6. / Brief resume of the intended work:
6.1 Need for the study
Inflammatory diseases including different types of rheumatoid diseases are very common throughout the world.Although rheumatism is one of the oldest known disease of mankind and affects the large population of the world , no substantial progress has been made in achieving permanent cure.The greatest disadvantage in the presently available potent synthetic drugs lies in every toxicity and reappearance of symptoms after discontinuation.Therefore,the search of screening and development of drugs for their anti-inflammatory activity is an unending problem.There is much hope of finding antirheumatic drug from indigenous plants. 1
Inflammation is a local response of living mammalian tissues to injury due to any agent with the signs of rubor,tumor,color and dolar.It is a body’ s defense reaction in order to eliminate or limit the spread of injurious agent as well as to remove the consequent necrosed cell and tissue. 2
Since olden days ,man has been traditionally using the herbs and plant products for combating the disease.In olden days, these plant products were used in crude form but,when synthetic chemistry started ;it lead to isolation of active principles.3
The direct utilization of plant material is a feature of the systems of traditional medicine not only in the developing world but, also in Europe, where one finds a considerable researches of faith in plant derived medicines. Preparation of decoction, tinctures ,galenicals and total extracts also of isolated pure phytoconstituents in medicine has been fully documented .For making synthetic drugs, the use of intermediates or starting materials may be based upon plant products like diosgenin , hecogenin, stigmasterol etc. 4
Roots of Glycosmis pentaphylla (Retz.) Correa , has been used traditionally for inflammation.5After doing literature survey, it has been found that work on anti-inflammatory activity on roots of Glycosmis pentaphylla have not been done yet .So to give a scientific background to the above traditional claim this work has been taken up.
6.2 Review of literature
INTRODUCTION TO THE PLANT:
Botanical Name : Glycosmis pentaphylla (Retz.) Correa .
Family : Rutaceae
Vernacular name : 5
Hindi : Ban-nimbu
Kannada : Gurodagida,Manikyan
Sanskrit : Ashvashakota,Vananimbuka ,Pathalagarudi
Tamil : Anam,Kula pannai
Telugu : Golugu,Gongi padu
Malayalam : Panal,Panchi
Marathi : Kirmira
Morphology: 6
Glycosmis pentaphylla ( Retz.) Correa is a shrub or a small tree.
Leaves: 3-5 foliolate,leaflets entire to sub-dentate to sub-crenate,lateral nerves to 12 pairs.
Flower: 5-merous, in axillary, elongate ,dense racemes or cymes.
Fruit : Globose to ellipsoid with glandular pericarp.
Distribution : Almost all districts .India,Sri Lanka to S.E.Asia and W.Malaysia .
CHEMICAL CONSTITUENTS : 6
Leaves :Contains quinolone alkaloid-glycolone.
Flowers:Contains alkaloids and an amide, alkaloids-arborine, arborinine, skimmianine, glycorine, glycosmicine benzamide-2-methylamino. Also contains carbazole alkaloid-mupamine
Roots:
Contains Dictamine, γ-fagarine, skimmianine, β-sitosterol, coumarin,
stigmasterol, myricylalcohol, base glyborine, triterpenes-arborinolA,
arborinolB, arborine, arborinine,carbazolealkaloid- Glycozolinol,Glycozolicine,
3-formylcarbazole and glycosinine,glycozolidol.Root bark contains Acridone
alkaloids-Noracronycine, de-methylacronycine and e-N-methylnoracronycine,
quinazoline alkaloid-Glycophymine,glycosolone,glycolone,amide-Glycomide
6.3 Traditional uses : 5
Ø  The plant is used in indigenous medicine for cough, rheumatism, anaemia and jaundice.
Ø  The juice of the leaves ,which is bitter, is used in fever, liver complaints and as vermifuge.
Ø  A paste of the leaves with ginger is applied in eczema and skin infections.
Ø  A decoction of the root is given for facial inflammations.
REPORTED WORKS:
1.Wang J, DiY, Yang X, Li S, Wang Y , Hao X, in 2006 reported , Hydroquinone diglycoside acyl esters from the stems ofGlycosmis pentaphylla .7 Four hydroquinone diglycoside acyl esters, glypentosides A–C (1–3) and seguinoside F (4), were isolated from the stems of Glycosmis pentaphylla.
