The flowers from plant for proposed study were collected from botanical garden of IFTM; District Moradabad, Uttar Pradesh India in the month of January 2010. At the time of flower collection, plant was of three and half month old. The specimens were identified, authenticated and voucher specimen of herbarium (ref. no. IFTM/Pharmacog/Auth/10/2 dated 29.1.2010) is preserved in Pharmacognosy division of IFTM, Moradabad.

Fresh calendula flowers were obtained from the botanical garden; the petals were separated and washed thoroughly under running water. The excess water was drained out completely and the petals were packed in distillation flask of Clavenger's apparatus with sufficient quantity of water and few pieces of porcelain chips to avoid bumping during distillation.

The extraction was continued for 8 h. The calendula oil was collected from graduated receiver and purified by anhydrous sodium sulphate for removing water traces. The yield of the oil obtained was found to be 1.25%.

Upon successful isolation of oil, the physico-chemical parameters for oil was evaluated which included specific gravity (0.795 g/ml at 25°C), viscosity by Brookfield viscometer (15 Cp) and ester value (113.43), acid number (3.37), saponification value (116.8).[16] For acid value determination, 1 g of calendula oil was taken in a dried conical flask containing 25 ml of absolute alcohol and added (2–3) drops of phenolphthalein and resulting mixture was heated with shaking on water bath for 10 min, then cooled and finally titrated the solution against 0.1 N KOH until pink color appears (end point). The calculation was done as per Eq. (1).

For saponification value determination, it was performed by taking approximately 1 g of the oil into a 250 ml round bottom flask. In this, 25 ml of alcoholic potassium hydroxide solution (0.5 N) added and fitted with reflux condenser. Finally, flask contents were heated on a boiling water bath for 1 h with occasional shaking. While the solution was still hot, added three drops of phenolphthalein indicator and titrated with the excess potassium hydroxide with the 0.5 N hydrochloric acid (V ml of hydrochloric acid at end point represented by C). By same procedure, without sample, titration was performed (V ml of hydrochloric acid at end point represented by B). The saponification value was calculated by formula stated in Eq. (2). The ester value is defined as the mg of KOH required to react with glycerin (glycerol/or glycerin) after saponify 1 g of fat. It is calculated from the saponification value and the acid value as formula in Eq. (3).

Where B is ml of HCl required by Blank, C is ml of HCl required by Sample.

Ester value = Saponification value - Acid value      (3)

GC–MS analyses were performed on a capillary gas chromatograph directly coupled with mass spectrometer having thermal desorption system (model Shimadzu QP-2010 TD 20). AB-Innowax column (60 m length × 0.25 mm id × 0.25 μm film thickness) was used under the following conditions: Column oven temp: 70.0°C, injection temp: 260.00°C, pressure 156.7 kPa, total flow: 40.5 ml/min, column flow: 1.21 ml/min, the volume of injected sample 0.1 μl of oil, split ratio: 30.0, ion source temperature: 250.00°C, interface temp: 260°C with scan m/z starts from 40.00 and end at m/z: 950.00. MS spectra of separated compounds were compared with one from Wiley 7 Nist 05 mass spectral database. The compounds characterized from isolated calendula oil by GC–MS are presented in Figure 1 and Table 2.

Reddening of the skin, as a direct result of UVB radiation, is an excellent warning sign to avoid further sun exposure.[17] As UVB is 1000 times more erythemogenic than UVA, SPF indicates chiefly the acute protection against UVB, but it predicts no indication of a product's protection against UVA.[18] Products with the same SPF may have quite different absorption spectral profiles and sunscreens with high SPF do not guarantee protection against UVA. In current market, there is a demand of such stable sunscreen cream but the same is a challenge because difficulty to stabilize the components, no irritancy, easy spreadibility, feel effect after application, etc. In the present study, for the formulation purpose, oil phase was comprised of stearyl alcohol, bees wax, sorbitan monooleate, whereas sorbitol solution, polysorbate 80, methyl paraben, propyl paraben, and deionized water constituted the aqueous phase. Calendula flower oil 5% was used as active sunscreening agent in cream formulation.

Cream formulation of isolated Calendula oil from flowers of Calendula officinalis were prepared using formula given in Table 3.

All the content of oil phase and water phase was heated up to 70°C separately. Afterwards the oil phase was added slowly to aqueous phase with continuous stirring to form a crude emulsion. It was cooled to about 55°C and homogenized. The resulting materials were cooled with agitation until congealed. The cream was stored at room temperature afterwards calendula oil was weighed (5%) and incorporated in the formed emulsion under constant homogenization. Whole formulation was stored in closed amber colored glass bottle.

The prepared formulation was evaluated for its sensory evaluation, pH, spreadibility, specific gravity, conductivity, zeta potential, poly dispersity index (PDI), globule size, and skin irritancy, as per official methods, and results are mentioned in [Table 4].

A quantity of 1.0 g of prepared cream was weighed, transferred to 100 ml volumetric flask, and finally diluted to volume with ethanol. Further, it was kept for ultrasonication for 5 min and filtered through cotton filter, discarding the ten first ml. A 5.0 ml aliquot was transferred to 25 ml volumetric flask and the volume was adjusted with ethanol.[19]

The absorbance spectra of sample in solution form were obtained in range of 290–320 nm, every 5 nm interval, and three determinations were made at each point, followed by application of Mansur equation.