INTRODUCTION:

Sunlight is an important factor in environmental systems. However, harmful effects of solar radiation are caused by the ultraviolet (UV) region of the electromagnetic spectrum. UV radiation is divided into three wavelength ranges: UV-C (200-280nm) that is absorbed by the ozone layer, so cannot reach earth, UV-B (280-320nm) and UV-A (320-400nm)¹. About 95% of UV radiation that reaches earth is UV-A and only 5% is UV-B².

However, UV-B is responsible for many harmful effects on the skin such as, sunburns, erythema, delayed tanning, DNA damage and non-melanoma cancer^3,4. UV-A causes the generation of reactive oxygen species (ROS), acute tanning, and skin aging^2,5.

Sunscreens are considered as one of the photoprotection tools against UV radiation⁶. They contain organic compenents such as aminobenzoic acid, cinnamates, benzophenones, and inorganic filters such as metal oxides (zinc oxide)⁷. Currently many studies discuss their side effects on humans and environment⁸. For example, organic compenents used as topical photoprotective agents have been shown to cause photoallergic contact dermatitis (PACD)^9,10. Another adverse effect of sunscreens is endocrine disruption due to their varying effects on estrogen, androgen, and progesterone receptors, which can cause some reproductive disorders¹¹, in addition to their effect on thyroid hormones causing a decrease of T4 levels in plasma¹². There is a growing interest in using herbal substances such as polyphenols to avoid these harmful effects^13. Polyphenols can absorb UV radiation in wavelengths between 200-400nm¹⁴. They have a lot of benefits in skin care such as whitening effect, antiaging effect, and anti-photocancerogenic effect, due to their antioxidant and anti-inflammatory properties^15,16,17.

Traditionally Viola odorata has been used, as herbal medicine, for the treatment of many skin diseases like eczema, acne, and upper respiratory diseases¹⁸. In this study, we evaluated SPF values and antioxidant activity of Viola odorata flower extracts using three solvents (chloroform, ethyl acetate and ethanol). The correlation of these properties with TPC was also investigated to verify the possibility of using these extracts as sunscreen agents depending on their polyphenols content.

MATRIAL AND METHODS:

Plant material:

Viola odorata flowers were collected from Syrian coast villages during the flowering stage of the plant. Part of flowers was stored in a freezer for 4 months at -18⁰C, another part was air-dried and the rest was used fresh.

Chemicals and reagents:

The reagents used in this study, including Folin-Ciocalteu reagent, 1,1-diphenyl-2-picrylhydrazyl (DPPH), were purchased from Merk (Germany). Sodium carbonate was from Himedia (India). Four different solvents were used: chloroform and ethyl acetate were obtained from LOBA Chemie, ethanol and methanol was obtained from Fisher (UK).

Extraction of polyphenols:

Fresh and frozen flowers were cut while dried flowers were crushed. Ten grams of each sample (fresh, frozen, and dried) was extracted with sequential extraction using 100ml of each solvent (chloroform, ethyl acetate, ethanol 50% respectively) by ultrasound for 30 min at room temperature, then filtered through filter paper to remove the debris. The supernatant was concentrated using a rotary evaporator to remove the solvent. The extracts were collected and freeze-dried.

Determination of total phenolic content:

The total phenolic content of each sample was determined using the Folin-Ciocalteu assay according to Singleton et al., with some modification¹⁹. 0.1ml of Folin-Ciocalteu reagent (diluted 10 times with distilled water) was mixed with 0.1ml of extract dissolved in methanol, for 5 min, and then 2ml of sodium carbonate 2% was added. After 30 min of incubation at room temperature in the dark, the absorbance was measured at 760nm against a blank (methanol) using UV-VIS spectrophotometer (DRAWELL). A calibration curve was prepared using a standard solution of Gallic acid (25, 50, 100, 150µg/ml).The results were expressed as milligrams of gallic acid equivalents per 1g of crude extract.

