Available Online at http://www.recentscientific.com International Journal of CODEN: IJRSFP (USA) Recent Scientific

International Journal of Recent Scientific Research Research Vol. 11, Issue, 1 (A), pp. 36717-36722, January, 2020 ISSN: 0976-3031 DOI: 10.24327/IJRSR Research Article

PHOTOPROTECTIVE AND ANTIOXIDATIVE POTENTIAL OF METHANOLIC LEAF EXTRACT OF lanzan SPRENG

Banerjee, S and *Bandyopadhyay, A

Department of Botany, the University of Burdwan, Burdwan, West Bengal, India

DOI: http://dx.doi.org/10.24327/ijrsr.2020.1101.4980

ARTICLE INFO ABSTRACT

Article History: UV-B radiation mainly causes photo-oxidative damage resulting DNA damage and skin cancer. Various chemical formulations are available as photoprotective agents. The successes of such Received 14th October, 2019 formulations are either not fully understood or they produce toxicity at certain levels. Hence, herbal Received in revised form 29th formulations with photoprotective activity with promising antioxidant effects are gaining popularity. November, 2019 In the following study, we have investigated methanolic leaf extract of Buchanania lanzan (BMLE) Accepted 05th December, 2019 for its photoprotective property with antioxidant activity by different assay methods including FT-IR Published online 28th January, 2020 and HPLC, to identify probable components of photoprotection of BMLE.

Key Words:

Buchanania lanzan, BMLE, SPF, Antioxidant, Quercetin

Copyright © Banerjee, S and Bandyopadhyay, A, 2020, this is an open-access article distributed under the terms of the

Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided

the original work is properly cited.

INTRODUCTION more sun burn than UV-A, and is highly genotoxic (Balakrishnan & Narayanaswamy, 2011). Thus photoprotection Long term exposure of human skin to solar ultraviolet radiation is necessary. Photoprotection is achieved by utilizing effective (UVR) induces the oxidative stress by generating the reactive sunscreen and sun protective clothing. Sunscreen products offer oxygen species (ROS). ROS triggers skin cancer (Patil, Fegade, sun protection against both UV-A and UV-B radiations. Based Zamindar, & Bhaskar, 2015)and also several skin damages on working principle of Sunscreens are classified into two such as sunburn, skin cancer, oxidative stress and also types (i) absorptive and (ii) scattering/reflective. There are photoaging depending on the amount and form of the UV many potent physical (Zinc oxide, Titanium oxide) and radiation and on the type of the individual exposed (Mauricio, chemical UV blockers (Oxybenzone, Avobenzone) along with 1996). Skin is considered as the most exposed part of the body natural substances (like flavonoids and phenolics), which are easily affected by direct exposure to the solar radiation popularly used for photoprotection. At present sunscreen including other environmental factors which leads to product contains both chemical and physical agents for sun photodamage (Imam, Azhar, & Mehmood, 2015). protection (Gabros & Zito, 2019). Physical and chemical

Based on wavelength UV radiations are classified into three blocker possesses harmful effects as they induce the production categories namely. UV-A (400nm to 320nm), UV-B (320nm to of reactive oxygen species (ROS). Whereas, botanical extract 280nm) and UV-C (280nm to 100nm).Among these three based sunscreens with their inherent antioxidant properties help radiations UV-C is fatal to all living organisms, but luckily to mitigate the oxidative damages (Kaur & Saraf, 2009) caused blocked by the ozone layer located in the stratosphere. by stressors and other factors.

Stratosphere also inhibits UV-B radiations to some extent but Modern pharmaceutical approach focuses on developing due to ozone layer is depletion the rate of UV-B radiation effective formulations based on combination of antioxidative increased; as a result protection from UV-B radiation is and photoprotective ability of natural products. A large number attaining most importance (Ranjithkumar, Sameesh, & of Indian possess remarkable antioxidant activities and Ramakrishnan, 2016). UV-A is able to penetrate deep in skin thus have become focus of intense research of this field (Patra, induce sun burn or erythema, phototoxic reactions, Pareta, Harwansh, & Kumar, 2011) (Siddiqui, Chowdhury, & photoallergy, photosensitivity, Photoaging, skin cancer and Prasad, 2015). In spite of having much literature on antioxidant immunosuppression. The UV-B radiation causes 1000 times activity of the selected , no absolute data were available

