Rajiv Gandhi University of Health Sciences s7

“EVALUATION OF AQUEOUS AND ALCOHOLIC EXTRACTS OF BARK OF Acacia Catechu FOR WOUND HEALING ACTIVITY IN RATS”

SYNOPSIS FOR M.PHARM DISSERTATION

SUBMITTED TO

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA

BY M.BASWANTH KUMAR REDDY I M.PHARM DEPARTMENT OF PHARMACOLOGY PES COLLEGE OF PHARMACY BENGALOORU-560050 (2009-10) RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA, BENGALOORU -560050 ANNEXURE-I PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1 Name of the candidate and address M.BASWANTH KUMAR REDDY

LOCAL ADDRESS P.E.S. COLLEGE OF PHARMACY HANUMANTHNAGAR, 50 FT. ROAD, BENGALOORU -560050.

PERMANENT ADDRESS F.No-102,RADHA ENCLAVE, SNEHAPURICOLONY,NACHARAM, HYDERABAD-500076, ANDHRA PRADESH.

2 Name of the institution P.E.S. COLLEGE OF PHARMACY HANUMANTHNAGAR, 50 FT. ROAD, BENGALOORU -560050.

3 Course of study and subject MASTER OF PHARMACY IN PHARMACOLOGY

4 Date of the admission 15th JUNE 2009

5 Title of the topic:

“Evaluation of aqueous and alcoholic extracts of bark of acacia catechu for wound healing activity in rats”

6.0 Brief resume of the intended work: 6.1 Need for the study:

Plants and their extracts have immense potential for the management and treatment of wound. Wound is defined simply as the disruptions of the cellular and anatomic continuity of a tissue integrity .Wounds are physical injuries that result in an opening or breaking of the skin. The process of wound healing consists of integrated cellular and biochemical events leading to reestablishment of structural and functional integrity with regain of strength of injured tissue. The aim of treating a wound is to either shorten the time required for healing or to minimize the undesired consequences. Attention should be directed towards discovering an agent, which will accelerate wound healing. Several drugs from plants are known to possess wound healing activity. Wound healing herbals encourage blood clotting, fight infection and accelerate the healing of wounds. Some of these plants have been screened scientifically for evaluation of their wound healing activity in different pharmacological models and patients, but the potential of most remains unexplored .1 Research on wound healing drugs is a developing area in modern biomedical sciences. Scientists who are trying to develop newer drugs from natural resources are looking towards the Ayurveda. Several drugs of plant, mineral, and animal origin are described in the Ayurveda for their wound healing properties under the term Vranaropaka. One of the plants traditionally used for treatment of wounds is Acacia catechu belonging to the family mimosaceae. The bark of the plant is made into a paste and applied over the wounds. It is believed that the plant bark helps in faster healing of wounds.

Since Acacia catechu is traditionally used for treatment of wound healing2,3, it was thought worthwhile to investigate this plant for wound healing activity.

6.2 Review of the Literature:

Introduction:-

Acacia catechu commonly known as cutch tree or black catechu, is a sized deciduous tree, commonly found in Indian forests unto an elevation of 15m with dark grayish or brown rough bark and hooked short spines, leaves bi-pinately compound, leaflets :30-50 pairs, main rachis pubescent with a large conspicuous gland near the middle of the rachis; flowers pale yellow, sessile in peduncled axillary spikes; fruits flat brown pods, shiny and with a triangular beak at the apex and narrowed at the base; seeds 3-10 per pod. It is widely used in skin related disorders. It is a good anti-inflammatory herb. Helps to cure pile when taken with lemon regularly.

3 Common names :- English : betel-nut palm, black cutch, catechu tree, cutch tree, heartwood; Hindi : khair, supari, sundra, cachu,koir, tallatuma; Nepali: khair, khaira; Sanskrit : khadir; Tamil :karungali, kamugu, paku, baga; kannada : kalu; Telugu : podalimanu; Malayalam : karinnali 4

Botanical classification: Kingdom : Plantae Division : Magnioliophyta Class : Magniolipsida Family : Mimosaceae Genus : Acacia Species : catechu

Role in the habitat:- Common in forests of India and Burma.Lighter ,Variety of catechu is an imported one from Malaya and Singapore. 5

Description:-A moderate sized deciduous tree, with dark grayish or brown rough bark.

