Evaluation of Wound Healing and Anti-Oxidant Property of Seed Oil of Morinda citrifolia L (Noni) in Wistar Rats

 

Khuntia Tapas Kumar1*, Panda D.S.2 and Khuntia S.3

 

1Dept. of Pharmacology, K.L.R. Pharmacy College, Paloncha, A.P., India

2Dept. of Pharmaceutics, Institute of Pharmacy and Tech., Salipur, Orissa

3College of Pharmaceutical Sciences, Puri, Orissa

 

ABSTRACT:

Wound healing is a complex pathway that is energy dependent. Non-healing wounds frequently require the use of physical agents to achieve healing. Wound healing disorders present a serious clinical problem and are likely to increase since they are associated with disease such as diabetes, hypertension and obesity. Additionally, increasing life expectancies will cause more people to face such disorders and further aggravate problem thus several animal models have been established to serve as an experimental basis to determine molecular and cellular mechanisms underlying and controlling an undisturbed healing process. Morinda citrifolia L. (Noni) (Rubiaceae) has been used in folk remedies by Polynesians, Indians for over 2000 years, and is reported to have a broad range of therapeutic effects, including antibacterial, antiviral, antifungal, antitumor, antihelmintic, analgesic, hypotensive, anti-inflammatory, and immune enhancing effects.

 

This paper describes a common biochemical pathway that helps to understand, at a molecular level, how the transfer of energy to a wound can result in positive results. The mechanism of action for seed oil of Morinda citrifolia L (Noni) is reviewed along with biochemical estimations. Based on our study we conclude that test compound is showing potent anti-oxidant and wound healing activity when compared with positive control i.e. Allanzyme and the biochemical estimations also favoured to us in this case.

 

KEYWORDS: Wound healing, Morinda citrifolia L (Noni), antifungal, antitumor, analgesic, hypotensive, anti-inflammatory, anti-oxidant

 

INTRODUCTION:

Wound is defined as any injury/damage occurring to any tissue or organ. For treating this external or internal medication is used.  Many dosage forms are available in the market. But there is need of new drugs or combinations to treat wounds effectively; hence wound healing studies are required. Wound healing disorders present a serious clinical problem and are likely to increase since they are associated with disease such as diabetes, hypertension and obesity. Additionally, increasing life expectancies will cause more people to face such disorders and further aggravate problem thus several animal models have been established to serve as an experimental basis to determine molecular and cellular mechanisms underlying and controlling an undisturbed healing process.


Hence describing a model of excisional skin wounding in Rats that can be used to assess molecular, cellular, and tissue movements in healthy female Rats. Morinda citrifolia L (Noni) (Rubiaceae) has been used in folk remedies by Polynesians, Indians for over 2000 years, and is reported to have a broad range of therapeutic effects, including antibacterial, antiviral, antifungal, antitumor, analgesic, hypotensive, anti-inflammatory, and immune enhancing effects.1

 

The present study was undertaken to establish the wound healing and anti-oxidant property of seed oil of Morinda citrifolia L (Noni) in Wistar female Rats.

 

MATERIALS AND METHODS:

Experimental Animals:

Animal care and handling was carried out according to Institutional Animal Ethics Committee (IAEC). Eight to ten weeks old Female Wistar rats weighing (120±40 g) were selected from the in-bred colony maintained under controlled conditions of temperature (22±3oC) humidity (40-70%) and light (12 and 12h of light and dark, respectively). They were feed with standard rodent chew, Nutrilab Rodent (Tetragon Chemie Pvt. Ltd., Bangalore) and purified drinking water. The animals had free access to food and water. Four animals were housed in a polypropylene cage containing paper bedding.

 

Chemicals:

Anesthetic ether from Kabra Drugs Limited, INDORE, Absolute alcohol, Hayman, ENGLAND and Sterillium disinfectant solution was procured from Bode Chemie Hamburg, GERMANY.

 

Instruments and General Requirements:

UV spectrophotometer, Anaesthesia chamber, Scissors, Forceps, Cotton, Hair clipper, Sterile cages with paper bedding, Camera.

