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Year : 2016  |  Volume : 8  |  Issue : 3  |  Page : 160-168  

Evaluation of skin anti-aging potential of Citrus reticulata blanco peel

1 Department of Biological Sciences, Sunandan Divatia School of Science, SVKM's NMIMS, Vile-Parle (W), Mumbai, Maharashtra, India
2 Department of Chemistry, Sunandan Divatia School of Science, SVKM's NMIMS, Vile-Parle (W), Mumbai, Maharashtra, India

Date of Web Publication24-May-2016

Correspondence Address:
Vinita D Apraj
Department of Biological Sciences, Sunandan Divatia School of Science, SVKM's NMIMS, Vile-Parle (W), Mumbai - 400 056 , Maharashtra
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0974-8490.182913

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Background: The peel of Citrus reticulata Blanco is traditionally used as tonic, stomachic, astringent, and carminative. It is also useful in skin care. Objective: To study the anti-aging potential of alcoholic extracts of C. reticulata Blanco peel using in vitro antioxidant and anti-enzyme assays. Materials and Methods: Plant extracts were obtained by Soxhlation (CR HAE- Hot Alcoholic Extract of Citrus reticulata) and maceration method (CR CAE- Cold Alcoholic Extract of Citrus reticulata). Qualitative and quantitative phytochemical analysis was performed. Further, in vitro antioxidant, anti-enzyme, and gas chromatography-mass spectrometry (GC-MS) analyses were performed. Results: Total phenolic and flavonoid contents of CR HAE were found to be higher than CR CAE. EC50value of CR HAE and CR CAE for 1,1-Diphenyl-2-picrylhydrazyl, Superoxide anion, and 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) assays were 250.33 ± 40.16 μg/ml and 254.73 ± 15.78 μg/ml, 221.27 ± 11.25 μg/ml and 354.20 ± 23.79 μg/ml, and 59.16 ± 2.17 μg/ml and 59.12 ± 6.21 μg/ml, respectively. Oxygen radical absorbance capacity values for CR HAE and CR CAE were found to be 1243 and 1063 μmoles 6-hydroxy-2,5,7,8-tetra methylchromane-2-carboxylic acid equivalent/g of substance, respectively. Anti-collagenase and anti-elastase activities were evaluated for both CR HAE and CR CAE. EC50values of CR HAE and CR CAE for anti-collagenase and anti-elastase were 329.33 ± 6.38 μg/ml, 466.93 ± 8.04 μg/ml and 3.22 ± 0.24 mg/ml, 5.09 ± 0.30 mg/ml, respectively. CR HAE exhibited stronger anti-collagenase and anti-elastase activity than CR CAE. GC-MS analysis of CR HAE was carried out because CR HAE exhibited higher antioxidant and anti-enzyme potential than CR CAE. Conclusion: C. reticulata peel can be utilized in anti-wrinkle skin care formulations.

  • Skin anti-aging potential of Citrus reticulata Blanco peel was evaluated through
  • In vitro antioxidant and anti-enzyme assays
  • Two types of extraction were performed and extracts were subjected to qualitative and quantitative phytochemical analysis. Extract obtained by Soxhlation (CR HAE) showed higher total phenolic and flavonoid contents than extract obtained by maceration (CR CAE)
  • CR HAE demonstrated strong DPPH and Superoxide free radical scavenging activity whereas, ABTS scavenging activity of both the extracts were found to be similar. Oxygen Radical Absorbance Capacity (ORAC) of CR HAE was found to be more; indicating its strong antioxidant potential
  • In vitro collagenase and elastase enzyme inhibition activities were evaluated for both the extracts and CR HAE showed strong anti-collagenase and anti-elastase potential indicating its anti-aging ability
  • GC-MS analysis of CR HAE revealed the presence of various compounds mainly including Polymethoxyflavones. CR HAE exhibited promising antioxidant and anti-enzymatic activity and can be used as a potent anti-wrinkle agent in anti-aging skin care formulations.