Determinaton of antioxidant activity:

The antioxidant capacity of flower extracts was determined using DPPH assay according to modified method of Brand William et al.²⁰. 0.2ml of methanolic extract solution at different concentrations (0.1, 0.25, 0.5, 1, and 2mg/ml) was mixed with 2ml of DPPH solution (0.1mM). After 30 min of incubation at room temperature in the dark, the absorbance decrease was measured at 520nm using UV-VIS spectrophotometer (DRAWELL). The inhibition of free radical DPPH in percent (Inh%) was calculated using the following equation:

Inh% = [(A_control- A_sample)/ A_control]x100

Where A_control is the absorbance of DPPH solution without sample.

In vitro Dertermination of sun protection factor SPF:

Five concentrations (0.1, 0.25, 0.5, 1, 2mg/ml) of each extract were dissolved in methanol. UV absorption was measured using UV-VIS spectrophotometer between 290-320 nm. SPF was calculated by the spectrophotometric method developed by Mansur et al. using the following equation²¹:

₃₂₀

SPF = CF x ∑ x EE(λ) x I(λ) x Abs(λ)

²⁹⁰

Where CF is a correction factor (CF= 10), EE(λ) is erythmogenic effect at wavelength λ, I is solar intensity spectrum, abs is the absorbance of sample at wavelength λ, EE x I value is constant according to Mansure.

Statistical analysis:

Data were analyzed with COSTAT 6.4 and SPSS 20, using ANOVA test to compare the different parameters between the three flowers states in used solvents. Person's correlation test was used to calculate the correlation coefficient between theses parameters. The level of significant was set at p < 0.05

RESULTS:

Total phenolic content:

Total phenolic content of different extract fractions is presented in Table 1. It was determined using a linear gallic acid standard curve (y =0.0022x+0.2738; R²=0.949). TPC ranged from 1.039 to 18.575mg GAE/g crud extract. There was a significant difference in TPC among the three states of flowers (fresh, frozen and dried) for the same solvent, except between extracts of dried and frozen flowers in chloroform fraction. A significant difference in TPC was also found among extract fractions in the three solvents (chloroform, ethyl acetate, and ethanol) for the same state, except between chloroform and ethyl acetate fraction from the dried flowers. As shown in figure 1, the highest polyphenols content was seen in the ethanolic extract fraction from the three states of flowers, followed by ethyl acetate fraction and then chloroform fraction. Furthermore, fresh flowers showed the highest content of polyphenols in all solvents.

Antioxidant activity:

The ability of Viola odorata flowers extract fractions at the different concentrations (0.1, 0.25, 0.5, 1, and 2 mg/ml) to quench reactive species by hydrogen donation (% of DPPH inhibition) is presented in table 2. Values ranged from 0.49% -89%. A significant difference was found among extracts in the three states using the three solvents (chloroform, ethyl acetate and ethanol) at the most of extract concentrations. The ethanolic extract fraction showed the highest ability of scavenging DPPH radical, while chloroform extract fraction was the lowest. There was also a strong correlation between extract concentration and the ability of scavenging DPPH in all solvents. We obtained the highest antioxidant activity at the concentration of 2 mg/ml of the extract for all states of Viola odorata flowers in all solvents. At this concentration, DPPH inhibition exceed 80% in ethanolic extract fraction in the three flowers states.

Table1. Total phenolic content in Viola odorata flowers extract fractions in the three states (fresh, dried, and frozen) according to the three used solvents (chloroform, ethyl acetate, and ethanol).

Fraction	Flowers states	TPC (mg GAE/g crud extraction)	p-value
Chloroform	Fresh	3.414^d	0.011
	Dried	1.039^ef
	Frozen	0.532^f
Ethyl acetate	Fresh	4.921^c	0.004
	Dried	0.769^f
	Frozen	1.850^e
Ethanol	Fresh	18.575^a	0.014
	Dried	15.06^b
	Frozen	8.186^g

Means with different letters (a-e) within the same coloumn are significantly differents (P<0.05)

Fig.1: Total phenolic content in Viola odorata flowers extract fractions using the three solvents (chloroform, ethyl acetate, and ethanol) according to the three flowers states (fresh, dried, and frozen).

