In Vitro Sun Protection Factor Determination of Selected Medicinal Plants and Formulation of Sunscreen Cream

Bindu Poude^*, Akash Gurung, Hari Prasad Subedi, Sagar Babu KC and Anita Tiwari Kalpana Parajuli ^*Correspondence: Bindu Poude, Department of Pharmaceutical Sciences, School of Health and Allied Sciences, Pokhara University, Pokhara, Nepal, Email:

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Introduction

Before the introduction of orthodox drugs, different plant extracts have been used for the treatment of several diseases. Due to the presence of secondary metabolites, plants poses antifungal, antimicrobial, anticancer, antidiabetic and other pharmacological activities (Larbie CO, et al., 2019). Similarly, natural ingredients have been traditionally used for skin care purposes. Source of natural ingredients can be herbs, flowers, fruits, roots and leaves (Rebeiro AS, et al., 2015). Natural ingredients are gaining more popularity as consumers seek more natural ingredients in their personal care product because they are less hypo allergic compared to synthetic cosmeceutical ingredients and consumer need not to worry about skin irritations. Cosmeceutical preparations with natural ingredients supply the skin with nutrients like vitamin A, vitamin C, vitamin E, phenolic compounds, flavonoids and terpenoids which act as antioxidant, anti-inflammatory, antiaging and anti-melanogenic effect on skin and enhance the skin health (Lohani A et al., 2018).

Exposure to Ultraviolet (UV) radiation triggers the rapid generation and accumulation of Reactive Oxygen Species (ROS) in the skin. When there is imbalance between ROS and antioxidants, they can also take part in the pathological process known as oxidative stress and leads to cell damage and aging (Petruk G et al., 2018). UV radiation consists of three components, UVA, UVB and UVC. Among these components UVA and UVB reach the earth surface in sufficient amount to damage the skin whereas UVC is almost completely absorbed by the ozone layer (Yang Y and Li S, 2015). UVB is responsible for erythema and sunburn. In contrast to UVB, UVA is more efficient in inducing immediate and delayed pigment darkening and delayed tanning. UVA induces several adverse effects including immunosuppression, photoaging, ocular damage and skin cancer. UV radiations also have beneficial effect as it increases synthesis of vitamin D in skin. There are a lot of different types of sunscreen products (oils, sticks, gels, creams, lotions) which absorb UV rays and prevents them from penetrating the skin (Korac RR and Kambholja KM, 2011).

Different class of antioxidants such as flavonoids, polyphenols, carotenoids and vitamins show protectant activity against UV radiation. Different bioactive compounds responsible for skin protection against UV radiation are quercetin, resveratrol, lycopene, beta-carotene, vitamin C and vitamin E (Petruk G et al., 2018). Phenolic compounds act as antioxidant and protect leaf from photodamage. Different physical sunblock such as zinc oxide, titanium dioxide are also used. Besides that, the skin’s natural sun blockers are proteins, lipids and nucleotides (Korac RR and Kambholja KM, 2011).

Sun protection factor is defined as the UV energy required to produce a Minimal Erythema Dose (MED) on protected skin divided by the UV energy required producing a minimal erythema dose on unprotected skin. MED is defined as the dose of UV irradiation sufficient to produce a minimal erythema on unprotected skin. The higher the SPF, more effective is the product in preventing sunburn. Photo protection afforded by sunscreen against solar radiation exposure can be determined by photo testing in human volunteer (Dutra EA, et al., 2004).

Topical cream formulations are considered to be more acceptable to patients as they can be effortlessly applied on various part of the skin without imparting the greasy feel. Creams are more potent and occlusive for long period of time as compared to lotions. Topical creams are prepared by the fusion process wherein the oil phase and the water phase components are separately heated and mixed with continuous agitation (Mendonsa NS, et al., 2019). Creams and ointments containing plant extract are being formulated due to rapid expansion of demand for herbal cosmetics (El-Gied AA, et al., 2015). Plants or their parts used in herbal cosmetic preparation should have various properties like antioxidant, anti-inflammatory, antiseptic, emollient and antibacterial (Aswal A, et al., 2013) (Table 1).