Abbreviation Used: ECM: Extracellular matrix, UV: Ultra violet, ROS: Reactive Oxygen Species, MMP: Matrix metalloproteinase, Chc: Clostridium histolyticum collagenase, DPPH: 2, 2-diphenyl-1-picrylhydrazyl, GC-MS: Gas Chromatography- Mass Spectroscopy, RT: Room Temperature, μg GAE/ mg: Microgram Gallic acid equivalent / milligram, W/V: Weight by Volume, μg QE/ mg: Microgram Quercetin equivalent / milligram, CR HAE: Hot Alcoholic Extract of Citrus reticulata Blanco, CR CAE: Cold Alcoholic Extract of Citrus reticulata Blanco, EC50: Half Maximal Effective Concentration, PMS NADH: Phenazine methosulfate nicotinamide adenine dinucleotide, NBT: Nitroblue tetrazolium, DMSO: Dimethyl sulfoxide, APS: Ammonium Persulphate, AAPH: 2,2 -azobis(2-amidino-propane) dihydrochloride, TROLOX: (±) 6-hydroxy-2,5,7,8-tetramethyl chromane-2-carboxylic acid, ORAC: Oxygen Radical Absorbance Capacity, FALGPA: N-[3-(2-Furyl) acryloyl)]-Leu-Gly-Pro-Ala, SANA: Succinyl-Ala-Ala-Ala-p-nitroanilide, Rf: Retardation Factor, MSD: Mass Selective Detector

Keywords: 1,1-Diphenyl-2-picrylhydrazyl, 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), anti-wrinkle, elastase, gas chromatography-mass spectrometry, oxygen radical absorbance capacity

How to cite this article:
Apraj VD, Pandita NS. Evaluation of skin anti-aging potential of Citrus reticulata blanco peel. Phcog Res 2016;8:160-8

How to cite this URL:
Apraj VD, Pandita NS. Evaluation of skin anti-aging potential of Citrus reticulata blanco peel. Phcog Res [serial online] 2016 [cited 2021 Apr 21];8:160-8. Available from: http://www.phcogres.com/text.asp?2016/8/3/160/182913

   Introduction Top

The most important role of skin for human being is to create a barrier between inside and outside environment of the body. Internally, the skin shelters and protects the entire physiochemical phenomenon necessary for life, externally it is a barrier against mechanical forces. Skin is the largest organ of the body.[1] It is a very sensitive organ and can easily get damage by infection and diseases.

Skin problems can arise due to various factors such as environmental pollution, over exposure to ultraviolet rays, age of individual, harmful microorganisms, eating habits, stress, and chemicals. Skin treatment and care is essential not only to have a healthy skin, but also for the overall well-being of the person.[2]

One of the major skin problems in today's world is premature skin aging. The process of skin aging has been divided into two categories: Intrinsic and extrinsic aging. Intrinsic skin aging or natural aging is caused by changes in elasticity of the skin over time. Extrinsic skin aging is predominately a result of exposure to solar radiation (photo aging). Free radicals formed due to oxidative stress, play a major role in the course of both intrinsic and extrinsic aging.[3] Skin is made up of three main layers; epidermis, dermis, and subcutis. The major components of dermis are collagen and elastin fibers. Reduction in collagen and elastin leads to the wrinkle formation.[4] The main reason that skin starts to sag and form wrinkles is the breakdown in elastin and collagen. Collagenase and elastase enzymes are responsible for breakdown of various components of the extracellular matrix (ECM), i.e., collagen and elastin. Elastin is a protein that helps the skin stay supple and firm. When your skin is stretched, it is the elastin that returns it to its normal position. Collagen is a fibrous protein. Collagen is special among proteins because of its great tensile strength providing firmness to the skin. Wrinkles result from a combination of intrinsic and extrinsic aging as well as due increased level of collagenase and elastase enzymes.[5]

