Formulation and In-Vitro Evaluation of Sun Protection Factor of Methanolic Extract of Zanthoxylum rhetsa DC. Sunscreen lotion.

 

Shantanu S. Kale1*, Amol H. Rajmane2. Vaibhav C. Urunkar2. Megha K. Gaikwad2 and Snehal B. Bhandare2

1Department of Pharmacognosy and Phytochemistry, M.G.V. Pharmacy College, Mumbai-Agra Road, Panchavati, Nasik 422001

2Institutional affiliations: Mahatma Gandhi Vidyamandir’s Pharmacy College, University of Pune, Pune

 

ABSTRACT:

Sunscreens are cosmetic formulations that block ultraviolet (UV) rays. Sunscreens are assigned sun protection factors, (SPF), ratings that are supposed to indicate the level of protection from UV radiation. In addition due to high cost and time consumption of in vivo SPF determination methodologies, in vitro SPF determination is gaining more importance. In the present study efforts were made to formulate sunscreen lotion using methanolic extracted Zanthoxylum rhetsa DC seed. Evaluation of same was done for sunscreen activity by using in vitro SPF method. SPF of formulation was found to be 1.09 with ultra boot star rating 2 which approaches toward sunscreen activity.

 

KEYWORDS: Zanthoxylum rhetsa, Tirphal, SPF, Sunscreen activity

 

 

INTRODUCTION:

Sunlight in the broad sense is the total frequency spectrum of electromagnetic radiation given off by the Sun. On Earth, sunlight is filtered through the Earth's atmosphere, and solar radiation is obvious as daylight when the Sun is above the horizon. The Sun emit X-rays, ultraviolet, visible light, infrared, and even radio waves.18 A measure of the power of light perceived by the human eye is expressed in SI derived unit called lumen. Direct sunlight has a luminous efficacy of about 93 lumens per watt of radiant flux, which includes infrared, visible, and ultraviolet light. At lower portion of the stratosphere from approximately 13 to 40 kilometers above Earth lies the protective ozone layer which absorbs 97–99% of the Sun's high frequency ultraviolet light preventing its damaging effects to life on Earth.19 When ultraviolet radiation is haphazardly absorbed by the atmosphere or other protective coating like ozone layer due to ozone depletion, it can cause damage to the skin known as sunburn or trigger an adaptive change in human skin pigmentation. The UV radiation part of spectrum of electromagnetic can be divided into three regions in increasing order of wavelengths. Ultraviolet C or (UV-C) range, which spans a range of 100 to 290 nm, Ultraviolet B or (UV-B) range spans 290 to 320 nm and Ultraviolet A or (UV-A) spans 320 to 400 nm. Fortunately owing to absorption by the ozone layer in lower portion of the stratosphere very little UV-C reaches the Earth's Lithosphere. On one hand exposure to UV-A radiation results in damage to the elastic and collagen fibers of connective tissue of skin, which causes photo-ageing, on the other hand UV-A radiation used in tanning and PUVA therapy (Psoralen and UV-A radiation) for psoriasis being less damaging to the DNA. UV-B radiations bring about acute inflammation (sun burn) and intensification of photo-ageing. 18