Table 2. Comparative analysis of DPPH inhibition of Viola odorata flowers extracts at different concentrations (0.1, 0.25, 0.5, 1, and 2 mg/ml) in the three states (fresh, dried and frozen) using the three solvents (chloroform, ethyl acetate and ethanol).

		Dried flowers			Fresh flowers			Frozen flowers
Solvent	Con. mg/ml	DPPH inhibition%	P-value	R	DPPH inhibition%	P-value	R	DPPH inhibition%	P-value	R
Chloroform	0.1	1.74^d	0.0144	0.948^***	6^c	0.0032	0.881^**	0.49^d	0.009	0.97^***
	0.25	2.81^d			12.5^b			3.08^c
	0.5	4.03^c			10.3^b			3.68^c
	1	5.5^b			11.2^b			5.02^b
	2	6.88^a			17.02^a			8.27^a
Ethyl acetate	0.1	5.89^e	0.0063	0.989^***	2.35^d	0.005	0.98^***	0.85^e	0.0093	0.948^***
	0.25	13.86^d			8.55^c			9.96^d
	0.5	30.95^c			7.77^c			28.11^c
	1	52.27^b			38.32^b			61.07^b
	2	85.73^a			64.16^a			77.13^a
Ethanol	0.1	11.41^d	0.0087	0.901^***	12.04^c	0.0048	0.953^***	17.26^d	0.0105	0.837^**
	0.25	27.94^c			11.71^c			27.68^c
	0.5	38.03^b			5.34^d			56.66^b
	1	80.9^a			52.15^b			89.96^a
	2	85.32^a			84.11^a			85.87^a

Means with different letters (a-e) within the same column are significantly different (P<0.05)

Table 3. Comparative analysis of SPF values of Viola odorata flowers extracts at different concentrations (0.1, 0.25, 0.5, 1, and 2 mg/ml) in the three states (fresh, dried and frozen) using the three solvents (chloroform, ethyl acetate and ethanol).

		Dried flowers			Fresh flowers			Frozen flowers
solvent	Con. mg/ml	SPF	P-value	R	SPF	P-value	R	SPF	P-value	R
Chloroform	0.1	0.136^d	0.0052	0.996^***	0.79^c	0.0055	0.878^**	0.12^d	0.0052	0.998^***
	0.25	0.402^cd			1.54^c			0.36^d
	0.5	0.699^c			3.58^b			0.73^c
	1	1.678^b			12.85^a			1.53^b
	2	3.094^a			12.59^a			2.96^a
Ethyl acetate	0.1	1.03^d	0.0072	0.914^***	1.051^d	0.0053	0.997^***	0.99^e	0.0059	0.978^***
	0.25	4.66^c			1.847^d			2.266^d
	0.5	10.5^b			3.967^c			5.34^c
	1	18.45^a			8.439^b			11.047^b
	2	21.11^a			16.121^a			16.622^a
Ethanol	0.1	5.825^c	0.015	0.828^**	3.761^e	0.0092	0.93^**	6.068^c	0.0112	0.829^**
	0.25	17.098^b			7.636^d			7.972^c
	0.5	17.695^b			14.357^c			20.247^b
	1	27.19^a			22.514^b			27.073^b
	2	27.978^a			26.907^a			27.249^a

Means with different letters (a-e) within the same column are significantly different (P<0.05)

Correlation assay:

The previous results clearly indicated that the quantitative estimation of TPC, DPPH, and SPF values is influenced by two variables that are the state of flowers and the extracting solvent. Therefore, the correlation analyses between the studied parameters were analyzed within the extracts according to each variable. As it is shown in Figure 2, there was a strong positive correlation between SPF values and antioxidant activity of Viola odorata flowers extracts in different states using ethyl acetate and ethanol as extracting solvent (R=0.902, R=0.858 respectively). However, this correlation was moderate in chloroform extract fraction (R=0.622).