Cosmetics are used regularly and universally in different forms to enhance beauty. Skin care cosmetics treat the surface layer of the skin by providing better protection against the environment than skin left untreated.[6] There is an increasing demand for facial skin care cosmetics. According to data monitor, global spending on skin care products in 2012 was 82 billion $, where two-thirds of spending comprised facial skin care. A report by research and markets, expects the global skin care products industry revenue to cross 100 billion $ in 2018. Facial care segment is expected to continue to dominate the market. The increasing demand for anti-aging products and growing concern for the use of natural and organic skin care products are the major factors driving the skin care industry.[7]

Cosmetics alone are not sufficient to take care of skin and body parts; it requires association of active ingredients to check the damage and aging of the skin. These herbal actives which contain active ingredients are very essential to maintain the skin health. Cosmetics with herbal actives are now emerging as an appropriate solution to the current problem.[8]

In the present study, skin anti-aging potential of alcoholic extracts of Citrus reticulata Blanco peel were evaluated by in vitro antioxidant, anti-collagenase, and anti-elastase assays. C. reticulata Blanco (Rutaceae) is commonly known as Narangi or Santra (Orange). The fruit is laxative, aphrodisiac, astringent, tonic, and relieves vomiting.[9] The fruit peel is traditionally used as tonic, stomachic, astringent, carminative, and anti-scorbutic. Citrus peel has been regarded as a by-product of the citrus fruit industry, but it has been reported to contain several active components at far higher concentration than the pulp.[10] The fruit peel is also useful in skin care.

   Materials and Methods Top

Chemicals and reagents

Ascorbic acid, quercetin, ferric chloride, sodium carbonate (Na2 CO3), sodium nitrite (NaNO2), sodium hydroxide (NaOH), aluminum chloride (AlCl3), 1,1-Diphenyl-2-picrylhydrazyl (DPPH), porcine pancreatic elastase (EC., Succinyl-Ala-Ala-Ala-p-nitroanilide (SANA), Clostridium histolyticum collagenase (ChC) (EC., N-(3-[2-Furyl]acryIoyl)-Leu-Gly-Pro-Ala (FALGPA), fluorescein sodium salt, 2,2-azobis (2-methyl propionamidine) dihydrochloride (AAPH), and gallic acid were purchased from Sigma Chemical Co. (St. Louis, USA.) 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), (±) 6-hydroxy-2, 5, 7, 8-tetra methylchromane-2-carboxylic acid (TROLOX) acquired from Fluka, USA. Nitro blue tetrazolium (NBT), phenazine methosulfate (PMS), and nicotinamide adenine dinucleotide - reduced disodium salt (NADH) were obtained from SRL Pvt. Ltd., Mumbai, India. Ammonium per sulfate (APS) was purchased from Rankem, India.

Plant material collection

Fruits of C. reticulata Blanco, Rutaceae were obtained in January 2013 from Mumbai (India). The taxonomic identification of plant material was confirmed by Dr. C. S. Latto, University of Mumbai. Authentication was done at Agharkar Research Institute, Pune.

Extraction of plant material

Fruits were washed thoroughly under water. Peels were separated from fruits and dried under shade. Then, the dried material was subjected to the powder form and stored in an airtight container at room temperature. About 50 g of dried powder was extracted with 300 ml methanol. Plant extracts were obtained by Soxhlation (CR HAE- Hot Alcoholic Extract of Citrus reticulata) and maceration method (CR CAE- Cold Alcoholic Extract of Citrus reticulata). Soxhlet extraction was carried out at 65°C for 10–12 h. Cold maceration was carried out by constant shaking; where the mixture of plant powder and methanol was kept on a rotary shaker (REMI CIS-24 PLUS) and was set at 100 rpm at RT for 72 h. The extracts were filtered using Whatman filter paper no. 1, and the filtrates were then evaporated under reduced pressure and dried using a rotary evaporator (R-205, Buchi Laboratory Equipment, Flawil, Switzerland) set at 50°C. Dried extracts were stored at 4°C, in labeled, sterile, capped bottles until further use.

