Anti-inflammatory and wound healing activity of Eupatorium odoratum Linn. Leaves

Prabhudutta Panda^*, Arpita Ghosh and Maitrayee Panda

College of Pharmaceutical Sciences, Mohuda, Berhampur-760002, Orissa, India

ABSTRACT:

The present study was carried out to elucidate the potential of methanol extract of Eupatorium odoratum L (Family- Asteraceae) leaves on anti-inflammatory and wound healing effects using various animal models. The dried, powdered leaves of E. odoratum were extracted successively with petroleum ether (60–80⁰C), chloroform and methanol in soxhlet apparatus. The methanol extract (yield 23.6 % w/w with respected to dry powdered plant material) was selected for all experimental procedure. Anti-inflammatory effect were investigated by employing the carrageenan induced rat paw edema test in adult Wister albino rats and the wound healing activity in Swiss Albino rats. Results were revealed that the methanol extract was found significant anti-inflammatory effect (P<0.001) at the dose levels of 100,200 and 400 mg/kg, orally in mice. Ointment and gel was prepared from the methanol extract of E. odoratum and tested for pH, viscosity, spreadability, drug contents uniformity. The formulations were evaluated for its acute skin irritancy, wound healing activity in Swiss Albino rats. The formulations did not produce any skin irritation for about a week when applied over the skin. The formulations and the normal methanol extracts of E. odoratum showed significant (P< 0.001) wound healing activity by excision wound model and comparable with that of the reference standards and control bases. The measurement of the wound areas were taken on 3^rd, 6^th, 9^th, 12^th, 15^th and 18^th days and the percentages of wound closures were calculated. It concludes that, methanol extract possessed remarkable anti-inflammatory and wound healing activity effect on animal models.

KEYWORDS: Eupatorium odoratum leaves, methanol extract, Anti-inflammatory, wound healing

INTRODUCTION:

Eupatorium odoratum L. Syn: Eupatorium conyzoides Vahl. (Family-Asteraceae) is a Christmas bush, also known as bitter bush, Siam weed, baby tea, Santa María, is a scrambling shrub. It may reach 1m or more as a free standing shrub and 4m or more when climbing into trees or shrubs. Stems reach 2cm in diameter. The plants are maintained by a system of abundant, yellowish, fine lateral roots. Multiple sprouts arise from the root crown and lower stems. The individual branches are long with relatively few branches. The opposite, three-nerved leaves are deltoid to ovate-lanciolate, usually with a dentate margin and a long pointed tip. The leaves are aromatic when crushed. The inflorescences are corymbs of cylindrical heads located on the terminals of lateral branches. There are 15 to 25 tubular florets per head, white, lavender, pink, or blue in color. The seeds are a brownish gray to black achene that is 4mm long with a pale brown pappus 5 or 6 mm long ^{1, 2}.

E. odoratum L. has acquired a reputation as a medicinal herb for a variety of ailments including malaria, fever, and the aqueous leaf extract of the plant is used as antiseptic for wound dressing. The decoction of the leaf was used as a cough remedy and the stem decoction can be used in pulmonary hemorrhage³. The phytochemical investigation revealed the presence of α-pinene, β- pinene, myreene, limonene, β- caryophyllene⁴, d-eupatene, eupatol, lupenol, β-sitosterol, β-amyrin, intermedine, pinderine⁵, tamarixetin, salvigenin, odoratin, rhamnetin^6-8. The literature reveals that the E. odoratum leaves are used orally against wounds, inflammation in traditional system.

Hence, an effort has been made to establish the scientific validity to investigate the possible anti-inflammatory and wound healing activity made from the dried methanol extract of E. odoratum L. leaves in animal models.

MATERIALS AND METHODS:

Plant material and extraction:

E. odoratum L. leaves were collected during the month of August from the rural village of Mohuda in Ganjam District, Orissa, India, identified and authenticated by Prof. S.K. Dash, Head, PG Department of Bioscience, College of Pharmaceutical Sciences, Mohuda; comparing with the voucher specimen (EOL-I) present in the herbarium, has been kept in the laboratory for future references. The collected plants were washed and air-dried under the shade, powdered by a mechanical grinder and passed through 40-mesh sieve and stored in a closed vessel. The powdered leaves of E. odoratum L. (1kg) were extracted successively with 1200ml of petroleum ether (60–80⁰C) and 1200ml of chloroform in soxhlet apparatus. A dark greenish black coloured petroleum ether extract was obtained. Powdered leaves (marc), after proper drying, were extracted with chloroform to produce a greenish brown extract. The same powdered leaves after proper drying were again extracted with methanol (18h) to produce a greenish brown extract. The extractions were carried out with fresh solvent until the extract became colorless. These extracts were again dried and concentrated by evaporating the solvent completely under vacuum at the range of boiling points of solvent (Methanol at 78.4 °C) using rotatory evaporator (Jain Scientific glass works, DTC 201, Ambala Cantt, India) The Methanol extract (yield 23.6 % w/w with respect to dry powdered plant material) was selected for all experimental procedure. The chemical constituents of the extract was identified by qualitative analysis and confirmed by the thin layer chromatography (i.e. hR_f values).

Preliminary phytochemical analysis:

The methanol extract of E. odoratum was subjected to preliminary phytochemical screening for detection of major chemical groups. In each case test 10% w/v solution of the extract in methanol was used and unless otherwise mentioned in individual test⁹. Results of different chemical tests on the chloroform extract of E. odoratum showed the presence of phytoconstituents viz., steroids, triterpene, flavonoids, alkaloids, flavonoids.

Animals:

Swiss Albino rats of either sex weighing 150-200g obtained from M/s Ghosh and Ghosh Enterprises., Kolkata, India, were housed in standard polypropylene cages at room temperature of 30 ± 2 ⁰C and 60-65% relative humidity and had free access to food and water ad libitum. The animals were fed with a commercial diet (Hindustan Lever Ltd., Bangalore).

Acute Toxicity analysis:

Toxicity study of the E. odoratum methanol extract (EOME) was performed to get the information, how safe is this extract for the therapeutic use. The LD₅₀value of EOME was derived by the method of Litchfield and Wilcoxon¹⁰. The maximum non-lethal dose was found to be 4000mg/kg body weight, orally. The 0.025% CMC was used as a vehicle and showed no mortality. The determination of acute toxicity by adopting fixed dose the guideline of CPCSEA and 1/10^th of LD₅₀cut off values¹¹ of the extracts were taken as screening dose. i.e. 100,200, 400 mg/kg for subsequent studies.

Anti-inflammatory activity:

Anti-inflammatory activity was evaluated using the carrageenan-induced edema in rat paw according to the technique of Winter¹² and Satyanarayana¹³. After16h. fasted rats were divided into four groups of six each. Group-I, served as a control, received 0.025% w/v CMC at the dose level of 10 ml/kg, orally, Group-II to IV, animals received EOME at dose of 100, 200 and 400mg/kg, orally, Group-V, animals were treated with standard drug diclofenac sodium at the dose level of 10mg/kg, orally. Acute inflammation was induced by carrageenan in sub planter side of the right hind paw in rats. The paw was marked with ink at the level of the lateral malleolus and dipped in Perpex cell up to this mark. The measurement of the paw volume was carried out by means of Ugo Basile Plethysmograph model 7150, before and after 4h after carrageenan injection ¹⁴. Percentage inhibition of edema was calculated using formula¹⁵ i.e. % Pain Inhibition = (1- Vt/Vc) x 100. Where, Vt = Increase in paw volume in drug treated rats. Vc = Increase in paw volume in control group treated rats.

Formulations:

The dried methanol extracts of E. odoratum was taken for the preparation of ointment and gel. Formulations were prepared using an ointment base and gel base according to the formula given in the Table-1^{16, 17}. Appropriate standard methods of fusion were adopted, where the solid fats were melted and mixed, and trituration was followed for preparation of the ointment and gel^18,19. The methanol extract of E. odoratum was incorporated in the bases to get (10%) concentrations. Formulations were packed in wide-mouthed plastic jars with screw-capped lid.

Acute skin irritation study:

The primary skin irritation test was performed on albino rats and weighing about 150-200 gm. The animals were maintained on standard animal feed and had free access to water ad libitum. The animals were kept under standard laboratory condition. The total mass was divided into four batches, each batch containing seven animals. Two batches of each were used for control and test. Dorsal hairs at the back of the rats were clipped off one day prior to the commencement of the study. Animals showing normal skin texture were housed individually in cages with copography meshes to avoid contact with the bedding. 50mg of the each formulation was applied over one square centimeter area of intact and abraded skin to different animals. Aqueous solution of 0.8% formalin was applied as standard irritant. The animals were observed for seven days for any signs of oedema and erythema^{20, 21}.