Leaves:- Leaves bipinnately compound, with 9-30 pairs of pinnae and a glandular rachis; leaflets 16-50 pairs, oblong-linear, 2-6 mm long, glabrous or pubescent.

Flowers :- Flowers in 5-10 cm long axillary spikes, pentamerous, white to pale yellow, with a campanulate calyx, 1-1.5 mm long, and a corolla 2.5-3 mm long; stamens numerous, far exserted from the corolla, with white or yellowish white filaments.

Fruits :- Fruit a strap-shaped pod, 5-8.5 cm x 1-1.5 cm, flat, tapering at both ends, shiny, brown, dehiscent, 3-10 seeded; seeds broadly ovoid. Seed :- 3-10 per pod.4

Chemical constituents: -

The chief constituents of the heartwood are catechin and catechu tannic acid. The wood contains epicatechin, atzelchin, catechin tetramer, dicatechin, gallochin, gossypetin, isorhamn.etin, kaempferol. 3

Uses:-

Khersal, a crystalline form of cutch sometimes found deposited in cavities of the wood is used medicinally for the treatment of coughs and sore throat. The bark is said to be effective against dysentery, diarrhoea and in healing of wounds. The seeds have been reported to have an antibacterial action. In East Africa, the powdered bark, mixed with sulphate of copper and egg yolk, is applied to cancerous growths.

4 Traditional medicinal uses:- It is useful in passive diarrhea, high blood pressure, dysentery, colitis, gastric problems, bronchial asthma, cough, leucorrhoea and leprosy. It is used as a mouthwash for mouth, gum, sore throat, gingivitis, dental and oral infections It is useful in cough, diarrhea, ulcers, boils and eruptions of the skin.3

SOME OF THE SCIENTIFIC REPORTS OF Acacia catechu

An aqueous extract of acacia catechu exhibited Immunomodulatory effect on mice. 6 It showed significant effect on both cell mediated and humoral immunity. Antipyretic, antidiarrhoeal, hypoglycaemic and hepatoprotective effects of the ethyl acetate extract of Acacia catechu in albino rats 7. It showed anti diarrheic 8,antioxidant 9,anti microbial activity 10 experimental animal models.

SOME OF THE HERBS HAVING WOUND HEALING ACTIVITY

The best known wound healing agents from plant source are Aloe vera and Centella Asiatica. 11,12 The search of a potent wound healing agent from plant source is continuing and different plants used traditionally in the treatment of wounds are being screened for wound healing activity. Recently, it was reported that methanolic extract of Tectona grandis leaves used traditionally in Mangalore region of Karnataka possess good wound healing activity 13.

Chowdary reported the wound healing activity of ethanol extract of Terminalia bellirica Roxb. fruit in albino rats. 14` In another study Dnyaneshwar et al have reported the wound healing activity of methanolic extract of root of Mimosa pudica.15 Root bark of Calotropis gigantea accelerated wound healing in rats and supported its traditional use as reported by Pradeep et al.16 H.K Kumara Swamy et al have reported the wound healing activity of Embelia ribes Burm. on rats.17

6.3 Objectives of the study

1. Preparation of alcoholic and aqueous extract of bark Acacia catechu.

2. To investigate preliminary phytochemical constituents of, alcoholic extract, aqueous extract of bark Acacia catechu.

3. To establish the pharmacological profile of prepared extracts for its wound healing activity.

5 7. Materials and methods

7.1 Source of data Whole work is planned to generate data from laboratory studies i.e.; experiments are performed as described in reference, experimental studies in journals and in textbooks available with college and various institutions.

IISc library, Bengalooru. PESCP library, Bengalooru. RGUHS digital library (Helinet), Bengalooru. Websites: www.sciencedirect.com www.pubmed.com www.google.com www.jhetchem.com www.ijp-online.com

Will be used to obtain related information regarding this research protocol.

7.2 Method of collection of data The whole study is divided in the following three phases.

Phase I: Collection of plant material.

The plant material will be procured from the authenticated supplier from Bengalooru market in the month of February and March and it will be authenticated by taxonomist, horticulture Department Bengalooru.

Phase II: Preparations of extracts.

Alcoholic extract and aqueous extract of bark of Acacia catechu. The alcoholic extract is prepared by successively extraction with soxhelet apparatus. The marc obtained after alcoholic extraction was macerated with water to obtain an aqueous extract.

