“Phytochemical investigation of anthocephalus CadambaMiq.Bark and in-vitro immunomodulatory activity on Human Neutrophils”

M. Pharm Dissertation Protocol

Submitted to the

RajivGandhiUniversity Of Health Sciences, Karnataka, Bangalore

By

Datar Subrahmanyam Anantrao

Under the guidance of

Dr. Arunkumar Beknal

M.Pharm., Ph.D

DEPARTMENT OF pharmacognosy

H.K.E.SCOLLEGE OF PHARMACY,

GULBARGA – 585105

2009-10

RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES, KARNATAKA

BANGALORE

ANNEXURE – II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1 / Name of the candidate ( in block letters) / : / Datar Subrahmanyam Anantrao
Permanent address / : /
C/o A.S. Datar H.No. 1109 “Indira” Shri Ram Nagar, Jalgaon Tq: Dapoli Dist: Ratnagiri
Maharashtra State – 415712
2 / Name of the institution / : / H.K.E Society’s College of Pharmacy, Sedam Road, Gulbarga– 585105
3 / Course of study and subjects / : / Master of Pharmacy in pharmacognosy
4 / Date of admission to the course / : / 16th June 2009
5 / Title of Topic / : / “Phytochemical investigation of anthocephalus cadambaMiq.Bark and in-vitro immunomodulatory activity on Human Neutrophils”
6 / Brief Resume of the intended work
6.1 / Need for the study
Immunostimulation or immunomodulation constitutes an alternative to conventional chemotherapy and prophylaxis of infection, especially when the host defence mechanism has to be activated under conditions of impaired immune responsiveness1.
Chemotherapeutic agents available today have mainly immunosuppressive activity, most of them are cytotoxic and exert a variety of side effects. The metabolism and clinical safety has not been clearly established. This has given rise to carry out a systematic and multidisciplinary study in research of immunomodulatory agents from natural source and to unfold many efficacious drugs for use in clinical practice2.
The stem bark of the Anthocephalus cadamba mainly contains saponins A, B, C, D cadambagenic acid, D-glucose, L-rhamnose, L-fructose, Oleanolic acid and various important chemical constituents3.
The bark of Anthocephaluscadamba is used traditionally as febrifuge and tonic it is also used in cough and fever4.
However detailed pharmacological investigation of Anthocephalus cadamba bark to explore possibility of immunomodulatory activity is required. Hence, the bark of Anthocephaluscadamba has been selected for phytochemical investigation and immunomodulatory activity.
6.2 / Review of Literature
Anthocephalus cadamba Miq.
Synonyms:Anthocephalus indicus Rich, Neolamarkia Cadamba Bosser.
Family: Rubiaceae
Vernacular names
Sanskrit : Halipriya, Neepa, Vruttapushpa
Hindi: Kadam
Kannada: Kaad Vailu, Kadaga
Marathi : Kadamb, Kadam, Nhiv, Rajkadam
English : Wild Cinchona
Origin And Habitat
The plant is found in moist and warm type of deciduous and evergreen forests. It is found in the sub-Himalayan tract from Nepal eastwards on the lower hills of Dargeeling terai, in West Bengal, in west coasts of Karnataka, Kerala and Western ghats3.
Description
It is medium sized tree attaining 2m.girth and 18m.hight. Branches spreading horizontally and slightly enlarged at their junction with the main stem. Bark is dark brown, roughish, with shallow fissures, exfoliating in small irregular woody scales4.
Chemical constituents
The stem bark contains saponins – A, B,C and D cadambagenic acid, D-glucose, L-rhamnose, L-fructose, oleanolic acid, quinovic acid, β-sitosterol, cadambine cinchonine and dihydrocinchonine.
The leaves contain glycoalkaloids as 3 β-dihydrocadambine,nonglucosidic isomeric indole alkaloids as kadambine, 3-a- dihydrocadambine,glycoside alkaloids as isodihydrocadambine, β- sitosterol,cadamine, 3α-isodihydrocadambine and 3α-dihydrocadambine3.
The various parts of the plant Anthocephalus cadamba namely, bark, leaves, fruits and roots traditionally claimed for the following uses
  • The bark3 acts as anti-inflammatory, antiemetic, antibilious, carminative, digestive, expectorant, galactagogue and tonic.
  • Decoction of Leaves4 is used as gargle in case of apthae and stomatitis.
  • Juice of Fruit5 is given to childrens with cumin and sugar in gastric irritability and it is given in fever.
  • The roots3 are diuretic and used in calculus and gravels.
  • The antidiarrhoeal property of the hydroethanolic extract of the flowering tops of Anthocephalus cadamba was assessed on experimental animals. The dry hydroethnolic extract (250-500 mg/kg body mass, p.o) exhibited a dose-dependent decrease in the total number of faecal droppings in castor oil-induced diarrhoea in mice6.
  • Antimicrobial potential of Anthocephalus cadamba against a wide range of microorganism was studied. To validate the ethnotherapeutic claims of the plant in skin diseases, wound healing activity was studied, besides antioxidant activity to understand the mechanism of wound healing. The alcoholic and aqueous extract of this plant showed significant antibacterial and antifungal activity against almost all the organisms. The results indicated that Anthocephalus cadamba extract possesses potent antioxidant activity7.
The bark of the plant has been claimed to be used as tonic4 and until now there is no scientific validation for the claimed use. Hence, here is an attempt to prove it’s tonic property as immunomodulatory activity on human neutrophils.
6.3 / Objectives of the study
The overall aim of proposed study is to explore the application of traditional medicinal plants (bark) for immunomodulatory activity. The specific objectives aimed in the present work are:
  1. Extraction of Anthocephalus cadamba bark by successive solvent extraction by using different solvents.
  2. Preliminary phytochemical investigation and TLC studies.
  3. Isolation of important phytoconstituents by different chromatographic techniques.
  4. To perform detailed study of immunomodulatory activity of Anthocephalus cadamba bark on human neutrophils using different parameters.

