Anti Inflammatory Activity In Vivo In Rats Biology Essay

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Abstract: The aim of this work was the evaluation of the preparation of matrine ethosome and the percutaneous permeation in vitro and the anti-inflammatory activity in vivo in the rat skin. The matrine ethosome was prepared by ethanol injection-sonication method. The particle size of the ethosome was measured by a laser particle size analyzer and the entrapment efficiency was detected by ultracentrifugation. The anti-inflammatory activity in vivo of the formulation containing matrine was determined by a reflection spectrophotometer. The average particle size of matrine ethosome was in the range of 50-200 nm with a narrow distribution, and the entrapment efficiency was in the range of 40%-90%. The erythema-index variation of the empty ethosome had no significant difference with that of physiological saline solution (p>0.05), however, there were significant differences between water-ethanol solution and empty ethosome or physiological saline solution (p < 0.01). Compared with other formulations, the matrine ethosome had the largest 24 h accumulative permeation quantity (60.5%) and with no permeation time delay. The matrine ethosome was able to make the induced erythema disappear more rapidly than the non-ethosome formulations of matrine. The average particle size of matrine ethosome decreases with the increase of ethanol concentration, and increases with the increase of phospholipid concentration; while the entrapment efficiency increases with the increase of the concentration of both ethanol and phospholipid. The matrine ethosome can increase the percutaneous permeation of matrine in the experiment in vitro. And it also can improve the anti- inflammatory activity of matrine in vivo in the rat skin.

Keywords: ethosome, matrine, percutaneous permeation in vitro, anti-inflammatory activity in vivo

1. Introduction

Matrine is a major active component of Sophora flavescens alkaloids, with comprehensive pharmacological effects (Wang Junxue et al. 2000; Liang Yugang, 2005), and is widely applied for various inflammatory dermatoses, arthritis and tumors. The formula of Matrine is C15H24N2O, and its molecular weight is 248.37. Ethosome as a new liposome in drug delivery system has high deformability and high entrapment efficiency, and can penetrate through the skin completely and improve the drug delivery through the skin. Compared to other liposomes, the physical and chemical properties of ethosome make it deliver the drug through the stratum corneum into a deeper skin layer efficiently or even into the blood circulation (Dayan et al., 2000). It is very important in the design of the topical drug carrier and transdermal delivery system. Moreover, the ethosome carrier also can provide an efficient intracellular delivery for both hydrophilic and lipophilic drugs (Touitou et al., 2001), and as a result, it permits the antibacterial peptide to penetrate into the fibrocyte easily (Godin et al., 2004). In this paper, the matrine ethosome was prepared by using lecithin and ethanol, and its percutaneous permeation in vitro and anti-inflammatory activity in vivo in rats was also studied.

2. Materials

Soybean Lecithin was supplied by Avanti polar lipids inc., USA, and methylnicotinate (MN, purity >99%) was purchased from Sigma-Aldrich, Germany. Matrine standard was obtained from national institute for the control of pharmaceutical and biological products. Raw material of matrine was from Shanxi Huanpeng natural production Co. Ltd. All other agents were of analytical grade. Double distilled water was produced in the lab. Sonifier Probe type ultrasonic instrument was obtained from Branson Corporation, USA. High performance liquid chromatography (HPLC) equipped with C18 column (150mm-4.6mm, 5μm) was purchased from Waters Company, USA. SP60 reflection spectrophotometer was from X-Rite Incorporated, USA, and Hill Top chambers were from Hill Top Research, Inc. USA. Zetasizer 2000 Laser particle size analyzer was purchased from Malvern Instruments Ltd., England, and Sigma 3K30 desktop high-speed freezing centrifuge was from Sigma Company, Germany.

22 healthy SD rats aged 4-6 months of ordinary grade with weight of 250-300 g were supplied by the experimental animal center in Central South University, and there is no limit to the sex. The experimental process for the treatment of animals measures up to the standards of animal ethics.

3. Methods

3.1 Preparation of ethosome

Modified method of ethanol injection combined with sonication was applied for the preparation of ethosome. The ethosome comprised 1-3 g/L lecithin, 30%-45% (v/v) ethanol, the drug and water. Lecithin was dissolved in ethanol in a glass bottle, and matrine or methylnicotinate was dissolved in double distilled water and stirred by magnetic stirrer. The glass bottle was connected to a syringe hermetically, which permitted the supplement of ethanol but avoids evaporation. After dissolution of the drug, lecithin-ethanol solution was added into the drug solution at a flow rate of 200 μl·min-1, and then the mixture was finely homogenized by sonication for 5 min. The obtained solution was filtered through 0.22 μm disposable filter and then the ethosome was obtained. All the processes were carried out under the protection of nitrogen at room temperature. The final concentration of the drug is 10 g/L (matrine) or 2 g/L (methylnicotinate) by adjusting the addition of the drug.

