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Cosmeceutical potential of geranium and calendula essential oil: Determination of antioxidant activity and in vitro sun protection factor

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The present investigation was aimed to find out the sun protection factor (SPF) and antioxidant potential of geranium essential oil (GEO) and calendula essential oil (CEO) because having a combination of these two properties moves up the oils as an
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  ORIGINAL CONTRIBUTION Cosmeceutical potential of geranium and calendula essentialoil: Determination of antioxidant activity and in vitro sunprotection factor   Alka Lohani MPharm  |  Arun Kumar Mishra PhD  |  Anurag Verma PhD Faculty of Pharmacy,IFTM University,Moradabad,India Correspondence Alka Lohani, School of PharmaceuticalSciences, IFTM University, Moradabad, UttarPradesh, India.Email: alkalohani06@gmail.com Summary  The present investigation was aimed to find out the sun protection factor (SPF) andantioxidant potential of geranium essential oil (GEO) and calendula essential oil(CEO) because having a combination of these two properties moves up the oils asan active ingredient of various cosmeceutical formulations for their preventive andprotective properties. Essential oils were obtained by hydrodistillation of  Pelargo-nium graveolens  leaves (GEO) and  Calendula officinalis  flowers (CEO). The composi-tion and identification of chemical constituents of oils were determined by GCMSanalysis. Free radical scavenging activity was measured by nitric oxide scavengingactivity and 1,1 ‐ diphenyl ‐ 2 ‐ picrylhydrazyl (DPPH) radical scavenging activity. It wasobserved that both GEO and CEO have the potential to reduce or prevent oxidativestress and can be used in skin care regimen to slow down skin aging via its antioxi-dant properties. In vitro SPF was determined by a very simple and rapid spectro-scopic method. SPF value of GEO and CEO was found to 6.45 and 8.36,respectively. The SPF of CEO was higher than GEO, and the results of SPF showthat these essential oils can be employed in sunscreen formulations to protect theskin from sunburn. From the results, it can be concluded that the combined antioxi-dant and SPF property of GEO and CEO can provide synergistic photoprotectiveeffect or lift up the additional value of the cosmeceutical formulation. KEYWORDS antioxidant, calendula oil, essential oil, geranium oil, sun protection factor 1  |  INTRODUCTION From time immortal, natural ingredients, such as plant and flowerextracts, have been used traditionally in cosmetics for skin care.These breakthroughs are gaining more popularity as worldwide con-sumers seek more natural ingredients in their personal care products.Unlike synthetic cosmeceutical ingredients, the natural ingredientsare generally hypoallergenic and consumers need not to worry aboutexperiencing skin irritation. 1 Cosmeceutical preparations based onnatural ingredients supply the skin with nutrients and enhance skinhealth. For example, vitamins A, C, E, and phenolic compounds suchas flavonoids, tannins, and lignins, found in plant extracts, act asantioxidants 2 – 4 ; terpene  /  terpenoids present in essential oils abundantin flowers, leaves, seeds, rhizomes, and barks possess anti ‐ inflamma-tory, antimicrobial, antioxidant, antiaging, and anti ‐ melanogeniceffects on skin 5,6 Essential oils have been around for centuries inmany cultures for their medicinal and therapeutic advantages andhave enhanced lives of thousands of years, offering a variety of ben-efits from medicinal cosmeceuticals and dietary purposes to religioususe. Chemical constituents of an essential oil are one of the factorsthat determine the purity and therapeutic value of oil. Being a com-plicated mixture of chemical components, each biological responseshown by an essential oil is on account of the actions of one or Received: 19 December 2017 |  Revised: 20 May 2018 |  Accepted: 15 August 2018DOI: 10.1111/jocd.12789  J Cosmet Dermatol.  