phytochemical and wound healing studies
on leaves of calycopteris floribunda lam.
Protocol of Dissertation Submitted (2012-2013) by
Miss. Vidhya Balasaheb Dorage
To
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
BANGALORE, KARNATAKA

Under the guidance of
Dr. Gaviraj. E. N.
Professor & Head, Department of Pharmacognosy
BLDEA’S COLLEGE OF PHARMACY,
BLDE UNIVERSITY CAMPUS, BIJAPUR-586 103
ANNEXURE - II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1. / Name of the Candidate & Address
(In block letters) / : /
Miss. Vidhya Balasaheb Dorage
DEPARTMENT OF PHARMACOGNOSY,
B.L.D.E.A’s. COLLEGE OF PHARMACY,
B.L.D.E. UNIVERSITY CAMPUS,
BIJAPUR- 586 103
2. / Name of the Institution / : / B.L.D.E.A’S. COLLEGE OF PHARMACY,
BIJAPUR- 586 103
3. / Course of Study and Subject / : / M. PharmACY
(PHARMACOGNOSY)
4. / Date of Admission to Course / : / 06/02/2012
5. / Title of the Topic / : / phytochemical and wound healing STUDIES ON leaves of calycopteris floribunda lam.
BRIEF RESUME OF THE INTENDED WORK:
6.1 Need for the study:
Wounds are physical injuries that result in an opening or break of the skin. Wound may be produced by physical, chemical, thermal, microbial or immunological insult to the tissue. Healing is a complex and intricate process initiated in response to an injury that restores the function and integrity of damaged tissues. Wound healing involves continuous cell–cell and cell–matrix interactions that allow the process to proceed in three overlapping phases viz. inflammation (0–3 days), cellular proliferation (3–12 days) and remodeling (3–6 months)[1].
In spite of tremendous advances in the pharmaceutical drug industry, the availability of drugs capable of stimulating the process of wound repair is still limited [2]. Moreover, the management of chronic wounds is another major problem due to the high cost of therapy and the presence of unwanted side effects [3, 4].
Medicinal plants have been used since time immemorial for treatment of various ailments of skin and dermatological disorders especially cuts, wounds and burns. In developed countries too, people are seeking alternative to modern therapies of wound healing like antibiotics, corticosteroids, etc. obviously due to its side effects [1]. Greater understanding is required in case of pathogenesis of chronic wounds, which fails to heal. The two inseparable aspects namely pathogenesis and failure to heal have intensified the use of herbal drugs as wound healing agents. In this context, the phytochemical and wound healing studies on leaves of widely used folklore plant, Calycopteris floribunda is planned to be investigated.
Introduction to the Plant:
Calycopteris floribunda, commonly known asUkshi in Marathi,Kokkarai in Hindi, and Enjir in Kannada belongs to family Combretaceae [5, 6]. It is a large climbing shrubwhich is 5-10 m long, with vinesthat are about 5–10 cm indiameter, the stemand leavesare said to have medicinal properties. Ukshi is found extensively in the low-lying tropical evergreen forests of theWestern Ghats.
It bearsgrey barkand tenuousbrancheswith thick fluff on the surface. The keratinous leaves, ovoid or oval, are 5–12 cm long. New branches are hairy and rust coloured. The flowersoccur in dense clusters are the end of branches. The bractsof the small flowers are ovoid or oval, with thick fluffs on the surface. Petalsare absent and the 10 stamensare arranged in 2 cycles.
Thefruitinception bears 1ventricleand 3 pendulous ovulesinside. The fluffy sham-winged fruit, which is about 8 mm long, has 5 edges and 5 persistentcalyxeswhich enlarge into the fluffy wing 10–14 mm in length. The sepals are prominent, hairy and green.
The leaf of the plant Calycopteris floribunda reported to contain tannins, flavanol, Octacesanol, sitosterol, 3'0-methylcalycopterin, 4-0-methylcalycopterin, ellagic acid, gossoypol, quercetin, proanthocyanidin, calycopterin, calycopterin methyl ester and oxymethyl calycopterin [7].
The plant parts especially leaves being used medicinally for various complications such as intestinal worms, colic, leprosy, malarial fever, dysentery, ulcers and vomiting. The fruits are useful in jaundice, ulcers, pruritus and skin diseases [8].