Preliminary phytochemical analysis of extracts

The extracts as mentioned above were subjected to various qualitative phytochemical tests for identification of chemical constituents present in the plant material according to the described methods.[11]

Determination of total phenolic content

Total phenolic content (TPC) were determined with the Folin - Ciocalteu reagent using the given method.[12] To 0.5 ml of each sample, 2.5 ml 1:10 dilution of Folin - Ciocalteau's reagent and 2 ml of Na2 CO3 (7.5% w/v) were added and incubated at 765 nm using a ultraviolet-visible spectrophotometer. Results were expressed as µg of gallic acid equivalent per mg of extract (µg GAE/mg extract).

Determination of total flavonoid content

Total flavonoid content (TFC) was measured by the AlCl3 colorimetric assay.[13]

An aliquot (1 ml) of extracts or standard solutions of quercetin (20, 40, 60, 80, and 100 μg/ml) was added to a 10 ml volumetric flask containing 4 ml of distilled water. To the flask, 0.30 ml of 5% NaNO2 was added and after 5 min, 0.3 ml of 10% AlCl3 was added. After 5 min, 2 ml of

1M NaOH was added and the volume was made up to 10 ml with distilled water. The solution was mixed and absorbance was measured against the blank at 510 nm. The TFC was expressed as µg of quercetin equivalent per mg of extract (µg QE/mg extract).

Antioxidant assays

1, 1-Diphenyl-2-picrylhydrazyl free radical scavenging assay

The free radical scavenging activity was evaluated by given method with some modifications.[14] The free radical scavenging activity was evaluated by given method with some modifications. A volume of 1 ml of the different concentrations (100–800 µg/ml) of test samples was mixed with 200 μl of (0.36 mg/ml) DPPH methanol solution. After shaking, the mixture was incubated in dark at room temperature for 30 min, and then the absorbance was measured at 517 nm. Ascorbic acid was used as a positive control.

Phenazine methosulfate-NADH system superoxide anion scavenging assay

The superoxide scavenging activity was evaluated by given method.[15] Tris HCl buffer (3 ml, 16 mM, pH 8.0) containing 0.75 ml NBT (50 µM) solution, 0.75 ml NADH (78 µM) solution, and 0.3 ml sample solution of extract (50–350 µg/ml) in methanol were mixed. The reaction was started when 0.75 ml of PMS solution (10 µM) was added to the mixture. The reaction mixture was incubated at 25°C for 5 min, and the absorbance was read at 560 nm against the corresponding blank samples. Ascorbic acid was used as a standard.

2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging assay

The assay is performed as per the given method.[16] ABTS radical cations were produced by reacting ABTS and APS and incubating the mixture at room temperature in dark for 16 h. In brief, the total reaction volume contained 10 mM PBS pH 7.4 (positive control or test solutions) of various concentrations. ABTS radical solution was added to a final concentration of 0.219 mM. The reaction mixture was mixed and immediately read at 734 nm using microplate reader (VERSA max, Molecular devices, USA). A control reaction was carried out without the test sample. Gallic acid was used as a positive control.

The ability of the extracts to scavenge DPPH, PMS-NADH radicals, and ABTS free radical was calculated by the following formula:

Radical scavenging activity (%) = ([Abs of control − Abs of sample]/Abs of control) ×100, where Abs is the absorbance at mentioned wavelength in the presence or absence of the test extracts.

Oxygen radical absorbance capacity assay

This assay was performed as per the method described.[17]

A preincubation mixture of 140 µl contained − 20 µl of test solution or TROLOX of various concentrations. 75 mM sodium phosphate buffer, pH 7.4; 120 µl of sodium fluorescein (117 nM) mixed and incubated at 37°C for 10 min. Following preincubation, 60 µl of AAPH (40 mM) was added and mixed for 15 s. The reaction was carried out for 90 min at 37°C. The fluorescence measurements were taken at 485 nm excitation and 520 nm emission filters.