2018;1 – 8. wileyonlinelibrary.com/journal/jocd  ©  2018 Wiley Periodicals, Inc.  |  1  more of its chemical components. The versatile therapeutic potentialof essential oils has drawn the attention of investigators to testthem for various therapeutic activities. Essential oils have beenshown to possess antifungal, 7 antimicrobial, 8 antiviral, 9 anticancer, 10 and antioxidant 11 activities and can treat skin inflammation, dispensewith scarring, decrease imperfections, smooth wrinkles, and makeage marks vanish. 6,12 Generally, these oils can be added to creams,lotions, or carrier oils to be used on the skin. If used every day,these oils have the ability to treat dermatitis, psoriasis, aggravation,skin inflammation, staining, sunburns, etc. These versatile activitiesof essential oils formed the basis of diverse potential applications,especially in aromatherapy, cosmeceuticals, and complementary med-icines.The antioxidant capacity of essential oils is one of the biologicalproperties of highly considerable interest in cosmeceuticals. Antioxi-dants are useful in two ways: On one hand, they prevent the degrada-tion of active ingredient in the cosmeceuticals product, and on theother hand, antioxidants protect the skin damage and slow down theskin aging process by enhancing skin glow, minimize age spots, sunspots, fine lines, and wrinkles. 13 Use of essential oils as natural antioxi-dant is of the great interest in cosmeceuticals since most commonlyused synthetic antioxidants (such as butylhydroxytoluene and buty-lated hydroxyanisole) suspected to be nocuous to human health. 14,15 Essential oils have also been claimed to provide protection againstthe ultraviolet light coming from the sun. Prolonged skin exposure toultraviolet (UV) light causes various skin problems (such as prematureskin aging and skin cancer). 16 Solar UV radiation is divided into wave-length ranges recognized as UV  ‐ A (315 ‐ 400 nm), UV  ‐ B (280 ‐ 315 nm),and UV  ‐ C (100 ‐ 280 nm). Of the UV radiation, 95% of UV  ‐ A and 5% ofUV  ‐ B penetrate the atmosphere and no measurable UV  ‐ C reaches theearth ' s surface. 17 Both UV  ‐ A and UV  ‐ B play a major role in skin dam-age. 18 UV  ‐ B radiation is able to penetrate the epidermis and is thechief cause of skin reddening and sunburn, and tends to damage theskin ' s more superficial epidermal layers. It plays a key role in the devel-opment of skin cancer and a contributory role in tanning and photoag-ing. 19 UV  ‐ A rays are 30 ‐ 50 times more prevalent than UV  ‐ B rays andare able to penetrate more deeply into the dermis, releasing free radi-cals, diminish protective antioxidants in the skin, and causes DNA changes. 20 UV  ‐ A rays not only harm epidermis, but also damage elastinand collagen in the dermis, leads to photoaging, including wrinkles, pig-mentation, dark spots, rough skin texture, and loss of elasticity. Pro-longed exposure to these harmful radiations causes skin cancer. 