Research Article | Volume: 8, Issue: 6, Nov-Dec, 2020

Screening of in vitro sun protection factor of some medicinal plant extracts by ultraviolet spectroscopy method

Manisha Pralhad Sutar Sanjay Ravindra Chaudhari   

Open Access   

Published:  Nov 25, 2020

DOI: 10.7324/JABB.2020.80608
Abstract

The proposed work is aimed to investigate the ultraviolet (UV) light absorption capacity and sun protection factor (SPF) of selected plant extracts. Chemical agents are protecting against UV radiations but long exposure may cause allergies to the skin. The alternative is required to overcome such skin allergies, hyperpigmentation, sunburn, photoaging, and skin irritation. The plant products and their phytochemicals have recently been used for their antioxidant property, which can be used for UV absorbance ability and photoprotective property. The methanol extracts of flowers of Butea monosperma, leaves of Neolamarckia cadamba, peel of Punica granatum and leaves of Cymbopogon citratus were prepared by continuous Soxhlet extraction method and their UV absorbance was measured between 200 and 400 nm using UV spectrophotometer at 30μg/ml concentration. Further extracts were subjected to in vitro SPF determination at 20–40 μg/ml concentration by using the Mansur equation between 290 and 320 nm range using a spectrophotometer. All extracts showed UV absorbance capacity in 200–400 nm range and the P. granatum extracts having higher SPF. The SPF for B. monosperma flowers, N. cadamba leaves, P. granatum, and C. citratus leaves were found to be 2.1430 ± 0.0271, 2.2892 ± 0.0287, 4.1401 ± 0.0551, 0.8751 ± 0.0112 at 20 μg/ml, 2.3824 ± 0.0301, 2.3020 ± 0.0284, 4.3373 ± 0.0566, and 1.0940 ± 0.0140 at 30μg/ml and 2.5953 ± 0.0328, 2.7789 ± 0.0345, 6.0643 ± 0.0804, and 1.8765 ± 0.0239 at 40 μg/ml, respectively. The initial study proved the UV absorbing and Sun protecting capacity of selected plant extracts. This finding suggests that the extracts can be considered to develop photoprotective formulation in different combinations and proportions, which will provide an additive or synergetic effect. The proposed spectrophotometric method is simple, convenient and cost-effective for SPF determination.


Keyword:     UV radiation Spectrophotometer Photoprotective Plant extracts Sun protection factor.


Citation:

Sutar MP, Chaudhari SR. Screening of in vitro sun protection factor of some medicinal plant extracts by ultraviolet spectroscopy method. J App Biol Biotech. 2020;8(6):48-53. https://dx.doi.org/10.7324/JABB.2020.80608

Copyright: Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license.

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1. INTRODUCTION

Solar ultraviolet (UV) radiation is divided into three regions, UV-C 290–200 nm, UV-B 320–290 nm, and UV-A 400–320 nm [1]. Scientific studies specified that exposure to solar radiation damages the skin in different ways. UV-C get filtered through stratospheric layer and not causing any more harmful effect on the skin. UV-B radiations are not completely filtered out by the ozone layer, are one thousand times more responsible to cause sunburn; DNA absorbs it and initiates carcinogenic processes. UV-A are long-wave UV-A or I (340–400 nm) and short-wave UV-A or II (320–340 nm). UV-A radiations produce immediate tanning effect and darkening of melanin in the epidermis. It causes premature photoaging, suppression of immunological functions and necrosis of endothelial cells. UV-A radiations reaches the deeper layers of the epidermis, dermis, and generates free radicals [2,3]. The oxygenated molecules such as superoxide anions, hydroxyl radical, singlet oxygen, hydrogen peroxide, ferric ion, and nitric oxide damage the skin. The diseases associated with the reactive oxygen species (ROSs) or free radicals mainly depend on the balance of the pro-oxidant and antioxidant concentration in the body [4]. Sunscreens are chemicals that protect against the adverse effects of solar radiation. Phytoconstituents extracted from plants have been recently considered as potential sunscreen resources because of their UV ray absorption capacity in the UV regions and their antioxidant property. Green tea polyphenols, aloe extract, aromatic compounds isolated from lichens, and glycosides are examples of natural substances evaluated for their sunscreen properties. There are reviews about the photoprotective effects of some naturally occurring herbal polyphenols and phenolic compound rich extracts in the skin damage induced by UV irradiation. Several studies have shown the flavonoids and phenolics act as free radical scavengers and enzyme inhibition causes oxidation [5]. Natural compounds act as catalysts in the light phase of photosynthesis and protect plant cells from scavenging ROS especially the antioxidants such as Vitamin C, Vitamin E, flavonoids, carotenoids, and phenolic acids [6].