7 / Materials and methods
7.1 / Source of data
The required data will be obtained from:
  • Electronic data (Internet)
  • Published research paper
  • Review article from journal
  • Library of HKE’s College of Pharmacy, GulbargaUniversity, Gulbarga
  • R & D and pharmaceutical industries.

7.2 / Methods of collection of data (including sampling procedure, if any)
The bark of Anthocephalus cadamba is collected from area named Dapoli Dist. Ratnagiri in MaharashtraState, and the same is anthenticated at the Botanical Survey of India, Western Regional Centre. Koregaon Road, Pune-411011.
A. Chemical Studies
Successive solvent extraction
The air-dried powdered bark will be extracted in soxhlet assembly successively with the solvents of increasing polarity. Each time before extracting with next solvent the powdered material will be air dried in hot air oven below 500C8.
Each extract will be concentrated by distilling off the solvent and then evaporated to dryness on water bath. The extract obtained with each solvent will be weighed. Its percentage will be calculated interms of air dried weight of plant material.
The obtained extractswill be subjected for ,
  1. Identification of phytoconstituents by using various qualitative tests and chromatographic techniques8.
  2. Separation and isolation of phytoconstitutents by column chromatography technique.
  3. Subjecting isolated compounds for spectral studies.
  4. Immunomodulatory activity of obtained extracts.
B. Pharmacological studies
In-vitro immunomodulatory activity on human neutrophils such as9:
  1. Nitroblue tetrazolium assay for respiratory burst activity (Quantitative method)
  2. Granulocyte phagocytosis assay – using killed candida albicans organisms.
  3. Candidicidal assay
  4. Chemotaxis assay (micropore filter method)
Other parameters like determination of total and absolute neutrophil count, plasma cortisol estimation etc.
7.3 / Does the study require any investigation or intervention to be conducted on patients or other humans or animals? if so please describe briefly
In-vitro investigation on neutrophils of healthy human volunteers will be undertaken.
7.4 / Has ethical clearance been obtained from your institution in case of 7.3 ?
The study has been cleared from ethical committee of institution
8 / List of References
  1. Hennessey LR, Barkar JR. Basic and clinical immunology edited by Stites D.P, Terr, A.I, Praslow T.G, 8th ed; Lange International, New Jersy 1994; 784.
  2. Breakmann I.I., Dardymov I.V, New Substances of plant origin which increase non specific resistance, Ann. Rev. Pharmacol 1986; 9: 419.
  3. Deshpande DJ. A Hand book of Medicinal Herbs, Agrobio of India, Agro house behind Nusrani Cinema Chopa Sani road Jodhpur; 2006 Page No. 150 to 152.
  4. Kirtikar KR.and Basu BD. Indian Medical plants, Vol.II Page No.1251-1252.
  5. Nadkarni KM “ Indian Materia Medica”. Popular Prakashan, Bombay, Revised and Enlarged edition Vol. No.I Page. No.118 to 119.
  6. ALAM M. Ashraful et al. Antidarrhoael property of the hydroethanolic extract of the flowering tops of Anthocephalus cadamba. Rev. bras. Farmacogn. (online). 2008, Vol.18, n-2, pp.155-159.
  7. Umachigi et al., Antimicrobial, Wound healing and antioxidant activities of Anthocephalus cadamba. Afr. J. Trad. CAM (2007) 4 (4): 481-487.
  8. Kokate CK and Purohit “Practical Pharmacognosy” Nirali Prakashan: Vallabh Prakashan 1994, 4th Edition.Page.No. 107-111.
  9. Wilkinson P.C. “Neutrophil Leucocyte Function test” Chapter 10, In ‘Techniques in clinical immunology’, Edited by R.A.Thompson, Second Edition, Oxford, London, Boston, Melbourne Blackwell Scientific Publication 1977, 201-218pp.

9 / Signature of Candidate
10 / Remarks of guide / The above study genuinely contributes to the scientific information and hence permitted.
11 / Name and designation of the
11.1 / Guide / Dr. Arunkumar Beknal
M.Pharm., Ph.D
PROFESSOR,
Dept. OF Pharmacognosy,
H.K.E.SCollege of Pharmacy,
Gulbarga - 585105
11.2 / Signature
11.3 / Co- guide (if any)
11.4 / Signature
11.5 / Head of the Department / Dr. M.A. Halkai
M.Pharm., Ph.D
PROFESSOR andHead
Dept. OF Pharmacognosy,
H.K.E.SCollege of Pharmacy,
Gulbarga - 585105
11.6 / Signature
12 / 12.1 / Remarks of the Chairman and Principal
12.2 / Signature