Table 1: List of selected plants and parts used for the study

Materials and Methods

Plant collection and identification

All the plants were collected from Rupatal, Deurali, and Khudi of Kaski district at an altitude ranging from 800-1200 m. The herbaria of collected plants sample were prepared. Then it was identified by comparing the morphological characteristics and was confirmed by taxonomist at National Herbarium and Vegetation laboratories, Godawari, Lalitpur, Nepal. All the plant samples were made clean from mud, dirt, or lichens present and then cut into pieces with the help of plant cutter and knives and the left for shade drying until dried completely before extraction started.

Extraction of plants

Bark, leaves, roots, bulb and peels of the selected plants were separated, dried in shade at room temperature and grinded to coarse powder using blender. The 100 gram dried powder of each plant was extracted with 800 ml ethanol for 24 hours. Then the residue obtained from the initial extraction was again extracted with 800 ml ethanol for 24 hours. All the extract was concentrated to dryness, under reduced pressure and controlled temperature, using rotary evaporator.

Phytochemical screening

The phytochemical analysis was performed for testing the different chemical groups present in the ethanolic extract of various plants. The presence of phytochemical like alkaloids, flavonoids and phenols was evaluated according to methods described by Zhang J, et al., 2011 with some modifications.

Reagents used for phytochemical screening

Mayer’s reagent: About 1.358 gram of HgCl2 was dissolved in 60 ml of water and it was mixed with a solution of 5 g KI in 10 ml water and volume was adjusted upto 100 ml by water.

Wagner’s reagent: Potassium iodide of about 16.6 gram of was dissolved in 100 ml of distilled water and few crystals of iodine were added to the solution and stirred properly.

Ferric chloride solution: Fifteen grams of ferric chloride hexahydrate was dissolved in 100 ml of distilled water.

Lead acetate solution: Ten grams of lead acetate was dissolved in 100 ml of carbondioxide free water.

Sodium hydroxide solution: Twenty grams of NaOH was dissolved in 100 ml of distilled water.

Phytochemical tests

Alkaloids test: Each of the sample extracts were dissolved individually in dilute hydrochloric acid and filtered. Then the filtrates were treated with

Mayer’s reagent and Wagner’s reagent to test for the presence of alkaloids.

Phenolic test: All extracts were treated with 5 ml FeCl3 solution.

Flavonoids test: In alkaline test, all extracts were treated with 5 ml of sodium hydroxide and observed. In lead acetate test, all extracts were treated with 5 ml of lead acetate solution and observed.

Determination of total phenolic content

The total phenolic content of plant extract was determined using a spectrophotometric method according to Zhang J, et al., 2011 with slight modifications.

Preparation of gallic acid solutions and test samples

Gallic acid was taken as standard phenolic compound. Different concentrations of gallic acid (7.82125 µg/ml, 15.625 µg/ml, 31.25 µg/ml, 62.5 µg/ ml, 125 µg/ml, 250 µg/ml, 500 µg/ml and 1000 µg/ml) were prepared. From dried extract of plants 1 mg/ml concentration was prepared as a test sample.

Preparation of 10% sodium carbonate solution: Ten grams of sodium carbonate was dissolved in distilled water and diluted up to 50 ml to make 10% of solution (Indian Pharmacopoeia).

Determination of total phenolic content

About 1 ml of test sample was added to 1 ml of 2 N Folin reagent followed by the addition of 5 ml of distilled water and left for five minutes. Then, 1 ml of 10% NaCO₃ was added and incubated at room temperature for 1 hour. Finally, the absorbance of the reaction mixture was measured at 760 nm against blank ethanol. The total phenol content was expressed as µg gallic acid equivalents (GAE)/mg of extract, using the calibration curve of gallic acid (Figure 1) (7.8125-1000 µg/ml) standards. All the determinations were carried in triplicate.

Figure 1: Calibration curve of gallic acid

Determination of total flavonoid content

The content of flavonoids in plant extracts was determined using a spectrophotometric method according to Zhishen J, et al., 1999 with slight modifications.

Preparation of quercetin and test samples

Quercetin was taken as standard flavonoid compound. Different concentrations of quercetin (7.8125 µg/ml, 15.625 µg/ ml, 31.25 µg/ ml, 62.5 µg/ ml, 125 µg/ml, 250 µg/ml, 500 µg/ml and 1000 µg/ml) were prepared. From the stock solution 1 mg/ml concentration of extract were prepared as test samples.