Recent research focuses on the use of UV light absorbing property of plant extracts which are previously proved for their antioxidant activity and the presence of polyphenolic compounds and their therapeutic values [7,8]. Butea flower known as Palash was studied for antioxidant activity by different in vitro assay [9] and also proven for the presence of flavonoid and phenolic content in ethanolic extract [10]. Ethanolic flower extract also proved for its sunscreen activity in a cream formulation [11]. Neolamarckia cadamba is also known as cadama plant having free radical scavenging property when studied for the methanolic extract of the leaf. It also shows the presence of phenolic compounds [12,13]. The plant bark with other plant extracts studied for its antioxidant assay [14]. The methanolic extract of the peel of Punica granatum possess better antioxidant activity, and phenolic content compared to aqueous and ether extract [15-17]. Ethanolic extract of P. granatum was investigated for its sunscreen activity [18]. The chloroform, methanol and water extract of Cymbopogon citratus leaf is shown potential in vitro antioxidant activity [19] and leaf shown the presence of flavonoids, tannins, and phenolic compounds [20].


2. MATERIALS AND METHODS

2.1. Chemicals and Reagents

Methanol and petroleum ether (60–80°C) were of analytical grades and procured from Merck, India Ltd, Mumbai, India.

2.2. Collection and Authentication of Plants

The flowers of B. monosperma and fresh leaves of N. cadamba (Roxb.) were collected from Ahmedabad, Gujarat. The peels of P. granatum (Linn) and the fresh leaves of C. citratus, (Stapf) were collected from the medicinal plant garden of Alard College of Pharmacy, Pune. The plant specimens were identified at the Botanical Survey of India, Western Regional Centre, Pune. Specimen Voucher No. for B. monosperma (Lam.) family Fabaceae was BSI/WRC/IDEN.CER/2016/664, N. cadamba (Roxb.), family Rubiaceae was BSI /WRC/IDEN.CER. /2016/666, P. granatum (Linn), family Punicaceae was BSI/WRC/IDEN.CER/2016/665, and C. citratus (Stapf) family Gramineae was BSI/WRC/IDEN.CER. /2016/662.

2.3. Extraction of Plant Material

50 g of coarse powders were passed through a 40?mesh sieve, defatted with petroleum ether (60–80°C) and dried marc was extracted with 200 ml of methanol for 36 h using continuous hot extraction method in Soxhlet apparatus [21,22]. Extracts were concentrated under reduced pressure and at the temperature of 40°C using a rotary evaporator. The concentrated extracts were placed in the desiccator to remove the traces of the solvent. The percentage of yields were calculated and recorded. The extracts were named BM for methanolic extract of B. monosperma, NC for methanolic extract N. cadamba, PG for methanolic extract P. granatum, and CC for methanolic extract C. citratus.

2.4. Spectra and Absorbance Measurement of Extracts in UV Region

Photoprotective are the chemicals that have specific absorbance in some parts of the UV spectrum and few have absorbance over the full range of UV. This property is important for any product to be considered as a sun protective agent. Plant extracts with a wide range of natural compounds usually cover UV range in 200–400 nm wavelength [23]. In recent years, naturally occurring compounds gaining attention as protective agents [24]. To examine the UV absorbing property of the plant extracts are required to expose to the UV region 200–400 nm using a spectrophotometer. The UV spectra were measured at 30 mg/ml concentration of each extract diluted with solvent methanol [25].

2.4.1. Sample preparation

10 mg of each extract dissolved in 100 ml of methanol to get 100 mg/ml of concentration, subjected to ultrasonication for 10 min and filtered through Whatman filter paper. 30 mg/ml concentration of each extract was scanned at over the wave length range from 200 nm to 400 nm using UV-visible a spectrophotometer (Make- JASCO and model V-630) in triplicate. The absorption spectra measured in 1 cm quartz cell using “Spectra Measurement” mode employing a reference cell containing methanol as pure solvent [26]. The absorption spectra and characteristic peaks of B. monosperma flower, N. cadamba leaves, P. granatum peel, and C. citratus leaves extracts were recorded.

2.5. Determination In Vitro Sun Protection Factor (SPF) of Extracts

There is an immense need to explore the sunburn protective properties of plants with proven medicinal value and a rich source of phytoconstituents. The efficacy of sunscreen agent is usually expressed by the SPF, which is defined as the UV energy required to produce a minimal erythema dose (MED) on protected skin, divided by the UV energy required to produce a MED on unprotected skin [27,28].