In addition to these, UV-B radiations are also reported to induce immune-suppression which reduces normal immunological defense mechanisms of the skin, therefore chances of development of malignant tumor increases. The application of sun care products like sunscreens is an easiest, commercially viable and efficient method of protecting skin against damaging effects of UV radiations. Sunscreen 20 is a lotion, spray, gel or other topical products like cream that absorbs or reflects some of the sun's ultraviolet (UV) radiation on the skin exposed to sunlight and thus helps protect against sunburn. The level of sun protection sunscreens offer has traditionally been estimated using the sun protection factor, The SPF is a numerical rating system to indicate the degree of protection provided by a sun care products like sunscreen. 5 The spectral limits conventionally accepted by photo-biologists and dermatologists for SPF determinations are UVB: (290nm - 320nm); UVA: (320nm - 400nm), UVA II: (320-340nm) and UVA I: (340-400nm.) SPF is defined as the ratio of the minimal erythema dose (MED) of solar radiation measured in the presence and in the absence of a sunscreen agent. 7 Regulatory agencies like the US-FDA and COLIPA (The Comité de Liaison de la Parfumerie in Europe) has made in vivo testing on human volunteers using an erythemal endpoint to determine the SPF of topical cream mandatory. 7Although it is a recommended and recognized method by US FDA and COLIPA, it has several disadvantages like being expensive, time-consuming and is potentially hazardous to human clinical subjects. Having said this, there are still many questions left unanswered about both the scientific accuracy and reproducibility of in vivo measurements of SPF, whereas, an in vitro measurement has the advantage of not exposing human subjects to harmful UV radiation, is cost-effective and provides us with statistically significant data which helps us to develop an effective sunscreen product. Thus, for economical, practical and ethical considerations a suitable method for in vitro determination of SPF is used more often.8 Sunscreens incorporate a wide variety of chemicals like Organic Compounds and their derivatives, Organic Esters, Salt and inorganic compounds, (Mineral) (Table I) 21 which have particular absorbance. The principal organic ingredients in sunscreens are usually aromatic molecules conjugated with carbonyl groups. This general structure allows the molecule to absorb high-energy ultraviolet rays and release the energy as lower-energy rays, thereby preventing the skin-damaging ultraviolet rays from reaching the skin. The inorganic materials like Titanium dioxide 22 incorporated in formulation as principle sunscreen agent reflect and scatter ultraviolet and visible radiation from a film of inert metal particle which forms an opaque barrier, they are photo stable, do not react with organic sunscreens and due to their light scattering properties there is less variability in the photo-protective effect of inorganic agents as compared to organic agents. In order to get a broad spectrum UV protection, more than one active sunscreen ingredients are added in the sunscreens. The EU norms has regularly listed 27 different organic and inorganic sunscreen ingredients since two decades, which are approved by Australian Government’s Department of Health and Ageing, Therapeutic Goods Administration (TGA) for use in Australia whereas only 16 ingredients are listed in US-FDA monograph, out of which Avobenzone and Zinc oxide are used frequently since 1978.6 Inorganic sunscreens are cosmetically unacceptable because of their opaque quality and being occlusive. Organic sunscreen have been increasingly reported for allergic and contact dermatitis, phototoxic and photo-allergic reactions, contact urticaria producing skin rash notable for pale red, raised, itchy bumps and even solitary cases of severe anaphylactic reactions.  In nutshell principle sunscreen agents listed in table I have following broad areas of concern:

·        Potentially carcinogenic properties of some sunscreen ingredients

·        Vitamin D deficiency caused by reduced exposure to ultraviolet light

·        Incomplete protection against the full ultraviolet spectrum combined with increased time spent in the sun

Despite the availability of diverse choices of sunscreens, the search for new and improved sunscreens still continues. Therefore, the researchers have turned their attention towards developing herbal sunscreen agents which are effective with less or no side effects. In present study we have made an attempt to formulate the topical sunscreen lotion from methanolic extract of Zanthoxylum rhetsa seeds and in-Vitro evaluation of Sun Protection Factor of formulated topical sunscreen lotion.

