Evaluation of Anti-Inflammatory, Antipyretic and Wound Healing Activity of Curcuma Inodora (Zingibaraceae)

Deepak Bharati, Sonawane SA, Kanase KG, Undale VR, Abhynkar MM and Bhosale AV

Department of Pharmacology, PDEA’s SGRS College of Pharmacy; Saswad, Tal- Purandar. Dist-Pune, Maharashtra, India 412301

ABSTRACT

Curcuma inodora is small Zingibarous herb and is used by the tribal’s as a hair tonic and for cure of wounds. The objective of the present investigation was to study the selected dose of dried methanol extract of rhizomes of Curcuma inodora (200mg/kg i.p.) toward possible anti-inflammatory, antipyretic and wound healing potential in experimental animal models. Anti-inflammatory potential of methanol extract was evaluated by carrageenan induced paw oedema and formalin induced paw oedema in rats. Also antipyretic and wound healing activity was also tested on animal models. The studies were conducted on Wistar rats of either sex (160-180 g). The change in oedema volume of the rat hind paw was measured using plethysmometer. The dried methanol extract of curcuma inodora (L.) inhibited the formation of paw oedema to significant levels in rats treated either with carrageenan or formalin. At a dose of 200 mg/kg orally, the dried methanol extract produced 74% inhibition in case of the carrageenan-induced oedema (P<0.01), and there was 79.61% inhibition in formalin-induced oedema (P<0.01).

Dried methanol extract of Curcuma inodora was also evaluated for antipyretic activity on animals as per Vogel’s method. Curcuma inodora elicited a dose dependant inhibition of rectal temperature compared with control group. Dried methanol extract of Curcuma inodora produced 38.31 % inhibition at 200 mg/kg dose with a maximal inhibition 44.06 % at same dose which was compared with standard inhibition at 100 mg/kg p.o.

In excision wound model, 10% ointment of ethanol extract of C. inodora was evaluated for wound healing activity. The result showed that ethanol extract ointment possesses a definite pro-healing action. This was demonstrated by a significant increase in the rate of wound contraction and by enhanced epithelialization.

The results indicated that the dried methanol extract of the rhizomes was active against all the experimentally induced laboratory models of inflammation, pyrexia and wound healing.

INTRODUCTION

As man began to acquire closer acquaintance with his environment he was not only able to sort out as to which plants served for eating and which did not, but he went beyond and began to associate curative characteristics with certain plants, classifying them as pain killer, febrifuges, antiphlogistics, soporifics and so on. This must have involved, no doubt a good deal of trial and errors and possibly some deaths in beginning also, but as it happened antidotes against poison were also discovered. In other words it can be said that the curative nature of herbs and plants have always shown the way to medicinal discoveries and the facts can hardly be denied that any efforts to eliminate man from nature will make his body system a playground for disease.

Although several species of genus Curcuma family- Zingiberaceae, are utilized for the cure of various diseases.

Fig. 1. Photograph showing excision wounds

Curcuma Longa (Turmeric) is useful in cough and bronchitis. It has great reputation in Ayurveda and is used in diabetes and liver disease. It is used as choleretic and cholagogue.1 Turmeric is used primarily as a coloring agent and condiment.² Turmeric is used in different ayurvedic preparation to cure eczema, Itching and parasitic skin diseases. It is and ingredient of ointment used for piles. It is used in urinary diseases.³

Roots of Curcuma zedoaria (kachur) are used in flatulence, dyspepsia, bronchitis, fever and skin diseases caused by impure blood. It is also used as expectorant, carminative, diuretic and rubeficient3. Curcuma caesia is chiefly used in cosmetics. Its paste is applied to bruises and rheumatic pains. Curcuma angustifolia commonly called as East Indian arrowroot is an excellent diet in dysentery and gonorrhea.³

MATERIALS AND METHODS:

Plant material:

