www.ijpsonline.
com
medium and DMF was used as control. The zone of REFERENCES
inhibition was compared with standard drugs after
24 h of incubation at 25° for antibacterial activity 1. Khalaj A, Abdollahi M, Kebriaeezadeh A, Adibpour N, Pandi Z,
Rasoulamini S. The antinociceptive and antiinflammatory activities
and 48 h at 30° for antifungal activity. The zone and lack of ulcerogenicity of a benzodioxin-4-one and its analog
of inhibition obtained for the test compounds and benzoxazine As cyclic acetal-like derivatives of salicylic acid and
reference compounds are presented in Table 2 and salicylamide in mice and rats. Indian J Pharmacol 2002;34:184-8.
2. Mashevakaya IV, Anikina LV, Vikharev YB, Safin VA, Koltsova SV,
fig. 2. Maslivets AN. Synthesis and antiinflammatory activity of the products
of interaction of 4-aroyl-1,2-dihydro-4h-pyrrolo[5,1-c][1,4]benzoxazine-
Compounds tested for antiinflammatory activity 1,2,4-triones with 4-amino-1,2,4-triazole. Pharm Chem J 2001;35,414-7.
3. Gokhan N, Erdogan H, Durlu NT, Demirdamar R. Analgesic activity
showed inhibition of edema induced by carrageenan of acylated 2-benzoxazolinone derivatives. Il Farmaco 1999;54:112-5.
injection ranging from 25 to 83.3%. Compound 4. Teller J. In: Schaumann E, editor. Houben-Weyl Methods of Organic
4j was found to be most potent compound with Chemistry, Hetarenes IV. Six-membered and larger hetero-rings with
maximum unsaturation. Stuttgart: Georg Thieme; 1997. p. 141-77.
83.3% inhibition of edema, which is more than the 5. Beach M, Frechette R. 2-Substituted amino and thioalkylbenzoxazine
standard (79.5%) and 4e was found equipotent to antimicrobial agents. WO Patent Appl 1997;9728167.
the standard. The compounds tested for analgesic 6. Waisser K, Gregor J, Dostal H, Kunes J, Kubicova L, Klimesova V,
et al. Influence of the replacement of the oxo function with the thioxo
activity showed protection in the range of 53–66%, group on the antimycobacterial activity of 3-aryl-6,8-dichloro-2H-1,3-
and the compound 4j was the most active (65.35%) benzoxazine-2,4 (3H) diones and 3-arylquinazoline-2,4(1H,3H)-diones.
among all. indomethacin was used as standard and Il Farmaco 2001;56:803-7.
7. Waisser K, Perina M, Kunes J, Klimesova V, Kaustova J. 3-Benzyl-
the percent protection observed was 66.25%. All the 2H-1,3-benzoxazine-2,4(3H)-diones, a new group of antimycobacterial
synthesized compounds were tested for antibacterial compounds against potentially pathogenic strains. Il Farmaco
and antifungal activity. All the test compounds 2003;58:1137-49.
8. Largeron M, Mesples B, Gressens P, Cecchelli R, Spedding M,
inhibited E. coli, S. aureus and A. niger but only Ridant AL, et al. The neuroprotective activity of 8-alkylamino-1,
compound (4c, 4e, 4j) were potent in inibiting growth 4-benzoxazine antioxidants. Eur J Pharmacol 2001;424:189-94.
of B. subtilis. Compound (4c, 4h, 4j) showed zone of 9. Ilas J, Anderluh PS, Dolenc MS, Kikelj D. Recent advances in the
synthesis of 2H-1,4-benzoxazin-3-(4H)-ones and 3,4-dihydro-2H-1,4
inhibition 19, 25 and 20 mm, 18, 20, 15 mm and 20, benzoxazines. Tetrahedron 2005;61:7325-48.