19,20 The use of sunscreen in skin care cosmeceuticals is an effectiveapproach for reducing UV  ‐ generated reactive oxygen species (ROS) ‐ mediated skin damage. The effectiveness of sunscreen or sunblocks isexpressed by the sun protection factor (SPF). According to FDA, SPF isa measure of how much UV radiation is required to produce sunburnon the protected skin (with sunscreen) relative to the amount of UV radiation required to produce sunburn on unprotected skin. 21 HigherSPF value of a product indicates higher effectiveness in sunburn pro-tection.However, there is a need for good scientific research to back upthe efficacy of essential oils, so it is not all marketing smoke andmirrors. In light of the above, in the present investigation, an attempthas been made to explore the antioxidant and sun protection potentialof two plant ‐ derived essential oils, viz., geranium essential oil (GEO)and calendula essential oil (CEO). Geranium essential oil (GEO) isextracted from  Pelargonium graveolens  (Family: Geraniaceae) alsoknown as geranium. It has a wonderful uplifting, soothing, and floweryaroma and is an important floral component in cosmeceuticals and aro-matherapy. It is widely used for treating wounds. 22 The antiseptic,antifungal, and antibacterial properties of GEO 23 – 26 can be utilized towipe out bacteria responsible for acne. Skin cleansing action of GEOhelp to purge toxins, dirt, excess sebum, and dead cells from skinpores. 27 The anti ‐ inflammatory and skin soothing properties of GEOcalm down the skin rashes and provide relief in inflammatory skin con-ditions. 28 Calendula officinalis (  genus Calendula), is a self ‐ sowing annualplant that belongs to the family Asteraceae. Calendula is widely usedin skin care cosmeceuticals because it is beneficial in wound healing,inflammation, and cell rejuvenation and makes the skin smooth andsoft. 29 Calendula essential oil (CEO) has a great potential to destroyfree radicals, and its advantage as an antioxidant in cosmeceuticalscannot be ignored. Rich in antioxidants, GEO, and CEO has a greatpotential to destroy free radicals, 27,29 and its advantage as an antioxi-dant in cosmeceuticals cannot be ignored. To the best of our knowl-edge, there have been no reports on the simultaneous determinationof antioxidant capacity and SPF of both the essential oils. Therefore,the paucity of such important information leads us to attempt the pre-sent research work. 2  |  MATERIAL Plant material (leaves) of  P. graveolens  and flowers of  C. officinalis were collected from the local garden in Nainital District, Uttarak-hand, India. 1,1 ‐ Diphenyl ‐ 2 ‐ picrylhydrazyl (DPPH) was obtained as agift sample from Himedia, India. Napthylethylenediamine dihy-drochloride and L ‐ ascorbic acid were obtained from Sigma ‐ Aldrich,Bangalore, India. All the chemicals used were of analytical grade. 2.1  |  Extraction of essential oil The leaves ( P. graveolens ) and flowers ( C. officinalis ) were cleaned toremove dust and dirt, separated from stems, and cut into smallpieces. Clevenger ‐ type apparatus was used for the extraction ofessential oil. Plant leaves or flowers were placed into the round bot-tom flask of Clevenger apparatus and soaked with sufficient quantityof water. Few porcelain chips were added to the flask to avoidbumping during the distillation process. The extraction process wascontinued for 8 ‐ 12 hours. The extracted oil was separated fromwater and collected in small tubes, sealed, labeled, and stored inlight ‐ resistant vials at 4°C for further use. 2.2  |  Characterization of oil Oil was characterized for its physical characters, that is, color, odor,density, saponification value, and acid number. 2 |  LOHANI  ET AL .  For the determination of saponification value, 1 g oil sample wasadded to 50 mL alcoholic KOH (0.5N) and refluxed for 30 minutesfor perfect dissolution of the sample. 1mL of phenolphthalein indica-tor was added and the content and was titrated with 0.5N HCl toan endpoint. The same process was repeated using blank sample. 