Phase III: Preliminary Phytochemical investigation.

Preliminary Phytochemical investigation will be done as described in Practical Pharmacognosy-techniques and experiments.18

Phase IV: Acute oral toxicity.

Female Swiss albino mice (18-20 g) are individually identified and allowed to acclimate to the laboratory condition for 7 days before the start of the study. Only one mouse receives single dose at a particular time. First animal receives a dose of 175 mg/kg and is observed for any toxicity signs, survival or death up to 48 hrs. If the first animal died or appeared moribund, the second animal receives a lower dose (55mg/kg). The dose progression or reduction factor is 3.2 times of the previous dose. If no mortality is

6 observed in the first animal then the second animal receives a higher dose (55 mg/kg). Dosing of the next animal is continued depending on the outcome of the previously dose for a fixed time interval (48 hours). The test is stopped when one of the stopping criteria is observed. 5 reversals occur in any 6 consecutive animals tested. 3 consecutive animals died at one dose level. Survived animals are observed for long-term outcomes for a period of 14 days. The acute oral toxicity values are calculated using AOT 425 software (Environmental Protection Agency, USA) based on the short term (48 hours) and long term out come (14 days).19

Phase V: Pharmacological evaluation.

For determination of wound healing activity the following experiment are to be performed. All the models used in the pharmacological experiments will consist of the below 6 groups consisting of 6 animals in each group. Separate set of animals shall be used for individual test (or) models.

GROUP CLASSIFICATION : -

TREATMENT DRUG DOSE

Group-1 Control Ointment base

Group-2 Nitrofurazone ointment (Standard) 0.2%w/w

Group-3 Alcoholic extract of Acacia catechu Low dose

Group-4 Alcoholic extract of Acacia catechu High dose

Group-5 Aqueous extract of Acacia catechu Low dose

Group -6 Aqueous extract of Acacia catechu High dose

Standard to be used :- 1) Excision wound model - Nitrofurazone ointment 2) Incision wound model - Nitrofurazone ointment

7 ASSESSMENT OF WOUND HEALING ACTIVITY

Model 1: Excision wound model 17 The rats will be inflicted with excision wound under light ether anesthesia. The skin area will be excised to full thickness on the dorsal thoracic region of the rats to obtain a wound area of about 500mm2. The drugs will be topically applied daily till the complete epithelialization starting from the day of operation. The wounds will be traced on mm2 graph paper on the days of 4th, 8th, 12th and 16th. The wound closure will be measured to see the percentage of wound closure and epithelialization time that indicates the formation of new epithelial tissue to cover the wound. The number of days required for falling of the scar without any residual of the raw wound will give the period of epithelialization.

Model 2: Incision wound model 17

In incision wound model, 6 cm long paravertebral incisions will be made through full thickness of the skin on either side of the vertebral column of the rat. The incision should be at least 1 cm lateral to vertebral column. The wounds will be closed with interrupted sutures of 1 cm apart using a surgical thread (No. 000) and curved needle (No. 11). The wounds will be left undressed and drugs are topically applied to the wound once a day, till complete healing.

Model 3: Determination of Tensile strength 20

Tensile strength of the wound is determined by using a Tensiometer. The tensile strength increment indicates better wound healing stimulation by the applied drug.

STATISTICAL ANALYSIS

Values will be expressed as mean ± SEM from 6 animals. Statistical difference in mean will be analyzed using one way ANOVA (analysis of variance) followed by suitable posthoc test. p<0.05 will be considerable significant.

7.3 Does the study require any investigation to be conducted on patients Or other humans or animals?

Yes, the experimental models require usage of laboratory animals.

8 7.4 Has ethical clearance been obtained from your institution in case of 7.3?

Yes, ethical clearance has been obtained [copy of IAEC clearance has been attached]

9 REFERENCES:

1. Biswas TK, Mukherjee B. Plant medicines of Indian origin for wound healing activity. Int J low Extrem Wounds. 2003;2:25-39. 2. Vaidyarantnam PS. Indian Medicinal Plants. Chennai: Orient Longman Private Limited; 1994. 3. Yoganarasimhan SN. Medicinal Plant of India (Karnataka). Bangalore: Interline Publishing Pvt Ltd; 1996. 4. Krithikar KR, Basu BD. Indian medicinal plants.2nd ed. New Delhi: Periodical Experts Book Agency; 1991.