3.2 Analysis of ethosome particle size

Analysis of the ethosome particle size was performed by a laser particle size analyzer. The water-ethanol solution (filtered through 0.22 μm filter) of the same proportion for the preparation of ethosome was used as sample diluents. The particle size analysis was carried out as soon as the ethosome have been prepared. The ethosome particle size was determined for 5 batches to calculate the average value.

3.3 Entrapment efficiency of ethosome

Ultracentrifugation (Dayan et al., 2000) was applied to separate the unencapsulated portion of the matrine ethosome to determine the entrapment efficiency. The solution was stored overnight at 4 °C and then poured into a centrifuge tube to be centrifugated at 4°C at 56000 -g for 45 min by a high-speed freezing centrifuge. Then the ethosome pills were obtained by removing the supernatant. The content of the drug was determined by HPLC. The entrapment efficiency was calculated by the following equation:

Where DE is the content of the drug detected in the ethosome pills, and DS is the content of drug detected in the supernatant. The result was expressed by the average value of the ethosomes from 3 different batches ± standard deviation.

3.4 The percutaneous permeation in vitro of ethosome

The skin samples were obtained after hair removal of the rats by the method described in (Cevc et al. 2001). The rat skin was applied in the percutaneous permeation experiment once they were obtained. A self-made Franz diffusion cell was used for the percutaneous permeation study. The diffusion cell comprises a supplying cell and a receiving cell, and the diffusion area 0.75 cm2. The rat skin was supported horizontally, and the stratum corneum side was contacted with the medication agent. The receiving cell was filled with isotonic phosphate-buffered solution (pH 7.4). Each formulation (200 μL) was used on the rat skin. The experiment duration is 24 hours. Total solution in the receiving cell was collected and analyzed by HPLC to determine the permeation amount of the drug, and then the receiving cell was filled again with fresh medium at the same volume. Six different percutaneous permeation experiments were performed for each formulation, and the result was expressed by average and standard deviation.

3.5 Determination of the drug by HPLC

Methylnicotinate was analyzed by HPLC at room temperature at the detective wavelength of 218 nm, and the mobile phase was a mixture of acetonitrile and water (35:65 v/v) at the flow rate of 0.6 mL·min-1. Matrine was analyzed by HPLC at room temperature at 220 nm, and the mobile phase was a mixture of methanol-water- triethylamine (54:46:0.02) at the flow rate of 1 mL·min-1.The injection volume was 20 μL.

3.6 The anti-inflammatory activity in vivo of the ethosome formulation on rats

Cutaneous tolerability of empty formulation for topic application in rats (9 rats) was detected by a reflection spectrophotometer, which is a kind of non-invasive technique and doesn't cause any damages to the skin. Light source C and 2° standard were applied by the reflection spectrophotometer, reflection spectrum in the range of 400-700 nm could be obtained by observers. Six sites are randomly located on the dorsal skin of the rats with round template (1 cm2), and the rats were divided into three groups according to different application time (6, 24, 48 h), and as a result, there are three rats in each group, and two sites on each rat were used for the same formulation. The distance between each point was at least 2 cm in order to avoid any possible disturbances, and to monitor the appearance of induced erythema. The baseline value of the erythema index (EI) was calculated before the ethosome was applied on each site, and then the formulations were applied on the skin sites of the rats with Hill Top chambers. Before reading from the reflection spectrophotometer, the Hill Top chambers was removed and then the rat skin surface was softly washed by water to remove the formulation used, and the rat skin was dried for 15 min. The possible induced erythema was detected after the formulations applied for 6 h, 24 h or 48 h, and EI was calculated by the following equation (Dawson et al., 1980):

EI=100[log1/R560+1.5(log1/R540+log1/R580) ?2(log1/R510+log1/R610)],

Where 1/R was the reciprocal of the reflectance at certain wavelength (510, 540, 560, 580, 610), and ΔEI was the variation between the EI value and the baseline value.

The evaluation of the induced erythema by methylnicotinate (MN) in vivo: to compare 2 g/L MN ethosome with MN water solution, MN water-ethanol solution and empty ethosome H, the dorsal skins of 6 rats (four administration sites were designed for each rat) were treated by four topical formulations for 15 min to monitor the appearance of chemically induced erythema and the EI variation.

