and Asperagillus niger

2.4%, whereas chloroform had a minimum value of Elaeocarpus sphaericus. Phytother Res 2000;14:36-9. 0.5%. Moisture content was found to be 9.7%. In 8. Singh RK, Bhattacharya SK, Acharya, SB. Studies on extracts of Elaeocarpus sphaericus fruits on in vitro rat mast cells. Phytomed phytochemical investigations, PE showed the presence 2000;7:205-7. of phytosterols along with fats and fixed oils. CE 9. Sarkar PK, Sengupta SS, Bhattacharya SS. Effect of Elaeocarpus had phytosterols. EE gave the tests for alkaloids, ganitrus Roxb. seeds on blood pressure. Indian J Pharma 1972;4: 128-35. flavonoids, carbohydrates, proteins and tannins. 10. Sarkar PK, Sengupta SS. Studies with ethylacetate extract of WE showed the presence of proteins, tannins and Elaeocarpus ganitrus seeds on mammalian heart-intact and isolated carbohydrates. All the extracts (PE, CE, EE and WE) preparations. Indian J Pharm 1972;4:129-37. 11. Sarkar PK, Sengupta SS, Bhattacharya SS. Further observations with were evaluated for the antifungal activity on different Elaeocarpus ganitrus on normal and hypodynamic hearts. Indian J fungal strains. The maximum inhibition (MIC 1.5 mg/ Pharm 1973;5:252-8. ml) was observed for CE against C. albicans. The CE 12. Bhattacharya SK, Debnath PK, Pandey VB, Sanyal AK. Pharmacological investigations on Elaeocarpus ganitrus. Planta Medica and EE showed maximum inhibitory potential (MIC 1975;28:174-7. 3.0 mg/ml) on A. niger. In conclusion the CE and EE 13. Dasgupta A, Agarwal, SS, Basu DK. Anticonvulsant activity of were found to be more active antifungals. The activity the mixed fatty acids of Elaeocarpus ganitrus roxb. (rudraksh). Indian J Physiol Pharmacol 1984;28:245-6. guided fractionation of active extracts may be taken 14. Acharya RS, editors. In: Shiv Purana. Vol 1. Barely: Sanskrit Institute; up for further development. 1976. p. 170. 15. Singh RK, Nath G. Antimicrobial activity of Elaeocarpus sphaericus. Phytother Res 1999;13:448-50. REFERENCES 16. The Indian Pharmacopoeia, Vol. 2. New Delhi: Controller of Publications; 1996. p. A48-99. 1. Pandey G editor. Dravyaguna Vijnana (Materia Medica- vegetable 17. WHO guidelines. Quality control methods for medicinal plant materials. drugs). Krishna Das Ayurvedic series 48. Part III. Varanasi: Geneva: 1998. p. 30-1. Chowkhamba Krishanadas Academy; 2004. p. 261-2. 18. Evans WC, editor. Alkaloids. Trease and Evans Pharmacognosy. 14th 2. Asolkar LV, Kakkar KK, Chakre OJ. editors. Second supplement to ed. Noida: Gopsons Papers Limited; 1996. p. 340. glossary of Indian medicinal plant with active principles. Vol. 1, suppl 19. Farnsworth NR. Biological and phytochemical screening of . J 2. New Delhi; Publication and Information Directorate, CSIR; 1992. Pharm Sci 1966;55:225-86. p. 177. 20. Evans WC, editor. Carbohydrates. Trease and Evans Pharmacognosy, 3. Available from: http://www.devshoppe.com/RudrakshaTherapy.html. 14th ed. Noida: Gopsons Papers Limited; 1996. p. 191. [cited in 2009]. 21. Evans WC, editor. Phenols and phenolic glycosides. Trease and Evans 4. Satyavati GV, Raina MK, Sharma, M, editors. Medicinal Plants of Pharmacognosy, 14th ed. Noida: Gopsons Papers Limited; 1996. p. 218. India. New Delhi: Indian Council of Medical Research; 1976. p. 370-1. 22. Khandelwal KR. editor. Preliminary Phytochemical Screening. Practical 5. Singh RK, Pandey BL. Anti-inflammatory activity of Elaeocarpus Pharmacognosy, 12th ed. Pune: Niralli Parkashan; 2004. p. 149-56. sphaericus fruit extract in rats. J Med Arom Plant Sci 1999;21:1030-2. 23. Johansen DA. Plant Microtechnique. 1st ed. New York: McGraw-Hill; 6. Katavic PL, Venables DA, Rali T, Carroll AR. Indolizidine alkaloids 1940. p. 523. with delta-opioid receptor binding affinity from the leaves of 24. O’Brien TP, Feder N, Mccull ME. Polychromatic Staining of Plant cell Elaeocarpus fuscoides. J Nat Prod 2007;69:1295-9. walls by toluidine blue O. Protoplasma 1965;59:368-73. 7. Singh RK, Acharya SB, Bhattacharya SK. Pharmacological activity of 25. Patel RP, Trivedi BM. The in vitro antibacterial activity of some