 

Zanthoxylum rhetsa DC. (Rutaceae) tree (‘Tirphal’) plant is native in rugged section of the western coastline of India from Mangalore to Raigad of western ghats of Sahyadri mountains range situated along the western side of India which runs north to south starting near the border of Gujarat and Maharashtra, south of the river Tapti, and runs approximately 1600 km through the states of Maharashtra, Goa, Karnataka, Tamil Nadu and Kerala ending at Kanyakumari, at the southern tip of India. It is also found in evergreen monsoon forests of the foothills of Assam and Meghalaya. Zanthoxylum rhetsa (Rutaceae) is a lofty, deciduous tree, up to 35 m. tall, The seed carpel yield an essential oil called ‘Mullilam oil’, which is obtained by steam distillation of the dried ripe seed carpel.1 The oil has pleasant odor resembling that of sweet orange and tangerine. It is used in the indigenous system of medicine for the treatment of cholera.9The oil is also used as an antiseptic, a disinfectant, and for the treatment of asthma, toothache and rheumatism.4The dried fruits are fibrous in nature and yield 14% oleoresin and 3% of essential oil.2Owning to the property of plant lipids sun-screening capability there has been growing interest in herbal extracts of seeds as they contain fat and oil and its lipid constituents, which confers desirable properties for cosmetic, nutritional and pharmaceutical purposes. In addition to this seed extract containing plant lipids also act as a solvent for dissolving fat-soluble aromatics, steroids or dyes, such as gamma oryzanol, vitamins and phospholipids in respective fields.

 

 


Table1: FDA Approved Sunscreen Agents 21 and there classification

Sr. No.

Chemicals

Compounds

1.

Organic Compounds and their derivatives

 

·     PABA and 4-aminobenzoic acid derivative (Padimate O)

·     Phenylbenzimidazole sulfonic acid

·     Derivative of benzophenone i.e. benzophenone-8, Benzophenone-3 and benzophenone-4

·     A dibenzoylmethane derivative (Butyl Methoxydibenzoylmethane)i.e Avobenzone

·     Benzylidene camphor derivative (terephthalylidene dicamphor sulfonic acid)Ecamsule

·     Organic camphor derivative 4-Methylbenzylidene camphor (4-MBC)

·     a benzotriazole-based organic compound (Bisoctrizole) Tinosorb M and S

·     Lipophilic benzotriazole derivative (Drometrizole trisiloxane)

·     Disodium phenyl dibenzimidazole tetrasulfonate (Bisdisulizole disodium)

·     Ethylhexyl triazone

·     Diethylamino hydroxybenzoyl hexyl benzoate

·     Diethylhexyl butamido triazone

2.

Organic Esters

·     Ester of methoxycinnamic acid i.e.2-Ethoxyethyl p-methoxycinnamate (Cinoxate)

·     A derivative of cyclohexanol  an ester formed from salicylic acid and 3,3,5-trimethylcyclohexanol, i.e. Homosalate

·     Ester formed by the condensation of a diphenylcyanoacrylate with 2-ethylhexanol.i.e Octocrylene

·     Ester formed from methoxycinnamic acid and 2-ethylhexanol.i.e. Octyl methoxycinnamate

·     Ester formed by the condensation of a salicylic acid with 2-ethylhexanol.i.e. Octyl salicylate

3.

Salt

Salt formed between triethanolamine and salicylic acid. Trolamine salicylate

4.

In organic compounds, (Mineral)

·     Titanium dioxide, also known as titanium(IV) oxide

·     Zinc oxide

 

MATERIALS AND METHODS:

Plant material:

Plant material of Zanthoxylum rhetsa was collected from Western Ghats, India. Authentication of plant was done by Dr. P.G. Diwakar, Joint Director, Botanical Survey of India, Koregaon road, Pune. The herbarium of plant specimen has been deposited at B.S.I., Pune, with the voucher specimen number: ZARAMR1 and the reference number: BSI/WRC/Tech./2010 The seeds were cleaned, dried under direct sunlight and powdered by a mechanical grinder.

 

Seed methanolic extraction:

Dried powder seeds of Zanthoxylum rhetsa (50 g) were Soxhlet Extracted in methanol. Then after solvent was rotary evaporated to get the extract

 

Sunscreen lotion formulation

Part A (Table No.2)

Sr. No.