The evaluation of the anti-inflammatory activity of the matrine ethosome: to compare the regression effect of the induced erythema of the matrine ethosome with that of matrine water solution or water-ethanol solution, four sites were randomly located on the dorsal skin of each rat (totally 6 rats), after all the sites were treated with 100 μL 2 g/L MN for 15 min, the skin surface was softly washed by water to remove the MN formulation, and 200 μL of various matrine formulations such as 10 g/L matrine ethanol solution, water or water-ethanol solution were applied. 200 μL physiological saline solution was used as control. Another rat was used for the study of anti-inflammatory activity of the empty ethosome (six sites were randomly located on the dorsal skin). Timing was started from the application of various matrine or control formulations and the chemically induced erythema had been monitored.

3.7 Statistic analysis

Statistic analysis was carried out by software SPSS14.0 in one-way ANOVA method. P<0.05 was regarded as significant difference. All the results were expressed by average ± standard deviation.

4. Results

4.1 The particle size and entrapment efficiency of the matrine ethosome

The ethosomes with different proportions of ethanol and soybean phospholipid were firstly prepared in this study, and the effects of different proportions in various prescriptions on the average particle sizes and their distribution were evaluated. The result listed in table 1 shows the ethosomes, especially the one prepared by ethanol with maximum concentration, had a comparatively narrow distribution. The particle size of the ethosome decreased with the increase of ethanol concentration, and increased with the increase of phospholipid concentration. Specially, the particle size of formulation C (178 nm) was the largest, while that of formulation G and H (about 50 nm) was the smallest. The particle size and distribution of matrine ethosome prepared according to the composition H (Table 1) is reported in Fig. 1, the size is 50.8 nm and poly-dispersity index is 0.095. At the same time, the content of ethanol and phospholipid had a significantly positive effect on the entrapment efficiency of the carriers, namely, the higher the contents of ethanol and phospholipid were, the more matrine was entrapped in the ethosome. The entrapment efficiencies of formulation H and I were the largest (more than 80%), while that of formulation A was the smallest (about 40%). The loading capacity and average particle size of the carrier are important indexes affecting the medicine efficacy. Because of the small particle size, uniform distribution of the particle size and high entrapment efficiency, the ethosome H was chosen for the percutaneous permeation in vitro and the in vivo anti-inflammatory experiment.

4.2 The percutaneous permeation in vitro of the matrine ethosome

To evaluate the percutaneous permeation of the ethosome, the percutaneous permeation experiment was carried out by the permeation of various methylnicotinate (MN) and matrine ethosome formulations through the rat skin into a self-made Franz diffusion cell. The effect of organic solvents on percutaneous permeation was evaluated by comparing the percutaneous permeation curve of the MN ethosome with that of MN water solution and MN water-ethanol solution. Fig.2 shows the percutaneous permeation through the rat skin of various formulations containing MN at the concentration of 0.2% (w/v). The results reveal that the 24 h accumulative permeation quantity of MN ethosome (77.9% of the dosage applied) > empty ethosome/ MN water-ethanol solution (62.2%) > methylnicotinate water-ethanol solution (39.3%) > MN water solution (24.7%), and the permeation time delay of MN water solution (3 h) > MN water-ethanol solution (2 h) > empty ethosome/ MN water-ethanol solution (l h) > MN ethosome (null). Statistical analysis result shows that there were significant differences between the 24 h accumulative permeation quantities of four formulations containing MN compared, wherein F=491.6,P<0.01; All the P values in the comparisons between every two formulations were smaller than 0.01, indicating significant differences. Fig.3 shows the in vitro percutaneous permeation through the rat skin of various formulations containing matrine at a concentration of 1% (w/v). The percutaneous permeation results were similar to those of MN formulations. The results reveal the 24 h accumulative permeation quantity of matrine ethosome (60.5% of the dosage applied) > empty ethosome/ matrine water-ethanol solution (46.9%) > matrine water-ethanol solution (21.5%) > matrine water solution (7.9%), and the permeation time delay of matrine water solution (3 h) > matrine water-ethanol solution (2 h) > empty ethosome/ matrine water-ethanol solution (l h) > matrine ethosome (null). Statistical analysis result shows that there were significant differences between the 24 h accumulative permeation quantities of four formulations containing matrine wherein F=947.9,P<0.01; All the P values in the comparisons between every two formulations were smaller than 0.01, indicating significant differences.