6.2 Literature review
Tall and Pentela studied the hepatoprotective activity of the leaves of C. floribunda[9]. Adult male wistar rats were rendered hepatotoxic by Rifampicin-Isoniazid(50mg/kg each) by intraperitonially for 14days while leaf extract (100mg/kg and 200mg/kg) was administered orally for 14 days. Administration of leaf extract (100mg/kg and 200mg/kg) resulted in a significant (p<0.01) increased in plasma and hepatic lipid profiles. The extract suppresses cytochrome P-450 activity. Decreased levels of transaminases indicate stabilization of plasma membrane and protection of hepatocytes against damage caused by hepatotoxin. The levels of SOD, catalase, GSH significantly decreased along with concentration of malondialdehyde in these groups indicating increased lipid peroxidation. Histomorphological findings also supported the biochemical findings. The present study demonstrated that administration of leaf extract (100mg/kg and 200mg/kg) had significant hepatoprotective activity as evidenced by the biochemical, functional and histopathological parameters.
Chinna Eswaraiah and Satyanarayana reported the hepatoprotective activity of the stems of C. floribunda[10].They evaluated the hepatoprotective activity of chloroform and methanol fractions from stem against carbon tetrachloride (CCl4) induced liver damage in Wistar albino rats. Chloroform fraction and methanolic extract (100mg/kg, 200mg/kg .p.o.), were administerd respectively, Silymarin (25 mg/kg.p.o.) was given as reference standard. The stem extracts were effective in protecting the liver against the injury induced by CCl4 in animals. This was evident from significant reduction in serum enzyme, Serum glutamic oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT), Alkaline phosphatase (ALP) and Total bilirubin (TB).Various pathological changes like centribular necrosis and vacuolization were observed in CCL4 treated rats, which were significant protective activity in groups treated with C. floribunda and silymarin.
Bhat et al. [8] reported antimicrobial activity of the leaves of C. floribunda. The antimicrobial activities of leaf extracts of Calycopteris floribunda in three different solvents such as diethyl ether-methanol, aqueous 90% methanol extract and petroleum ether-butanol extract were tested against Bacillus cereus, Bacillus subtilis and Staphylococcus aureus. The diethyl ether-methanol extract of the leaves and its petroleum ether-butanol fraction showed significant antibacterial activity. The antioxidant property was maximum in petroleum ether-butanol extract and was minimum in diethyl ether-methanol extract.
Dey et al. conducted experiments on antibacterial activity and antioxidant property in C. floribunda using leaf extracts in different solvents [11].
Sreekanth et al. in their studies on leaf extracts of C. floribunda revealed that the extracts were toxic to calf, rabbits and rat [12].
Bhat et al. conducted ethnomedicinal survey in Uttara Kannada district of Karnataka. The study revealed uses of 106 plants in traditional practices for curing various types of wounds [13].The highest Use value is forCalycopterisfloribunda(1.80), followed by Rauvolfia serpentina and Achyranthes aspera (1.67).
Ali et al. isolated pachypodol (5,4'-dihydroxy-3,7,3'-trimethoxyflavone) from the leaves of C.floribundaby repeated column chromatography on silica gel [14]. While the general toxicity of pachypodol was determined by the brine shrimp lethality assay, the cytotoxic potential of this flavonoid has been evaluated by the Promega's CellTiter 96 Non-Radioactive Cell Proliferation Assay using the CaCo-2 colon cancer cell line (IC(50) = 185.6 microM).
Mayer isolated five biflavonoids fromCalycopterisfloribunda[15]. The structures of five biflavonoids, 6"-demethoxyneocalycopterone (1), calyflorenone C (2), 6"-epi-calyflorenone B (3), 6"-epi-calyflorenone C (4) and calyflorenone D (5) from the green parts ofCalycopterisfloribundawere established by NMR and MS. Their NMR and chiroptical properties (CD, [alpha]20D ) were compared with those of the known C. f. biflavonoids 6-11. Compound 1 represents a calycopterone derivative, 2-5 have a calyflorenone skeleton. With regard to one chiral center (C-6"), 4 and 3 are the respective epimers of 2 and 11.
Mayer isolated neocalycopterone (1) and its methyl ether (2), along with two new biflavonoids, calyflorenones A (3) and B (4), from dried leaves ofCalycopterisfloribunda(Combretaceae) [16]. Cytotoxicity test results of 1 and 2 are reported. The monoflavonoid penduletin was isolated as a minor component from C.floribundafor the first time.
