INTRODUCTION:

Dermatologists now strongly recommend to apply a topical sunscreen formulation (which absorb, reflect or scatter suns radiation) with a sun protective factor (SPF) value of 15 (threshold value) or greater, preferably year round, to protect the skin and hair against harmful UV rays, especially, UV B rays^1,2.

In fact, sunscreen application began in early 20^th century³. Currently, there are several sunscreen formulations in the form of creams, oils, gels, ointments, lotions, balms, waxes or butters^1,4,5,6. Some of these sunscreens are synthetic and others are natural and herbal^1,2,4,6,7. Synthetic sunscreens are generally fast acting, efficacious and provide broad spectrum UV protection^{1,2,4,7,9,10.}However, these are relatively expensive and often induce undesirable side effects, such as contact and/or irritant dermatitis, hypersensitivity, allergies, whitening of skin, vitamin D deficiencies and even skin cancers^7,8,9. Moreover, some of these are not user friendly because of their opaque quality, occlusiveness, comedogenicity and tendency to stain cloths^1,7,8,9. Yet, an another point of particular concern is that it is claimed that mothers with high blood levels of certain synthetic sunscreen ingredients are more likely to give birth to underweight babies if applied during pregnancy period¹¹. Conversely, herbal sunscreens are claimed to be ecofriendly, safe, efficacious, relatively cheap, noncomedogenic and devoid of undesirable side effects^4,5,9,10,11. Accordingly, at present, there is an unprecedental and imperative need to develop novel sunscreens from herbal sources. In this context, we have initiated programme of research to investigate in vitro sunscreen activity in terms of sun protection factor (SPF) of Sri Lankan herbs. As of now, we have evaluated the sun protection activity of orthodox black tea made from leaves of Camellia sinensis¹² from salt marshy plants, Suaeda monoica, Suaeda maritima, Halosarcica indica¹³ and Salicornia brachiata¹⁴ leaves of seashore plants. Ipomoea Pes-capprae¹⁵, pericarp of Gracinia mangostana¹⁶, leaves of Flueggea leucopyrus¹⁷ and leaves of Codiaeum veriegatum and its two varieties¹⁸.

In this study, we report, for the first time, in vitro sun protective activity of roots of Heteropogon contortus (Eathana in Sinhala and Arukkam-pillu in Tamil), a member of Family Poaceae by assessing the SPF value using UV spectroscopic technique and Mansur equation^2,8,12,19.

H. contortus is a perennial grass which grows in Sri Lanka, India, Africa, Southern Asia, Northern Australia and parts of Oceania^20,21. The grass has become a naturalised weed in tropical and subtropical regions in Asia. The plant is 0.5 to 1.5 m tall. Leaves and sheaths are green to grey-green in colour. Leaf blades are linear 3-30 cm long, 2-8 mm wide, abruptly narrowed at the tip. Basal sheaths are laterally compressed^20,21.

Ethnomedically, H. contortus roots are claimed to treat eye diseases, vomiting, fever, dysentery, epilepsy, swelling, asthma, bronchial diseases, jaundice, phyelitis, gnorrhoea, strangury, wounds, ulcers, diarrhoea, eazemas, diabetes mellitus, body heat conditions and cystitis^20,21. Roots are also claimed to be diuretic and aphrodisiae²¹. Experimentally, methanolic extract of all plant is shown to have bronchorelaxent and antiinflammatory activities²².

MATERIALS AND METHODS:

Collection of the root (H. contortus):

Fresh matured roots of the plant H. contortus were collected from Galle district (6.0535° N; 80.2210° E), Sri Lanka in June, 2017.

Identification of plant material:

The plant material was identified by Dr. Hashendra, Department of Plant Sciences, Faculty of Sciences, University of Colombo, Sri Lanka. A voucher specimen has been deposited at the Department of Basic Sciences, Faculty of Allied Health Sciences, General Sir John Kotelawala Defence University, Werahera, Sri Lanka.

Preparation of methanolic extract of H. contortus:

Matured roots of H. contortus were taken and washed twice in running tap water. Roots were then cut into small pieces and air dried for a week until a constant weight was obtained. The dried roots were powdered using a domestic blender. Powdered roots (16.5 g) were macerated for 7 days in 150 mL of distilled methanol. The resulting brown coloured extract was filtered through a double layer of cotton and then filtered again through a Whatman filter paper (No: 1). The filtrate was evaporated in a water bath set at 70⁰C until dryness (Yield: 13.63%). The brown coloured product was stored in a sealed beaker and kept in a refrigerator (4⁰C) until use.

Phytochemical analysis of H. contortus methanolic extract:

Methanolic root extract of H. contortus was subjected to following phytochemical screening. Qualitative analysis was performed for alkaloids using Meyer’s and Wagner’s tests. Test for phenols was achieved via Libermann’s test and FeCl₃test. Alkaline reagent test was carried out for flavonoids. Additionally, foam test was performed for the analysis of saponins. Molish test and xanthoproteic test were performed for the investigation of carbohydrate and proteins, respectively. Gelatin test was carried out for tannins.

