GC-MS Chemical Profile, Antioxidant activity, and Sun Protection Factor of Essential oil of Tea Tree (Melaleuca alternifolia) and Rosemary (Rosmarinus officinalis L.)

The present investigation aimed to determine the oxidative potential, sun protection factor value, and half-maximal inhibitory concentration of rosemary essential oil and tea tree essential oil. These two essential oils have gained popularity as active ingredients in many cosmetic preparations due to their antioxidant activity, whether used individually or in combination. Gas Chromatography-mass spectroscopy is used to identify the presence of different phytochemical constituents in essential oils. The GC-MS (Gas chromatography-mass spectroscopy) chemical analysis of tea tree oil revealed 34 and rosemary oil revealed 35 volatile chemical components with sesquiterpene hydrocarbon, monoterpenes (42.77%), and alcohols (41.01%) as major detected classes. The 2, 2-diphenylpicrylhydrazyl (DPPH) and nitric oxide-free scavenging activity techniques were used to investigate the antioxidant capacity of these oils. It was found that both tea tree and rosemary oil have the potential to slow down skin aging through their anti-oxidative properties using the approach of free radical scavenging activity. The UV spectroscopy method was used to determine the sun protection factor, and the sun protection values of rosemary and tea tree oil were found to be 8.45 and 6.85, respectively. Rosemary oil was an extremely promising tea tree essential oil for anti-aging and sunburn prevention. The study's findings indicated that rosemary and tea tree essential oil can both offer a synergistic sun protection factor effect, antioxidant property, and anti-aging or extra activity of cosmetic preparations.

(BHT) are suspected to be harmful side effects on human health 5 . The antioxidants are beneficial in two ways: on the one hand, they aid in the declination of active constituents in cosmeceuticals products; on the other hand, antioxidants protect skin damage and slow the aging process by improving skin gleam and minimizing age spots, sun spots, fine lines, and wrinkles 4 . It would seem that vitamins and nutritional factors have an effect on the skin's antioxidative protection and that combining multiple antioxidants at the same time has a synergistic effect 6,7,8 . Sunburn, photodermatoses, hyperpigmentation, photoaging of the skin, and precancerous lesions and cancers are all side effects of UV exposure. The mechanisms discussed in this paper are involved in the formation of these clinical changes in the skin 9 . UV exposure causes skin side effects such as sunburn, photodermatoses, hyperpigmentation, photoaging of the skin, and precancerous lesions and cancers. The mechanisms discussed in this paper are involved in the formation of these clinical differences in the skin 10 .
Rosemary (Rosmarinus officinalis L.), from a botanical perspective, this plant is expressive because it contains over 240 active pharmacological and nutritional composites. Antifungal properties of the Rosemary plant have been discovered through pharmacological research. Gas chromatographymass spectrometry was used to find out the chemical constituents of essential oil (EOs) of rosemary upstanding region collected in Kerman province. Kerman province's rosemary essential yield was calculated to be 3.2 percent. The essential oil included 35 composites that accounted for 98.74% of the total oils 11 . The principal constituents were α-pinene (14.62%), camphor (12.67%), verbenone (10.19%), and 1, 8-cineole (10.63 %) 12 . Carnosic acid, carnosol, rosmarinic acid, and hesperidin dominate these plants' polyphenolic histories. The antioxidant components of rosemary have been identified as cyclic diterpene diphenols, carnosolic acid, and carnosol 2. The antioxidant activity of essential oil is one of the biological properties of highly considerable interest in cosmeceutical formulation.
Tea tree oil (Melaleuca alternifolia) is used principally to supplement endeavors to validate its use, and its reputed therapeutic antioxidant properties have been studied In vitro and, in some cases, In vivo. It is a volatile essential oil derived primarily from the Australian native plant Melaleuca alternifolia 13 . TTO is used as the main ingredient in many topical preparations used to treat cutaneous infections due to its anti-aging properties 14,15 .
The essential oil is high in triglycerides, free fatty acids, tocopherols, sterols, phospholipids, waxes, squalene, and phenolic compounds. According to top dermatologists, facial oils are the missing piece in traditional beauty routines. Skin moisture levels decline with age, causing dehydration and making fine lines and wrinkles more visible. Oils not only hydrate the skin, but their high antioxidant content protects cells from free radical damage, preventing further ageing 16 . Oils are excellent for delivering desired skin benefits in spot applications 17 .
The antioxidant capacity and sun protection factor of tea tree and rosemary oil are primarily attributed to terpinen-4-ol, a major constituent of the EOs (essential oils). As a result, the terpinen-4-ol content was limited to 30% with no upper limit to maximize anti-aging activity. In contrast, a 15% upper limit and no less significant boundary was established for 1,8-cineole, though the reasoning behind this may not have been entirely sound 18 . For many years, cineole was mistakenly thought to be a skin and mucous membrane irritant, which fueled efforts to reduce its concentration in TTO (tea tree oil). This reputation will be built on anecdotal evidence from the past and unsubstantiated statements 8 .