30 The saponification value was calculated using the formula:saponification value ¼ 56 : 1   Nweight of sample ð V  2  V  1 Þ where 56.1 = molecular weight of KOH, N = normality of the KOH, V  1  = titer value for the sample, and  V  2  = titer value for blank.For the estimation of acid number, 1 g oil sample was weighedadded to a mixture of 25 mL diethyl ether and 25 mL ethanol. Onemilliliter of phenolphthalein was added, and the solution was titratedwith 0.1N KOH until a pink color (which persists for 15 seconds)was obtained. 30 The acid value was calculated asAcid value ¼ 56 : 1  N   V M where 56.1 = molecular weight of KOH, N = normality of KOH, V   = volume of KOH used, and  M  = mass of the sample. 2.3  |  GCMS analysis of essential oil The identification of the components of the essential oil sampleswas carried out by gas chromatography equipment (Shimadzu QP ‐ 2010 Plus with Thermal Desorption System TD 20, Shimadzu, Kyoto,Japan); AB ‐ INNOWax column (60 m length  ×  0.25 mm id  ×  0.25  μ mthickness) was used under the conditions: column oven temperaturewas 50.0°C, injection temperature was 260.0°C, pressure was69.0 kPa, total flow was 125.2 mL  /  min, and column flow was1.21 mL  /  min. Injected oil sample volume was 0.1  μ L, split ratio was100.0, ion source temperature was 230.00°C, interface temperaturewas 270.0°C, and the mass spectrometer was scanned over the 40 ‐ 650  m  /   z . Identification of individual oil component was done bycomparing the mass data of individual oil component peaks with thestandard library database (Wiley 7 NIST 05 mass spectral database). 2.4  |  Determination of antioxidant capacity  The antioxidant capacity of GEO and CEO was determined by thefollowing methods: 2.4.1  |  Nitric oxide scavenging activity  Sodium nitroprusside decomposes in aqueous physiological solution(pH ‐ 7.4) and generates nitric oxide. Nitric oxide reacts with oxygenunder aerobic conditions and generated nitrite ions that can be esti-mated by using Griess reagent (1 part of 0.1% napthylethylene dia-mine dihydrochloride in distilled water and 1 part of 1% sulfanilamidein 5% phosphoric acid). 31 Nitric oxide scavengers compete with oxy-gen leading to reduced production of nitrite ions. In this method,10 mmol  /  L sodium nitroprusside was prepared in phosphate buffer(pH 7.4) and 2 mL of this solution was added to oil sample and stan-dard (ascorbic acid) dilutions (10 ‐ 250  μ g  /  mL) in methanol. The tubeswere incubated at 25°C for 2 hours. After incubation, 0.5 mL Griessreagent was added to the incubated tubes and absorbance was mea-sured at 546 nm using UV  ‐ Visible Spectrophotometer. The amount ofnitric oxide radical was calculated by following the equation: 32 % Inhibition ¼ AbC  AbSAbC   100where AbC is absorbance of control and AbS is absorbance of sam-ple  /  standard.% Inhibition of each sample dilution was calculated and a graphwas plotted to obtain a linear regression equation, by taking concen-tration ( μ g  /  mL) on  x  ‐ axis and % Inhibition on  y  ‐ axis. From the linearregression equation, IC 50  value was calculated. IC 50  value is the con-centration of the sample required to scavenge 50% nitric oxide ‐ freeradical. The experiment was done in triplicate. 2.4.2  |  1,1 ‐ Diphenyl ‐ 2 ‐ picrylhydrazyl (DPPH) radicalscavenging activity  To evaluate the antioxidant capacity of GEO and CEO, the oil sampleswere allowed to react with methanolic solution of DPPH. DPPH is astable nitrogen ‐ centered ‐ free radical which changes the color fromdark violet to yellow in the presence of compounds that are capableof either donating hydrogen or transferring an electron. 33 During thereaction, the reduction in DPPH can be monitored by the decrease inits absorbance at a characteristic wavelength (  λ max : 515 ‐ 517 nm). 