We choose the concentration 2mg/ml of extracts that gave the best SPF value to optimize the correlation between TPC, SPF and DPPH. There was a moderate to good significant correlation between DPPH and TPC and between SPF and TPC (R=0.5, R= 0.748) respectively (data not presented).

Fig 2: Correlation between antioxidant activity and SPF values of Viola odorata flowers extracts at different concentrations (0.1, 0.25, 0.5, 1, and 2 mg/ml) in the three states (fresh, dried and frozen) using the three solvents (chloroform, ethyl acetate and ethanol).

DISCUSSION:

Nowadays, a special interest is dedicated to the usage of herbal products for skincare thanks to their different properties like photo-protection, anti-aging, moisturizing, antioxidant, astringent, anti-irritant, and antimicrobial activity. These properties often correlate with each other¹⁸. In our study, we choose Viola odorata flowers because it is known to be a rich source of polyphenols²². The content of polyphenols is largely influenced by their solubility in a solvent. According to the literature, solvents such as methanol, ethanol, ethyl acetate, and propanol have been used to extract polyphenols²³. In this study, we found that ethanol 50% was the best solvent with the three flower states (fresh, frozen, and dried) to extract polyphenols. Many studies agree with this result. Boussetta et al. found that ethanol -in- water solutions (50%) were more effective than pure ethanol in the extraction of phenolic acids and flavonoid glucosides from flax seeds²⁴. This result could be explained by the polarity of polyphenols contained in Viola odorata flowers such as anthocyanins, flavonoids (rutin), tannins and phenolic acids¹⁸, beside the diversity of polyphenols that water-ethanol mixtures can dissolve ²⁵.

According to our results, fresh flowers extract showed the highest TPC in the three solvents, while frozen flowers extract showed the lowest TPC. This result means that freezing negatively affected polyphenols stability, which can be explained by the damage of cells caused by crystallization of water through freezing and then thawing, allowing enzymatic reactions to occur²⁶, in addition to the effect of storage temperature and time on polyphenols stability²⁷. According to previous studies, rutin, monomeric anthocyanines, epicatchins, and phenolic acids are exposed to degradation through freezing storage²⁶.

As it is known, UV radiation can induce oxidative stress in the skin, which is known to be associated with carcinogenic processes and skin aging^28,29,30. To avoid UV disorders, the usage of topical formulations containing antioxidants is necessary. Our study showed that the ethanolic extract fraction had the highest capacity of DPPH radical scavenging (about 85%) in comparison to other extracts. A similar result was reported by Baldissetto et al. with hydroalcoholic Moringa oleifera leaf extracts³. Moreover, antioxidant activity increased with the increase of extract concentration. This result comes in accordance with the studies by Liu et al. and khan et al. for Zea mays and Sonchus asper respectively^31,32.

Sunscreens are substances that protect skin against the damaging effects of UV. Recently, plant extracts have been recognized as good sunscreen agents due to their ability to absorb UV rays and their antioxidant activity ^14,33. In vitro SPF is one of universal measurements used to predict the ability of a substance to protect against UV radiation ³⁴. FDA recommends the use of broad- spectrum sunscreen with SPF values of 15 or higher³⁵. In our study Viola odorata flower extracts showed good SPF values. At the concentration of 2mg/ml of ethanolic extract, the SPF value almost reached the value of 27 with the three flowers states. SPF was about 21 for ethyl acetate extract fraction of dried flowers, but it was lower for fresh and frozen flowers (closed to 16). In a similar contest, Khazaeli et al. reported that ethyl acetate extract of Viola odorata flowers had an SPF value close to 25.69³⁶. Furthermore, our SPF results of Viola odorata flowers was higher than SPF values given by plants mixture which ranged between 2.14 and 12.67 for a chain of concentrations of the mixture³⁷.

Various biological activities and factors influence the efficacy of a natural product as photoprotector, such as, antioxidant activity, filtering activity against UV radiation, anticancer properties, as well as its chemical composition³⁸. At the concentration of 2mg/ml of Viola odorata flower extract, which had the highest SPF value, we found a strong correlation between SPF values and TPC in the extracts of flowers in the three states using the three solvents. This could means that photoprotection activity of Viola odorata flower extracts is attributed to polyphenols content. Polyphenols are known by their ability to absorb UV radiation in wavelengths range between 200-400 nm, associated with their aromatic system and ᴨ electron system³⁹.

