ANTI-INFLAMMATORY EFFECT OF CREAM CONTAINING CALENDULA OFFICINALIS LEAF EXTRACT ON CARRAGEENAN-INDUCED MICE PAW EDEMA MODEL

Hoang Le Son*, Nguyen Van Hoang, Tran Van Minh

International University, Vietnam National University Ho Chi Minh City

*Corresponding  author: hlson@hcmiu.edu.vn

(Received August 26^th, 2013)

Summary

Anti-Inflammatory Effect of Cream Containing Calendula officinalis Leaf Extract on

Carrageenan-Induced Mice Paw Edema Model

Calendula officinalis leaf extract-loaded cream was prepared and assayed for anti-inflammatory activity on carrageenan-induced acute inflammation mouse model. Properties of anti-inflammation were evaluated on creams impregnated various concentrations of Calendula officinalisleaf extract. During the experiment, acute inflammation in form of paw edema was induced in Swiss albino mice divided into seven groups with five mice of each group. Group I (negative control) received no treatment at all. Group II, III, IV, V, and VI received cream containing the leaf extract of Calendula officinalis at the concentration of 0.0%, 1.0%, 2.0%, 3.0%, and 4.0%, respectively. Group VII (positive control) received standard medication, 1.5% calamine. The results showed that groups I-VII exhibited significantly different anti-inflammatory effect on carrageenan-induced paw edema in mice with the healing times of 25.30 ± 0.30, 23.60 ± 0.46, 22.00 ± 0.45, 20.80 ± 0.40, 13.10 ± 0.28, 15.60 ± 0.20, and 16.90 ± 0.48 days, respectively. Meanwhile the healing percentage after 8-day treatment was 11.70 ± 0.04, 14.00 ± 0.07, 15.80 ± 0.09, 34.10 ± 0.01, 80.00 ± 0.03, 71.80 ± 0.04, and 51.74 ± 0.06 %, respectively. These findings suggest that the cream containing 3% of the leaf extract of Calendula  officinalis is the best recipe among the tested formulations and could be considered for further investigation related to clinical trials.

Keywords: Calendula officinalis, Carrageenan, Cream, Anti-Inflammatory, Healing.

1. Introduction

Inflammation is defined as the local response of living mammalian tissues to injury due to certain agents. It is a body defense reaction to prevent the spread of injurious agents and to remove the necrosis cells and tissues. The inflammatory response can be provoked by physical, chemical and biological agents, including mechanical trauma, exposure to excessive amounts of sunlight, x-rays and radioactive materials, corrosive chemicals, extremes of heat and cold, and infectious agents such as bacteria, viruses and fungi [1]. Symptoms of inflammation include redness, swelling, heat, pain, loss of function. There are two types of inflammation, acute and chronic. Acute inflammation is of short duration and represents the early body reaction, resolves quickly and is usually followed by healing. Chronic inflammation is defined as prolonged process in which tissue destruction and inflammation occur at the same time [2]. Inflammatory responses occur in three different phases, each apparently mediated by different mechanisms: 1) An acute transient phase characterized by local vasodilation and increased capillary permeability; 2) A sub-acute phase, characterized by infiltration of leukocytes and phagocyte cells; and 3) A chronic proliferative phase, in which tissue degeneration and fibrosis occur.

Calendula officinalis (C. officinalis) is commonly referred to as marigold. This annual herbal plant belongs to the family Asteraceae. The plant is native to central, eastern and southern Europe. C. officinalis is characterized by orange-yellow petals having a spicy, pungent smell and the leaves have a bitter after taste. Their stem grows to a height of about 30-60 cm. The name Calendula originates from the Latin word calend meaning the first day of the month; this was believed to be the time that C. officinalis plant captures the energy of the sun and blossoms in the morning. C. officinalis has been used for centuries as a local remedy. Its action is stimulant and diaphoretic. C. officinalis was taken internally to reduce fevers, promote perspiration and treat cancer [3,4,5]. The flowers were prepared into extracts, lotions, and ointments and applied directly to the skin to promote wound healing and to soothe inflamed and damaged skin.

C. officinalis is mainly comprised of triterpenes, sterols, flavonoids, saponins, tannins, carbohydrates and amino acids. As this plant possesses powerful healing and anti-inflammatory properties [6], the aim of the present study was to prepare C. officinalis leaf extract-loaded cream and test its anti -inflammatory activity on carrageenan -induced paw edema in mice.

2. Materials and methods

Chemicals: emulsifier glycerol monostearate (GMS) – E471 was obtained from Henan Aowei International Trading Co., Ltd. Carrageenan was procured from Sigma-Aldrich (Singapore). Glycerol and ethanol were purchased from Shanghai Demand Chemical Co., Ltd. Shea butter and coconut oil were bought from Natural Essential Oil Co., Ltd of Ho Chi Minh City. 1.5% calamine cream was obtained from Medinova Co., Ltd. Distilled water was used throughout.

Collection of plant material and extraction: leaves of C. officinalis were collected from Da Lat City. Selected fresh leaves were cleaned and shade-dried for 3 days and the dried leaves were pulverized by a mechanical grinder and passed through a 20-mesh sieve. The dried leaf powder was successively extracted with 80% ethanol using a Soxhlet apparatus. The extractive was filtered and the solvent was removed under reduced pressure with a rotatory evaporator and concentrated at 35°C. The extract was then collected and stored in a sealed jar               at 4ºC for preparing formulations and pharmacological assay.