4.3 The anti-inflammatory activity in vivo of the matrine ethosome in the rat skin

One of the important properties of the topical medicine is the skin tolerability. In this study, the possible erythema induced by topical empty ethosome was evaluated by a reflection spectrophotometry (see Fig.4), the results reveal that there was no significant difference between ΔEI of the empty ethosome and physiological saline solution (control), but the differences between ΔEI of empty water-ethanol solution and empty ethosome or physiological saline solution were significant (F values of 6 h, 24 h, and 48 were 1324.9, 873.1, and 2237.7, respectively. All the P values were smaller than 0.01). Fig. 5 shows the erythema index variations of the rat skin in vivo after the topical application of various MN and control formulations. As compared MN ethosome with MN water solution or MN water-ethanol solution, the significantly high ΔEI was as its characterization, strong erythema appeared in early stage, and a peak appeared at 1 h of the erythema index variation curve of methylnicotinate ethosome, and high ΔEI kept constant for a long period (10 h). To evaluate the effect of matrine ethosome on the improvement of medicinal anti-inflammatory activity, erythema was induced at different skin sites by the treatment of a formulation containing MN (according to the result of the erythema index variation mentioned above after the application of various formulations containing MN, the MN ethosome H of unseparated free drug was chosen as the erythema induction formulation in this experiment), then the formulation containing matrine was used and then the erythema started to regress (see Fig. 6). The results reveal that matrine ethosome was able to make the erythema disappear more rapidly as compared with the non-ethosome formulations of matrine. Especially, the induced erythema at the site treated by topical MN formulation followed by the matrine ethosome was completely disappeared, while the induced erythema was still presented at the site treated by matrine water solution or matrine water-ethanol solution. In this study, empty ethosome H was used to verify the possible effect of the components in the empty ethosome on the chemically induced erythema, the result reveals that the empty ethosome had no effect of either erythema induction or anti-inflammatory activity (see Fig.4, Fig.5 and Fig.6).