The role of polyphenols in photoprotection was discussed by several studies. For example, the effect was seen with anthocianins and rutin existing in Viola odorata flowers, but also with elagic acid, chlorogenic acid, feluric acid and rutin in Moringa oleifera leaf extract^{3,39, 40}.

The antioxidant activity of the studied extracts is also correlated with the TPC, that could explained by the presence of hydroxyl groups in the structure of these polyphenols and their hydrogen donating ability⁴¹. Moreover, the strong correlation that is found between SPF values and antioxidant activity could propose that polyphenols existing in Viola odorata flowers have these both properties.

CONCLUSION:

Our results showed that Viola odorata flowers has a high content of polyphenols, good antioxidant properties, and possess the ability to protect against harmful solar radiation. Therefore, it is a promising candidate that can potentially be used as a natural ingredient in sunscreen and anti-aging formulations. Further studies must be conducted to assess the in vivo sun-protection effects of Viola odorata flowers extract.

REFERENCES:

1. Neelima G, et al., Formulation and Evaluation of Sun Protection Factor of Poly Herbal Sunscreen Cream. Research Journal of Topical and Cosmetic Sciences. 2016 June 7(1): 09-14. Doi: 10.5958/2321-5844.2016.00002.9

2. Greinert, R., et al., European Code against Cancer 4th Edition: Ultraviolet radiation and cancer. The International Journal of Cancer Epidemiology,Detection, and Prevention. 2015 Dec 395: S75-S83. Doi: 10.1016/j.canep.2014.12.014

3. Baldisserotto, A., et al., Moringa oleifera leaf extracts as multifunctional ingredients for “natural and organic” sunscreens and photoprotective preparations. Molecules, 2018 Mar 23(3): 664-680. Doi: 10.3390/molecules23030664

4. Saha, D, et al., Skin cancer: dance of death. Asian Journal of Pharmceutical Research. 2011 June 1(2): 34-36. Doi: 10.5958/2231–5691

5. Panich, U., et al., Ultraviolet radiation-induced skin aging: the role of DNA damage and oxidative stress in epidermal stem cell damage mediated skin aging. Stem Cells International, 2016 Apr 2016: doi: 7370642

6. Patil, S., et al., Formulation of gel and its UV protective study of some medicinal flowers. Research Journal of Topical and Cosmetic Science. 2011 Jul-Dec. 2(2): 64-65

7. Nash, J.F., Human safety and efficacy of ultraviolet filters and sunscreen products. Dermatol Clin, 2006 Jan. 24(1): 35-51. Doi: 10.1016/j.det.2005.09.006

8. Siller, A., et al., Update about the effects of the sunscreen ingredients oxybenzone and octinoxate on humans and the environment. Plastic Surgical Nursing Journal, 2018 Oct-Dec. 38(4): 157-160. Doi: 10.1097

9. Russo, J., et al., Photoallergic Contact Dermatitis to Sunscreens Containing Oxybenzone in La Plata, Argentina. Actas Dermo-Sifiliográficas (English Edition), 2018 Jul-Aug. 109(6): 521-528. Doi: 10.1016/j.adengel.2018.05

10. Ratnasooriya, W., et al., In vitro sunscreen activity of Methanolic root extract of a Sri Lankan grass Heteropogon contortus. Asian Journal of Pharmaceutical Analysis, 2018 Apr-Jun. 8(2): 65-68. Doi: 10.5958/2231-5675.2018.00012.1

11. Maipas, S. and P. Nicolopoulou-Stamati, Sun lotion chemicals as endocrine disruptors. Hormones, 2015 Apr. 14(1): 32-46. Doi: 10.14310/horm.2002.1572

12. Schmutzler, C., et al., The ultraviolet filter benzophenone 2 interferes with the thyroid hormone axis in rats and is a potent in vitro inhibitor of human recombinant thyroid peroxidase. Endocrinology, 2007 June. 148(6): 2835-2844. Doi: 10.1210/en.2006-1280

13. Roy, A. and R.K. Sahu, Formulation and Development of Herbal Sunscreen Cream. Research Journal of Topical and Cosmetic Sciences, 2014 Jan-June. 5(1): 12-14.

