PHYTOCHEMICAL INVESTIGATION AND EVALUATION FOR ANTHELMINTIC ACTIVITY OF ETHANOLIC EXTRACT OF
CINNAMOMUM TAMALA NEES EBERM LEAVES
M.PHARM DISSERTATION PROTOCOL
SUBMITTED TO
Rajiv Gandhi University of Health Science, Karnataka Bangalore
BY
LOVHARE RAHUL BATUSING B.Pharm
Under the guidance of
Dr. G. K. KAPSE M.Pharm., Ph.D
PROFESSOR
DEPARTMENT OF PHARMACEUTICAL CHEMISTRY
LUQMAN COLLEGE OF PHARMACY, GULBARGA-585102
2011-2012
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA, BANGALORE
ANNEXURE II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
1. / Name of the candidateAnd address
/ MR. lOVHARE RAHUL BATUSINGc/o mr.b.c.johari (postman-shahada)
NITIN NAGAR , PLOT NO.08, INFRONT OF
MAHAVIR HOSPITAL, SHAHADA
post: shahada-425409. dist: nandurbar, [MAHARASHTRA]
2. / Name of the institution / Luqman college of pharmacy,
old jewargi road,
gulbarga-585102.
3. / Course of study and subject / m.pharm
(Pharmaceutical Chemistry)
4. / date of adimission to course /
08 -06-2011
5. / TITLE OF THE TOPIC: / PHYTOCHEMICAL INVESTIGATION AND EVALUATION FOR ANTHELMINTIC ACTIVITY OF ETHANOLIC EXTRACT OF
CINNAMOMUM TAMALA NEES EBERM LEAVES
6 / Brief Resume of the intended work:
6.1 Introduction
Helminthiasis or worm infestation is one of the most prevalent disease and one of the most serious public health problems in the world. Hundreds of millions if not billions of human infections by helminthes exist worldwide and with increased world travel and immigration from the developing countries1.
Antihelmintics are the drugs that expel parasitic worms (helminthes) from the body by either stunning (vermifuges) or by killing (vermicides) them. For the treatment of helminthiasis, there are allopathic drugs like Albendazole, Mebendazole, Piperazine, Levamisole, Dethyl carbamazine are available. Also, there are herbs used are Artemisia absinthium, Adiantum capillus, Cedrus deodara, Nerium indicum, Cassia angustifolia etc2.
6.2 Need for the study:
There are different approaches for the selection of plants that may contain new biologically active compounds3. One of the approaches used is the ethno medical data approach, in which the selection of a plant is based on the prior information on the folk medicinal use of the plant. It is generally known that ethno medical data provides substantially increased chance of finding active plants relative to random approach4.
One of the such finding is that reveals the use of plant Cinnamomum tamala Nees & Eberm which has been used in Indian folk medicine as tonic to brain, anthelmintic, diuretic and treating diseases of the anus and rectum5.
Cinnamomum tamala Nees & Eberm belonging to the family Lauraceae, commonly known as Tejpat, is a small evergreen tree found throughout India along the North-Western Himalayas, Sikkim, Assam, Meghalaya, Central and Southern India. Plant is highly distributed sub tropical Himalaya and cultivated as spice in India6.
They are habitat in tropical & subtropical Himalayas (3000-7800 ft) and in Sylhet and Khasia hills (3000-4000 ft)7. Bark of the plant is aromatic, stimulant and is also used to treat rheumatism, colic diarrhea and scorpion sting. Leaves are tonic to brain, diuretic, hypoglycemic and also used to treat respiratory tract infections5.
The present study is an attempt to investigate the phytochemical composition, isolation and evaluation for the anthelmintic activity of ethanolic extract and its isolated compounds of Cinnamomum tamala Nees & Eberm leaves.