Sample collection and processing
The samples were collected for RMO (Rosemary oil) and TTO (Tea tree oil) testing, and their active chemical ingredients piqued the experimenters' interest due to their antioxidative activity. The essential oils of the tea tree and rosemary plant (leaves) were purchased from natural aroma product private limited New Delhi. As a free experimental sample from Shanghai, China, DPPH (2,2-diphenylpicrylhydrazyl) was obtained. Sigma-Aldrich Chemicals Pvt. Ltd. The naphthyl ethylenediamine dihydrochloride and L-ascorbic acid was given by Limited, USA. All chemicals and solvents were of the highest quality.

Characterization of RMO (Rosemary oil)/TTO (Tea tree oil)
The physical properties of an oil, such as appearance (color), aroma (odor), specific gravity, saponification number, and acid number, were used to classify it 5 .

Saponification number
To determine the saponification value, add one ml of essential oil to 50 mL of ethanolmixed KOH(potassium hydroxide) solution (0.5N) and reflux for thirty minutes to ensure complete dissolution of all chemicals. After adding one mL of phenolphthalein indicator, the sample solution was titrated with 0.5 N Hydrochloric acid solutions until the solution's color changed. A similar procedure was followed with a blank sample solution. The saponification number was then calculated using the following formula: Formula for Saponification number=56.1xNormality/ Weight (sample) (Vs-Vb) Where, Molecular weight of potassium hydroxide=56.1, Vs= Sample titer value, and Vb=blank titer value.

Acid value
One g of EOs (essential oils) was accurately measured and added to a solution mixture of 25 milliliters of diethyl ether and 25 milliliters of C 2 H 5 OH (ethyl alcohol) to calculate the acid value. After adding 1 mL of the indicator phenolphthalein, the solution was titrated with 0.1 N potassium hydroxide solution and filled in the burette until the color was obtained 19 . The acid number was determined as the following formula: Formula for Acid value = 56.1(molecular weight of KOH) x Normality x Volume/Mass

GC-MS analysis of TTO/RMO
The chemical constituents present in the EOs (essential oils) samples were identified using GCMS apparatus (Agilent-5973, Aligent Technologies Inc., USA); an ABINNO Wax capillary Colum (30 m length, Inner diameter 0.25mm & 0.25mm film consistency) was used under the following conditions column roaster temperature was 70.0 o C & sample injection heat was 250.0 o C The gas chromatography operating conditions were as follows: gas carrier flow rate 2.0 mL/min, column temperature programming from 70 o C to 270 o C at 4 o C/min, injector temperatures of 216 and 275 o C, respectively, and mass spectroscopy operating process was scanned over the 40.850 m/z range. To differentiate individual oil constituents, the chromatogram data of both EOs element peaks were compared to the standard database from (Wiley 8 database library).