5. Discussions

Matrine has the wide varieties of pharmacological actions and efficacies including anti-bacteria, anti-inflammatory, anti-rheumatism, anti-tumor, anti-allergy, anti-virus, antiarrhythmic, and the regulation effect of immunity and biological reactions (Huang Zhiping et al., 2006; Li Wanyu et al., 2006; Jia Yubo, 2007; Jiang Hezhong, 2007; Li Baoxin et al., 2007; Zhang Ying et al., 2007; Yang Xueyun et al., 2008). At present, the formulations containing matrine mainly include the tablet, capsule, injection, liposome, foam agent, suppository, etc (Che Jun et al.,2007; Chen Rui et al., 2007; Li Chengwang., 2007; Tian Juan et al., 2007; Wu Guifang et al., 2007; Zhang Xiuming et al., 2007). Ethosome as a new liposome is especially suitable to be a topical administration carrier and a transdermal administration carrier (Jain et al., 2007; Fang YP et al., 2008). Matrine is a water soluble drug, and generally the entrapment efficiency of the ethosome of water soluble drug is higher than that of the vesicle formulation (Furneri et al., 2000), so matrine ethosome was prepared firstly in this study, then its percutaneous permeation in vitro and the anti-inflammatory activity in vivo in the rat skin were also researched. It is found that the particle size of matrine ethosome decreased with the increase of ethanol concentration, and increased with the increase of phospholipid concentration. The contents of ethanol and phospholipid had a significantly positive effect on the entrapment efficiency of carriers. It is also found that for MN water solution as compared with other MN formulations, its permeation time delay was the longest, and the 24 h accumulative permeation quantity of MN was the smallest. On the contrast, there was no permeation time delay for MN ethosome, and the 24 h accumulative permeation quantity of MN was significantly higher than that of other MN formulations. At the same time, compared with MN water-solution, The 24 h accumulative permeation quantity of empty ethosome/MN water-ethanol solution was larger than that of MN water solution or MN water-ethanol solution, while smaller than that of MN ethosome. This reveals empty ethosome incorporated into MN water-ethanol solution could enhance the drug permeation significantly. It is perhaps as a result of the interaction of ethosome, ethanol and drug: on the one hand, the drug permeates easily into the ethosome with the help of ethanol, then the ethosome may enhance the drug permeation; on the other hand, ethanol also may enhance the drug permeation through the rat skin. Because of the flexility and penetrability of the ethosome (permitting fast diffusion of the drug from the environment to the ethosome), its real distribution is difficult to be detected in the skin, however, significantly positive effect of the drug entrapped into the ethosome on penetrability of molecules through the rat skin has been confirmed in this study. As for in vitro percutaneous permeation of various formulations containing matrine, matrine can permeate through the rat skin from the matrine water solution or matrine water-ethanol solution, and their percutaneous permeation curves are similar. However, the 24 h accumulative permeation quantity of matrine water solution was the smallest. The percutaneous permeation of empty ethosome incorporated into matrine water-ethanol solution indicates its enhancing permeation effect on matrine, which is similar to the percutaneous permeation of MN investigated. Compared with other formulations containing matrine, the matrine ethosome had the largest 24 h accumulative permeation quantity and with no permeation time delay. In statistical analysis, significant differences are observed in the comparisons of 24 h accumulative permeation quantity of four formulations containing matrine, and significant differences also exist in the comparison of every two formulations. The difference between the 24 h accumulative quantities of MN and matrine of the same formulation passing through the rat skin may be caused by the difference of the physical and chemical properties of two molecules. Ethanol may enhance permeation effect of both MN and matrine. However, the enhancing permeation effect of all the ethosomes is much larger than the expected effect of ethanol applied alone. This may be due to the synergistic effect of ethanol, phospholipid vesicle and lipid of the skin, especially due to this formulation itself of the ethosome. Generally, ethosome as a new carrier of percutaneous permeation is able to enhance drug percutaneous delivery and accumulation in the skin as compared with liposome and ethanol solution (Rao Yue-feng et al., 2004; Feng Xuan et al., 2008). The ethosome contains ethanol in a high concentration as compared with liposome: on the one hand, ethanol may enhance the deformability of lipidic bi-layer of the ethosome; on the other hand, the interaction of ethanol and lipid of the stratum corneum may decrease the transformation temperature of the latter, increase its fluidity and then enhance drug permeation. It is noteworthy that phospholipids are dissolved in ethanol easily, so the ethanol concentration is not suitable to be too high in the ethosome system, not higher than 45 % generally (Touitou et al., 2000); otherwise it may be again the formation of ethosome. In the experiment of long period skin tolerability, it is found that there was no significant difference between ΔEI of empty ethosome and that of physiological saline water; however, ΔEI of empty water-ethanol solution was significantly different from that of empty ethosome and physiological saline water, namely, the skin erythema could be induced significantly by empty water-ethanol solution; and it also means that ethanol in the ethosome had no induction effect on the skin erythema, although the concentration was high. There are two key factors for the induction of erythema by MN: the composition of the drug carrier and application duration. The intensity of the induction erythema is proportional to the content of MN in the rat skin. Therefore, all kinds of formulations applied to the rat skin must keep the same content of methylnicotinate, and the total dosages and durations of the formulations applied should be absolutely identical, so any differences of the erythemas induced by various formulations are caused by the carrier and the enhancing permeation effect. The results of the erythema index variation after various formulations of MN and control applied reveal that as compared MN ethosome with MN water solution or MN water-ethanol solution, the significantly high ΔEI was as its characterization, strong erythema appeared in early stage, and a peak appeared at 1 h of the erythema index variation curve of methylnicotinate ethosome, and high ΔEI kept constant for a long period (10 h). The results of the anti-inflammatory activity of various formulations containing matrine and the control in the rat skin reveal that the matrine ethosome could make the erythema disappear more rapidly as compared with the non-ethosome formulations of matrine, especially, the induced erythema at the site treated by the formulation of MN and followed by the matrine ethosome for 3 h was completely disappeared, while the induced erythema was still presented at the site treated by matrine water solution or matrine water-ethanol solution. In this study, it is also observed that the empty ethosome H had no effect of either erythema induction or anti-inflammatory activity. All the results confirm that the ethosome can improve the anti-inflammatory activity of matrine in the rat skin. The study has indicated that the ethosome is a kind of promising carrier for topical administration, and it can improve the drug delivery through the rat skin to achieve a better efficacy, and is worthy of being further studied.

  

6. Conclusions

The particle size of matrine ethosome depends on its composition, and it decreases with the increase of ethanol concentration and increases with the increase of phospholipid concentration. However, the entrapment efficiency increases with the increase of ethanol and phospholipid concentration. The empty ethosome shows a good tolerability when it is used as a delivery carrier in the rat skin. In the in vitro experiment, the matrine ethosome can improve the percutaneous permeation of matrine. And the matrine ethosome can enhance its anti-inflammatory activity to the in vivo induced erythema in the rat skin.

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