Evaluation of wound healing activity (Excision method):

The rats were inflicted with excision wounds as described by Morton and Malone ²². The rats were anaesthetized with ether solution prior to creation of the wounds. The dorsal thoracic region of the animal was shaved with electric clipper and the area of the wound to be created was outlined on the back of the animals with methylene blue using a circular stainless steel stencil. A full thickness of the excision wound of 500 mm² was created along the markings using toothed forceps, a surgical blade and pointed scissors. The entire wound left open to the atmosphere^{23, 24}. The animals were divided into seven groups of six each. The animals of Group I were applied with ointment base and considered as the control I, Group II received gel base and considered as control II, Group III served as reference standard I (Neosporin; Neomycin and Polymyxin B Sulfates and Bacitracin Zinc M/S Glaxo Smith Kline Pharmaceuticals Limited, Mumbai), Group IV served as reference standard II (Betadine; Povidone-Iodine IP 5% w/w, M/S Win-Medicare Pvt. Ltd, New Delhi). The animals of Group V, was treated with ointment 10%. The animals of Group VI, was treated with gel 10%. The animals of Group VII, were treated with pure extract with 1% caboxy methyl cellulose base (Ext-I). The ointment and gel were topically applied once in a day, starting from the day of the operation, till completion of epithelialisation. The measurement of the wound areas of the excision wound model were taken on 3^rd, 6^th, 9^th, 12^th, 15^th and 18^th days. Thereafter on alternate days until healing were complete; the percentage of wound closure was calculated. All the protocols were reviewed and permitted by the Animals Ethical Committee (Reference Code: 1170/ac/08/CPCSEA), College of Pharmaceutical Sciences, Mohuda, Berhampur, Orissa

Statistical analysis:

The experimental results were expressed as the Mean ± Standard error of mean (SEM) and the statistical significance was evaluated by One-way analysis of variance (ANOVA) followed by Dunnett’s t-test ²⁵.

RESULTS:

Preliminary phytochemical analysis:

Results of different chemical tests on the chloroform extract of E. odoratum showed the presence of phytoconstituents viz., steroids, triterpene, flavonoids, alkaloids, flavonoids.

Anti-inflammatory activity:

Indigenous drug systems can be a source of variety of new drugs, which can provide relief in inflammation but their claimed reputation has to be verified on scientific basis. The present investigation revealed that the anti-inflammatory activity of E. odoratum on carrageenan induced paw edema in rats is shown in Table-2. These results indicate that, EOME showed significant reduction (P<0.001) in edema volume at oral dose of 100, 200 and 400 mg/kg of body weight, which is comparable to the standard drug diclofenac sodium at the dose of 10mg/ kg in acute inflammatory model.

Wound healing activity:

The percentages closure of excision wound area in animals treated with Ointment and Gel were found to be 100% and 98.16%. The animals treated with normal methanol extract with 1% caboxy methyl cellulose base were found to be 96.56%.

DISCUSSION:

In acute toxicity study, oral administration of EOME did not produce any mortality in mice upto a dose level of 4g /kg. This may be due to broad non-toxic range of the plant, where the plant extract showed a high LD₅₀and relatively safety.

Edema represents the early phase of inflammation in carrageenan induced paw edema and is the simplest and most widely used acute inflammatory model for studying anti-inflammatory agent. The development of carrageenan-induced edema is believed to be biphasic of which the first phase is mediated by release of histamine, serotonin and kinine in the first hour after injection of carrageenan and the second phase is related to release of prostaglandin like substances in 2-3h^{26, 27, 28}. The EOME showed significant anti-inflammatory activity at 4h. against carrageenan injection suggesting that the extract predominantly inhibits the release of prostaglandin like substances from phlogenic stimuli. There are reports that flavonoid possesses anti-inflammatory activity^{29, 30, 31, 32} and some of them also act as phoshpholipase inhibitors ^{33, 34, 35}. Also, there are few reports on the experimental models; the non selective antagonist of opiod receptors apparently acts by antagonizing the action of endogenous opiods involved in pain or stress ³⁶. In the present study, the maximum anti-inflammatory effect of EOME may be attributed to presence of flavonoids as evident by preliminary phytochemical investigations.