The Cinnamomum tamala Nees & Eberm belongs to family Lauraceae, which has been claimed to possess anthelmintic activities also5. Literature survey reveals that a detailed chemical investigation on the leaves of Cinnamomum tamala Nees & Eberm and its evaluation for its biological activities is required which has not been explored completely yet7-11. Hence, thorough phytochemical and pharmacological studies can be carried out using this plant material. The investigations on these bioactive entities of Cinnamomum tamala Nees & Eberm leaves will yield information about chemical constituents and evaluation of the extracts and isolated compounds for the above mentioned activities that might be of great use for scientists involved in various facets of Pharmaceutical research7.
6.3 Objective of the study:
1. Identification and authentication of the plant Cinnamomum tamala Nees & Eberm.
2. Collection and shade drying of the plant material of Cinnamomum tamala Nees & Eberm leaves.
3. Soxhlet extraction of the leaves powder by sequential extraction with suitable solvents i.e. petroleum ether (40-450C), chloroform, ethanol (95%) followed by distilled water.
4. Preliminary phytochemical screening to investigate the chemical composition of all the above mentioned extracts.
5. Isolation and characterization of pure compounds of ethanolic extract of Cinnamomum tamala Nees & Eberm leaves.
6. Screening of the ethanolic extract and isolated compound(s) for their in vitro anthelmintic activity2.
6.4 Review of literature:
· Anand Murari Saxena et al., (2009)7 studied the antidiabetic potential of the 95% ethanolic extract Cinnamomum tamala Nees leaves was assessed in single oral dose of 250mg/kg body weight in normal fasted, fed, glucose loaded and Streptozotocin-induced diabetic male albino rats. Diabetes was confirmed by checking urine glucose by diastex after 48 hours of Streptozotocin injection. Further, effect of crude extract was studied on muscle glycogen content and histology of pancreatic beta-cells granularity of normal rats to probe in to its mechanism of action.
· Varsha Dhulasavant et al., (2011)8 carried out the investigation on the hypolipidemic effect of Cinnamomum tamala Nees leaves extracts in high cholesterol diet induced male albino rat (wister strain) to cause hyperlipidemia. Aqueous and ethanolic extracts of leaves of Cinnamomum tamala Nees were administered in doses of 400mg/kg /day p.o. each for 10 days. Simultaneous administration of Cinnamomum tamala Nees leaves extracts were shown to be significantly (p<0.001) and prevent the rise in serum levels of total cholesterol, triglyceride, LDL-C, VLDL-C and atherogenic index was significant (p<0.01) in increasing the level of HDL-C.
· Kapoor I P S, Singh B et al., (2009)9 studied antioxidant & antimicrobial potential of extract of methanol, ethanol, iso-octane and carbon tetrachloride oleoresins of Tejpat leaf and reported that they exhibited strong inhibition against E.coli at 3000ppm. Essential oil and oleoresins showed good to moderate activity against other tested bacteria i.e., Staphylococcus aureus, Pseudomonas aeruginosa, Protes vulgaris, Klebsiella pneumoniae and Bacillus cereus. And the study suggested that volatile oil and oleoresins of Tejpat leaf posses excellent antibacterial properties even at very low concentration.
· Eswaran M B et al., (2010)10 undertaken study to investigate the effect of Cinnamomum tamala Nees leaf extract has shown gastroprotective activity on experimental gastric ulcers in rats. Cinnamomum tamala leaf extract (CTE; 50,100 and 200mg/kg body weight) was administered orally, twice daily for 5 days for prevention from ethanol (EtOH)-, cold-restraint stress (CRS)- and pylorus ligation (PL)-induced ulcers. Estimation of H(+)K(+) ATPase activity and gastric wall mucous were performed in EtOH-induced ulcer model, antioxidant enzyme activities was carried out in CRS-induced ulcer model and various gastric secretion parameters like volume of gastric juice, acid output, and pH value were estimated in PL-induced ulcer mode. A significant reduction in lesion index was observed in ulcer-induced animals treated with CTE at different doses when compared with ulcerated rats in all models.