Rajasekaran and Periasamyinvestigated ethanolic leaf extract of Calycopteris for hepatoprotective activity against cadmium induced liver damage [7]. Various biochemical parameters, serum glutamate oxalo acetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), serum alkaline phosphatase and total protein were determined to assess the effect of the leaf extract on the cadmium induced hepatic damage. The animals treated with cadmium recorded elevated concentration indicating severe hepatic damage by cadmium, whereas the blood samples from the animals treated with 200 mg/kg (b.w) and 400 mg/kg (b.w) of ethanolic leaf extract of Calycopteris floribunda showed significant reduction in the serum markers indicating the effect of the leaf extract in restoring the normal functional ability of the hepatocytes. Silymarin (100 mg/kg, p.o.) was given as reference drug. The present study concluded that the ethanolic leaf extract of Calycopteris floribunda showed significant hepatoprotection against cadmium‐induced hepatocellular injury.
Sujit et al. [17] performed studies on dichloromethane-methanol extract of leaves of Calycopteris floribunda and its aqueous 90% methanol soluble fractions. These showed significant antibacterial activity against Bacillus subtilis, Streptococcus pyogen, Staphyloccus aureus and Salmonella typhi. The aqueous 90% methanol and 1-butanol soluble fractions of the leaves showed significant β-glucuronidase inhibition and antioxidant activity. Two pure compounds, 3,8-di-O-methyl ellagic and 2,3,7-tri-O-methyl ellagic acids were isolated from the 1-butanol soluble fraction of the parent extract.
Satyanarayana and Chinna Eswaraiahstudied the in vitro antioxidant methods like superoxide radical, hydroxyl radical, lipid peroxidation and DPPH radical methods for chloroform and methanol extracts of Calycopteris floribunda stem [18]. The results of antioxidant activity revealed that, the chloroform extract has lower IC50 values than the methanolic extract of Calycopteris floribunda. The lower IC50 value indicates the higher free radical scavenging ability. So, the chloroform extract has better antioxidant activity than methanolic extract. The results were compared with the standard ascorbic acid. The plant contains phytosterols, triterpenoids, alkaloids, saponins, flavonoids, glycosides and tannins. These active constituents alone or in combination may be responsible for the observed antioxidant activity.
6.3 The objectives of the present study are:
  • Extraction of Calycopteris floribundaLam. leaves using 70% ethanol.
  • Phytochemical investigation of the extract.
  • Acute toxicity study of the test extract
  • Evaluation of the extract for wound healing activity.

MATERIALS AND METHODS:
7.1 Source of data
  • Performed experiments
  • Published research papers.
  • Review articles from Journals.
  • Electronic databases.
  • Library.
7.2 Method of collection of data
Collection:The plant Calycopteris floribunda Lam.will be collected from the forests of Uttara kannada district of Karnataka.
Authentication: Botanist will authenticate the plant using standard procedures.
Phytochemical studies:
For the present study, leaves of Calycopteris floribunda Lam.will be shade dried and coarsely powdered. Powdered drug will be extracted using soxhlet apparatus with70% ethanol. Extract will be concentrated and further subjected to chemical analysis and chromatographic studies.
Evaluation of wound healing activity:
Albino rats of either sex weighing 150-200 g will be selected, and divided into four groups of five each. I group serves as control, II and III group will be treated with test extract at two doses and IV will serve as standard. Animals will be depilated at the desired site before wounding. They will be housed individually with free access to food and water, the basal food intake and body weights to the nearest gram will be noted.An excision wound model will be used for studying wound healing activity as described by Nagappa et al., 2001 with some modifications [19]. The incision wound model will be used for studying wound healing activity as described by Govindarajan et al. [20].
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, in the present study wound healing activity of the test extract will be demonstrated in rats, 20 each for two different models.
7.4 Has ethical clearance been obtained from your institution in case of 7.3?
The study protocol has been submitted for approval by IAEC and permission will be obtained in the forthcoming meeting.
9. / SIGNATURE OF CANDIDATE /
(Miss Vidhya Balasaheb Dorage )
10. / REMARKS OF THE GUIDE / The present study has been proposed on the basis of literature review. This study will be carried out by Miss Vidhya Balasaheb Dorage
11. / NAME AND DESIGNATION OF
THE GUIDE / Dr. Gaviraj E. N.
Professor & Head, Dept. of Pharmacognosy
12. / SIGNATURE
13. / CO-GUIDE / Dr. Shivakumar Hugar
Professor & Head, Dept. of Pharmacology
14. / SIGNATURE
15. / HEAD OF THE DEPARTMENT / Dr. Gaviraj E. N.
Professor & Head, Dept. of PharmacognosyB.L.D.E.A’S College of Pharmacy,
Bijapur-586 103
16. / SIGNATURE
17. / REMARKS OF THE PRINCIPAL / The above mentioned information is correct and I recommend the same for approval.