Content

Chemical description

Use /Role

Pecentage  (% w/w)

1

Liquid Paraffine B.P

Mineral (Paraffinum liquidum)oil

 

Emollient

 

10

2

Super Hartolan

Lanolin Alcohol

A natural lipid rich in sterols W/O emulsifier and excellent emulsion stabilizer

 

2

3

Cetyle alcohol

1-Hexadecanol

Emollient,

Coemulsifier

2

4

Stearic Acid .

Octadecanoic acid

Emollient,

Co-emulsifier

20

5

Arlacel 83

Sorbitan Sesquioleate

Sorbitan sesquiester W/O emulsifier, particularly recommended for unsaturated lipid components such as oleyl alcohol or vegetable oils

2

 

 

Part B - (Table No.3)

Sr. No.

Content

Chemical description

Use /Role

Pecentage (% w/w)

1

Glycerin

Glycerin

Emollient

7.5

2

Sorbitol 70% Solution

Sorbitol

Moisture stabilizer

2.5

3

Propylene Glycol

Propylene Glycol

Humectants

5

4

Deionised Water

Water, Aqua

Vehicle

up to 100%

5

Triethanolamine

Nitrilotriethanol

Surface active agent

5

6

Methanolic Extract of Zanthoxylum rhetsa DC

Active Ingredient

Active Ingredient

5

Method of preparation:

1. Heat (A) and (B) independently to 75 0 C.

2. Add (B) to (A) slowly with continuous stirring.

3. Cool to 35 0 C, add  active  ingredient.

 


Determination of In-vitro SPF of sunscreen lotion

Method

Approximately 100 mg of the investigational sample was applied and spread on 50 sq.cm area to obtain a sample film thickness of 2 μl /cm2 on Transpore surgical tape to get an even film as suggested  in the operation  manual of  Optometric LLC SPF-290S for the sample preparation and application technique. The samples thus prepared were exposed to Xenon arc lamp with UV range 290nm to 400nm for determining the SPF. WIN SPF has used the following equation for calculating SPF value.

Where,

MPFλ = Scan MPF (Monochromatic Protection Factor) value, = Spectral irradiance of terrestrial sunlight under controlled conditions, = Erythemal effectiveness The SPF-290 software used Trapezoidal Approx. calculating technique to approximate the integral for SPF and  Erythemal UVA protection factor. These include UVA/UVB ratio, critical wavelength, cumulative absorbance, etc. The Average Absorbance method is used for calculating average protection factor

 

Table 4: Results of SPF and other parameters of Zanthoxylum rhetsa DC. Methanolic extract  sunscreen lotion

Sr. no.

Parameter

Scan I

Scan II

Scan III

Average value

1

SPF

1.09

1.09

 

1. 09

2

Standard deviation

0.03

0.03

0.02

0.03

3

UVA/UVB ratio

0.391

0.403

0.406

0.400

4

Critical wavelength

368.9

373.1

372.5

371.5

5

Boot star rating

1

2

2

2

 

Table 5: Boot star rating system

Mean UVA/UVB Ratio

Star Rating Category

Star Rating Designation

0 to 0.2

-

No Claim

0.21 to 0.4

*

Minimum

0.41 to 0.6

**

Moderate

0.61 to 0.8

***

Good

0.81 to 0.9

****

Superior

0.91 and above

*****

Ultra

 

RESULTS:

Sunscreen lotion was formulated using methanolic extract of seeds of Zanthoxylum rhetsa [Table 2 and 3]. Formulated lotion was evaluated for sunscreen activity using in vitro SPF method. SPF value of sunscreen lotion was found to be 1.09 [Table 4, Figure 1, 2, 3] with Boot star rating 2 [Table 4 and 5]. Sunscreen lotion formulated using methanolic extract of seeds of Zanthoxylum rhetsa shown the UV-A/UV-B ratio 0.400, indicating that it has moderate UV-A protection. The critical wavelength standard is a measure of UVA-performance in relation to its UVB-performance. It is given as the upper limit of the spectral range from 290 nm on, within which 90% of the area under the extinction curve of the whole UV-range between 290 nm and 400 nm is covered. Sunscreen lotion formulated using methanolic extract of seeds of Zanthoxylum rhetsa shown critical wavelength 371.5 indicating it as a "broad spectrum". SPF value for sunscreen above 2 is considered as having good sunscreen activity. It indicates that formulated sunscreen cream was found near the range of good sunscreen activity.