Estimation of oxidative activities for TTO (Tea tree oil)/RMO (Rosemary oil)
TTO/RMO antioxidant activity was examined using the following methods: Nitric oxide free radical scavenging activity: The ability of antioxidant-containing samples to scavenge nitric oxide free radicals is measured by this assay. At physiological pH, sodium nitroprusside (SNP) spontaneously initiates the formation of nitric oxide, which then reacts with oxygen to form nitrite ions, as measured by the Griess Illosvoy reaction. As a positive control, standard ascorbic acid was dissolved in methanol, just like essential oils. Oil samples and standard concentrations ranging from 10 to 1000 g/mL were mixed with 2 mL of a 10 mM SNP solution prepared in 0.5 mM PBS at pH 7.4. The final solution was incubated at 25°C for 120 minutes. Following incubation, 0.5 mL of oil sample and 0.5 mL of Griess reagent were mixed. In a UV-Visible double beam spectrophotometer, the abs of the colored solution were measured at 546nm in comparison to the control (10 mmol/L SNP in phosphate buffer solution without essential oil or standard sample). The rate of inhibition of nitric oxide free radical scavenging activity by essential oil was calculated using the equation below 20 .

ABS-Absorbance
The percentage inhibition was calculated for each test solution dilution, and a chart with concentration (g/mL) and free radical inhibit percentage was generated to obtain a linear equation. The half-maximal inhibitory concentration value was calculated using the linear equation. The IC 50 value is defined as the sample concentration required for scavenging 50% of the nitric oxide free radical. The experiment was carried out three times.

2,2-diphenylpicrylhydrazyl (DPPH) free radical scavenging activity
After a few minor adjustments, the DPPH (2, 2-diphenylpicrylhydrazyl) free radical scavenging assay was used to assess DPPH activities in a 96well microtiter plate. A sample stock solution volume of 100L produced final concentrations of 500, 250, 125, 63, 16,8,4, and 2 g/mL after being diluted twice. The positive control was ascorbic acid. Following that, each well received 100 L of DPPH (2, 2-diphenylpicrylhydrazyl) at a concentration of 0.04 percent (w/v). The mixture was blended and incubated for 30 min at room temperature in the dark. A microplate reader was used to calculate the abs value at 515nm. The methanol sample serves as a blank for the test sample. The control well contained a DPPH solution made of methanol. Each well's total volume is 200 L. On each sample, three copies of each test were run. The percentage of inhibition was calculated 21 .
The rate of inhibition was calculated for each test solution dilution, and a chart was built to achieve a linear equation, with concentration and rate of inhibition, and the half maximal inhibitory concentration was measured using the equation. The IC 50 (half maximal inhibitory concentration) value is the sample concentration needed to scavenge 50% of the NO (nitric oxide) assay.

Estimation of SPF (sun protection factor) value
There are two types of In vitro SPF techniques. A very simple mathematical equation was developed to replace the In vitro method used in methods that measure UV absorption or transmission through hydrogel product films in quartz plates or biomembranes, as well as methods that rely on spectrophotometric analysis of diluted solutions to determine the absorption properties of essential oil agents 22 .
The COPILA standard was used to calculate in vitro SPF, which involves calculating the percentage of a product's transmittance over the UV spectra, subjective by the EE weighting factor at a range of absorbance 23 . SPF spectrophotometric =(CorrectionFactor), 10 x ∑ 290 320 EE(λ) x Intensity (λ) x absorbance (λ) Where, EE=Erythemogenic Effect of radiation at a wavelength (λ), I (λ)=Intensity of solar light at wavelength, abs=Absorbanc by a test sample of the standard solution. It was calculated that a standard microemulsion preparation containing 8% homosalate presented an SPF value of 4, calculated by a UV spectrophotometer.
The values for the term (I) λ x (EE) λ are constant, which were calculated, and are shown in Table 1. (I) λ is the sunlight spectra intensity, (EE) λ is the erythemal action spectrum the intensity spectrum & erythemal spectrum are constant The sample (1 g) was weighed, transferred to a volumetric flask of 100 mL, filled to volume with ethanol, mixed for 15 min, and then filtered through Whatman filters. A 5.0 mL sample was transferred to a 25 mL volumetric flask after being diluted to volume with methanol. The absorption values were measured between 290 and 320nm (every 5nm). Each measurement was taken three times, and the final number represents the average of those three measurements. The Mansur equation was then used to calculate the sun protection factor values for the formulations. The In vitro method is replaced by a combination of two relatively simple mathematical equations 24