18. / NAME OF THE PRINCIPAL / Dr. N.V.KALYANE
Professor and Principal,
B.L.D.E.A’s College of Pharmacy,
Bijapur-586 103
19. / SIGNATURE

BIBLIOGRAPHY

  1. Kumar B, Vijayakumar M, Govindarajan R, Pushpangadan P, Ethnopharmacological approaches to wound healing-Exploring medicinal plants of India, J Ethnopharmacol, 114 (2007) 103-13
  2. Udupa AL, Kulkarni DR, Udupa SL, Effect of Tridax procumbens extracts on wound healing, Int J Pharmacog, 33 (1995) 37–40
  3. Porras-Reyes BH, Lewis WH, Roman J, Simchowitz L, Mustoe TA, Enhancement of wound healing by the alkaloid taspine defining mechanism of action. Soc Expt Biol Med, 203 (1993) 18–25
  4. Suh DD, Schwartz IP, Canning DA, Snyder HM, Zderic SA, Kirsch AJ, Comparison of dermal and epithelial approaches to laser tissue soldering for skin flap closure. Lasers Surgery Medicine 22 (1998) 268–274
  5. Nadakarni AK, Indian Materia Medica, Bombay Popular Prakashan, Reprint 2000
  6. Rajasekaran A and Periasamy M, Hepatoprotective effect of ethanolic leaf extract of Calycopteris floribunda Lam on cadmium induced hepatotoxicity in rats, Research Journal of Pharmaceutical, Biological and Chemical Sciences 3 (2012) 382-390
  7. Rama Bhat P, Prajna PS, Kumar V, Adarsh Hegde M and Singh L, Antimicrobial properties of leaves of Calycopteris floribunda Lam., Journal of Medicinal Plants Research, 5 (2011) 2448-2451
  8. Thalla S and Pentela B, Hepatoprotective effect of hydroalcoholic extract of Calycopteris floribunda leaves on rifampicin-isoniazid induced rats, International Journal of Chemical and Pharmaceutical Sciences, 2 (2011) 15-21.
  9. Chinna Eswaraiah M and Satyanarayana T, Hepatoprotective activity of extracts from stem of Calycopteris floribunda Lam. against carbon tetrachloride induecd toxicity in rats, International Journal of Pharmacognosy and Phytochemical Research, 2 (2010) 53-57
  10. Dey SK, Shoeb Mohammed, Rob Tamanna, Nilafar Nahar, Mosihuzzaman M, Nasin Sultana Biological and chemical studies of Calycopteris floribunda leaves. The Dhaka Univ. J. Pharm. Sci., 4 ((2005) 1816-1839
  11. Sreekanth P, Narayana K, Sridhar NB, Avinash Bhat Toxicity studies of Calycopteris floribunda in calf, rabbit and rat. Journal of Ethnopharmacol., 107((2007) 229 – 233
  12. Bhat P, Hegde G, Hegde GR., Ethnomedicinal practices in different communities of Uttara Kannada district of Karnataka for treatment of wounds, Journal of Ethnopharmacolgy, 143 (2012) 501-514
  13. Ali HA, Chowdary Ak, Rahman AK, Borkowski T, Nahar L, Sarker SD, Pachypodol, a flavonol from the leaves ofCalycopterisfloribunda, inhibits the growth of CaCo 2 colon cancer cell line in vitro, Phytotherapy Research, 22 (2008) 1684-87
  14. Mayer R,Five biflavonoids fromCalycopterisfloribunda(Combretaceae). Phytochemistry, 65(2004) 593-601
  15. Mayer R, Calycopterones and calyflorenones, novel biflavonoids fromCalycopterisfloribunda, J Nat Prod, 62 (1999) 1274-8.
  16. Sujit K. Dey, Mohammad Shoeb, Tamanna Rob, Nilufar Nahar1 M. Mosihuzzaman and Nasim Sultana , Biological and Chemical Studies on Calycopteris floribunda leavesDhaka Univ. J. Pharm. Sci. 4(2005) 103-106
  17. Satyanarayana T, Chinna Eswaraiah M,In-vitro antioxidant and free radical scavenging potential of stem of Calycopteris floribunda lam.Research Journal of Pharmaceutical, Biological and Chemical Sciences 1 (2010) 117-123.
  18. Nagappa AN and Cheriyan Binu Wound healing activity of the aqueous extract of Thespesia populnea fruit. Fitotherapia, 72 (2001) 503-506
  19. Govindarajan R, Vijayakumar M, Rao CV, Shirwaikar A, Mehrotra S and Puspangadan P (2004). Healing potential of Anogeissus latifolia for dermal wound in rats. Acta Pharm.,54: 331-338