*Corresponding author: Bandyopadhyay, A Department of Botany, the University of Burdwan, Burdwan, West Bengal, India Banerjee, S and Bandyopadhyay, A., Photoprotective and Antioxidative Potential of Methanolic leaf Extract of Buchanania Lanzan Spreng on SPF. Moreover, there exists confusion about degree of was plotted and the total phenol was expressed as mg of gallic protection offered by topical sunscreens and antioxidants solely acid equivalent weight (GAE)/mg of dry mass. on the basis of SPF level (De Galvez, 2010) The efficacy of a Quantitative estimation of total Flavonoid sunscreen is generally expressed by Sun Protection Factor (SPF), is the ratio of UV energy required to produce a minimal Total flavonoid was estimated with the AlCl3 method erythemal dose (MED) in protected skin to unprotected skin. (Lamaison & Carnet, 1990) with slight modifications. For this, Recent researches on developing new compounds with 1ml BMLE aliquot was added with 4ml of distilled water and photoprotective property includes otocrylene analogues, 300µl of 5% sodium nitrite. Then 300µl of 10% Aluminium (Polonini, et al., 2014) bis(indolyl)methane derivatives found chloride was added to the mixture and 10 minutes incubation in cruciferous and marine sources, (Ergindemir, Aker, period in room temperature was given. After incubation 2ml of Hamitbeyli, & Ocal, 2016) bile acids/azastilbenes conjugates, 1M NaOH solution added and the volume of the mixture was (Santos, Polonini, Suzuki, & Raposo, 2015) N-acyl hydrazone made upto 10ml with distilled water and the absorbance was compounds (Reis, Corrêa, Chung, & Santos, 2014) and measured at 510nm spectrophotometrically. Quercetin was benzophenone and lactone derivatives (Gonçalves, et al., used as the standard. 2018). Quercetin and Rutin incorporated in oil-in-water Quantitative estimation of total tannin emulsion shows sun protection factor at a concentration of 10% (w/w). When these two flavonoids were used in association Total tannin was estimated according to vanillin hydrochloride with titanium dioxide the SPF value found to increase to 30 method (Burns, 1971). In a test tube to 1 ml extract 5ml of (Choquenet, Couteau, Paparis, & Coiffard, 2008). Furthermore, vanillin hydrochloride reagent was mixed (which was made the photostabilizing and antioxidant properties of quercetin just before using by mixing 5ml of 8% hydrochloric acid in makes it suitable for formulation of broad-spectrum sunscreen methanol with 4% vanillin in methanol keeping in mind that having butyl methoxy dibenzoyl methane and octyl methoxy the reagent must not be colored). Then the mixture was cinnamate (Santo & Matteo, 2010). Gonçalves et. al reported a incubated in room temperature for 20 minutes followed by new quercetin derivative (Quercetin 3,7,3',4'-tetraethyl ether) measurement of absorbance at 500nm using spectrophotometer. which showed improved skin penetration in sunscreen The blank was prepared with vanillin hydrochloride reagent formulation compared to quercetin (Gonçalves, et al., 2019) only. The standard curve was prepared using phloroglucinol and the present tannin was calculated as the phloroglucinol This paper attempts to conduct an evaluation of antioxidant and equivalent. photoprotective property of methanolic leaf extract of Buchnania lanzan and also to identify the chemical nature of Assessment of Sun Protection Factor the major components of BMLE through FT-IR and HPLC The SPF factor was measured by the equation of Mansur et. al analyses. 1986 (Mansur, Breder, Mansur, & Azulay, 1986). The MATERIALS AND METHODS absorption spectra of BMLE (concentration 500µg/ml) were recorded from the range of 290nm to 320nm in the interval of Plant material collection and extraction every 5nm. Three replicates were taken. The equation is as