TTO & RMO physicochemical characterization
Tea tree oil yield was discovered to be between one and two percent. Tea tree oil, also known as Melaleuca alternifolia oil, is a water-soluble essential oil with a distinct camphoraceous aroma and a yellowish to nearly clear color. At 25°C, the acid and saponification numbers of the EOs were 1.01-1.22 (mg KOH/g), 177.56-200.45 (mg KOH/g), and 0.8950-0.9050 g/mL, respectively. Rosemary oil yield was found to be between 0.5 and 2%. Rosmarinus officinalis Linn, a member of the Lamiaceae family, flowering tops and leaves were used to create rosemary essential oil. Colorless to pale yellow transparent liquid of herbal camphoraceous minty aromatic woody balsamic medicinal phenolic essential oil. At 25°C, the acid values of the oil were 0.5-1.12 (mg KOH/g), 185-195 (mg KOH/g), and 0.908g/mL, respectively.

(Gas chromatography-mass spectroscopy) Analysis
A complete GCMS analysis of tea tree and rosemary essential oils was performed on the obtained essential oil.

GCMS analysis of RMO
The gas chromatogram of rosemary oil is shown in Fig. 1(A), and its chemical composition is shown in Table 2. RMO's gas chromatographymass spectroscopy analysis revealed 35 chemical constituents. Carnosol (28%), terpinen-4-ol (20.6%), β-pinene (10.55%), caryophyllene (2.54%), epi-camphor (9.97%), camphor (7.26%), α-pinene (1.9%), and 1,8-cineole were the most prevalent components of RMO oil. Because of their age-defying and antioxidant properties, these chemicals can reduce or prevent oxidative stress, and they can be used in skincare routines to slow skin aging. As a result, monoterpenes became prevalent, which is why they are important in cosmeceuticals and perfumes 25 . Figure 1(B) depicts the gas chromatogram of rosemary oil, and Table 3 lists its chemical components. TTO's gas chromatogram revealed a total of 34 chemical constituents. The majority of the oil (44.5%) was composed of Cineole-1, 8 (4.51%) Cyclohexanol, 8.78% Terpineol, 2.65% Terpineol-4-ol, 12.11% (10.5%) α-pinene, β-pinene (5.81%) Terpinolene. Because of their anti-aging and antioxidant properties, these substances can be used in skincare regimens to delay skin aging and possibly reduce or prevent oxidative stress. Other elements were present in lesser amounts. The primary goal of the GCMS study was to detect the presence of monoterpenes, oxygenated monoterpenes, alcohols, sesquiterpene hydrocarbons, and diterpenes in essential oils because they are primarily responsible for antioxidant activity, sun protection factors, and skin aging 26 .  . 1(A). Gas chromatogram of RMO by GCMS analysis Fig. 1 (B). Gas chromatogram of TTO by GCMS analysis

Determination of In vitro antioxidant capacity
Significant quantities of essential oils are naturally responsible for antioxidant capacity. We used two techniques in this investigation: To ascertain the antioxidative potency of rosemary oil and tea tree oil

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Nitric oxide assay • 2, 2-diphenyl-1-picryl-hydrazyl-hydrate assay Nitric oxide free radical scavenging activity Table 4 and Fig. 2 (A) show that the nitric oxide radical scavenging of oils ranges between 23.13 and 81.57% and 36.55 and 90.04%, respectively. Tea tree essential oil (23.13%) and rosemary essential oil (36.55%) had the lowest activity at 0.10 g/mL, while at 1.0 mg/mL, tea tree essential oil (81.57%) and rosemary essential oil (90.04%) had the highest activity. At this dose, the EOs have greater than 50% scavenge efficacy, with an IC 50 value of 0.50 mg/mL (62.76%) 2 . The essential oils TTO and RMO have oxidation properties, as evidenced by the decreased absorbance of the reaction mixture.