 

 

Figure 1: SPF-290 Graph Report of Zanthoxylum rhetsa DC. Methanolic extract  sunscreen lotion ,Scan1

 

Figure 2: SPF-290 Graph Report of Zanthoxylum rhetsa DC. Methanolic extract  sunscreen lotion, Scan 2

 

Figure 3: SPF-290 Graph Report of Zanthoxylum rhetsa DC. Methanolic extract sunscreen lotion, Scan 3

 

DISCUSSION:

The Boot star rating is based on the average of UVA/UVB ratio. The UV-A/UV-B ratio defines the performance of a sunscreen in the UV-A range (320 – 400 nm) in relation to its performance in the UV-B range (290 – 320 nm). It is calculated as the ratio between the areas under the UV-A and UV-B parts of the extinction curve, both areas are normalized to the range of wavelengths involved. A UVA/UVB ratio goes from 0 to 1. The lower the ratio, the feeble the UVA protection, the closer the UV-A/UV-B ratio approaches 1, the better the sunscreen will protect against UVA radiation. Determination of critical wavelength by means of UV spectrophotometry provides a rapid, inexpensive, and reliable measure of broad-spectrum protection, which is largely independent of SPF, yet ensures long-wavelength UVA protection commensurate with SPF. The procedure provides a routine, sensitive means of differentiating and classifying sunscreen products and, importantly, reduces the need to subject volunteers to acute exposures of high-dose, nonterrestrial UV, the health risks of which are still poorly understood. Zanthoxylum rhetsa methanolic seed extract may be considered as good candidate for sunscreen or cosmeceutical purposes.

 

CONCLUSION:

SPF value for sunscreen above 2 is considered as having good sunscreen activity. In present study formulated sunscreen cream was found near the range of good sunscreen activity and hence Zanthoxylum rhetsa DC. Methanolic extract may be considered as good candidate for sunscreen or cosmaceutical purposes.

 

ACKNOWLEDGEMENTS:

We express our sincere thanks to Mr. Milind and Anjali Katariya of Reva Pharma Pvt. Ltd., Sinnar, Nashik and Mr. Rahul Phate for his kind guidance to carry out formulation. Dr. Chandrashekhar, Dr. Anupama and Miss. Priyanka of Kelkar Lab, Mulund for their kind help.

 

Abbreviations

Table.No.8 Abbreviations

Sr.

No.

Abbreviation

Meaning

1

UV

Ultra Violet

2

SPF

Sun Protection Factor

3

IR

Infrared

4

MED

Minimal erythemal Dose

5

US-FDA

United State-Food & Drug Administration

6

COLIPA

The Comité de Liaison de la Parfumerie in Europe

 

REFERENCES:

1.       L. Jirovetz , G. Buchbauer , M. P. Shafi , A. Saidutty, Short communication ,Analysis of the aroma compounds of the essential oil of seeds of the spiceplant Zanthoxylum rhetsa from southern India, Springer-Verlag, Z Lebensm Unters Forsch A,2006; 1998: 228±229.

2.       M.Paridhavi,S.S.Agarwal, Isolation,Characterization and Antiinflamatory Activity of compounds isolated from seeds of Zanthoxylum almatum. Indian Journal Of Natural Products, 23(1), 2006; 18.

3.       Lalitsingh Ranawat, Jigar Bhatt, Jagruti Patel. Hepatoprotective activity of ethanolic extract of bark of Zanthoxylum armatum DC in CCl4induced hepatic damage in rats. Journal of Ethnopharmacology,2010, 127; pp.777-780.