· Manmeet S Saluja et al., (2010)11 performed this particular study for the explosion of antitumor activity of acetone and ethanol extracts from the leaves of Cinnamomum tamala Nees against the Ehrlich Ascites Carcinoma(EAC) in mice. This particular activity was assessed by using survival time, peritoneal cell count, hematological studies, solid tumor mass, histopathological studies and in vitro cytotoxicity. In peritoneal cell count, acetone and ethanolic extracts is indirectly towards the tumor cell. In hematological studies the blood was drawn from each mouse by the rectro orbital plexus method and the WBCs, RBCs, Hb, PCV count shows reasonable effects. Wise in the solid tumor mass the mice divided into 3 groups and the tumor cells injected into the right hind limb i.e. thigh region of mice by I.M. route.
7. / Materials and Method
7.1 Method of collection of data:
· From library based books.
· From available literature survey.
· Web sites :
i) www.pubmed.com,
ii) www.scirus.com,
iii) www.herbmed.com,
iv) http://jgate-helinet.informindia.co.in
7.2 Operational definition:
7.2-1 Collection of plant material:
· The plant specimen will be identified and authenticated by taxonomist. The leaves are collected, shade dried and then powdered to the 22 mesh size and stored in an airtight container.
7.2-2 Method of extraction, isolation and characterization:
· The powder of dried leaves of Cinnamomum tamala Nees & Eberm was extracted using Soxhlet extraction apparatus. In the extraction procedure a total amount of 2000gm powdered leaves are extracted with each solvent. The extraction of the powder of leaves of Cinnamomum tamala Nees & Eberm would be carried out using the solvents petroleum ether followed by chloroform, ethanol (95%) and finally water sequentially according to the increase in their polarity. Each solvent will be run for 20 cycles and each extract is filtered and then concentrated by removing the excess of the solvent by distillation to obtain crude extract. It is then dried in rotary evaporator. On successive extraction of leaves of Cinnamomum tamala Nees & Eberm with different solvents differing in their polarity, results in separation of constituent(s) of different polarity range in different solvent fractions.
The extracts thus obtained will be concentrated and evaporated under reduced pressure and controlled temperature. The ethanolic extract is subjected to column chromatography packed with the silica gel C as adsorbent in the column. The column will be eluted with the 100% petroleum ether, petroleum ether : benzene, 100%benzene, benzene : chloroform, 100%chloroform, chloroform: methanol, 100%methenol within their polarity (95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35.60:40, 55:45, 50:50, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, 5:95).
7.2-3 Analysis of compound
· U.V., I.R. absorption studies
· Mass, NMR spectral studies
7.2-4 In vitro Anthelmintic activity studies:
· Healthy adult Indian earthworm, Pheretima posthuma (Annelida, Megascolecidae) will be used for evaluating the anthelmintic activity due to its anatomical and physiological resemblance with the intestinal round worm parasites of human beings 12.
· The anthelmintic activity will be evaluated using required number of groups, each consisting of six approximately equal sized Indian earthworms.
· The various extracts would be evaluated for anthelmintic activity at the dose levels of 2.5, 5, 10, 25 and 50mg/ml concentrations13. This wide range of dose is taken to establish the relationship between dose and pharmacological activity and also to find out the minimum and maximum dose that can be better therapeutically effective in comparison to the standard drug.
· During the study, observations will be made for the time taken for paralysis and/or death of individual worms. Paralysis is said to occur when the worms do not revive even in normal saline water. Death is ascertained when the worms lose their motility followed with fading away of their body color.
· Piperazine citrate (15 mg/ml) is used as a reference standard for comparison of the anthelmintic activity.
· Assessment of anthelmintic activity by statistical analysis:
The data on biological study are reported as mean ± Standard deviation (n=5).
For determining statistical significance, standard error mean and analysis of variance
(ANOVA) at 5% level is employed. P values <0.05 will be considered as significant14.