34 The free radical scavenging activity of GEO and CEO was estimatedby DPPH using the method described by Elmastas et al 35 Differentdilutions of oil samples and standard (ascorbic acid) in the concentra-tion range of 10 ‐ 250  μ g  /  mL were prepared in methanol. 0.1 mmol  /  Lsolution of DPPH was prepared in methanol, and 3 mL of this solu-tion was added to the equal volume of diluted sample and standardsolutions, mixed well, and the tubes were incubated for 30 minute indark at 30°C. The absorbance of each dilution was measured at517 nm using UV  ‐ Visible Spectrophotometer. DPPH radical scaveng-ing capability was calculated using the following equation: % Inhibition ¼ AbC  AbSAbC   100where AbC is absorbance of control and AbS is absorbance of sam-ple  /  standard.% Inhibition of each sample dilution was calculated and a graphwas plotted to obtain a linear regression equation, by taking concen-tration ( μ g  /  mL) on  x  ‐ axis and % Inhibition on  y  ‐ axis. From the linearregression equation, IC 50  value was calculated. IC 50  value is the con-centration of the sample required to scavenge 50% DPPH ‐ free radi-cal. The experiment was done in triplicate. 2.5  |  In vitro sun protection factor determination The in vitro SPF measurement techniques represent an admissibleand fast tool for shortening in vivo experiment numbers and risks LOHANI  ET AL .  |  3  related to UV exposure of human subjects. In vitro SPF was deter-mined according to the COLIPA standards which include measure-ment of the percent transmittance of a sunscreen product across theUV spectrum weighted by the erythemal weighting factors at differ-ent wavelengths. 36 SPF spectrophotometric  ¼ CF  ∑ 320290 EE ð  λ Þ I ð  λ Þ AbSwhere CF = correction factor (10), EE (  λ ) = erythemal action spec-trum, I(  λ ) = solar intensity spectrum, and Abs (  λ ) = spectrophotomet-ric absorbance values at wavelength  λ . The values of EE (  λ )  ×  I(  λ ) areconstants and are given in Table 1. 37 For the determination of SPF, 1%w  /  v solution was prepared inethanol, and from this stock solution, 0.1% concentration was pre-pared with ethanol. The absorbance of the sample solution wastaken by UV  ‐ visible spectrophotometer in the range of 290 ‐ 320 nm,every 5 nm interval, using ethanol as a blank. A total of threereadings were taken at each point, followed by the calculation ofSPF using Mansur equation. 36 3  |  RESULT AND DISCUSSION3.1  |  Physicochemical characterization of GEO After the efficient extraction of GEO and CEO, the percent yieldwas found to be 1.5% and 1.2%, respectively. The color of GEOwas light greenish with a strong rose fragrance while CEO was yel-lowish brown. For GEO, density was found to be 0.897 ± 0.01 g  /  mL (at 25°C), saponification value was 110.90 ± 0.8, and acid num-ber was 2.58 ± 0.5, and for CEO, density was 0.795 g  /  mL (at 25°C),saponification value was 115.24 ± 0.06, and acid number was3.06 ± 0.80. 3.2  |  GCMS analysis The oil obtained by hydrodistillation of  P. graveolens  leaves and  C. of-ficinalis  flowers was subjected to detailed GCMS analysis. 3.2.1  |  GCMS analysis of GEO The gas chromatogram of GEO is shown in Figure 1A. A total of36 compounds were observed in the gas chromatogram of GEO.The highly abundant constituent of the oil was citronellol (37.01%)and geraniol (17.99%). Other constituents were citronellyl formate(5.51%), linalool (4.11%), rose oxide (2.40%), geranyl formate(2.19%), citronellyl propionate (1.90%), geranyl tiglate (1.59%),  α ‐ pinene (1.59%), geranyl propionate (1.10%), and limonene (1.02%; TABLE 1  Values of EE(  λ )  ×  I(  λ ) used for sun protection factorcalculation 37 SN Absorbance Value of EE(  λ )  ×  I(  λ ) 1 290 0.01502 295 0.08173 300 0.28744 305 0.32785 310 0.18646 315 0.08377 320 0.0180EE (  λ ) is erythemal action spectrum, and I (  λ ) is solar intensity spectrum.The EE (  λ ) and I (  λ ) are given as constants. FIGURE 1  Gas chromatogram; A, geranium essential oil and B, calendula essential oil 4 |  LOHANI  ET AL .  