4.       Randy S, Perry R. Multifunctional Cosmetics, Cosmetic Science and Technology Series: Mercel Dekker, Inc; Vol-26, New York; 2001:pp.148.

5.       Sayre R M,Desrochers D L, Wilson C J,Marlowe E, et al. Skintype,minimal.erythema dose(MED)& sunlight acclimatization, PubMed Indexed for MEDLINE;5(4): Oct 1981:439-43.

6.       Nash, JF. "Human Safety and Efficacy of Ultraviolet Filters and Sunscreen Products". Dermatologic Clinics, PubMed Indexed for MEDLINE 24 (1): 35–51,2006.

7.       COLIPA- Guidelines For The Colorimetric Determination of a Skin Color Typing and prediction of The Minimal Erythemal Dose(MED) Without UV Exposure, COLIPA Guidelines 2007: 1

8.       COLIPA- Method for The In Vitro Determination Of UVA Protection Provided By Sunscreen Products. COLIPA Guidelines, 2007a: 2-4

9.       National Institute of Science Communication and Information Resources and CSIR ,The Wealth of India- A Dictionary of Indian Raw Materials and Industrial Products, New Delhi 1956,First Supplement  Series (Raw Materials); Vol-5: R-Z:pp.378-379.

10.     Hilda B. Poucher’s Perfumes, Cosmetics and Soaps. Dordrecht: Kluwer Academic Publishers; 2000: 10th ed, pp.468.

11.     Nadkarni KM, Nadkarni AK. Indian Materia Medica, Bombay: Popular Prakashan Pvt. Ltd; Vol-1, 1976: 3rd ed,pp.1302-1304.

12.     A Husain, O.P.Virman,Directory of Indian Medicinal Plants, Control Institute of Medicinal and Ayurvedic Plants,1992, 499-500

13.     Wilkinson JB, Moore RJ. Harry’s Cosmeticology.. Britain: Longman Scientific and Technical Publishers; 1982, 7th ed: 222-258.

14.     Masson P, Merot F. Phototype and ITA° parameters for determination of MED and SPF in tanned or untanned subjects. Poster; Preprints 17th IFSCC congress, Yokohama. 1992:10.

15.     Michael Brown and Nicola Fardell,Butler H.(ed.). Sun damage and  sunscreen  preprations In Poucher’s  Perfumes,Cosmetics and soaps,.Springer,Kluwer Academic Publishers, Great Britain,10th  edn:467-503.

16.     Westerhof W. The relation between constitutional skin color and photosensitivity estimated from UV induced erythema and pigmentation dose-response curves. J Invest Dermatol 1990(94): 812-816.

17.     David AK. Sunscreens: Preparation And Evaluation, Cosmetics & Toiletries magazine, 2003.

18.     Review of electromagnetic spectrum by artwork by Randy Russell Available from: URL: http//www.windows2universe.org/sun/spectrum /multispectral_sun_overview.html

19.     The Ozone Layer, by author Author:Brien Sparling Available from: URL: athttp://www.nas.nasa.gov/About/Education/Ozone/ozonelayer.html

20.     “Preventing melanoma”Cancer Reseaech U.K.Retrieved 2009-09-22 Available from: URL: htpp://www.cancerhelp.org.uk/type/malanoma/about/preventing.melanoma

21.     FDA allowable ingradients Available from: URL: http://en.wickipedia.org/wiki/sunscreen#Differences_between_sunblock_and_Sunscreen.

22.     Titanium dioxide Available from: URL:  http://en.wikipedia.org/wiki/Titanium_dioxide

 

 

Received on 18.05.2011

Accepted on 10.06.2011     

© A&V Publication all right reserved

Research Journal of Pharmacognosy and Phytochemistry. 3(5): Sept.- Oct.2011, 206-210