Preparation of aluminium chloride (10%): Ten grams of aluminium chloride was dissolved in 100 ml of water to prepare 10% of aluminium chloride solution.

Determination of total flavonoid content

Total flavonoid content was determined by Aluminium chloride colorimetric method. 1 ml of each extract solution (1 µg/ml) was mixed with 4 ml of distilled water. Then, 0.3 ml of 5% sodium nitrate was added. After 5 minutes, 0.3 ml of 10% aluminium chloride was added and allowed to stand for 6 minutes. Then, 2 ml of 1 M sodium hydroxide was added and absorbance was measured at 510 nm using UV spectrophotometer. The calibration curve was prepared using quercetin as the standard (Figure 2). Total flavonoid content was calculated from the calibration curve and results were expressed as µg quercetin equivalent per gram dry extract weight.

Figure 2: Calibration curve of quercetin

Determination of sun protection factor

In vitro SPF was determined using spectrophotometric analysis. For the determination of SPF, 1% w/v solution was prepared in ethanol and from this 0.01% w/v concentration was prepared. The absorbance of the sample solutions were taken by UV-visible spectrophotometer in the range of 290- 320 nm, in every 5 nm interval using ethanol as a blank. Three readings were taken at every interval. The SPF value was calculated by following equation-

Where,

CF=Correlation factor

EE=Erythemogenic effect of radiation with wavelength

Abs=Spectrophotometric absorbance values of wavelength

The values of EE(λ) × I(λ) is constant at a fixed wavelength (3).

Formulation of cream

For the formulation of cream in laboratory method described by Farboud ES, et al., 2011 with slight modifications was used. Required amount of both aqueous phase and lipid phase were heated separately at 70 ± 2°C. The aqueous phase was added to the lipid phase, with continuous stirring, using magnetic stirrer until congealed. Four formulations, having concentrations of 0%, 1%, 2.5% and 5% plant extract, were prepared. All the formulations, containing certain level of plant extracts, were evaluated for different parameters. The formulation components, used during formulation of cream, are listed below (Table 2).

Table 2: List of components used during the formulation of the cream

Evaluation of pH of cream

The pH meter was calibrated using standard buffer solution. pH of 0.5 g was measured by digital pH meter (Aswal A, et al., 2013).

Appearance

The appearances of the creams were evaluated visually for their color and homogeneity (Joshi P, et al., 2019).

Evaluation of SPF of cream

Sunscreen samples were diluted using ethanol, final concentration of cream having 1 µg/ml, and analyzed by UV-Visible spectrophotometry ranging from 290 nm to 320 nm, at an interval of 5 nm.

Evaluation of acid value

Acid values of creams were determined according to method described by Aswal A, et al., 2013. Four grams of cream was dissolved in 20 ml mixture of equal volume of ether and ethanol and heated until sample dissolved. Five ml of this mixture was taken in a conical flask and titrated with 0.1 N NaOH until fairly pink color persisted for 30 seconds. 1 ml of phenol- phthalein was used as indicator.

where,

n=No.of ml of NaOH required

w=The weight of substance (Aswal A, et al., 2013).

Evaluation of saponification value

Saponification values of creams were determined according to method by Aswal A, et al., 2013. 2 g of cream was refluxed with 25 ml of 0.5 N of alcoholic KOH, for 30 minutes, and the sample was titrated with 0.5 N HCl, phenolphthalein as an indicator. Then, saponification value was calculated from following formula.

Where,

a=Volume of titrant (in ml)

b=Volume of titrate (in ml)

w=Weight of substance (in grams) (Aswal A, et al., 2013).

Stability testing

Formulated creams were stored at three different temperatures, 4°C, room temperature (21 ± 5°C) and 40 ± 2°C, and the stability study were carried out at 0^th, 7^th, 14^th, and 21^st day (Smaoui S, et al., 2019).

Results

Extraction yield value

Obtained yield values of each ethanolic extracts were expressed as extract yield percentage (Table 3).