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medicinal oils. Indian J Med Res 1962;50:211-22. traditionally for treatment of fungal infections. J Ethnopharmacol 26. Newton SM, Lau C, Gurcha, S, Besra, GS, Wright CW. The evaluation 2006;108:124-32. of forty three plant species for in vitro antimycobacterial activities; isolation of active constituents from Psoralea coryfolia and Sanguinaria canadenes. J Ethnopharmacol 2002;79:57-67. Accepted 24 March 2010 27. Collier HOJ, Potter MD, Taylor EP Antifungal activities Revised 23 November 2009 of bisisoquinolinium and bisquinolinium salts. Br J Pharmacol Received 7 July 2009 1955;10:343-9. Indian J. Pharm. Sci., 2010, 72 (2): 261-265 28. Hamza OJM. Antifungal activity of some Tanzanian plants used

Simultaneous Spectrophotometric Estimation of Haloperidol and Trihexyphenidyl in Tablets

S. P. WATE AND A. A. BORKAR* Sharad Pawar College of Pharmacy, Wanadongri, Hingna Road, Nagpur–441 110, India

Wate and Borkar: Simultaneous Estimation of Haloperidol and Trihexyphenidyl

The combination of haloperidol and trihexyphenidyl is a dosage form to be used as antidyskinetic agent. Literature revealed that there is no single method for the simultaneous estimation of these drugs in tablet dosage form, which prompted us to develop a simple, rapid, accurate, economical and sensitive spectrophotometric method. The simultaneous estimation method is based on the principle of additivity of absorbance, for the determination of haloperidol and trihexyphenidyl in tablet formulation. The absorption maxima of the drugs were found to be at 245.0 nm and 206.0 nm respectively for haloperidol and trihexyphenidyl in methanol and 0.1N HCl (90:10). The obeyance of Beer Lambert’s law was observed in the concentration range of 2.5-12.5 µg/ml for haloperidol and 1.0-5.0 µg/ml for trihexyphenidyl. The accuracy and reproducibility of the proposed method was statistically validated by recovery studies.

Key words: Haloperidol, trihexyphenidyl, simultaneous estimation, recovery study

Haloperidol (HP) is an antidyskinetic and spectrophotometric method. antipsychotic drug whose IUPAC name is 4-[4-(4-chlorophenyl)-4-hydroxy-1-piperidyl]-1-(4- Shimadzu 1700 UV/Vis spectrophotometer with fluorophenyl)-butan-1-one. Trihexyphenidyl (THP) matched cuvettes was used for the experimental is an antidyskinetic and antiparkinson drug whose work. The chemicals used were of analytical grade. IUPAC name is 1-cyclohexyl-1-phenyl-3-(1-piperidyl)- Commercially available tablets of HP and THP in 1-propanol. HP is offi cial in BP[1] and THP in IP[2]. combination were procured from the local pharmacy. BP suggests a titrimetric assay method for HP, Standard HP and THP were received as gift samples while IP suggest a titrimetric assay method for THP. from Stadmed Pvt. Ltd., Kolkata. Literature survey revealed that HPLC methods[3,4] have been reported for the estimation of HP and THP Standard stock solutions of HP and THP were individually and with other drugs in pharmaceutical prepared separately by dissolving 25 mg each of dosage forms. However, no method is reported standard HP and THP in methanol and 0.1N HCl for the simultaneous estimation of these drugs in (90:10) and making up the volume to 50 ml with combined dosage forms.This prompted us to develop same solvent. Standard solutions (25 µg/ml) HP and simple, rapid, accurate, economical and sensitive THP were further prepared by taking 2.5 ml of stock solution of each drug in two 50 ml volumetric fl asks *Address for correspondence separately and making up the volume to the mark E-mail: [email protected] with same solvent.

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