Table-1: Physicochemical evaluations of ointment and gel

Formulations

Ingredients

Concentration

(%, m/m)

Drug content (%)

Mean ± SD

Viscosity (cps)

Mean ± SD

Spreadability

(sec)

Oint-10%

Extract

Cetostearyl alcohol

PEG6000

Petroleum jelly

Liquid paraffin

Methylparaben

0.18

98.24±0.24

6.46

16.200±100

Gel-10%

Extract

Carbomer934P

Glycerin

Triethanolamine

Water

Methylparaben

0.5

0.18

98.44±0.26

5.98

13.600±100

Table-2. Anti-inflammatory activity of Eupatorium odoratum leaves on carrageenan induced paw edema in albino rats.

Treatment	Dose (Mg/kg)	Percentage of inhibition of paw edema after carrageen injection
Treatment	Dose (Mg/kg)	1h	2h	3h	4h
(Group-I) Control (0.025% CMC)	10 mg/kg)	14.10±1.96	36.21±4.08	42.31±4.16	42.23±5.26
(Group-II) EOME	100 mg/kg	32.72±2.76^a	72.62±9.64^a	102.52±8.49^b	112.44±10.68^b
(Group-III) EOME	200 mg/kg	25.60±4.22^a	47.22±1.21^a	71.45±7.84^a	82.24±4.46^a
(Group-IV) EOME	400 mg/kg	20.58±0.72^a	42.13±0.78^a	63.7±7.62^a	70.83±4.35^a
(Group-V) Diclofenac sodium	10mg/ kg	45.22±2.43^a	98.89±3.65^a	132.22±2.33^a	136.50±5.65^a

Results expressed as Mean  S.E.M. ^bP <0.01, ^a P< 0.001, significantly different from control; Paired t-test (n = 6)

Table-3: Topical application of ointments and gels from extract of Eupatorium odoratum on wound healing activity in rats. [% of wound healing = (1-t/c) x 100]

Group

Post wounding days

0 day

3^rd days

6^th days

9^th days

12^th days

15^th days

18^th days

Control I

510.88±0.97*

(0.00)

483.43±1.05*

(5.37)

401.59±1.30*

(21.39)

355.64±0.99*

(30.39)

272.84±0.89*

(46.59)

190.69±0.63*

(62.67)

88.43±0.57*

(82.69)

Control II

508.29±1.22*

(0.00)

482.75±0.99*

(5.02)

405.63±1.31*

(20.20)

364.46±0.78*

(28.30)

283.38±0.60*

(44.25)

202.36±0.47*

(60.19)

97.21±0.67*

(80.87)

Standard-I

508.79±1.52*

(0.00)

406.36±1.19*

(20.13)

323.50±1.63*

(36.42)

251.85±0.75*

(50.50)

135.49±0.53*

(73.37)

9.47±0.27*

(98.14)

(100)

Standard-II

510.46±1.63*

(0.00)

415.73±1.39*

(18.56)

333.39±1.29*

(34.69)

260.43±0.60*

(48.98)

144.37±0.56*

(71.72)

19.50±0.48*

(96.18)

(100)

Ointment

506.19±1.63*

(0.00)

441.24±1.35*

(12.83)

329.59±1.19*

(34.90)

267.29±0.80*

(47.19)

140.43±0.50*

(72.26)

18.52±0.46*

(96.34)

(100)

Gel

508.12±0.88*

(0.00)

455.24±0.74*

(10.40)

352.48±0.94*

(30.63)

288.40±0.66*

(43.24)

168.56±0.57*

(66.83)

72.52±0.52*

(85.72)

9.36±0.31*

(98.16)

Extract

506.22±1.12*

(0.00)

454.82±0.76*

(10.15)

360.40±0.52*

(28.80)

296.56±0.52*

(41.42)

178.54±0.46*

(64.73)

77.42±0.46*

(84.71)

17.42±0.43*

(96.56)

Results are expressed mean ± SEM of six readings; Significance evaluated by One-way analysis of variance (ANOVA) followed by Dennett’s t-test versus control group,*P < 0.001, (n = 6). Figures in parentheses indicate the percentage of wound contraction. The ointment comparable with the control I, standard-I and standard-II, similarly the gel and extract also comparable with the control II, standard-I and standard- II.