22, 18 mm against A. niger, E. coli and S. aureus, 10. Winter C, Risley E, Nuss G. Carrageenan-induced oedema in hind paw
respectively were found comparable to standard of rats as an assay for antiinflammatory drugs. Proc Soc Exp Biol Med
1962;111:544-7.
drugs with 22, 26 and 22 mm zone of inhibition
11. Seigmund E, Cadmus R, Lu G. A method for evaluating both non-
respectively. narcotic and narcotic analgesics. Proc Soc Exp Biol Med 1957;95:729-31.
12. Microbiological assay of antibiotics. In: Indian Pharmacopoeia. Vol. 2.
Delhi: The Controller of Publications; 1996. p. A-100.
ACKNOWLEDGEMENTS
One of the authors Mymooona Akhter is thankful to UGC Accepted 28 January 2011
Revised 20 November 2010
for financial support. The authors are grateful to Janab
Received 17 March 2010
Abdul Mueed Sb. for providing infrastructure and facility
Indian J. Pharm. Sci., 2011, 73 (1): 101-104
to carry out this work.
Determination of Anthelmintic Activity of the Leaf and Bark
Extract of Tamarindus Indica Linn
S. S. DAS1, MONALISHA DEY AND A. K. GHOSH2*
Department of Pharmacology, Gupta College of Technological Sciences, Ashram More, Asansol - 713 301, 1Laboratory
of Pharmacotherapy of Life-Style Related Diseases, Graduate School of Pharmaceutical Sciences, Tohoku University,
6-3 Aoba Aramaki, Aoba-Ku, Sendai, Miyagi 980 - 8578, Japan, 2School of Pharmaceutical Sciences, Vinayaka Mission
Sikkim University, Tadong, Gangtok - 737 542, East Sikkim, India
*Address for correspondence
E-mail: akg_mail@yahoo.com
104 Indian Journal of Pharmaceutical Sciences January - February 2011
www.ijpsonline.com
Das, et al.: Anthelmintic Activity of Tamarindus Indica Linn
The aim of the present study was to evaluate the anthelmintic activity of ethanolic and aqueous extract of leaves and
bark of Tamarindus indica Linn using Pheretima posthuma and Tubifex tubifex as test worms. The time of paralysis
and time of death were studied and the activity was compared with piperazine citrate as reference standard. The
alcohol and aqueous extract of bark of Tamarindus indica exhibited significant anthelmintic activity as evidenced by
decreased paralyzing time and death time. The results thus support the use of Tamarindus indica as an anthelmintic
agent.
Key words: Anthelmintic activity, Pheretima posthuma, piperazine citrate, Tamarindus indica, Tubifex tubifex
Helminth infections are among the commonest The shade-dried leaves and bark were pulverized
infections in man, affecting a large proportion of the into coarse particles and extracted with water by
world's population. In developing countries they pose maceration (5% chloroform water) and with absolute
a major threat to public health and contribute to the ethanol using Soxhlet extractor for 72 h. Both the
prevalence of malnutrition, anemia, eosinophilia, and aqueous and alcohol extract were concentrated in
pneumonia. Anthelmintics are drugs that either kill a rotary evaporator at a temperature less than 45°
or expel infesting helminths and the gastrointestinal and preserved in desiccators for further use. The
tract is the abode of many helminths, although some yield for alcoholic extract and aqueous extract were
also live in tissues, or their larvae migrate into 49.40% and 47.08%, respectively. The preliminary
tissues. They harm the host by depriving him of phytochemical analysis[5,6] were carried out to find
food, causing blood loss, injury to organs, intestinal out the phytoconstituents present in the crude
or lymphatic obstruction and by secreting toxins. extracts.