[Click here to view]

It was reported that topical sunscreen formulations or extracts were exposed to UV radiation for their in vivo or in vitro protection determination. Human volunteers are used for in vivo determination but it is a time-consuming process and causing harm to skin [29,30]. In vitro analysis is the best solution for the SPF determination, using the measurement of absorption or the transmission of UV radiations. The extracts were further processed to determine the SPF [31].

The stock of 100 mg/ml of concentration was used to determine SPF of different concentrations (20 mg/ml, 30 mg/ml, and 40 mg/ml) and measured at 290–320 nm at 5 nm interval. The measurements were performed in triplicate for each concentration using 1 cm quartz cell and methanol was used as blank. A very simple mathematical equation was developed by Mansur et al which substitutes the in vitro method proposed by Sayre et al [32] by using a UV spectrophotometer [33]. Given formula was used to calculate the SPF,

The Equation is,


[Click here to view]

Where, CF = Correction Factor (10)

EE (l) = Erythrogenic Effect of radiation

I (l) = Solar Intensity spectrum

Abs (l) = Spectrophotometric absorbance value.

The values of EE x I are constants. The results are shown in Table 1.

Table 1: Normalized product function used in calculation of SPF.

Sr. NoWavelength (λ)EE X I (normalized)
12900.0150
22950.0817
33000.2874
43050.3278
53100.1864
63150.0839
73200.0180
Total1

3. RESULTS AND DISCUSSION

3.1. Extraction of Plant Material

Percentage of yield for BM, NC, PG, and CC extract was found to be 2.2, 1.9, 5.7, and 1.7 % w/v, respectively. PG extract shown higher % of yield compared to other plant extracts.

3.2. Spectra and Absorbance Measurement of Extracts in UV Region

All extracts were subjected to spectra and absorbance measurement in the UV region which will be the indication of the UV absorbing or transmitting property of the extracts. The spectra and wavelength of 30 mg/ml of each solution were studied and maximum absorbance spectra with respective wavelengths were noted. All the extract exhibited significant absorption in the whole UV range (UV-A, UV-B, and UV-C). Natural substances investigated in plants have recently studied for their potential as sunscreen candidates because of their UV-ray absorbing property. Literature showed the flavonoids and other phenolic compounds having an antioxidant property and previously studied for the given extracts. This data make a platform to exploit extracts in combination as potential ingredients in sunscreen formulations. The wavelength and their respective absorbance for all the subjected extracts were reported in Table 2 and shown in Figure 1.

Table 2: Absorption ability of extracts.

Sr. NoWavelengthAbsorbance
BM extract
 13711.037
 23140.681
 32691.012
 42281.339
NC extract
 13260.315
 23090.294
 32840.435
 42640.253
PG extract
 13670.206
 23400.173
 32581.389
 42172.056
CC extract
 13860.119
 22790.620
 32650.576
 42121.663

Figure 1: Spectra of selected plant extracts for ultraviolet absorbing capacity



[Click here to view]

3.3. Determination of In Vitro SPF of Extracts

SPF numbers have become a worldwide standard for measuring the effectiveness of photoprotective products. The in vitro SPF determination is one of the useful tools for screening tests during product development, as a supplement base for in vivo studies. Whereas the in vivo test is time consuming and includes various degrees of variability. The SPF numbers of extracts were calculated by applying Mansur mathematical equation in the UV-B region, which is considered to be the region of highest incidence during the day and people are exposed for a longer time [34]. Whereas plant literature shows that natural substances extracted from plants have potential sunscreen activity as it gets absorbed in the UV region due to the presence of antioxidants [35].

The absorbance spectra indicated that the compound present in the extracts having the ability to absorb the UV radiation from the range of 200–400 nm and the values of SPF values determined with three different concentrations in the UV range from 290 to 320 nm. The absorbance values obtained were used to calculate the SPF and it was observed that there is an increase in SPF value with an increase in concentration. The current study was carried out at lower concentrations and shown appreciable SPF values. Hence, the higher concentration will definitely provide better sun protection values. The SFP values for 20, 30 and 40 mg/ml are shown in Tables 3-5, respectively. The highest SPF was observed in PG extract and lowest in CC extract as shown in Figure 2. The range of SPF values obtained was PG>NC>BM>CC.

Table 3: Determination of in vitro SPF at 20 μg/ml concentration.