Table 3: Yield value of the extract of selected plants

Phytochemical screening

Phytochemical screening of various plant extract confirmed absence or the presence of alkaloids, flavonoids and phenols. The results are as follows (Table 4).

Table 4: Phytochemical screening of selected plants

Total phenol content

The quantitative determination of total phenolic content was carried out using Folin-Ciocalteu reagent in terms of gallic acid equivalent (GAE). The calibration curve of gallic acid. Table 5shows total phenolic content expressed as µg gallic acid equivalent per milligram dry extract weight.

Table 5: Total phenolic content of selected plant samples

Total flavonoids content

Quantitative determination of total flavonoid was performed by precipitating with aluminium chloride in an alkalinized medium. The calibration curve with quercetin as the standard is shown in figure while Table 6shows the total flavonoid content of plant extracts

Table 6: Total flavonoid content of selected plant samples

Discussion

In our study, nine different plants were collected from Deurali, Rupatal, Rajakochautara and Bijaypur of Kaski district. Ethnobotanical information revealed that the selected plants in this study are traditionally used for various medicinal purposes and possess different pharmacological properties. The thesis work included phytochemical screening, determination of total phenol and flavonoid content, in vitro SPF of selected nine plants, formulation and evaluation of sunscreen cream from most potent plant extract. Phytochemical screening was performed for different phytochemical constituents such as alkaloids, phenols and flavonoids. Ethanolic extract of all the plants showed the presence of phenols and flavonoids. According to Mustapha AA, et al., 2014, M. indica showed the presence of phenols, flavonoids and alkaloids which supports this research work. According to Makhija IK, et al., 2010, Ficus religiosa showed the presence of flavonoids which supports this research work.

M. indica showed highest amount of phenolic content. Total phenolic content of M. indica was found to be in the range of 63.89 to 116.80 mg GAE/g dry weight of extract in 80% methanol (Sultana B, et al., 2012), but in this research work total phenolic content of this plant was found to be 182 mg GAE/g dry weight of extract in ethanol. This variation may be due to variation in solvent. The plant having high phenolic content showed the highest SPF value (Ebrahimzadeh MA, et al., 2014), which supports our study. Flavonoids do not seem to be simply detectable by any method therefore AlCl3 was used as a complexing reagent. The method is based on the formation of a stable complex between AlCl3 keto and hydroxyl group of flavones and flavonoids (Hassan SM, et al., 2013). M. indica showed the highest flavonoid content. Total phenolic content of the M. indica was found to be 925.55 CE/g dry weight of extract in 80% methanol (Sultana et al., 2012). In this research, total flavonoids content of ethanolic extract of M. indica bark was found to be 2949 mg QE/g dry weight of extract. This may be due to variation in solvent and may be due to variation in comparison with standard where catechin was used in previous work and quercetin in this work. The plant having high flavonoid content showed the highest SPF value (Costa SC, et al., 2015). From above discussion we can conclude that there may be correlation between phenolic flavonoid compounds and SPF value. Flavonoids and phenols have been reported as an important functional compound in plants which play a vital role in management of inflammation due to solar radiation (Imam S, et al., 2015).

There is growing demand for herbal cosmetics in the world (Aswal A, et al., 2013). Therefore, an herbal sunscreen cream containing ethanolic extract of M. indica was prepared. Four formulations were prepared and evaluated for various parameters such as pH, acid value, saponification value, in vitro SPF and stored at three different temperatures for their stability testing (Table 7). The measurement of SPF is an ultimate way to determine the effectiveness of the sunscreen formulation. The higher the SPF the more protection the sunscreen offers against UV light. Sunscreen is used to aid the body’s defense mechanism to protect against harmful UV-radiation from the Sun (Lohani A, et al., 2018). Here, in this research work, the formulation containing 5% plant extract showed the highest SPF value. Therefore, this formulation may protect skin against UV-radiation. Acid value is the mass of KOH in mg that is required to neutralize one gram of chemical substance. Higher the acid value, it will cause irritation to skin after application (Fatima S, et al., 2017; Swarnlata S, et al., 2011). Saponification value number represents the number of mg of KOH required to saponify 1g of fat. Saponification value influences pH and stability of cream. Higher saponification value means higher amount of free fatty acid which are prone to hydrolysis and can cause rancidification (Saraf S, et al., 2011). In our research work it is found that the saponification value is decreasing on increasing the concentration of plant extract.