The rhizomes of Curcuma inodora were collected in the botanical garden of PDEA’S SGRS College of Pharmacy, Saswad, Pune, and Maharashtra in September 2007. The plant material was identified and authenticated Agherkar Research Institute, Pune, India

Animals:

Wister rats and Wistar strain mice were obtained from the institutional animal house and they were kept in the departmental animal house at 25±2 ◦C and relative humidity 45–51.5%, light and dark cycles of 10 and 14 h, respectively for 1 week before and during the experiments for acclimatization. The animals were provided with standard rodent pellet diet and water was allowed ad labium. Rearing up of animals in the experimental period and there upkeep during the entire experimental span conformed to the norms of Institutional Animal Ethical Committee (IAEC), India and ethical guidelines for investigations of experimental pain in conscious animals.

Preparation of extracts:

Rhizomes of Curcuma inodora were washed with distilled water to remove dirt and soil, and shade dried. Routine pharmacognostic studies including organoleptic tests, macroscopic and microscopic observations were carried out to confirm the identity of the materials. The dried materials were processed as per our earlier described process that is powdered and passed through a 10-mesh sieve. The coarsely powdered material (500gm) was extracted thrice with ethanol. The extracts were filtered, pooled and concentrated at reduced temperature (−5 ◦C) and then freeze-dried at high vacuum and at temperature −40±2 ◦C ⁴

Fig-2: Effect of methanol extract of C. inodora on Carrageenan induced inflammation

Table 1:Anti-inflammatory effect of methanol extract of rhizomes of curcuma inodora by carrageenan induced inflammation

Drug dose / route	Oedema Volume (ML)	% Inhibition
Control	1.22+0.22	--
Aspirin (150mg/kg, oral)	0.26+0.13**	78.79%
Methanol extract of C. inodora(200mg/kg, oral)	0.32+0.09**	74.00%

Values are mean + SEM; n=6animals in each group. **P≤0.01 as compared to control (Dunnett’s test).

Preparation of formulation for wound healing activity:

A 10% (w/w) ointment was formulated using soft white paraffin obtained from S.K. Chem, India ⁵.

Stability of formulation:

Stability of ointment formulations prepared was evaluated in terms of the changes in physical and chemical parameters, which were likely to affect the stability and acceptability of the formulations.

Physical stability:

Ointment formulations were evaluated in terms of physical changes like phase separation and changes in color, odour, consistency etc. of the formulations thereby affecting their stability and other desired formulation properties. Samples of the ointment formulations were kept at different temperature conditions like 40 ◦C, 37 ◦C and room temperature for 45 days. They were periodically observed for physical changes like phase separation and development of objectionable color and odour, etc.

Centrifugation:

Centrifugation is believed to be an excellent tool for the evaluation of accelerated deterioration of ointments. Stability of formulated ointments to centrifugation was determined in 10 ml-graduated cylinder at 10,000 rpm for 10 min using a sigma centrifuge as described by Cockton and Wynn (1952)⁶. The formulations, which were resistant towards centrifugation were selected for further evaluation.

Spreadability

Spreadability was determined by modified wooden block and glass slide apparatus. The apparatus consisted of a wooden block, with fixed glass slide and a pulley. A pan was attached to another glass slide (movable) with the help of a string. For the determination of spreadability measured amount of ointments were placed in the fixed glass slides, the movable glass slide with a pan attached to it, was placed over the fixed glass slide, such that the ointments were sandwiched between the two slides for 5 min. The weight was continuously removed. Now about

50 g of weight was added to the pan. Time taken for the slides to separate was noted.

Fig-3: Effect of methanol extract of C. inodora on Formalin induced inflammation

Carrageenan induced paw oedema in rats:

In carrageenan- induced paw odema model, groups of rats were orally administered with the methanol extract C. inodora (100 mg/kg, 200mg/kg bw)⁷, Aspirin (150 mg/kg) or saline, 1 h before administration of an intradermal injection of carrageenan (0.1 ml of a 1% in 0.9% saline)⁸, into the plantar surface of the right hind paw.