14. Cefali, L.C., et al., Flavonoid-Enriched Plant-Extract-Loaded Emulsion: A Novel Phytocosmetic Sunscreen Formulation with Antioxidant Properties. Antioxidants, 2019 Oct. 8(10): 443. doi: 10.3390/antiox8100443

15. Dudonné, S., et al., Phenolic composition and antioxidant properties of poplar bud (Populus nigra) extract: individual antioxidant contribution of phenolics and transcriptional effect on skin aging. Journal of Agricultural and Food Chemistry, 2011 Mar. 59(9): 4527-4536. Doi: 10.1021/jf104791t

16. Xue, Y.-L., et al., Isolation and tyrosinase inhibitory effects of polyphenols from the leaves of persimmon, Diospyros kaki. Journal of Agricultural and Food Chemistry, 2011 May. 59(11): 6011-6017. Doi: 10.1021/jf200940h

17. Rugină, D., et al., Antiproliferative and apoptotic potential of cyanidin-based anthocyanins on melanoma cells. International Journal of Molecular Sciences, 2017 Apr. 18(5): 949. Doi: 10.3390/ijms18050949

18. Mittal, P., et al., Phytochemical and pharmacological potential of viola odorata. International Journal of Pharmacognosy (IJP), 2015 May.2(5): 215-220. Doi: 10.13040/IJPSR.0975-8232.IJP.2(5).215-20

19. Tiwari, P. and R.K. Patel, Estimation of Total Phenolics and Flavonoids and Antioxidant Potential of Drakshasava Prepared by Traditional and Modern Methods. Asian Journal of Research in Chemistry, 2013 Mar. 6(3): 204-208.

20. Brand-Williams, W., M.-E. Cuvelier, and C. Berset, Use of a free radical method to evaluate antioxidant activity. LWT-Food Science and Technology, 1995 June. 28(1): 25-30.

21. Lokapure, S.G., et al., In vitro Evaluation of Sun Protection factor and Diffusion study of Hibiscus rosa-sinensis L. flower Extract Gel. Research Journal of Pharmacy and Technology, 2014 Jun. 7(6): 643-647

22. Gonçalves, A.F.K., et al., Anti-oxidant capacity, total phenolic contents and HPLC determination of rutin in Viola tricolor (L) flowers. Free Radicals and Antioxidants, 2012 Oct-Dec. 2(4): 32-37. Doi: 10.5530/ax.2012.4.6

23. Omsone, L., Z. Kruma, and R. Galoburda, Comparison of different solvents and extraction methods for isolation of phenolic compounds from horseradish roots (Armoracia rusticana). International Journal of Biological, Biomolecular, Agricultural, Food and Biotechnological Engineering, 2012 . (6)4: 236-241.

24. Boussetta, N., et al., Valorization of oilseed residues: Extraction of polyphenols from flaxseed hulls by pulsed electric fields. Industrial Crops and Products, 2014 Jan. 52: 347-353. Doi: 10.1016/j.indcrop.2013.10.048

25. Durling, N.E., et al., Extraction of phenolics and essential oil from dried sage (Salvia officinalis) using ethanol–water mixtures. Food chemistry, 2007. 101(4): 1417-1424. Doi: 10.1016/j.foodchem.2006.03.050

26. Santarelli, V., et al., Response of organic and conventional apples to freezing and freezing pre-treatments: Focus on polyphenols content and antioxidant activity. Food chemistry, 2020 Mar. 308(125570) . Doi: 10.1016/j.foodchem.2019.125570