 

PREPARATION OF DRUG AND ADMINISTRATION:

Plant Material:

The seeds of the plant was collected from Cuttack district, India and taxonomically identified by Department of Pharmacognosy I.P.T., Salipur, Cuttack. Seed oil was collected from the seeds through seed oil extractor (Yield 50%). Test Drug (seed oil) was applied topically to the animals.

 

Experimental Protocol:

In-bread Wistar rats were procured from Institute of Pharmacy and Technology, Salipur.  Rats were kept in experimental room for one week for acclimatization. 51 female rats were selected within the weight range of 120±40 and animals were randomly divided into 3 groups of 17 animals each. The fur of the dorsum of each animal was removed with electric hair clipper after 24h the animals were sedated using anesthetic ether and the dorsal surface of skin was marked with a sterile circular stainless steel stencil. A full-thickness wound (600 mm2) was created by excising the skin including panniculus carnosus in an aseptic environment using sterile scissors and forceps.2

 

Study design:

After the creation of wounds to the entire animals Test group receives seed oil of Morinda citrifolia L (Noni), Positive control group animals receives Allanzyme were as control group receives normal saline. Mode of application of substance is topical and single application per day. (Table-1)

 

Animal distribution for biochemical analysis:

Animals were sacrificed at different time intervals and granulation tissue was collected for biochemical and histopathological analysis. (Table-2)

 

Wound area measurement:

Animals were anesthetized using anesthetic ether and wound measurement was taken on day 3, 7,9,14,17,21,24 and 28. Wound area was assessed by using transparent paper and permanent marker for tracing the wound. During the wound area measurement on day 3, 7, 9 and 14, three animals from each group were sacrificed and granulation tissue was collected for biochemical and histopathological studies.

 

Granulation tissue collection for Biochemical studies:

On day 3, 7, 9 and 14 three animals were sacrificed from each group and the granulation tissue was collected in vials and labeled carefully and stored at –20o C immediately.

 

NITRIC OXIDE ESTIMATION:

Reagents preparation:

Sodium nitropruside:

Part A: Sodium nitropruside (100 mM) Stock solution.

Preparation: Weigh 0.298g of Sodium nitropruside, dissolved in 10 mL of water.

 

Part B: sodium nitropruside (10 mM): From part A stock, 100 µL was added to final volume.

 

Greiss reagent:

Part A: 1% sulphanilamide: 1g sulphanilamide dissolved in 100 mL of 2% phosphoric acid.

 

Part B: 0.1% naphthlethylene diamine dihydrochloride (NDD): 0.1 g NDD dissolved in 100 mL of water. Mix part A and part B in equal volumes.

 

Test substance preparation:

Scar tissue were collected from the wounds of the animals at the different time intervals during the experiment and stored at -20o C. 2 g of tissue was weighed from each sample and soaked in the 6N HCl for the overnight. After that the tissue was homogenised with the help of mortar and pestle for Control and treatment groups.

 

Table-1. Study design for Morinda citrifolia L (Noni) research.

Test group

Number of animals

Drug

Quantity mL

Route of  administration

Number of applications/ day

Control

17

Saline

0.5

External

Single

P. Control

17

Allanzyme

 

External

Single

Test

17

Morinda citrifolia L

0.5

External

Single

 

Table-2.  Animal distribution for biochemical analysis:

Group

No of Animals

Number of animals sacrificed on post wounding day

Day 3

Day 7

Day 9

Day 14

After complete wound closure

Control

17

3

3

3

3

5

P. Control

17

3

3

3

3

5

Test

17

3

3

3

3

5

 

Procedure:

The Nitric Oxide assay was measured with the following reaction mixture in a final volume of 2 ml contains, Sodium nitropruside and test compound. The reaction mixture was incubated at 37o C for 30 minutes. 0.5 ml of incubated sample was removed and equal volume of Greiss reagent was added. The absorbance of the chromophore formed was measured at 546nm. The inhibition of the Nitric Oxide generated was measured by comparing the absorbance values of Control, Positive control and Test.3

 

% of inhibition=

   Control value -Treated value ÷ Control × 100

 

STATISTICAL ANALYSIS:

All the results were analysed by One-way ANOVA followed by Dunnett’s test. The level of significance was set at P<0.05.