Disease free mature leaves of Buchanania lanzan were follows collected from Ausgram (Lat: 23.232437 N Long: 87.863724 E) forest of Burdwan district of West Bengal, India. The leaves spectrophotometri = × EE(λ) × I(λ) × Abs(λ) were thoroughly washed with running tap water shade dried and grinded to a fine power for extraction in organic solvents. Where: EE (l) – erythemal effect spectrum; I (l) – solar An amount of 5g of leaf dust was taken and defattened using n- intensity spectrum; Abs (l)- absorbance of sunscreen product ; Hexane and extracted in HPLC grade methanol in sohxlet CF – correction factor (= 10). The values of EE x I are apparatus at 50°C for 72 hrs. The methanolic leaf extract of constants. They were determined by Sayre et al. (1979) (Sayre, Buchanania lanzan (BMLE) was filtered using Whatman filter Agin, Levee, & Marlowe, 1979) paper no. 1 and concentrated in rotary evaporator and stored in Table1 Normalized product function used in the calculation of SPF 4°C under desiccation for further experimental studies. Wavelength () EE × I (normalized) Quantitative estimation of total Phenol 290 0.0150 Total phenolic content was measured by Folin-ciocalteu’s 295 0.0817 300 0.2874 method (Singleton & Rossi, 1965). For this, 1ml of aliquots 305 0.3278 and standard gallic acid (10, 20, 40, 60, 80, 100 µg/ml) were 310 0.1864 kept in test tubes and 10 ml of distilled water and 1ml of Folin- 315 0.0839 ciocalteu’s reagent (diluted 1:10 with water) were mixed 320 0.0180 Total 1 subsequently. After 5 minutes, 10ml of 7% sodium carbonate solution was added and the volume was made upto 25ml. The EE - erythemal effect spectrum, I - solar intensity spectrum mixture is then incubated for 90 minutes at room temperature. Antioxidant activity The intensity of the color was measured at 760nm with spectrophotometer (Lasany). The experiment was performed in DPPH radical-scavenging activity triplicates. The blank was prepared using reagent blank with The DPPH assay of the BMLE was carried out as described solvent using gallic acid as the standard. The calibration curve previously (Ebrahimzadeh, Hosseinimehr, Hamidinia, & Jafari, 2008). A solution of 0.1 Mm DPPH in methanol was prepared 36718 | P a g e International Journal of Recent Scientific Research Vol. 11, Issue, 1 (A), pp. 36717-36722, January, 2020 and 1.0 mL of this solution was mixed with 1.0 mL of BMLE methanol and water (Merck) in a ratio of 80:20 with a flow rate with concentration 10, 30, 50, 100, 200, 400 µg/mL and of 1ml/min. The stop time and detecting wavelength were set at commercial antioxidant ascorbic acid. The mixture was left in 20 minutes and 252nm respectively. An amount of 20 μl of the dark at 25 °C for 30 min and the absorbance was measured BMLE (1mg/ml) was carefully filtered with a syringe filter at 517 nm. The ability of BMLE to scavenge DPPH radical was (porosity: 0.22 µ, Himedia, SF- 160) and the filtrate was calculated as % inhibition by the following equation: injected manually in the injector port with Hamilton glass syringe. Standard Quercetin (0.2 µg/ml) was dissolved in %inhibition={(Absorbance −Absorbance )}/(Absorba control sample HPLC grade methanol, filtered and injected in the same way. ncecontrol) × 100

Where Absorbance is the absorbance of the DPPH radical RESULTS AND DISCUSSION control + methanol; Absorbancesample is the absorbance of DPPH Quantification of Phenol, Flavonoid and Tannin radical + BMLE or ascorbic acid. IC50 values denote the concentration of the sample or standard, which is required to Quantitative estimation of three important phytometabolites scavenge 50% of DPPH free radicals. present in BMLE namely phenol, flavonoid and tannin were carried out according to the above mentioned protocols. The Reducing power determination results thus obtained are detailed below-