27. Ghasemzadeh, A., H.Z. Jaafar, and A. Rahmat, Changes in antioxidant and antibacterial activities as well as phytochemical constituents associated with ginger storage and polyphenol oxidase activity. BMC Complementary and Alternative Medicine, 2016 Sep. 16, 382(2016). Doi: 10.1186/s12906-016-1352-1

28. Narayanan, D.L., R.N. Saladi, and J.L. Fox, Ultraviolet radiation and skin cancer. International Journal of Dermatology, 2010 Sep. 49(9): 978-986. Doi: 10.1111/j.1365-4632.2010.04474.x

29. Khelker, T., N. Haque, and A. Agrawal, Ultraviolet Protection potential of Curcuma longa L. and Citrus sinensis (L.) Osbeck. Research Journal of Pharmacy and Technology, 2017 Dec. 10(12): 4282-4284. Doi: 10.5958/0974-360X.2017.00784.3

30. Rangasamy, P., et al., Phytochemical Analysis and Evaluation of In vitro Antioxidant and Anti-urolithiatic Potential of various fractions of Clitoria ternatea L. Blue Flowered Leaves. Asian Journal of Pharmaceutical Analysis, 2019 Apr- Jun. 9(2):67-79. Doi: 10.5958/2231-5675.2019.00014.0

31. Liu, J., et al., The antioxidant and free-radical scavenging activities of extract and fractions from corn silk (Zea mays L.) and related flavone glycosides. Food Chemistry, 2011 May. 126(1): 261-269.

32. Khan, R.A., et al., Evaluation of phenolic contents and antioxidant activity of various solvent extracts of Sonchus asper (L.) Hill. Chemistry Central Journal, 2012 Feb. 6(1): doi: 10.1186/1752-153X-6-12

33. Cefali, L., et al., Plant‐based active photoprotectants for sunscreens. International Journal of Cosmetic Science, 2016 Aug. 38(4): 346-353. Doi: 10.1111/ics.12316

34. Hupel, M., N. Poupart, and E.A. Gall, Development of a new in vitro method to evaluate the photoprotective sunscreen activity of plant extracts against high UV-B radiation. Talanta, 2011 Sep. 86: 362-371. Doi: 10.1016/j.talanta.2011.09.029

35. Nunes, A.R., et al., Photoprotective potential of medicinal plants from Cerrado biome (Brazil) in relation to phenolic content and antioxidant activity. Journal of Photochemistry and Photobiology B: Biology, 2018 Dec. 189: 119-123. Doi: 10.1016/j.jphotobiol.2018.10.013

36. Khazaeli, P. and M. Mehrabani, Screening of sun protective activity of the ethyl acetate extracts of some medicinal plants. Iranian Journal of Pharmaceutical Research, 2010 7(1): 5-9. Doi: 10.22037/ijpr.2010.738

37. Singh, M. and V. Sharma, Spectrophotometric determination of Sun Protection Factor and antioxidant potential of an herbal mixture. Biotechnology Journal International, 2015 10(3): 1-8. Doi: 10.9734/BBJ/2016/21434

38. Philip F. BuldersHerbal Medicine. IntechOpen. UK. 2017

39. Cockell, C.S. and J. Knowland, Ultraviolet radiation screening compounds. Biological Reviews, 1999. 74(3): 311-345. Doi: 10.1111/j.1469-185X.1999.tb00189.x

40. Radice, M., et al., Herbal extracts, lichens and biomolecules as natural photo-protection alternatives to synthetic UV filters. A systematic review. Fitoterapia, 2016. Oct. 114: 144-162. Doi: 0.1016/j.fitote.2016.09.003

41. Samydurai, P. and M. Saradha, Effects of Various Solvent on the Extraction of Antimicrobial, Antioxidant Phenolics from the Stem Bark of Decalepis hamiltonii Wight and Arn. Asian J. Res. Pharm, 2016. 6(2): 129-134. Doi: 10.5958/2231-5659.2016.00018.7

Received on 05.04.2021 Modified on 27.07.2021

Research J. Pharm.and Tech 2022; 15(2):655-660.

DOI: 10.52711/0974-360X.2022.00108