Sr.NoWave length (λ)EExIBM extractNC extractPG extractCC extract




Conc. 20 µg/mlConc. 20 µg/mlConc. 20 µg/mlConc. 20 µg/ml




AbsEExIx AbsAbsEExIx AbsAbsEExIx AbsAbsEExIx Abs
12900.0150.22280.00330.30650.00450.64390.00960.10240.0015
22950.08170.20590.01680.26420.02180.55020.04490.09430.0077
33000.28740.20900.06000.23850.06850.47370.13610.09070.0261
43050.32780.21640.07090.22290.07300.40390.13240.08730.0286
53100.18650.22000.04100.21400.03990.33830.06300.08380.0156
63150.08390.21870.01830.20840.01740.28050.02350.07910.0066
73200.0180.20810.00370.20620.00370.23510.00420.07220.0013
SPF2.1430±0.02712.2892±0.02874.1401±0.05510.8751±0.0112

*Values are calculated (n=3) as mean±SD

Table 4: Determination of in vitro SPF at 30 μg/ml concentration

Sr.NoWave length (λ)EExIBM extractNC extractPG extractCC extract




Conc. 30 µg/mlConc. 30 µg/mlConc. 30 µg/mlConc. 30 µg/ml




AbsEExIx AbsAbsEExIx AbsAbsEExIx AbsAbsEExIx Abs
12900.0150.26160.00390.33420.00500.60220.00900.12920.0019
22950.08170.24420.01990.27280.02220.53370.04360.11900.0097
33000.28740.23970.06880.23970.06880.47680.13700.11300.0325
43050.32780.23800.07800.22130.07250.42610.13970.10910.0358
53100.18650.23610.04400.20900.03890.37880.07070.10480.0195
63150.08390.23170.01940.22370.01870.33660.02820.09920.0083
73200.0180.22250.00400.24280.22850.30360.00550.09070.0016
SPF 2.3824±0.03012.3020±0.02844.3373±0.05661.0940±0.0140

*Values are calculated (n=3) as mean±SD

Table 5: Determination of in vitro SPF at 40 μG/ML concentration

Sr.NoWave length (λ)EExIBM extractNC extractPG extractCC extract




Conc. 40 µg/mlConc. 40 µg/mlConc. 40 µg/mlConc. 40 µg/ml




AbsEExIx AbsAbsEEx I x AbsAbsEEx I x AbsAbsEExIx Abs
12900.0150.26370.00390.41020.00610.92670.01390.22120.0033
22950.08170.24520.02000.33170.02710.79910.06530.20660.0169
33000.28740.25110.07210.29020.08340.69170.19880.19530.0561
43050.32780.26280.08610.26850.08800.59310.19440.18570.0609
53100.18650.26890.05010.25500.04750.49860.09300.17820.0332
63150.08390.26790.02240.25160.02110.41450.03480.17060.0143
73200.0180.25520.00450.25230.00450.34710.00620.16160.0029
SPF 2.5953±0.03282.7789±0.03456.0643±0.08041.8765±0.0239

*Values are calculated (n=3) as mean±SD

Figure 2: Graph for sun protection factor of plant extracts



[Click here to view]

CONCLUSION

This primary study revealed the UV absorbing capacity and sun protection property of the plant extracts. Along with their many therapeutic benefits, these botanicals could become beneficial, cost effective, and easily available ingredients to prove sun protection property. These plants were studied for various bioactive components such as alkaloids, tannins, phenols, and flavonoids which have known as a free-radical scavenger and strong antioxidants and useful to protect the skin from sun radiation induced damage. In past years, the plant of Boerhavia diffusa shown SPF values as 3.539–7.174 [36], fresh Aloe vera gel shown SPF of 0.0995 [37], the aqueous and methanolic extracts of Zingiber officinale at the lower concentration shown SPF 1.44–1.82 and 1.48–1.99, respectively [38]. Some studies were conducted on the mixture of herbs to get good amount of SPF value and considered for the development of topical formulations [39]. The undertaken study could be a useful tool for in-process quality control of formulation, product analysis and helpful to provide information before proceeding to in vivo studies. In future, these plant extracts in alone or in combinations with other additives can used to develop a photoprotective formulation such as creams and lotions with higher SPF.


4. ACKNOWLEDGMENT

The authors are thankful to Alard College of Pharmacy, Pune, for providing the infrastructure facility to conduct the research work. Also, I am thankful to Ms. Priyanka A. Ingle scientist from Botanical Survey of India, Western Regional Centre, Pune, for the authentication of plant material.


Conflict of interest

Authors declared that they do not have any conflicts of interest.


Financial support and sponsorship:

None.

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