Table 2). All the other components were present in the amountlower than 1.0%. From the results, it was observed that oxygenatedmonoterpenes were present in the majority, that is, the main rea-son that GEO is characterized by its sweet rose ‐ like (citronellol)and flowery rose ‐ like odor (geraniol) with important demand in per-fumery. Previous researchers reported citronellol and geraniol asthe main constituent of GEO, 27,38 and it may be said that themajor activities of GEO are due to the presence of its major con-stituents. 3.2.2  |  GCMS analysis of CEO The gas chromatogram and chemical compositions of CEO areshown in Figure 1B and Table 3. The eminently ample chemical con-stituent present in CEO was  trans ‐ β ‐ ocimene (48.28%), dihydrotage-tone (25.46%),  cis ‐ tagetone (4.62%), neo ‐ allo ‐ ocimene (3.79%), 1,8cineole (3.72%),  α ‐ pinene (2.68%), and artemisia ketone (1.02%).Other compounds were present in the smaller percentage. It wasobserved that monoterpenes are present in the majority. 3.3  |  Determination of antioxidant capacity  Many essential oils contain naturally occurring antioxidants. In thepresent investigation, two methods, viz., nitric oxide scavengingactivity and DPPH radical scavenging activity, were used to evaluatethe antioxidant potential of GEO and CEO. 3.3.1  |  Nitric oxide scavenging activity  The principle of this technique relies on the measurement of thecapacity of essential oil to trap nitric oxide, leading to a decreasedproduction of nitrite ions. The maximum nitric oxide scavengingactivity shown by ascorbic acid (standard) was 89.19 ± 0.05% at250  μ g  /  mL concentration. At the same concentration, GEO TABLE 2  Chemical composition of geranium essential oil obtainedby GCMS analysis S. No Retention time Name 1 7.80  α ‐ thujene2 8.091  α ‐ pinene3 8.674 Camphene4 9.782  β ‐ pinene5 10.321 Myrcene6 11.800 p ‐ cymene7 12.032 Limonene8 15.605 Linalool9 15.873 Rose oxide10 16.568  Cis ‐ rose oxide11 19.055 Terpinen ‐ 4 ‐ ol12 19.794  α ‐ terpineol13 21.992 Citronellol14 23.043 Geraniol15 23.545 Citronellyl formate16 24.566 Geranyl formate17 26.757 Citronellyl acetate18 27.129 Neryl acetate19 28.001 Geranyl acetate20 28.575 Phenethyl isobutyrate21 29.604 (E) ‐ caryophyllene22 30.616 Citronellyl propionate23 31.802 Geranyl propionate24 33.329 Geranyl isobutyrate25 34.045 Citronellyl butyrate26 34.587 E ‐ α ‐ bisabolene27 35.297 Geranyl butyrate28 35.495 E ‐ nerolidol29 35.920 Citronellyl valerate30 36.165 Epoxycaryophyllene31 36.255 2 ‐ phenylethyl tiglate32 37.113 Geranyl isovalerate33 39.291 E ‐ citronellyl tiglate34 40.579 Geranyl tiglate35 47.994 Citronellyl valerate36 49.073 Geranyl octanoate TABLE 3  Chemical composition of calendula essential oil obtainedby GCMS analysis S. No Retention time Name 1 5.882 Camphene2 6.136 Sabinene3 7.336 Limonene4 8.898  α ‐ pinene5 9.120 Verbenone6 9.575  Trans ‐ β ‐ ocimene7 11.492 Dihydrotagetone8 12.259 Carvenone9 12.559 1,8 cineole10 12.781  α ‐ pinene ‐ epoxide11 13.258 Neo ‐ allo ‐ ocimene12 15.945  Trans ‐ myoxide13 17.395  Cis ‐ tagetone14 17.675 Camphor15 18.153  α ‐ terpinolene16 19.936  Β ‐ caryophyllene17 21.526  Trans ‐ pinocarveol18 22.595 Artemisia ketone19 23.165  Trans ‐ ocimenone20 25.805 Isopiperitenone21 30.926 2 ‐ methyl ‐ 6 ‐ heptene ‐ 3 ‐ ol22 35.136 Spathulenol LOHANI  ET AL .  |  5
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