Cream preparation: the composition of the cream base is detailed in Table 1. In part A, glycerine was heated separately to 70°C. In part B, GMS was previously spread in a thin layer on the water surface, kept for 1 h until it was dissolved, and subsequently heated to 70°C. In part C, a mixture of cetyl alcohol, shea butter, and coconut oil was heated separately to 70°C with hotplate stirrer. Part B was then added to part C with stirring, and part A was added afterwards. The mixture was continuously stirred until the temperature of 70°C was reached. The cream was cooled with stirring to room temperature. Prepared cream base was stored at room temperature (25±2°C) in the tightly closed plastic containers protected from light.Formulation of the cream base containing C. officinalis leaf extract is presented in Table 2. C. officinalis leaf extract with different amounts was added to cream base making the formulations containing 0.0%, 1.0%, 2.0%, 3.0%, and 4.0% leaf extract, respectively. These formulations have carefully been preserved in tube at room temperature.

Table 1. Composition of the cream base

Table 2. Cream formulation containing

C. officinalis leaf extract

In vivo anti-inflammatory assay

Experimental animals: Healthy Swiss albino mice weighing 18–22 grams were purchased from Pasteur Institute of Ho Chi Minh City. They were bred in cages with free access to water and standard diet. Mice were divided into seven groups with five mice of each group. Group I (negative control) received no treatment at all. Group II, III, IV, V, and VI were applied cream containing the leaf extract at the concentration of 0.0%, 1.0%, 2.0%, 3.0%, and 4.0%, respectively. Group VII (positive control) received standard medication (1.5% calamine cream). All experiments were triplicate and conducted in accordance with animal use ethics as accepted internationally.

Establishment of acute inflammation mouse model: Carrageenan-induced mice paw edema was established based on the method described by Winteret al. (1962). This model is based on the principle of release of various inflammatory mediators by carrageenan and believed to be biphasic event. The early phase is mainly attributed to the release of histamine and serotonin. The late phase of edema is due to the release of prostaglandins, protease and lysosome.The process of inducing acute inflammatory mouse model was created in various stages. Dorsal surface of each mouse was clean shaved 24 hours before giving experiments. Shave area was disinfected with 70% ethanol, and paw edema was induced in Swiss albino mice by 1% carrageenan solution. Mice were challenged by a subcutaneous injection of 0.05 mL of 1% carrageenan solution into the sub-plantar side of the left hind paw. The paw volume was measured by plethysmometer before carrageenan injection and then 3 h after carrageenan injection. Assessment of inflammation degree was based on the observed variation in pilot sites as the inflammation size, color, and swelling. Inflammation size was measured on day 3, 5, 6, and 8 [7]. Paw edema was expressed as percentage of increase in paw volume measured at each time point relative to the initial paw volume (basal)

Assessment of anti-inflammatory effect of cream containing C. officinalis leaf extract on carrageenan-induced paw edema in mice: Mice were applied cream on inflammatory sites three times a day. The anti-inflammatory effect of each cream formulation was evaluated on inflammation appearance, healing percentage and healing time. Inflammation appearance was externally observed throughout the experiments. Skin color, edema and erythema were observed. The healing percentage was calculated by using the following formula:

Healing percentage (%) =x100%

Values represent the percentage of mean of triplicate ± SD. Mean values of samples followed by the same letter are not significantly different (P<0.05)

Healing time: As can be seen from Table 4, the longest healing time was noted in group I (negative control) (25.30 ± 0.30 days), the shortest one was observed in group V (13.10 ± 0.28 days). The group VII (positive control) need 16.90 ± 0.48 days to complete the healing. These results indicate that the cream impregnated 3% leaf extract of C. officinalis was the best dressing among the tested formulations.

Table 4. Healing time

Histological examination: The photomicrographs of inflammation from group V were taken at specific intervals for visual comparison as can be seen from Figure 1.

Figure 1(a,d) showed the structure of normal skin tissues composing of three main layers including epidermis, dermis and hypodermis. Sebaceous gland, hair follicles and muscle tissues were also observed clearly in normal skin tissues [Figure 1(a, P1, P2, P14) (d, P5)]. The presence of polymorphonuclear leukocytes [Figure 1(b, P3)] showed evidence of inflammation phase. It is known that inflammation is partly characterized by accumulation of leukocytes in inflammatory dermal sites. This was evidenced by a combination of vasodilation with thickening of the blood (due to fluids leaking out of the vessels) cause a slowing of flow rate, which encourages leukocytes stick to the side of the vessels.  Besides, mixed inflammatory cell infiltrate was noted as well as the presence of edema of tissue under skin [Figure 1(e, P8, P9), (f, P11)]. Based on the clinical features and the histological results, acute inflammation in form of paw edema was created successfully. Paw edema was typically characterized by the increased emigration of polymorphonuclear leukocytes (granulocytes) from blood into the damaged tissues.  The dermis and subcutaneous fat were partly damaged, resulting in local inflammatory response. During anti-inflammation process, the formation of cellular components such as lymphocytes, fibroblast, and fiber collagen marked a good restoration of tissue [Figure 1 (g, P12, P13)].

References

1. Harsh M. (2010), textbookof Pathology. 6 ^th Ed. New Delhi:  Jaypee Brothers Medical Publishers (P) LTD.

2. Jeffrey S. W., Peter A. W., CHAPTER 6: The inflammatory response Williams Hematology

http://medtextfree.wordpress.com/2011/12/20/chapter-6-the-inflammatory-response/.

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