The various physicochemical parameters utilized to evaluate the prepared ointment and gel are shown in Table-1. From the results, it was clearly evident that ointment and gel showed good homogeneity and extrudability. Formulations did not produce any skin irritation, i.e., erythema and edema for about a week when applied over the skin. The rheological behaviors of ointment and gel were studied with Rotational Brookfield Viscometer. The results indicated that the torque and shear stress increases where as viscosity decreases. The formulations and the normal methanol extracts of E. odoratum showed significant promotion of wound-healing activity with statistically significant (*P<0.001) in all the seven groups of animal which were depicted in the Table-3. The mean percentage closure of wound area was calculated on the 3^rd,6^th, 9^th, 12^th, 15^th and finally 18^th days. Post wounding days also shown in Table-3. The wound-healing activity was found to be comparable with that of the reference standards and control bases.

Both the ointment and gel formulations containing 10% methanol extract of E. odoratum showed significant wound healing activity and comparable with that of the commercial products of Neosporin and Betadine. Wound healing is a complex and dynamic process of restoring cellular structures and tissue layers in damaged tissue as closely as possible to its normal state. Wound contraction is a process that occurs throughout the healing process, commencing in the fibroblastic stage where by the area of the wound undergoes shrinkage. It has 3 phases; inflammatory, proliferative and maturational and is dependent upon the type and extent of damage, the general state of the host's health and the ability of the tissue to repair. The inflammatory phase is characterized by hemostasis and inflammation, followed by epithelization, angiogenesis and collagen deposition in the proliferative phase. In the maturational phase, the final phase of wound healing the wound undergoes contraction resulting in a smaller amount of apparent scar tissue. It mainly depends on the repairing ability of the tissue, type and extent of damage and general state of the health of the tissue. The granulation tissue of the wound is primarily composed of fibroblast, collagen, edema and small new blood vessel.

The undifferentiated mesenchymal cells of the wound margin modulate themselves into fibroblast, which start migrating into the wound gap along with the fibrin strands. The wound healing activity of two pharmaceutical formulations both the ointment and gel containing methanol extracts of E. odoratum was evaluated for its wound healing potentials in excision wound model in rats. Both the formulations responded significantly in this wound models tested. The results were also comparable to that of the standard drugs Neosporin and Betadine used as standard drugs for comparison in this present investigation. The results were also comparable in terms of wound contracting ability, epithelization period at the wound area. In the present investigation, preliminary phytochemical analysis of methanol leaf extract revealed the presence of steroids, triterpene, flavonoids, alkaloids, flavonoids. Flavonoids are known to reduce lipid peroxidation not only by preventing or slowing the onset of cell necrosis but also by improving vascularity. Hence, any drug that inhibits lipid peroxidation is believed to increase the viability of collagen fibrils by increasing the strength of collagen fibres, increasing the circulation, preventing the cell damage and by promoting the DNA synthesis³⁷ flavonoids³⁸ are also known to promote the wound healing process mainly due to their astringent and antimicrobial property, which seems to be responsible for wound contraction. The wound-healing property of E. odoratum may be attributed to the phytoconstituents present in the plant and the quicker process of wound healing could be a function of either the individual or the additive effects of the phytoconstituents. However, further phytochemical studies are needed to isolate the active compound(s) responsible for these pharmacological activities. Further studies with purified constituents are needed to understand the complete mechanism of wound healing activity of E. odoratum. Electron microscopic examination will yield the effect of the extract on angiogenesis, epithelialisation or collagen deposition. The data of this study indicated that the leaf extract of E. odoratum possess better wound healing activity and it can be used to treat different types of wounds in human beings too. Thus, this investigation confirms the use of the ointment and gel E. odoratum extract as a wound-healing agent as known from folklore medicine. Finally, it concluded that the EOME possess remarkable anti-inflammatory and wound healing activity. However, more detailed phytochemical studies are necessary to identify the active principles and exact mechanisms of action.

ACKNOWLEDGEMENT:

Author is thankful to the authority of College of Pharmaceutical sciences, Mohuda, Berhampur, India for laboratory facilities and also thankful to the Prof. S. K. Dash, Head, PG Department of Biosciences, College of Pharmaceutical sciences, Mohuda, Berhampur, for the identification of the plant.

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Received on 09.01.2010

Accepted on 24.03.2010

Research Journal of Pharmacognosy and Phytochemistry. 2(3): May-June 2010, 211-216

(Mg/kg)

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