Enzyme assays

Anti-collagenase assay

Collagenase inhibition assay was performed by the method described previously,[18] which is based on the hydrolysis of FALGPA by collagenase to produce FA-Leu and Gly-Pro-Ala. The assay was performed in

50 mM Tricine buffer (400 mM NaCl and 10 mM CaCl2, pH 7.5). ChC was dissolved in the buffer for use at an initial concentration of 0.8 units/ml. The synthetic substrate, FALGPA, was dissolved in the Tricine buffer to 2 mM. Sample extracts were incubated with the enzyme in the buffer for 15 min before adding substrate to start reaction. The final reaction mixture (75 μl total volume) contained 25 μl of 50 mM Tricine buffer, 25 μl of test extract (250–4000 μg/ml), and 25 μl of 0.1 units of enzyme ChC. Controls performed with 50 mM Tricine buffer as test extracts were dissolved in Tricine buffer (50 mM), while catechin was used as a positive control. After adding 50 μl of 2 mM FALGPA substrate, collagenase activity was measured immediately at 340 nm for 20 min using a 96 well micro plate reader (Bio-Tek M Quant, FLX 800).

Anti-elastase assay

The assay employed was based on methods from the literature.[19] This assay was performed in 0.2 mM Tris-HCL buffer (pH 8.0). Porcine pancreatic elastase (PE – E.C. was dissolved to make a 1 mg/ml stock solution in 0.2 mM Tris-HCL buffer. The substrate N-Succinyl-Ala-Ala-Ala-p-nitroanilide (SANA) was dissolved in buffer at 0.8 mM. The test extracts (0.156–10 mg/ml) were incubated with the enzyme for 20 min before adding substrate to begin the reaction. The final reaction mixture (Total 250 µl) contained 50 µl plant extract, 160 µl buffer, 20 µl enzyme, and 20 µl substrate. Catechin was used as a positive control. Negative controls were performed using Tris-HCL buffer. Absorbance was measured immediately at 410 nm and then continuously for 20 min using a 96 well micro plate reader (Bio-Tek M Quant, FLX 800).

The percentage inhibition for both of these assays is calculated by:

Enzyme inhibition activity (%) = (1− [B/A]) ×100

Where, A = Enzyme activity without sample, B = Activity in the presence of sample.

Gas chromatography-mass spectrometry analysis

Following conditions were adopted for gas chromatography-mass spectrometry (GC-MS) analysis.[20] The component identification was done using Agilent 7890 A gas chromatograph coupled with 5975 C inert Mass Selective Detector (MSD) with Triple Axis Detector. Samples were injected by Agilent 7693 auto sampler into Agilent 19091S-433: HP-5MS column (30 m × 250 μm × 0.25 μm). Helium was used as a carrier gas (1 ml/min). Injector and detector temperature were kept at 250°C. Column temperature was programed at 60°C for 3 min and then raised up to 160°C for 2 min at 10°C/min. Further temperature was raised up to 300°C (at 15°C/min). Mass spectra were acquired over 40–500 amu range in EI mode. The eluted compounds were identified by comparing mass spectral data with the standard data available in the library of National Institute of Standards and Technology. Main constituents from Soxhlet methanolic extract of C. reticulata Blanco peel (CR HAE) were identified by GC-MS analysis.

Statistical analysis

The EC50 values were expressed as mean ± standard deviation, statistical analysis was carried out by one-way analysis of variance followed by Tukey's multiple comparison test (P < 0.05) where, all samples, i.e., test extracts and positive standard were compared with each other. All calculations were performed using GraphPad Prism (version 5.0, GraphPad Software, USA).