5. Singh MP, Himdari P. Medicinal Herbals with their formulations. Delhi: Daya Publishing House; 2005. 6. Ismail S, Asad M. Immunomodulatory activity of acacia catechu. Indian J Physiol Pharmacol. 2009;53:25-33. 7. Ray D, Sharatchandra KH, Thokchom IS. Antipyretic, antidiarrhoel, hypoglycaemic and hepatoprotective activities of ethyl acetate extract of acacia catechu wild in albino rats. Indian J Pharmacol. 2006;38:408-13.

8. Nongyao S, Kitja S. The effects of extracts from anti-diarrheic Thai medicinal plants on the in vitro growth of the intestinal protozoa parasite: Blastocystis hominis. J Ethnopharmacol. 2005; 98:67-72.

9. Naik GH, Priyadarsini KI, Satav JG, Banavalikar MM, Sohoni DP, Biyani MK, et al. Comparative antioxidant activity of individual herbal components used in Ayurvedic medicine. Phytochemistry. 2003;63:97-104.

10. Rajni S, Astha J, Shikha P, Deepti G, Khare SK, 2005;66:99-102. Antimicrobial activity of some natural dyes. Dyes Pigments. 2005;66:99-102.

11. Heggers JP, Kucukcelebi A, Listengarten D, Stabenau J, Ko F, Broemeling LD, et al. Beneficial effect of Aloe on wound healing in an excisional wound model. J Altern Complement Med 1996;2:271-7.

12. Shukla A, Rasik AM, Jain GK, Shankar R, Kulshrestha DK, Dhawan BN, et al. In vitro and in vivo wound healing activity of asiaticoside isolated from Centella asiatica. J Ethnopharmacol 1999;5:1-11.

13. Majumdar M, Nayeem N, Kamath JV, Asad M . Evaluation of Tectona grandis leaves for wound healing activity. Pak J Pharm Sci. 2007;20:120-4.

10 14. Choudhary GP. Wound healing activity of the ethanol extract of Terminalia bellirica Roxb. fruits. Nat Prod Rad. 2008;7:19-21.

15. Dnyaneshwar DK, Rahul YM, Mandar BK, Minakshi NN, Prachi CM, HG. C, et al. Evaluation of wound healing activity of root of Mimosa pudica. J Ethnopharmacol. 2009;124:311-5.

16. Pradeep TD, Jennifer F, Akarte A, Emmanuel T. Wound healing activity of Calotropis gigantea root bark in rats. J Ethnopharmacol. 2009;125:178-81.

17. Kumara Swamy HM, Krishna V, Shankarmurthy K, Abdul RB, Mankani KL, Mahadevan KM, et al. Wound healing activity of embelin isolated from the ethanol extract of leaves of Embelia ribes Burm. J Ethnopharmacol. 2007;109:529-34.

18. Khandelwal KR. Practical Pharmacognosy-techniques and experiments. Pune, India: Nirali Prakashan; 1996.

19. OECD Guidelines for testing of chemicals [online] 2007 Aug 16; Available from: URL: http;//www.oecd.org/dataoecd/9/11/33663321.pdf.

20. Kakali S, Pulok KM, Das J, Pal M, Saha BP. Wound healing activity of Leucas lavandulaefolia Rees. J Ethnopharmacol. 1997;56:139-44. 12 9.0 SIGNATURE OF THE CANDIDATE

(BASWANTH KUMAR REDDY)

10.0 REMARKS OF THE GUIDE

11.1 NAME AND DESIGNATION OF THE Dr. Benson Mathai K. GUIDE Asst.Professor Department of Pharmacology P.E.S College of Pharmacy Bengalooru-560050.

11.2 SIGNATURE

11.3 CO-GUIDE NOT APPLICABLE 11.4 SIGNATURE

11.5 HEAD OF THE DEPARTMENT Mr. Srinath.R Asst.Professor & HOD Department of Pharmacology P.E.S College of Pharmacy Bengalooru-560050

11.6 SIGNATURE

12.0 REMARKS OF THE PRINCIPAL FORWARDED FOR APPROVAL

13.0 SIGNATURE OF THE PRINCIPAL

Prof.Dr.S.Mohan Principal & Director, PES College of Pharmacy Bengalooru-560050.