The compounds 1,8-cineole and α-pinene are reported as main components of Calendula essential oil and as having proven potential to scavenge free radicals, hence they are selected for the present study. The analysis of two main components of the Calendula oil by HPLC method demonstrated that peaks of 1,8-cineole and α-pinene were clearly resolved and peak symmetry was observed. The retention time (RT) for 1,8-cineole and α-pinene were 6.81 and 7.32min respectively ( and ). By employing HPLC method, the concentrations of 1,8-cineole and α-pinene in the essential oil were found to be 8.12±0.7 and 22.53±0.2%, respectively (). The analytical method was validated on parameters as linearity, LOD, LOQ, accuracy and precision (interday and intraday) ().
Chromatogram of 1,8-cineole and α-pinene as standard
Chromatogram of calendula oil with peaks of 1,8-cineole and α-pinene
Concentration of 1,8-cineole and α-pinene in Calendula essential oil by HPLC method
HPLC method validation parameters for quantization of 1,8-cineole and α-pinene in Calendula officinalis L. essential oil
The Calendula essential oil was used to prepare F4 and F5 formulations and then subjected to in-vivo study. To assess any possibility of interaction of formulation contents with Calendula oil components, recovery study was performed in triplicate by proposed HPLC method for the two formulations. The recovery of 1,8-cineole and α-pinene in both the formulations was above 95% which indicates no interaction (). Experimental doses of UV-B radiation on skin cells of rats was 500mJ/cm−2 with an exposure time 12 min per day (daily) for 1 month. The formulations dose 2.0mg cm−2 were applied daily 30 min before UV-B irradiation. The dosing of cream and UV-B irradiation was continued for 30 days and rats were sacrificed afterwards. We determined that in our conditions, increased level of end products of lipid peroxidation in skin samples of rats irradiated with UV-B radiation were observed. summarizes the increased level of MDA in skin tissue and suggests the increased lipid peroxidation leading to skin tissue damage and failure of defense strategy to prevent formation of ROS in presence of UV-B radiation. In the present work, it was observed that pretreatment of Calendula essential oil based cream formulations (F4 and F5) in the dose of 2mg cm−2/day topically decreased the level of MDA.
Recovery studies of 1,8-cineole and α-pinene from formulations
Effect of UV-B radiation and the formulations on MDA, SOD and CAT levels in rat skin
After UV-B irradiation, SOD level was lowered significantly (p<0.01) when compared with control, but in the case of Gr III and Gr IV, both the formulations significantly (p<0.01) increased the SOD level even more than the control group. There was no significant (p<0.01) difference between Gr I and Gr III SOD level. The F5 formulation was observed to be more significant in increasing the level of SOD. The level of CAT in UV-B treated group animals was found to be lower in comparison to the control group. The F4 and F5 formulation significantly (p<0.01) increased the CAT level but the tendency of F5 to increase the CATe level was more prominent. Also it was observed that the GSH level was significantly (p<0.01) decreased in UV-B induced oxidative stress but the level of GSH was significantly (p<0.05) increased after treatment with F4 and F4 cream. The F5 formulation increased the level of GSH more in comparison to F4 formulation. It was found that UV-B irradiation caused significant (p<0.01) reduction in ASC level when compared with control.
However, both the formulations (F4 and F5) increased the ASC level (). Decreased TP level revealed susceptibility to UV-B induced oxidation of protein content of skin tissue. Total protein content for UV-B treated group was observed to be significantly (p<0.01) lower as compared to the control group but the F4 and F5 formulations changed this condition (). There was no significant difference between TP value obtained for Gr I and Gr IV.