   Results and Discussion Top

Preliminary phytochemical analysis of extracts

Preliminary phytochemical analysis of methanolic extracts of C. reticulata Blanco indicates the presence of carbohydrates, amino acids, flavonoids, tannins and phenolic derivatives, steroids, etc., [Table 1].
Table 1: Preliminary phytochemical analysis of methanolic extracts of Citrus reticulata Blanco peel

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Determination of total phenolic content

The phenolics, particularly polyphenols exhibit a wide variety of beneficial biological activities in mammals, including antiviral, antibacterial, immune-stimulating, anti-allergic, anti-hypertensive, anti-ischemic, anti-arrhythmic, anti-thrombotic, hypocholesterolemic, anti-lipoperoxidant, hepatoprotective, anti-inflammatory, and anti-carcinogenic actions.[21],[22] Several studies have shown the flavonoids to act as scavengers of superoxide anions, singlet oxygen, hydroxyl radicals, and lipid peroxyl radicals.[23],[24] TPC and TFC were determined for both the extracts using standard curves [Figure 1]. CR HAE showed higher phenolic content, i.e., 187.93 ± 4.69 µg GAE/mg extract than CR CAE, i.e. 58.66 ± 2.40 µg GAE/mg extract. TFCs for CR HAE and CR CAE were found to be almost similar, i.e. 171.72 ± 4.13 µg QE/mg extract and 169.88 ± 9.79 µg QE/mg extract, respectively.
Figure 1: Standard curve for total phenolic and flavonoid content

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Determination of total flavonoid content

Antioxidant activity should not be concluded based on a single antioxidant test model. In practice, several in vitro test procedures are carried out for evaluating antioxidant activities with the samples of interest.[25] In the present study, we have evaluated antioxidant potential of CR HAE and CR CAE by various antioxidant assays including DPPH free radical scavenging assay, superoxide anion scavenging assay, ABTS radical scavenging assay, and oxygen radical absorbance capacity (ORAC) assay.

Antioxidant assays

1, 1-Diphenyl-2-picrylhydrazyl free radical scavenging assay

DPPH free radical scavenging assay is one of the most commonly used assays for testing preliminary radical scavenging activity of plant extracts. The antioxidant activity of plant extracts containing polyphenol components is due to their capacity to donate hydrogen atoms or electrons and to scavenge the free radicals.[26] Thus, the purple color of DPPH will be reduced to α, α- diphenyl-β-picrylhydrazine (yellow). Results indicate that CR HAE and CR CAE showed up to 85% of scavenging of DPPH free radicals [Figure 2]. Ascorbic acid was used as a positive control and showed scavenging activity up to 92%. CR HAE exhibited slightly higher antioxidant activity than CR CAE. EC50 values (µg/ml) were expressed in [Table 2].
Figure 2: 1,1-Diphenyl-2-picrylhydrazyl free radical scavenging activity of ascorbic acid, CR HAE, and CR CAE

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Table 2: EC50 value of methanolic extracts of Citrus reticulata Blanco Peel for DPPH, superoxide, ABTS, anti-collagenase, and anti-elastase assays

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Phenazine methosulfate-NADH system superoxide anion scavenging assay

Although superoxide anion is a weak oxidant, it ultimately produces powerful and dangerous hydroxyl radicals as well as singlet oxygen, both of which contribute to oxidative stress.[27] The superoxide radicals generated from dissolved oxygen by PMS-NADH coupling and can be measured by their ability to reduce NBT. The decrease in absorbance at 560 nm with the plant extract indicates their ability to quench superoxide radicals in the reaction mixture. CR HAE showed strong superoxide scavenging activity than CR CAE [Figure 3]. Ascorbic acid was used as a positive control and scavenges superoxide anion up to 52%. EC50 values (µg/ml) were calculated and expressed in [Table 2]. CR HAE was found to be more effective (P < 0.05) than CR CAE as it showed less EC50 value (µg/ml) and potent antioxidant activity.
Figure 3: Superoxide anion radical scavenging activity of ascorbic acid, CR HAE and CR CAE