8 / 7.3 Does the study require any investigations or interventions to be conducted on
Patients or humans or animals? If so, please describe briefly.
------No------
7.4 Has ethical clearance been obtained from your institution in case of 7.3?
------Not Applicable------
List of References:
1. Williams DA, Lemke TL. Parasitic Infections- Helminthes In: Foye’s Principles of Medicinal Chemistry. 5th Ed. New York: Lippincott William and Wilkins; 2002.
2. R.Ramasubramaniraja and M. Niranjan Babu. Anthelmintic Studies and Medicinal Herbs-An overview. Int. J.Pharmaceutical Sci. Review and Resarch;2010:vol5(3):007:39-47.
3. Chapuis J, Sordat B, Hostettmann K. J Ethanopharmacol. 1988, 23, 273-284.
4. Bradu BL, Sobti SN. Indian perfumer, 1988, 32, 4, 334-340.
5. Tripathi SN, Tiwari CM, Uphday BN, Singh RS. Screening of hypoglycemic action in certain indigenous drugs: short research communication. J.Res.Ind.Med.Yoga.Homeo:1979:14:3-4.
6. Ashok Majumadar. Home Remedies in Ayurveda. Amar Granth Publication. 363-364.
7. Anand Murari Saxena, Rahul Gupta. Hypoglycemic activity of ethanol extract of Cinnamomum tamala leaves in Normal and Streptozotocin Diabetic Rats. Iranian Journal of Pharmacology & therepeutics, 2009:(8)17-21.
8. Varsha Dhulasavant. Antihyperlipidemic activity of Cinnamomum tamala Nees on high cholesterol diet induced hyperlipidemia. Int. J. of Pharm. & Life Sci. Vol. 2, Issue 1: 2011, 506-510.
9. Kapoor IPS, Singh B, Singh G. Antimicrobial and antioxidant potential of Cinnamomum tamala Nees & Ebern.(Tejpat) essential oil and oleoresins. Natural product Radiance.2009:8(2):106-116.
10. Eswaran MB, Surendran S, Gastroprotective activity of Cinnamomum tamala leaves on experimental gastric ulcer in rats. Journal of Ethanopharmacology:2010:Mar.24;128(2):537-540.
11. Manmeet S Saluja, Sangameswaran B and Ajay Sharma. Cytotoxic activity of Cinnamomum tamala linn against Ehrlich Ascites Carcinoma (EAC) in mice. The Pharma Research, (2010), 3; 232-242.
12. Debidani Mishra et al. Phytochemical investigation and evaluation of extract from leaves of Eupatorium odoratum linn., Indian J Pharm. Education Research:44(4):369-374
13. Roshy Joseph C, Hanchezhian R, Patgiri Biswajyoti, Harish CR. Pharmacognostical study of Nagakeshara (Mesua Ferrea Linn.) – An ingredient in vyaghrihareetaki avalhea. International Journal of Research in Ayurveda & Pharmacy, 2010, 1 (2), 264-272.
14. Bolton S. Ind Pharmaceutical Statistics – Practical and clinical applications. New York: Morcel Dekker, 1997.
9. / SIGNATURE OF CANDIDATE / (LOVHARE RAHUL BATUSING)
10. / REMARKS OF THE GUIDE / The results of proposed investigation would be of significance for researchers. Hence, the proposed plan of work is recommended for registration
11.11.1 / NAME AND DESIGNATION OFGUIDE / Dr. G. K. KAPSE M.Pharm., Ph.D
PROFESSOR
11.2 / SIGNATURE
11.3 / CO-GUIDE / ------
11.4 / SIGNATURE / ------
11.5 / HEAD OF THE DEPARTMENT / Prof. G. SUDHEENDRA M.Pharm. (Ph.D.)
11.6 / SIGNATURE
12. / REMARKS OF THE CHAIRMAN AND PRINCIPAL / The necessary facilities will be provided in the institution. Hence, the proposed plan of work is recommended for registration.
SIGNATURE /