2. Pacher T, Bacher M , Hofer O , Greger H , in 2001 reported,
Stress induced carbazole phytoalexins in Glycosmis species .8
Induced formation of a series of carbazole alkaloids was observed in leaves of Glycosmis parviflora and G. pentaphylla after wounding, UV-irradiation, and particularly after inoculation with the fungus Botrytis cinerea. Detailed experiments with marked infection areas confirmed the restricted accumulation of carbazole derivatives which could not be detected in non-infected areas of the same leaf.
3. Quader MA., Nutan MTH , Rashid MA , in 1999 reported , Antitumor alkaloid from Glycosmis pentaphylla .9
Arborinine, an acridone alkaloid obtained from Glycosmis pentaphylla, exhibited significant inhibition of crown gall tumors produced by Agrobacterium tumefaciens in a potato disc bioassay.
4. Muthukrishnan J, Seifert K, Hoffmann KH , Lorenz MW,in 1999 reported, Inhibition of juvenile hormone biosynthesis in Gryllus bimaculatus by Glycosmis pentaphylla leaf compounds. 10
The EtOAc fraction of Glycosmis pentaphylla leaf extract inhibits the juvenile hormone III-biosynthesis in vitro of corpora allata from 3 day old females of the field cricket Gryllus bimaculatus. The bioactive compound which is responsible for this activity was identified as the quinazolone alkaloid arborine. This alkaloid showed also a larvicidal activity against the mosquito Culex quinquefasciatus
5. Bhattacharyya P, Chowdhury BK, in 1985 reported ,Glycolone, a quinolone alkaloid from Glycosmis pentaphylla11
Glycolone, a quinolone alkaloid has been isolated from the leaves of Glycosmis pentaphylla. The structure of the compound has been established as 4,8-dimethoxy-3-(3-methyl but-2-enyl)-2-quinolone from physical and chemical evidences.
6.Bhattacharyya P, Chakrabartty PK , Chowdhury BK ,in 1985 reported, Glycozolidol, an antibacterial carbazole alkaloid from Glycosmis pentaphylla.12
Glycozolidol, a new carbazole alkaloid, has been isolated from the roots of Glycosmis pentaphylla. Its structure has been established as 6-hydroxy-2-methoxy-3-methylcarbazole on the basis of physical and chemical evidence. The compound has been found to be active at some Gram-positive and Gram-negative bacteria.
7.  Mukherjee S, Mukherjee M, Ganguly SN , in 1983
reported , Glycozolinine, a carbazole derivative from Glycosmis
pentaphilla. 13.
A new carbazole derivative, glycozolinine, was
isolated from the seeds of Glycosmis pentaphylla. From physical and
chemical evidence its structure is 6-hydroxy-3-methylcarbazole.
6.3 OBJECTIVES OF THE PRESENT STUDY :
► Pharmacognostic evaluation Glycosmis pentaphylla (Retz.) Correa roots.
► Successive extraction using different solvents of increasing polarity.
► Phytochemical analysis of various extracts and isolation
and characterization of phytoconstituents by chromatography and
pectral studies respectively.
► Evaluation of extracts for anti-inflammatory activity.
7 / Materials and methods:
7.1 Source of data :
·  Published research papers.
·  Review articles from journals
·  Electronic data(internet)
·  Library of K.L.E.S’s College of pharmacy
7.2Method of collection of data:
Glycosmis pentaphylla(Retz.)Correa. roots will be collected from the
local areas and nearby states of Karnataka.
Authentication :
Renowned botanist will authenticate the plant.
Pharmacognostic studies:
For the present study the Glycosmis pentaphylla roots will be collected ,dried and coarsely powdered and subjected for Pharmacognostic evaluation including microscopic ,macroscopic and other physical constant.
The powdered drug will be extracted using soxhlet apparatus by Successive extraction using different solvents of increasing polarity.Extracts will be concentrated and further subjected to qualitative chemical tests,Chromatographic studies, Spectroscopic analysis and anti-inflammatory activity in proposed model.
Acute toxicity study : 14
The Albino mice of either sex will be used during investigation .The animals are fasted over night prior to the experimental procedure .The OECD guideline no-420 fixed dose method will be adopted and accordingly doses of different extracts will be calculated.
SCREENING OF ANTI-INFLAMMATORY ACTIVITY :
a.CARRAGEENIN INDUCED RAT PAW OEDEMA IN RATS :
Wister albino rats are divided into 6 groups ,of 6 animal in each.