Helminthiasis is rarely fatal, but is a major cause of
morbidity[1]. Indian earthworm Pheretima posthuma (Annelida)
were collected from the water logged areas of soil,
Tamarindus indica is a tree belonging to the family the average size of earthworm being 6-8 cm. They
Caesalpiniaceae whose different parts are used as were washed with tap water for the removal of
traditional medicine as analgesic, antiinflammatory, the adhering dirt. Aquarium worms Tubifex tubifex
diuretic, febrifuge, and anthelmintic, antifungal and (Annelida) were collected from the local market.
in gastrointestinal problems [2] . The anthelmintic The average sizes of the worms were 1-1.5 cm. The
activity of leaves has been reported by Sampat et anthelmintic assay was carried as per the method
al.[3] but the anthelmintic activity of the bark extracts of Ajayieoba et al[7]. with minor modifications. The
of Tamarindus indica has not been evaluated. assay was performed on adult Indian earthworm
Accordingly, this prompted us to investigate the Pheretima posthuma, due to its anatomical and
anthelmintic activity of Tamarindus indica bark physiological resemblance with the intestinal
extracts in comparison to the leaf extract. roundworm parasites of human beings. Pheretima
posthuma worms are easily available and used as a
The reference standard drug used in this experiment suitable model for screening of anthelmintic drug.
is piperazine citrate. It causes hyperpolarization The assay was also performed on the aquarium
of muscle by its GABA agonistic action opening worm, Tubifex tubifex, because they belong to same
Cl – channels that causes relaxation and depresses group of Annelida.
responsiveness to contractile action of acetylcholine
thereby flaccid paralysis occurs. The worms recover Briefly, 20 ml formulations containing three
if placed in a piperazine free medium[4]. different concentrations, each of crude alcoholic
and aqueous extract of bark and leaf (5, 10 and
The plant material was collected from the village 15 mg/ml in double distilled water) were prepared
Radhamadhabpur of District Bankura in September and six earthworms (same size) and approximately
during morning hours between 09.00 AM and 10.00 5 g of Tubifex worms were placed in it. Both
AM. They were identified at the Botanical Survey the test solution and standard drug solution were
of India, Shibpur, Howrah. Reference Number of the freshly prepared and ‘time for paralysis’ was noted
Authentication Letter is CNH/I-I/ (20)/2009/Tech.II/29. when no movement of any sort could be observed
January - February 2011 Indian Journal of Pharmaceutical Sciences 105
www.ijpsonline.com
TABLE 1: ANTHELMINTIC ACTIVITY OF ALCOHOLIC AND AQUEOUS EXTRACT OF LEAF AND BARK OF
TAMARINDUS INDICA
Groups Conc. Pheretima posthuma Tubifex tubifex
(mg/ml) Paralyzing time Death time Paralyzing time Death time
(min) (min) (min) (min)
Distilled water – – – – –
Bark extract (alcohol) 5 60.66±0.67* 80.66±0.67* 63.00±2.08* 75.33±1.45*
10 36.33±0.88* 66.33±0.88* 31.33±0.67* 39.33±0.88*
15 22.33±0.88ns 45.00±0.58* 14.66±0.88* 20.66±1.33*
Bark extract (aqueous) 5 >120 NA 67.66±2.60* 81.00±1.00*
10 89.33±1.76* 115.33±0.88* 53.00±2.52* 74.33±2.08*
15 58.33±1.45* 87.66±1.45* 23.00±1.73ns 28.00±1.15*
Leaf extract (alcohol) 5 >120 NA >120 NA
10 >120 NA >120 NA
15 >120 NA >120 NA
Leaf extract (aqueous) 5 >120 NA >120 NA
10 >120 NA >120 NA
15 >120 NA >120 NA
Piperazine citrate 10 25.00±1.16 64±0.88 22.66±1.76 45.33±1.20
NA=Not applicable. Results are expressed as mean±SEM. N=3; *P<0.05, as compared to standard, ns= not significant.
except when the worms were vigorously shaken. of paralysis and time of death as 25.00 and 64.00
The ‘time for death’ of worms was recorded after min, respectively. Considering the alcoholic extract
ascertaining that the worms neither moved when of bark showed comparable activity, it would be
shaken vigorously nor when dipped in warm water important to identify the key phytoconstituents.