Table 7: SPF value of selected plant samples

This study revealed that formulations I,II and III were found to be stable at all storage conditions except the formulation IV, which was unstable and resulted in breakdown of emulsion, at 40 ± 2°C from the 7th day. The formulation IV, which is kept at 40 ± 2°C, was found to be unstable i.e. liquefaction started at increased temperature along with increase in concentration of extract. Monitoring the pH of cream is necessary for determining the stability of pharmaceutical and cosmeceuticals (Table 8). Any changes in the pH of the product indicate the possible interaction which may provide an idea on the quality of the product (Smaoui S, et al., 2012). The pH of the human skin normally ranges from 4.5 to 6. Due to frequent washing and use of soap, the acidity of the skin is lost.

Table 8: Stability results of different creams at 3 different temperatures

Therefore, moisturizer has an acidic range which is used to normalize the skin. Acceptable pH range of the moisturizer should be between 5-8. The pH of the cream was found to be in the range 5.01-6.76. The result revealed that the pH of the cream was within the range (Saraf S, et al., 2011). The formulations have almost constant pH throughout the study. The appearance of the cream was not changed. In this study sunscreen cream containing 1% extract showed acid value and saponification value 8.6 ± 0.41 and 30.4 ± 0.83 respectively. In a previous research, antiaging facial cream containing 1% curcumin extract was formulated, which showed acid value and saponification value of 5.7 and 25.7 respectively (Panda S, et al., 2018). This difference in acid value and saponification value may be due to ingredients used in cream and chemical constituents present in extract (Table 9). According to Akter S, et al. 2013, on varying the concentration of stearic acid and cetyl alcohol, saponification value also varies. Saponification value goes on decreasing on increasing the concentration of those constituents. In this study, the saponification value goes on decreasing on increasing the concentration of extract.

Table 9: Acid value, saponification value and SPF of the cream stored at room temperature

According to Pratama G, et al., 2019, the cream having high amount of extract was very good for counteracting UV rays because it has high SPF value when compared to other formulations. Also, in this study, the formulation containing 5% extract showed the highest SPF i.e. 7.19 ± 0.12.

Conclusion

The study provided reasonable data to conclude that Magnifera indica has highest phenolic and flavonoid value which may have resulted in the highest SPF value among the nine medicinal plants taken during the study. The sunscreen cream, which is o/w type emulsion, containing different concentration yields good physical characteristics formulation. Method used for the evaluation of sunscreen cream, used in this work, is simple, fast, economical and easy to use.

Acknowledgement

We would like to express our humble gratitude to our supervisor, Kalpana Parajuli, Assistant Professor, School of Health and Allied Sciences, Pokhara University, for her keen and continuous interest, valuable suggestion and guidance throughout the course of research.

We would like to express our sincere thanks to Dr. Khem Raj Joshi, former Dean, School of Health and Allied Sciences, Pokhara University and Dr. Damaru Prasad Paneru, Director of School of Health and Allied Sciences, Mr. Nim Bahadur Dangi, Assistant Professor and program coordinator of B. Pharm. and Mr. Atismmodavardhana Kaundinnyayana, Assistant Professor and program coordinator of M. Pharm, Pokhara University for their continuous correspondence which helped us to work efficiently during our research.

We are thankful to Associate Professor Dr. Nirmala Jamarkattel, Assistant Professor Dr. Sushil Panta, Assistant Professor Dr. Shila Gurung, Assistant Professor Dr. Namraj Dhami, and all the faculty members of School of Health and Allied Sciences, Pokhara University, for their kind support and suggestions to perform our research work.

We are also thankful to Santosh Gurung and Durga Bahadur BK, Lab Assistants, and all the staffs of School of Health and Allied Sciences, Pokhara University, for their support and help.

Our best regard goes to investigation officers Krishna Dahal, Ganga Dutta Bhatta and their colleagues for helping us for the identification of plant species.

We are also thankful to our seniors and all our classmates for their kind support and guidance throughout our research work.

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