The doses of extracts were chosen, based on those used in an earlier study.The paw volume up to a fixed mark at the level of lateral malleolus, was measured by recording the volume displacement by digital plethysmometer (UGO-BASILE-7140 Barcelona,Italy), just before, and three hours after the injection of carrageenan. The average percent increase in paw volume of each group was calculated, and compared with that of the control (saline) and aspirin groups.

Formalin induced paw oedema in rats:

In formalin- induced paw odema model, groups of rats were orally administered with the methanol extract C. inodora (100 mg/kg, 200mg/kg bw), Aspirin (150 mg/kg) or saline, 1 h before administration of an intradermal injection of formalin (0.05 ml of 1% formalin), into the plantar surface of the right hind paw. The doses of extracts were chosen, based on those used in an earlier study.The paw volume up to a fixed mark at the level of lateral malleolus, was measured by recording the volume displacement by digital plethysmometer (UGO-BASILE-7140 Barcelona, Italy), just before, and three hours after the injection of formalin. The average percent increase in paw volume of each group was calculated, and compared with that of the control (saline) and aspirin groups.^7.8

Antipyretic testing in rats by Brewer’s yeast:

The procedure of vogel was used for the antipyretic studies⁸. The test was performed in rats by injecting 15% suspension of Brewer’s yeast in 0.9% saline of yeast to induce pyrexia. Rectal temperature of each animal was taken before and 18 h after the yeast injection using digital clinical thermometer. Animals that did not show a minimum increase of 0.5 ◦C in temperature 18h after yeast injection were discarded. The selected animals were grouped into five (n = 6) and treated with saline, extract or paracetamol⁹. The rectal temperature of each animal was again recorded at 0.5, 1, 1.5 and 2 h after treatment. Antipyretic effect was rated as the ability of test articles to reverse the induced pyrexia.

Fig. 4: Effect of methanol leaf extract of C. inodora on yeast induced pyrexia in rats. The results are presented as mean. Results were statistically compared between the saline (control) treatment and C.inodora methanol extract (MECI 200 mg/kg, ). ANOVA and Dunnett’s test were used as statistical methods.

Excision wounds:

An impression was made on the dorsal thoracic region 1 cm away from vertebral column and 5 cm away from ear using round seal of 2.5 cm diameter on the anaesthetized rat. The skin of impressed area was excised to the full thickness to obtain a wound area of about 500 mm².Fig.-1.¹⁰. Homeostasis was achieved by blotting the wound with cotton swab soaked in normal saline. Contractions, which contribute for wound closure in first 2 weeks, were studied by tracing the raw wound. Wound area was measured by retracing the wound on a millimeter scale graph paper. The degree of wound healing was calculated using formula: 1−(wound area on corresponding day/wound area on zero day)×100. The number of days for complete epithelization was noted.^{11, 12}

Table- 2: Anti-inflammatory effect of methanol extract of rhizomes of curcuma inodora by formalin induced inflammation

Drug dose/route	Oedema Volume(ML)	% Inhibition
Control	1.18+0.11	--
Aspirin (150mg/kg, oral)	0.16+0.14**	86.86%
Methanol extract of C.inodora(200mg/kg,oral)	0.28+0.09**	79.61%

Values are mean + SEM; n=6animals in each group . **P≤0.01 as compared to control (Dunnett’s test )

Rate of wound contraction:

The wound contraction rate was measured as percentage reduction in wound size at every 2 days interval. Progressive decrease in the wound size was monitored periodically by tracing the boundary and the area was accessed graphically.

Histopathological studies:

In excision model, the regenerated skin of wound was extracted out from rat after scarification of animal. Separated skin was preserved in 10% formalin solution to observe keratinisation, epithelization, fibrosis and angiogenesis. The preserved samples were sent to the pathology lab for pathology study.