The reducing power of BMLE was estimated according to Table2 Quantificatin of major phenolics of BMLE Bhalodia et. al, 2013 (Bhalodia, Nariya, Acharya, & Shukla, Amount (µg standard 2013). 1 ml of BMLE aliquots (10 – 100 µg/ml) were mixed Phenolic equivalent/mg of contents with 2.5 ml phosphate buffer (pH6.6) and 2.5 ml of 1% extract powder) potassium ferricyanide [K3Fe(CN)6]. It was then incubated at Phenol 97.50±0.953 50ºC for 20 min. After incubation the reaction was stopped by Flavonoid 71.33±1.630 Tannin 106.85±1.519 adding 10% 2.5ml (w/v) of trichloroacetic acid to it and centrifuged at 3000 rpm for 10 minutes. The supernatant was The BMLE contains total phenol 97.5±0.953 µg gallic acid mixed with 2.5 ml distilled water and 0.5 ml, 0.1% ferric equivalent/ mg of extract powder by reference to standard chloride. The absorbance of the mixture was taken at 700 nm. curve (y = 0.002x +0.090, R2 = 0.991). The total flavonoid Ascorbic acid was kept as positive control. content was 71.33 ± 1.630 µg quercetin equivqlent/ mg of extract powder by reference to standard curve (y = 0.003x – ABTS free radical assay 2 0.009, R = 0.982) and tannin content was 106.85±1.519 µg In the ABTS radical cation decolorization assay was measured phloroglucinol equivalent/ mg of extract by reference to by the method of Re et. al, 1999 (Re, Pellegrini, Proteggente, standard curve (y = 0.001x + 0.005, R2 = 0.997). Pannala, Yang, & Rice-Evans, 1999). The ABTS+ cation was Estimation of Sun Protection Factor generated by oxidation of 7 mM ABTS solution with 2.45 mM potassium persulfate solution in equal volume. This mixture The absorbance of BMLE (500 µg/ml) from 290nm to 320nm was kept in dark for 12-16 hrs at 25ºC for reaction. The showed the following SPF value. reaction mixture was then diluted with concentrated methanol Table 3 SPF value of BMLE (500 µg/ml) until the absorbance (734 nm) reached 0.700 ± 0.001 value. 1 mL of this solution was added to the (100-1000 µg/ml) of Wave SPF= ƩEE(λ) EE (λ) X I Absorbance EE X I X Length (λ X I(λ) X A X BMLE and ascorbic acid and absorbance measured at 734 nm. (λ) employed (A) A The percentage inhibition of ABTS radical by the extract was nm) 10 0.0198 ± 290 0.0150 1.318 ± 0.009 calculated following the equation mentioned in the DPPH 0.00015 assay. 0.1041 ± 295 0.0817 1.274 ± 0.007 0.00060 Fourier Transform Infrared (FT-IR) Spectrophotometric 0.3636 ± 300 0.2874 1.265 ± 0.006 assessment 0.00175 0.4147± 305 0.3278 1.266 ±0.009 FT-IR is the most effective method for identification of 0.00311 12.725± 0.083 0.2381 ± chemical bonds (functional groups) of compounds. BMLE was 310 0.1864 1.277 ±0.008 taken for FTIR analysis. The dried powder was mixed with 0.00162 0.1085± 315 0.0837 1.296 ±0.009 KBr pellet, and translucent sample disc was made. IR spectra 0.00080 and peaks were recorded on a Perkin Elmer FT-IR (model 0.0238 ± 320 0.0180 1.324 ± 0.012 RX1) spectrometer between 4000-400 cm-1. 0.00025

Analysis by High Performance Liquid Chromatography The presence of so many amounts of flavonoids helps the (HPLC) extract to absorb UV radiation and gives it the SPF value of 12.725 ± 0.083, which can effectively block 90-93% of UVR HPLC analyses of the leaf crude extract and Quercetin standard (according to Graph1). (Sigma, Q4951 – 10G) were done using a Quaternary Gradient Reverse Phase HPLC system (HITACHI Chromaster with 5160 Quaternary Gradient Pump and 5420 UV-Vis Detector). The column used for the experiment was HITACHI Cosmosil packed column (5C 18 – MS – II, 4.61D x 250mm). The mobile phase solvent system was a mixture of HPLC grade 36719 | P a g e Banerjee, S and Bandyopadhyay, A., Photoprotective and Antioxidative Potential of Methanolic leaf Extract of Buchanania Lanzan Spreng

Reducing Power Assay

Reducing power 2.5

SPF= 12.725±0.083 2

1.5

1 Ascorbic acid Absorbance 0.5 BMLE

0

0 50 100 150 Concentration (µg/ml) Graph1 Ultraviolet B protection level by sun protection factor (SPF) (Dale Wilson, Moon, & Armstrong, 2012)