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2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging assay

ABTS assay is based on the scavenging of light by ABTS radicals. An antioxidant with an ability to donate a hydrogen atom will quench the stable free radical, a process which is associated with a change in absorption, which can be followed spectrophotometrically.[28] ABTS activity was quantified in terms of percentage inhibition of the ABTS free radical cation by antioxidants in each sample. The ABTS values of the CR HAE, CR CAE, and gallic acid were presented in [Table 2]. CR HAE and CR CAE showed ABTS radical scavenging activity in concentration dependent manner and observed up to 59%. Gallic acid showed up to 79% of ABTS scavenging ability [Figure 4]. EC50 values (µg/ml) obtained for CR HAE and CR CAE indicated no significant difference (P < 0.05) and suggested that both the extracts have almost similar ABTS scavenging potential.
Figure 4: 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activity of Gallic acid, CR HAE and CR CAE

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Oxygen radical absorbance capacity assay

ORAC assay is considered to be a more biologically relevant assay than other methods of measuring antioxidant potency because it measures the hydrogen-atom transfer reactions and simulates in vivo antioxidant action.[29] It also measures how well water-soluble and lipid-soluble components of a natural substance protect a standardized target from oxidation by peroxyl nitrite, hydroxyl radicals, superoxide anion, and singlet oxygen, and generates a score based on comparison with an antioxidant control.[30] In the present study, we have measured ORAC values for CR HAE and CR CAE and expressed in terms of TROLOX (mmoles TROLOX Equivalent/g of substance) [Figure 5]. CR HAE showed higher ORAC value (1243 mmoles TROLOX Equivalent/g of substance) than CR CAE (1063 µmoles TROLOX Equivalent/g of substance) [Table 2].
Figure 5: Standard curve of 6-hydroxy-2,5,7,8-tetra methylchromane-2-carboxylic acid for oxygen radical absorbance capacity assay

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Enzyme assays

Anti-collagenase assay

Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that degrade various components of the ECM. Collagenase is one of the members of the MMP families and it is responsible for the degradation of collagen. Collagenase from the bacteria C. histolyticum (ChC)

(C is one of the few proteinases capable of degrading the triple-helical region of native collagen under physiological conditions and in vitro conditions using synthetic peptides as substrates. Protective effect of plant extracts against collagenase enzyme can be studied using ChC and synthetic substrate FALGPA.[31] In the present study, collagenase inhibition activity was evaluated for CR HAE and CR CAE. Catechin was used as a positive control. Results demonstrated that CR HAE extract was more effective in inhibiting collagenase enzyme than CR CAE and showed inhibition up to 76% [Figure 6]. Efficacy was measured in terms of EC50 value (µg/ml) and expressed in [Table 2].
Figure 6: Collagenase inhibition activity of catechin, CR HAE and CR CAE

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Anti-elastase assay

Elastase is the only enzyme capable of degrading elastin. Inhibition of elastase enzyme can retain the elasticity and suppleness of skin.[32] In terms of anti-aging, finding inhibitors of elastase enzymes can be useful to prevent loss of skin elasticity and thus skin sagging. In anti-elastase assay, porcine pancreatic elastase was assayed spectrophotometrically by using [N-Succ-(Ala) 3-p-nitroanilide] as the substrate, and the amount of p-nitroaniline was determined by measuring the absorbance at 410 nm. The inhibitory effects of CR HAE and CR CAE on elastase activity were investigated. Catechin was used as a positive control. CR HAE showed strong % elastase inhibition activity compared to CR CAE and up to 80% inhibition was observed [Figure 7]. EC50 values (mg/ml) were expressed in [Table 2] and it was observed that CR HAE was more potent than
Figure 7: Elastase inhibition activity of catechin, CR HAE and CR CAE

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CR CAE (P < 0.05). EC50 value obtained for catechin was found to be very less indicating its strong anti-elastase activity and its role as a strong anti-aging component. Kim et al., 2004 showed that the catechin and epigallocatechin gallate isolated from green tea (Camellia sinensis) were potent anti-elastase inhibitors.