Statistical analysis:

All the data were presented as mean ± S.E.M. and analysed by Dunnett’s test and unpaired Student’s t-test for the possible significant interrelation between the various groups. A value of P < 0.01 was considered statistically significant.

Results obtained from wound model have been expressed as mean ± SEM and were compared with the corresponding control (simple ointment) values. P- Values were calculated by Student’s t- test by comparing with control. The percentage of Wound Contraction was calculated as a percentage of the corresponding 0 day’s (original) wound area (mm²).

RESULTS:

Carrageenan induced paw oedema in rats:

The Curcuma inodora at the dose levels of 200mg/kg, exhibited resistance against Carrageenan induced pain after 30 min of the last dose and standard drug.

The average percent increase in paw volume indicates pain after treatment was significantly (P < 0.01) and dose dependently increased. The significant (P < 0.01) effect of standard drug aspirin at the dose

Formalin induced paw oedema in rats:

The Curcuma inodora at the dose levels of 200mg/kg, exhibited resistance against formalin (0.05 ml of 1% formalin) induced pain after 30 min of the last dose and standard drug.

Fig. 5- Excision model Histopathological characteristics of healed tissue by control group. Section studied show structure of skin with features consistent with good no. of fibroblasts, evidence of few blood vessels (angiogenesis) and moderate epithelization. Moderate amount of collagen proliferation also seen. Mild chronic inflammatory cells are seen. No infiltrate seen. Typical cells seen.

Antipyretic effect:

The results of the antipyretic study showed that intra-peritoneal administration of the rhizome extract at 100 mg/kg caused a significant (P < 0.05) while 200 mg/kg showed very significant (P < 0.01) inhibition of the pyrexia induced by yeast (Fig. 3). The antipyretic effect of 200 mg/kg rhizome extract was comparable to that of paracetamol (20 mg/kg i.p.) between 30 and 60 min after treatment.

Wound healing activity:

Stability of the formulations:

The stability of the ointment was evaluated with two parameters namely: (1) physical stability

There was no evidence of phase separation, development of objectionable odour or any other evidence of physical instability and effect on storage at varying temperature on spreadability of ointments. Spreadability of test formulation was compared to that of marketed formulation that is neomycin ointment. The readings indicate they are nearly same in the terms of applicability or spreading capacity. Storage even at accelerated stability conditions does not influence the stability of formulation. Thus it may be concluded that formulations are adequate and satisfactory as far as physical parameters are concerned.

Excision wound study:

The progress of the wound healing induced by Methanol extract of C.inodora ointment ( 10% w/w) treated groups, simple ointment (control) treated group and Neomycin (standard drug) treated group of animals are shown in Table 2. It is observed that the wound contracting ability of the extract ointment in different concentrations was significantly greater than that of the control (i.e. simple ointment treated group). The 10% (w/w) extract ointment treated groups showed significant wound healing from the fourth day onwards, which was comparable to that of the standard drug, i.e. neomycin ointment treated group of animals. The wound closure time was lesser, as well as

the percentage of wound contraction was much more with the 10% w/w extract ointment treated group.

Wound contraction studies

Wound contraction is a factor, which indicates rate of reduction of unhealed area during the course of treatment. Greater the reduction better is the efficacy of medication. In other words the wound will close at fast rate if the medication is more efficient. Table 2 records the reduction of wound area of the different groups over the period of 16 days. It may be seen that the fastest healing of wound took place in case of animals, which received ointment formulation containing plant material and standard neomycin ointment, complete healing was obtained within 10 days. The least rate of wound healing was seen in control group, which received no treatment. Wound failed to heal even after 14th day although the unhealed area was smaller

Fig. 6- Excision model Histopathological characteristics of healed tissue by treatment with 10% w/w ointment of powdered plant material. Section studied shows structure of skin with features consistent with good no of fibroblasts and angiogenesis with increased proliferation of collagen. There is increased epithelialization with good no. of chronic inflammatory cells. No atypical cells seen.