at 50°. A maximum time period of 120 min was
ascertained for the paralyzing as well as death time Phytochemical analysis of the crude extract revealed
of Pheretima posthuma and Tubifex tubifex worms. the presence of tannins along with other chemical
Piperazine citrate (10 mg/ml) was used as reference constituents contained within them. Tannins have
standard with distilled water as the vehicle control. been reported to produce anthelmintic activities[8,9], as
All experiments were repeated thrice. The mean they can bind to free proteins in the gastrointestinal
and SEM were analyzed statistically by ANOVA tract of host animal[10] or glycoprotein on the cuticle
followed by Dunnett’s test, P<0.05 being considered of the parasite and thereby cause deaths [11]. The
as significant. wormicidal activity of the aqueous and alcohol
extract against earthworms suggests that it is
From the observations made, a dose dependent effective against parasitic infections of humans[12]. It
paralytic effect much earlier and the time of death would be interesting to identify the active principle
was observed (Table 1). Although, both extracts responsible for the anthelmintic activity and to study
showed anthelmintic activity in a dose-dependent its further pharmacological actions.
manner but the alcoholic extract appeared to be
more effective for both types of worms. Evaluation
ACKNOWLEDGEMENTS
of anthelmintic activity was compared with reference
standard piperazine citrate. The alcohol extract of Authors thank the Chairman and Principal of Gupta
the bark of Tamarindus indica, caused paralysis College of Technological Sciences, Asansol for providing
at 22.33 min. and time of death at 45.00 min. for financial support for the entire project work.
Pheretima posthuma and 14.66 min as paralysis
time and 20.66 min as death time for Tubifex tubifex
REFERENCES
worms respectively. With the aqueous fractions
treatment of earthworm Pheretima posthuma and 1. Bundy DA. Immunoepidemiology of intestinal helmintic infection I:
worm Tubifex tubifex resulted in a paralysis time The global burden of intestinal nematode disease. Trans R Soc Trop
of 58.33 and 23.00 min respectively while time of Med Hyg 1994;8:259-61.
2. Ramankutty C, Nair RV, Warrier PK, Nambiar VP, Warrier PK, Sala
death was 87.66 and 28.00 min, respectively. The AV. Indian Medicinal Plants: A Compendium of 500 Species. Chennai
reference drug piperazine citrate showed the time India: Orient Longman Limited, Orient Blackswan; 1996. p. 5.
106 Indian Journal of Pharmaceutical Sciences January - February 2011
www.ijpsonline.com
3. Sampat VM, Mute VM, Patel KA, Sanghavi K, Mirchandani D, Forsk Leaves. Adv Pharmacol Toxicol 2009;10:13-6.
Babaria PC. Anthelmintic effect of Tamarindus indica Linn leaves 10. Athanasiadou S, Kyriazakis I, Jackson F, Coop RL. Direct anthelmintic
juice extract on Pheretima posthuma. Int J Pharm Res Dev 2009;7:1-7. effects of condensed tannins towards different gastrointestinal
4. Tripathi KD. Essentials of Medical Pharmacology. 5th ed. New Delhi nematodes of sheep: in vitro and in vivo studies. Vet Parasitol
(India): Jaypee Brothers (Medical Publishers); 2003. p. 4. 2001;99:205-19.
5. Kokate CK, Purohit AP, Gokhale SB. Pharmacognosy. 32nd ed. Pune 11. Thompson DP, Geary TG. The structure and function of helminth
(India): Nirali Prakashan; 2005. p. 1-624. surfaces. In: Marr JJ, editor. Biochemistry and Molecular Biology of
6. William CE, George ET, Daphne E. Trease and Evans Pharmacognosy. Parasites. 1st ed. New York: Academic Press; 1995. p. 203-32.
New York: W.B. Saunders; 2002. p. 214-393. 12. Chandrashekhar CH, Latha KP, Vagdevi HM, Vaidya VP. Anthelmintic
7. Ajaiyeoba EO, Onocha PA, Olarenwaju OT. In-vitro anthelmintic
activity of the crude extracts of Ficus racemosa. Int J Green Pharm
properties of Buchholzia coiaceae and Gynandropsis gynandra extract.