Gas chromatography-mass spectrometry analysis

CR HAE showed higher antioxidant and anti-aging abilities compared to CR CAE; hence, GC-MS analysis of CR HAE was carried out to understand the constituents present in it. A total of 20 different compounds were identified and presented in [Table 3]. The chromatogram showed 20 peaks in the retention time ranged from 2.95 min to 44.45 min [Figure 8]. The largest peak at 44.11 min with 44.37% area was identified as butylphosphonic acid, pentyl 4-(2-phenylprop-2-yl) phenyl ester. Zab et al., 2012[33] reported the antioxidant and anti-tumor activity of butylphosphonic acid, pentyl 4-(2-phenylprop-2-yl) phenyl ester present in the ethanolic extract of
Table 3: GC-MS analysis of Hot Alcoholic Extract (CR HAE) of Citrus reticulata Blanco

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Figure 8: Gas chromatography-mass spectrometry chromatogram of hot alcoholic extract of Citrus reticulata Blanco

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C. reticulata. Another major compound found was 4H-1-Benzopyran-4-one, 2-(3, 4-dihydro) or 4-Chromanone (21.43% area) which is a type of flavanone. Di Majo et al., 2005[34] isolated the flavanones from citrus fruits and showed structure-dependent antioxidant activity. Other compounds 3', 4', 5, 7, 8-Pentamethoxyflavone and 4', 5, 7, 8-Tetramethoxyflavone are types of polymethoxyflavone (PMF). PMF exhibit a broad spectrum of biological activities. Recently, Ho et al., 2012[35] isolated and identified hydroxylated PMFs from citrus peels and investigated their biological activities, including anti-inflammation and cancer chemopreventive property. Other compounds identified include D-Limonene (1.46% area), 4H-Pyran-4-one, 2, 3-dihydro-3, 5- dihydroxy-6-methyl (1.46% area), 2-Methoxy-4-vinylphenol (1.67% area), and n-Hexadecanoic acid (2.51% area) and could be related with the anti-aging potential of C. reticulata Blanco peel. Structures of all identified compounds were presented in [Figure 9].
Figure 9: Structures of compounds identified in gas chromatography-mass spectrometry analysis of hot alcoholic extract (CR HAE) of Citrus reticulata Blanco

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   Conclusion Top

C. reticulata Blanco extracts were evaluated against skin aging through in vitro antioxidant, anti-collagenase, and anti-elastase assays. The study showed that methanolic extract (Soxhlation) exhibited promising antioxidant and anti-enzymatic activity and can be used as a potent anti-wrinkle agent in anti-aging skin care formulations.


Authors are thankful to Underwriters Lab (UL), Bangalore, India, for GC-MS Analysis and Natural Remedies, Bangalore, India, for ORAC Analysis.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest

   References Top

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   Authors Top

Vinita D. Apraj, has completed Ph.D from Sunandan Divatia School of Science, NMIMS University, Mumbai in August 2015. The topic of research was 'Development and Evaluation of Herbal actives for skin care'. During her research work she has presented posters at various national conferences and won two prizes. Dr. Vinita Apraj has filed two patents based on her research work.

Nancy S. Pandita, is a Professor & HOD at the Department of Chemistry, Sunandan Divatia School of Science, NMIMS University, Mumbai. Her area of specialisation is Phytochemistry. She has 8 years of an academic and 22 years of an industry experience. Dr. Nancy Pandita has done work on number of plants/herbals/Ayurvedic drugs. She has expertise in isolation of chemical constituents from plants, their standardization, validation by HPLC and HPTLC. She has also worked on drug development and finding new drug candidates from herbals. She has developed a unique method for fingerprinting of plants/formulations by LC/MS/MS.


  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]

  [Table 1], [Table 2], [Table 3]


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