In that one group serves as control,second as standard and remaining for test drugs.Acute inflammation is produced by injecting 0.1 ml of 1%Carrageenin into sub-planter surface of rat hind paw. 15 The test extracts, reference standard drug,and control receives saline vehicle 30 min prior to carrageenin injection.The paw volume has to be measured at 0,0.5,1,2,3,4,5 & 6 hrs using plethysmograph apparatus
b.COTTON WOOL GRANAULOMA :
Male Wistar rats with an average weight of 200 gms are anesthetized with ether.The back skin is shaved and disinfected with 70% ethanol.An incision is made in the lumbar region.By a blunted forceps subcutaneous.Tunnel are formed and a sterilized cotton pellet is placed on both sides in the scapular region.The pellets are either standardized for use in dentistry weighing 20 mg or from raw cotton,which produce a more pronounced inflammation than bleached cotton.The animals are treated for 7 days subcutaneously or orally.Then the animals are sacrificed, the pellets prepared and dried until the weight remains constant.The net dry weight i.e. after subtracting the weight of the cotton pellet will be determined. 16
7.3  Does the study require any investigation or interventions to be conducted on the patients or other human/animals? If so, please describe briefly?
Yes,the above study requires investigations to be done on the albino rats and mice of Wistar strains for the determination of acute anti-inflammatory activity. The study will be planned in accordance with the procedure reported in literature.
7.4  Has ethical clearance been obtained from your institution in case of 7.3?
Applied for institutional ethical committee clearance clearance for use of animals.
8 / List of references.
1.Winter CA,Risely Anti-inflammatory and anti-pyretic activities of
indomethacin.J.Pharmacol.exp.ther.1963;141,396.
2. Harshmohan,Textbook of pathology,4 th Ed.Jeypee brother Medical
Publisher;2005.
3. Water JB.General pathology.NY 6 th ed.1987.
4. Derek A.Introdution inflammation Mediators and mechanisms.Brit.med.bull.
1987;43 (2):247-55.
5. Anonymous.The Wealth of India,A Dictionary of Indian Raw Materials
and Industrial Products Publication & Information Directorate,Vol- IV,
CSIR;2003.
6. Yoganarsimhan SN, Medicinal plants of India,Tamil Nadu volumeІI nd .
Srinivasan for interline Publishing Pvt Ltd,Bangalore;1996.
7. Wang J, Di Y, Yang X , Li S, Wang Y Hao X. Hydroquinone diglycoside
acyl esters from the stems of Glycosmis pentaphylla. Phytochemistry 2006
March;67(5): 486-91.
8 .Pacher T, Bacher M , Hofer O, Greger H. Stress induced
carbazole phytoalexins in Glycosmis species .
Phytochemistry 2001 September ;58(1):129-35.
9. Quader MA., Nutan MTH, Rashid MA. Antitumor alkaloid from
Glycosmis pentaphylla . Fitoterapia 1999 June 1; 70(3):305-07.
10. Muthukrishnan J, Seifert K, Hoffmann KH, Lorenz MW. Inhibition
of juvenile hormone biosynthesis in Gryllus bimaculatus by
Glycosmis pentaphylla leaf compounds. Phytochemistry 1999 Jan;50(2):
249-54.
11.Bhattacharyya P , Chowdhury BK. Glycolone, a quinolone alkaloid
From Glycosmis pentaphylla.Phytochemistry 1985;24(3): 634-35.
12. Bhattacharyya P, Chakrabartty PK, Chowdhury BK. Glycozolidol,
an antibacterial carbazole alkaloid from Glycosmis pentaphylla .
Phytochemistry1985;24(4):882-83.
13. Mukherjee S, Mukherjee M , Ganguly SN. Glycozolinine, a carbazole
derivative from Glycosmis pentaphilla. Phytochemistry 1983;22(4):1064-65.
14. OECD[Organisation for Economic Co-operation and Development].
Guideline 420:Acute oral toxicity- Fixed dose procedure,Paris:OECD ;1992.
15. Winter CA, Risely EA ,Nuss . Carrageenin Induced Oedema in Hind
paw of the Rat as Assay for Anti-inflammatory Drug.Proceedings of
Society of Experimental Biology Medicine 1962 ;111:544-47 .
16. Gerhard Vogel H, Wolf gang,Vogel H . Drug discovery and
Evaluation Pharmacological Assay.2 nd ed.Springer-Verlag,Heidel berg,
(NY): 2002.