 

RESULTS:

Wound healing is a complex and dynamic process with the wound environment changing with the changing health status of the individual. The knowledge of the physiology of the normal wound healing trajectory through the phases of hemostasis, inflammation, granulation and maturation provides a framework for an understanding of the basic principles of wound healing.

 

The preliminary phyto-chemical analysis of seed oil of Morinda citrifolia L (Noni) revealed the presence of flavonoids, saponins, alkaloids, triterpinoids, tannins and phenolics. (Table-3).

 

During the post wounding time, when we compare wound contraction between the treatment groups with control, (Noni) treatment resulted in a steady contraction of excision wounds with time and the contraction was statistically significant when compared to placebo control. A similar effect was observed with Allanzyme (Graph-1). The application of (Noni) reduced the mean wound healing time (MHT) by approximately 7 days when compared to control (Graph- 2).

 

 

Table-3: Qualitative Phytochemical Analysis of seed oil of Morinda citrifolia L (Noni). [4]

Plant constituents

Seed oil of Morinda citrifolia L (Noni)

Tests/Reagents used

 

1

Triterpenoids

 

(i) Tin & Thionyl chloride

+++

2

Saponins

 

(i)   Foam test

+

 

(ii)  Haemolysis test

+

3

Tannins and phenolics

 

(i) Ferric chloride solution

++

 

(ii) Lead acetate solution

+

 

(iii) Potassium dichromate sol.

+

 

(iv) Gelatin solution

+

4

Flavonoids

 

 

(i)   Magnesium and HCl

+++

 

(ii)  Lead acetate solution

++

 

(iii) NaOH & H2SO4

+

5

Alkaloids

 

 

(i)   Mayer’s test

+

 

(ii)  Wagner’s test

+

 

(iii) Hager’s test

+

 

(iv) Dragendorff’s test

+

+++  Phyto -constituent in high concentration, ++ Medium conc., +Low conc.

 

 

Graph-1: Mean wound contraction

 

Graph-2: Comparison of the Mean Wound Healing Time (Days)

 

Comparison of mean wound healing time (MHT) after topical application of (Noni) with Allanzyme in the Rat inflicted with excision wound.

 

Histology of granulation tissue obtained from the control group rats showing moderate aggregation of macrophages, granulation tissue of positive control animals showing decreased macrophage aggregation and those obtained from the test rats showing aggregation of macrophages. (Fig-1, Fig-2, Fig-3)

 

Fig-1 Histology of granulation tissue obtained from the control group rats showing moderate aggregation of macrophages. 

 

Fig-2 Histological section of granulation tissue of positive control animals showing decreased macrophage aggregation.

 

Fig-3 Histology of granulation tissue obtained from the test rats showing aggregation of macrophages.

 

The tensile strengths of the granuloma tissue were determined by the water flow technique. The seed oil of Morinda citrifolia L (Noni), showed highly significant increase in breaking strength (375.56±4.52, P<0.05), when compared to control (155.035±6.76, P<0.05). The Positive Control showed significant increase in breaking strength (283.85±4.44). (Table-4)

 

Table-4.  Influence of Morinda citrifolia on tensile strength of granuloma tissue. [5, 6]

GROUP

BREAKING STRENGTH (g)

Control

155.03±6.76

Seed oil of Morinda citrifolia

375.56±4.52*

Allanzyme

283.85±4.44*

* indicates significant difference at P<0.05 when compared to control. Values are Mean± SEM. Data analysed by One-way ANOVA followed by Dunnett’s test.

 

Nitric oxide is a potent pleiotropic mediator of physiological processes such as smooth muscle relaxation, neuronal signaling, inhibition of platelet aggregation and wound healing process. The seed oil of Morinda citrifolia L (Noni) showed a significant inhibition of nitric oxide level in the scar tissue when compared to the control and the positive control. (Graph-3)

 

DISCUSSION:

Granulation, collagen maturation and scar formation are some of the many phases of wound healing which run concurrently but independent of each other. The preliminary Phytochemical screening of Morinda citrifolia L (Noni) revealed the presence of flavonoids, saponins, alkaloids, tannins and phenolics and triterpinoids.