2, 2-diphenyl-1-picryl-hydrazyl-hydrate free radical scavenging activity
The free radical scavenging activity (FRSA) of essential oils ranges from 28.42 to 83.34% and 30.96 to 88.44%, according to TTO and RMO measurements. TTO has the lowest free radical scavenging activity of 28.42% at 0.10 mg/mL and the highest at 83.34% at 1.00 mg/ mL when compared to regular ascorbic acid. RMO has the lowest (30.96% at 0.10 mg/mL) and highest (88.44% at 1.00 mg/mL) free radical scavenging activity when compared to regular ascorbic acid. The IC 50 value was 0.25 mg/mL, and the free radical scavenging activity was 63.23%. The results shown in Table 5 and Fig. 2(B) show that the antioxidants' free radical scavenging abilities cause the constant and violet-blue color core to change to a yellowish color. When the results are compared, we can conclude that TTO and RMO essential oils both have beneficial antioxidant properties, and their concentration increases their ability to scavenge free radicals 25 .

FRSA-Free radical scavenging Activity
Using the DPPH assay technique, the IC 50 values for Tea tree essential oil and Rosemary essential oil are 0.25 g/mL and 0.25 g/mL, respectively. Tea tree essential oil has half maximal concentrations of 0.25 g/mL and 0.50 g/mL in the Nitric Oxide Scavenging technique. Both essential oils were 50% effective against free radicals at these concentrations 17 . When the results were compared, it was discovered that both rosemary and tea tree essential oils had potent antioxidant properties that increased with concentration.
on the growing conditions of the plants. Due to the conflicting information on essential oils, any SPF study would cost millions of dollars. This is one of the primary reasons why the scientific community has decided against funding essential oil SPF research. The TTO and RMO SPF values were 6.85 and 8.45, respectively, according to the current investigation (Table 4). It was discovered that the evaluated essential oils have low SPF ratings for the antioxidant agent primarily used for producing defense against oxidative stress and combating the bothersome free radicals that frequently accompany prolonged sun exposure. Even through this, they always provide numerous skin benefits 26 .

Determination of In vitro sun protection factor for TTO/RMO
Sun protection factor determination is a useful test for vetting substances when developing cosmeceutical products. The SPF rating demonstrates the efficacy of the microemulsion. The higher the sun protection factor, the better the formulation's UV protection against light. To be effective in preventing photoaging, sunburn, skin wrinkles, and other skin damage, essential oils must absorb a significant amount of UV-radiation (290-400nm). An examination of the literature revealed a scarcity of studies on how to calculate the SPF of essential oils. This is due to two factors. The first is that essential oils are highly flammable and have very different properties than thick, fatty vegetable oils; as a result, they do not provide adequate sun protection when used alone. Money is another consideration 23 . Plant-derived essential oils contain a wide range of chemical constituents. The percentage of these chemical components varies by harvest and batch depending

CONCLUSION
According to the present research work, tea tree and rosemary oil can prevent or reduce oxidative stress and can be used for anti-aging purposes due to their anti-oxidative properties. The essential oil of TTO and its majority chemical constituent 1, 8 cineole, terpineol-4-ol, β-pinene α-pinene, and above all rosemary essential oil and its chemical constituent's carnosol, camphor, terpinen-4-ol, are active against In vitro free radical scavenging activity & skin aging. The sun protection factor results show that tea tree oil/rosemary oil can be used to make microemulsions that protect the skin from sunburn. From the result, it can be concluded that Tea tree oil had a lower sun protection factor than rosemary essential oils. TTO and RMO essential oils have good antioxidant properties and their free radical scavenging activities (in DPPH and Nitric oxide scavenging methods) increase with concentration and have a synergistic photo-protective effect on exposure to UV light.