Effect of UV-B radiation and the formulations on GSH, ASC and TP levels in rat skin
Topical application or oral administration of antioxidants has been recently suggested as preventive therapy for skin photoaging and UV-B induced cancer. The level of biochemical parameters as MDA, SOD, CAT, ASC and TP level constitute the defense team against ROS generated in oxidative stress [15
]. Formation of ROS and subsequent lipid peroxidation is considered to be mechanism UV radiation induced photodamage. Recent studies have showed that the phytoconstituent, especially flavonoids and terpenoids found in Calendula herb, may be helpful to maintain the skin biological integrity [7
Studies conducted on essential oil mixtures of thyme or clove leaf with cinnamon leaf, rose, or parsley seed toward skin lipid, squalene oxidized by UV irradiation showed inhibitory activities toward malonaldehyde (MDA) formation [16
]. The conventional spectrophotometric methodology for MDA determination requires addition of TBA to the sample and heating at 90°C to form the MDA–(TBA)2
complex. Meanwhile, unsaturated fatty acids of biological sample also react with TBA to form colored substance, which also absorb at or near 535 nm. Thus, at the same wavelength, a higher amount of MDA is expected. MDA determination through formation of TBA complexes by the HPLC method also includes heat process and the same problem occurs there. However, sensitivity and specificity is more in the case of HPLC method. In the present study, MDA was estimated as lipid peroxidation marker by conventional spectrophotometric method. In our research, calendula oil based formulations (F4 and F5) when applied on rat skin prior to 30 min of UV-B exposure, caused a significant reduction in MDA level. It may be possible that Calendula based creams exhibit their effect due to the antioxidant effect. SOD, which is one of the cellular antioxidant enzymes, may play a key role as a defensive mechanism against oxidative damage because SOD catalyzes dismutation of O2−
. Recent studies on biochemistry of the skin tissue have shown marked decreases in SOD activity after exposure to UV-B radiation leaving the cell susceptible to oxidative damage [17
]. Increased levels of SOD in formulation treated groups (Gr III and Gr IV) indicated that F4 and F5 formulation may scavenge the ROS generated from UV-B radiation. Recently researchers have suggested that hydrogen peroxide is produced in the body organs naturally as well as by UV-B irradiation and catalase breaks it down [18
]. If there is a dip in catalase levels, hydrogen peroxide cannot be broken down. Hydrogen peroxide is a harmful by-product of many normal metabolic processes. To this end, catalase is frequently used by cells to rapidly catalyze the decomposition of hydrogen peroxide into less reactive gaseous oxygen and water molecules [19
]. In the present study, after UV-B irradiation, the level of catalase decreased but treatment of both the formulations reversed the condition and increased levels of catalase were observed. Other nonenzymatic antioxidant parameter Glutathione exists in reduced (GSH) and oxidized (GSSG) states. In the reduced state, the thiol group of cysteine is able to donate a reducing equivalent (H+
) to other unstable molecules, such as reactive oxygen species. GSH is considered to be a major endogenous antioxidant produced by the cells which participates directly in the neutralization of free radicals and reactive oxygen compounds, as well as maintains the exogenous antioxidants such as vitamins C and E in their reduced (active) forms [20
]. Decreased glutathione level indicates impaired antioxidant enzyme system of the skin cell [21
]. In the present investigation, decreased levels of GSH as obtained for Gr II, the formulation F4 and F5 treatment exhibited increased GSH levels. In earlier studies, it was observed that enhanced levels of ascorbic acid could fight against skin ageing [23
]. It means ascorbic acid levels of the skin can be severely depleted after UV irradiation. Considering this fact, the ascorbic acid (ASC) level in the present study was estimated. There was no significant difference in ASC level between group IV and group I. It may be due to the property of Calendula essential oil based cream that increases the ascorbic acid level. It is clear from these facts that increased ROS generation can overwhelm antioxidant defense mechanisms, resulting in oxidative stress and oxidative photo damage of proteins and other macromolecules in the skin.
Oxygen radicals generated from UV-B irradiation may cause modifications of the amino acids of proteins that frequently result in functional changes of structural or enzymatic proteins. It indicated that Calendula oil present in cream scavenges the free radicals by showing the increased level of total protein content as compared to UV-B induced oxidative stress. Recent studies on green tea polyphenol (GTP) revealed that GTP treatment inhibits the UV-B induced protein oxidation in vitro
in human skin fibroblast cells, which supports in vivo
The realm of possibilities in photo protection may include the development of sunscreens which remain at the surface of the skin for a longer time and may incorporate antioxidants that can neutralize ROS. Herbal compounds such as phenolic acids, flavonoids and polyphenols are very useful in eliminating the adverse effects of UV radiation on the skin. Antioxidants such as reduced glutathione (GSH) and enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) are known to attenuate the generation of ROS by removing potential oxidants or by transforming reactive oxygen species and reactive nitrogen species into stable compounds [25
]. Hydrophilic cream prepared from Calendula extract proved its significant antioxidant activity and suitable chemical and microbial stability [26
]. Findings of this research suggested that cream formulations based on Calendula essential oil have significant potential to protect and maintain the skin biochemical parameter in UV-B induced oxidative stress conditions. The findings also suggested that such formulations may prevent the oxidative decomposition which is responsible for the lipid peroxidation as well as formation of toxic components. The concentration of 1,8-cineole and α-pinene as major terpenoid content found in Calendula essential oil were 8.12 and 22.535 w/w, respectively, which may account for possible mechanism of balanced level of lipid peroxidation and antioxidant enzymes in UV-B induced oxidative damage in skin of albino rats.
In conclusion, the topical application of cream formulations containing 4% (F4) or 5% (F5) of Calendula essential oil before UV-B irradiation can significantly protect the skin from ROS generated from harmful radiation. Lowering in lipid peroxidation marker enzymes indicated the UV-B protective nature of Calendula essential oil based cream. The parameters included in the present investigation cannot reflect the complete damage induced by UV-B radiation. In the future, it will be necessary to conduct studies aiming at the evaluation of systemic effects of topically applied Calendula essential oil based cream will be required.