 

Flavonoids and triterpinoids are known to promote the wound healing process, mainly due to their astringent and anti microbial property, which seems to be responsible for wound contraction and increased rate of epithelialisation.7, 8

 

Tannins the main components of many plant extracts, act as free scavengers.9 Research in to the role of anti-oxidants from plant extracts in wound healing has been published widely.10 Hence the wound healing property may be due to the anti-oxidant activity of seed oil of Morinda citrifolia L (Noni). The wound breaking strength is determined by the rate of collagen synthesis and more so by the maturation process. In our study there is a significant (P<0.05) increase in the breaking strength in seed oil of (Noni) treated group, hence we assume that Morinda citrifolia might have increased the collagen and also probably have altered the maturation process, by affecting the cross inking of collagen or by affecting the quality of the collagen fibrils.

 

Graph-3. Nitric-oxide Estimation:

 

CONCLUSION:

The use of Morinda citrifolia L (Noni) in Indian traditional systems of medicine for various skin diseases has been justified by this work. Although a number of drugs available in the market, still there is some gap to be considered. Still people are facing difficulty with the chronic wounds.  These findings could justify, at least partially, the inclusion of this plant in the management of wound healing in folk medicine. This study is a trail for curing such chronic wounds. Since the role of free radicals and anti-oxidants in wound healing are clearly defined, wound healing potential of seed oil of Morinda citrifolia L (Noni) may be due to the potent antioxidant activity of the plant. Based on our study we conclude that test compound is showing potent activity when compared with positive control i.e. Allanzyme and the biochemical estimations also favoured to us in this case. Hence, the present findings provide scientific evidence that the seed oil of Morinda citrifolia L (Noni) is having capacity to cure the chronic wounds.

 

 

REFERENCE:

1.        Wang Mian-Ying et al., Morinda citrifolia (Noni): A literature review and recent advances in Noni research. Acta Pharmacol Sin 2002; 23: 1127 -1141

2.        Shanbhag Tara V. et al., Wound healing activity of Alcoholic extract of Kaempferia galanga in wistar rats. Indian J Physiol Pharmacol 2006; 50: 384–390

3.        Jitoe A, Masuda T, Tengah IGP, Suprapta DN, Gara IW, Nakatani N. Antioxidant activity of tropical ginger extract and analysis of the contained curcuminoids. J Agric Fd Che 1992; 40: 1337–1340.

4.        Odebiyi O.O. et al., Phytochemical screening of Nigerian Medicinal plants, Lloydia, 1978; 41: 234-246.

5.        Chitra Shenoy et al., Antibacterial and wound healing activity of the leaves of Annona squamosa Linn. (Annonaceae). Research J. of Pharmacognosy and Phyto. 2009; 1: 44-50

6.        Lee K.H. et al., Studies on the mechanism of action of salicylate retardation of wound healing by aspirin. J. Pharma Sci. 1968; 57: 1042-1043.

7.        Scortichini M, Pia RM. Preliminary in vitro evaluation of the anti microbial activity of terpenes and terpenoids towards Erwinia amylovora (Burill). J Appl Bacteriol.1991; 71: 109-112

8.        Tsuchiya H. et al., Comparative study on the anti bacterial activity of phytochemical flavonones against methicillin resistant Staphylococcus aureus, J Ehenophamacl. 1996; 50: 27.

9.        Kapoor L.D. et al., Survey of Indian plants for Saponin, alkaloid and flavonoid, l. Lloydia, 1969; 32: 297-304.

10.     Hwang J.K. et al., Alpha glycosidase inhibitory activity of hexagalloyl glucose from the galls of Quercus infectoria. Planta Med. 2000; 66: 273-274.

 

Received on 05.01.2010

Accepted on 24.03.2010        

© A&V Publication all right reserved

Research Journal of Pharmacognosy  and Phytochemistry. 2(3): May-June 2010, 203-207