Histopathological evaluation:

The multiple sections studied in Histopathological examination of the tissues of the wound area treated with the ointment formulation of plant material (10% w/w), 0.5% w:w neomycin ointment, and simple ointment (control) treated groups are shown in Figs.5-7 . The

histological examination showed that the original tissue regeneration was much greater in the skin wound treated with test ointments (Fig. 6) and neomycin ointment treated group (Fig. 7) without any oedema, congestion or inflammatory changes. More relative fibrosis were observed in the neomycin treated wound with flattened rete ridges in the epidermis comparing to the skin wound treated with either 10% of the test ointment.

DISCUSSION:

This study has investigated the scientific reasons behind the folkloric use of C.inodora zingi. in the management of inflammatory conditions, pyrexia and wound healing activity. The results indicated that the methanol extract rhizomes of the plant were active against all the experimentally induced laboratory models of inflammation, pyrexia and wound.

The dried methanol extract of curcuma inodora (L.) inhibited the formation of paw oedema to significant levels in rats treated either with Carrageenan or formalin. At a dose of 200 mg/kg orally, the dried methanol extract produced 74% inhibition in case of the Carrageenan-induced oedema (P<0.01), and there was 79.61% inhibition in formalin-induced oedema (P<0.01). Dried methanol extract of Curcuma inodora produced 38.31 % inhibition at 200 mg/kg dose with a maximal inhibition 44.06 % at same dose which was compared with standard inhibition at 100 mg/kg p.o. From that data we can conclude that methanol extract of Curcuma inodora inhibits the release of serotonin and bradykinin (2h) and prostaglandin (3h) .As the mechanism of carrageenan is it induces release of histamine (1h), serotonin and bradykinin (2h), prostaglandin (3h). ¹³

Wound healing process consists of different phases such as granulation, collagenation, collagen maturation and scar maturation which are concurrent but independent to each other. Hence in this study excision wound model was used to assess the effect of herbal ointment on various phases. The result showed that ethanol extract ointment possesses a definite pro-healing action. This was demonstrated by a significant increase in the rate of wound contraction and by enhanced epithelialization.^11.12

The results indicated that the dried methanol extract of the rhizomes was active against all the experimentally induced laboratory models of inflammation, pyrexia and wound healing. The Antinociceptive, antipyretic and wound healing activities may be attributed to the presence of alkaloids, phenols, polyphenols, saponins, tannins, anthraquinones, steroids in the dried methanol extract of rhizomes of Curcuma inodora.^14,15,16

ACKNOWLEDGMENT:

Author is thankful to Poona District Education Association and SGRS College of Pharmacy, Saswad, Pune for providing all the facilities and platform to complete this project.

Fig 7- Excision model Histopathological characteristics of healed tissue by treatment with 0.5% w/w ointment of neomycin studied shows structure of skin with feature of good epithelialization and fibroblast proliferation. Good no of blood vessels with increased collagen synthesis is also evident. Few chronic inflammatory cells seen. No atypical cells seen.

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Received on 30.04.2009

Accepted on 14.05.2009

Research Journal of Pharmacognosy and Phytochemistry. 1(1): July.-Aug. 2009, 35-40

Post wounding days	Wound area (mm2) (mean ±SEM) and percentage of wound contraction
	Simple ointment	Neomycin ointment(0.5%)	Plant material ointment(10%)
0	496.33+2.40	493.16+2.25	502.2+4.77
2	489.83+0.98	484.16+1.16*	483.5+2.18*
4	461.5+3.01	448.16+2.35*	427+2.76*
6	419.5+2.40	419.5+2.43	338.16+3.32*
8	378.16+3.21	373+1.63	277.88+3.83*
10	349.33+2.30	320.66+1.45*	255.66+3.48*
12	280.66+1.25	251+1.63*	181.83+1.12*
14	213.5+5.75	190.33+1.43*	173.16+1.53*
16	207.66+3.82	144.5+3.73	119.83+2.75*