Antioxidant Assay 120 DPPH Assay 100 120 80

100 60 Ascorbic acid

40 BMLE nging 80 20 60 Ascorbic acid 0 40 BMLE IC50 0f Reducing power DPPH % scava % DPPH 20

Graph 3 Plot showing Reducing power of Ascorbic acid and BMLE and the bar graph 0 represents the IC50 value of reducing power of ascorbic acid and BMLE to be 16.52 and 97.66 respectively 0 200 400 600 ABTS scavenging Assay Concentration (µg/ml)

ABTS scavanging activity 120 60 100 50 80 40 60 30 Ascorbic acid Ascorbic acid 40 BMLE 20 BMLE

ABTS % scavanging% ABTS 20

10 0 0 500 1000 1500 0 IC50 of DPPH Concentration (µg/ml)

400

Graph 2 Plot showing DPPH scavenging activity of Ascorbic acid and BMLE 350 and the bar graph represents the IC50 value of DPPH scavenging activity of 300 ascorbic acid and BMLE to be 5.33 and 50.35 respectively. 250 200 Ascorbic acid 150 BMLE 100 50 0 Concentration (µg/ml)

Graph 4 Plot showing ABTS scavenging activity of Ascorbic acid and BMLE and the bar graph represents the IC50 value of ABTS scavenging activity of ascorbic acid and BMLE to be 25.71 and 336.06 respectively 36720 | P a g e International Journal of Recent Scientific Research Vol. 11, Issue, 1 (A), pp. 36717-36722, January, 2020

damage and skin cancer (Strack, 1997). Antioxidant activity also helps in UV protection (Kittiwannachot, Borisut, Wanasawas, Ponpanich, Rattanasuk, & Chulasiri, 2008). It is reported that high SPF values in Dracocephalum moldavica and Viola tricolor (24.79 and 25.69 respectively) may be due to their high phenolic contents (Khazaeli & Mehrabani, 2008). Flavonoids are considered as the main group of antioxidant in Halimium halimifolium (Rebaya, et al., 2014). Quercetin, particularly Quercitrin (glycosylated form of Quercetin) effectively inhibits UV-B radiation induced oxidative damage

Graph 5 FT-IR spectrum of BMLE showing Phenolic O-H group, Methoxy, both under in vivo and in vitro conditions. Appreciable Methyl Ethar group, Aromatic ring stretch, Methyl C-H bend, Cyclohexane reduction of UV-B induced ROS generation in JB6 cell lines ring variations and Methylene group has been reported. The mechanism involved restoration of

The data of FTIR analysis showed similar pattern with phenolic catalase expression and GSH/GSSG ratio upon UV exposure, O-H group at wave number 3305.18 cm-1, Methylene group at which minimized oxidative DNA damage apoptosis and skin 2942.76 cm-1, Methoxy, Methyl Ether stretch at 2833.39 cm-1, inflammation. Thus Quercetin plays an important role not only Aromatic ring stretch at 1610.91 cm-1, Methyl C-H bend at as photoprotectant but also a potent antioxidant (Yin, et al., 1447.99 cm-1, and Cyclohexane ring stretch at 1003.39 cm-1. 2013). Earlier report claims that quercetin can also alleviate the 3570-3200 cm-1 broadly denotes the hydroxyl group and harmful effects of UV-A radiation (Inal, Kahraman, & Koken, 2001). alcoholic compounds (Coates, 2000).

CONCLUSION

From this study it can be concluded that, the methanolic leaf extract of Buchanania lanzan is an effective photoprotectant and can block 90-93% of UV-B radiation. Moreover, it possesses a significant amount of antioxidant activity. So, the leaf extract with high amount of quercitin can be used in different pharmaceutical formulations as potent anti- photoaging skin cream. Complete extraction, isolation and purification of specific phytometabolites endowed with UV- induced photoageing preventive activities need to be examined in detail before commercial application.

Acknowledgement

I hereby acknowledge The Head, Department of Botany, University of Burdwan for providing me infrastructural support and also recognize The Head, Department of Biotechnology, University of Burdwan for HPLC facility.

References

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