2008;2:100-3.
Pharm Biol 2001;39:217-20.
8. Niezen JH, Waghorn GC, Charleston WA. Growth and gastrointestinal
nematode parasitism in lambs grazing either Lucerne (Medicago sativa) Accepted 30 January 2011
or (Hedysarum coronarium), which contains condensed tannins. J Agri Revised 1 December 2010
Sci 1995;125:281-9. Received 15 February 2010
9. Shrestha B, Basnett H, Babu VD, Patel SS. Anthelmintic and
Antimicrobial activity of the chloroform extract of Pergularia Daemia Indian J. Pharm. Sci., 2011, 73 (1): 104-107
Determination of Alkyl Methanesulfonates in Doxazosin
Mesylate by Gas Chromatography-mass Spectrometer
C. SITARAM1, R. B. RUPAKULA*, B. N. REDDY1 AND C. S. P. SASTRY2
Department of Chemistry, College of Science, GITAM University, Visakhapatnam - 530 045, 1Analytical Research
and Development Department, Ogene systems (I) Pvt. Ltd, Plot No. 11-6-56, GSR Estates, Balanagar,
Hyderabad - 500 037, 2Department of Chemistry, Andhra University, Visakhapatnam - 530 045, India
Sitaram, et al.: Methanesulfonic Esters in Doxazosin Mesylate
High sensitive rapid gas chromatography-mass spectrometry method for the determination of four carcinogenic alkyl
methanesulfonates viz. methyl methanesulfonate, ethyl methanesulfonate, isopropyl methanesulfonate and n-butyl
methanesulfonate in doxazosin mesylate has been presented by using selective ion monitoring mode. The optimum
separation was achieved between methyl methanesulfonate, ethyl methanesulfonate, isopropyl methanesulfonate and
n-butyl methanesulfonate on a DB-5 (30 m×0.32 mm×1.0 μm) capillary column under programming temperature.
Acetonitrile, water and ammonia (90:9:1 v/v/v) mixture was used as diluent. Various factors involved in the gas
chromatography-mass spectrometry method development are also presented. This method was validated as per
International Conference on Harmonization guidelines. The limit of quantitation of methyl methanesulfonate,
ethyl methanesulfonate, isopropyl methanesulfonate and n-butyl methanesulfonate is 6 ppm with respect to 30
mg/ml of doxazosin mesylate.
Key words: Alkyl methanesulfonate, doxazosin mesylate, genotoxic, method development, validation
Alkyl mesylate esters of short chain alcohols are Since, methanesulfonicacid (MSA) or mesylate is
reactive, genotoxic and possibly carcinogenic the counter ion of doxazosin mesylate (DZM), the
alkylating agents. Regulatory authorities made usage of methyl, ethyl, isopropyl or n-butyl alcohol
it mandatory to study and submit the impurity either at any stage of synthesis or crystallization,
profile for the active pharmaceutical ingredients leads to the formation of genotoxic methyl, ethyl,
(APIs). Doxazosin mesylate chemically described as isopropyl and n-butyl methanesulfonates, respectively.
piperazine, 1-(4-amino-6,7-dimethoxy-2-quinazolinyl)- As on today, a threshold of toxicological concern
4-[(2,3-dihydro-1,4-benzodioxin-2-yl)carbonyl]-, mono (TTC) value of 1.5 µg/day intake of a genotoxic
methanesulfonate is an antihypertensive agent[1-4] used impurity is permitted. The permitted quantity in
in the treatment of benign prostatic hypertrophy. ppm is the ratio of TTC in µg/day and dose in g/
day [5] . Since, 8 mg of DZM is administered per
*Address for correspondence day[6]. Permissible intake of genotoxic impurities is
E-mail: rrcbabu7@yahoo.in 188 ppm/day. Even though some analytical methods
January - February 2011 Indian Journal of Pharmaceutical Sciences 107