In vitro evaluation of sun protection factor of methanolic extract of H. contortus:

The brown colour solid of H. contortus methonolic extract was dissolved in methanol and solutions of 0.2 mg/mL, 0.1 mg/mL and 0.05 mg/mL were prepared. Similarly, Dermatone® was dissolved in methanol to obtain solutions of 0.2mg/mL, 0.1 mg/mL and 0.05 mg/mL. These solutions were then kept in water bath at 23⁰C for an hour. Absorbance was determined in 1 cm quartz cell for the above-mentioned concentrations for both methanolic extract of H. contortus and Dermatone® using UV-1800 SHIMADZU spectrophotometer. Absorbance was performed in triplicates for the methanolic extract of H. contortus and Dermatone®, from 290 nm to 320 nm, with 5 nm intervals using ethanol as a blank. SPF values were then calculated using the Mansur equation given below^2,8,12,19

SPF = CF x EE (λ) x I (λ) x Abs (λ)

Where: EE – Erythemal effect spectrum; I – Solar intensity spectrum; Abs – Absorbance of sunscreen product; CF – Correction factor (=10). The values of EE x I are constant and predetermined.

Statistical analysis:

The results are given as mean ± SEM. Dose dependencies were investigated using Pearsons’s correlation analysis. Significance was set at P<0.05.

RESULTS:

The results of SPF evaluation of methanolic root extract of H. contortus and reference agent, Dermatone® are depicted in Tables 1 and 2. As shown in Table 1, all the concentrations of H. contortus exhibited moderate sun protective activity in terms of SPF values. The highest SPF value (13.02 ± 0.03) was evident with 0.2 mg/mL concentration and the lowest (12.85 ± 0.87) with 0.1 mg/mL concentration. EC₅₀ value for H. contortus was 0.210 mg/mL. However, the sun protective activity of H. contortus extract was not dose dependent (r² = 0.725; P>0.05). In contrast, the highest dose of Dermatone® exhibited SPF value of 27.32 ± 0.12 displaying a strong sun protective activity. Lowest concentration (0.05 mg/mL) had a SPF value of 8.21 ± 0.01 which was 36.1% lower than SPF value for the lowest dose of extract. However, SPF values of mid and high concentrations of Dermatone® were 8.57% and 52.34% higher than the corresponding concentrations of the extracts. The sun protective activity of Dermatane® was dose-dependent (r² = 0.99; P<0.05). EC₅₀value for Dermatone® was 0.11 mg/mL. Phytochemical analysis revealed the presence of flavonoids, phenols, alkaloids, carbohydrates and proteins (see Table 3).

Table 1: SPF values for methanolic extract of H. contortus at 0.05, 0.1 and 0.2 mg/mL concentrations:

Methanolic extract of H. contortus	Concentration mg/mL)	SPF ± SEM
	0.05	12.85 ± 0.87
	0.1	12.80 ± 0.13
	0.2	13.02 ± 0.03

Table 2: SPF values for Dermatone® at 0.05, 0.1 and 0.2 mg/mL concentrations

Dermatone®	Concentration (mg/mL)	SPF ± SEM
	0.05	8.21 ± 0.01
	0.1	14.00 ± 0.05
	0.2	27.32 ± 0.12

Table 3: Phytochemical analysis of methanolic extract of H. contortus:

Chemical class	Test performed	Response
Chemical class	Test performed	Positive	Negative
Alkaloids	Meyer’s test	ü	-
Alkaloids	Wagner’s test	ü	-
Phenols	Libermann’s test	ü	-
Phenols	Neutral FeCl₃test	ü	-
Flavonoids	Alkaline reagent test	ü	-
Saponins	Foam test	-	ü
Carbohydrates	Molish test	ü	-
Proteins	Xanthoproteic test	ü	-
Tannins	Gelatin test	-	ü

DISCUSSION:

This study examined the in vitro sunscreen potential (in terms of SPF value) of methanolic root extract of H. contortus (Family: Poaceae), a common weed in Sri Lanka, using a spectroscopic technique and Mansur equation^2,8,12,19. This in vitro bioassay is well recognized, widely used, simple, quick, sensitive, reliable, validated, inexpensive technique to evaluate sunscreen activity of both synthetic and herbal products/formulations^4,6,8,23. Furthermore, this in vitro assay bypasses the variability and ethical issues encountered with humans and animals as experimental subjects²⁴. It is also noteworthy that the experimental conditions used in this study were similar to those used by several other investigators^4,5,6,8,11 and by us^{13,14,15,16,17,18}.

The results convincingly show, for the first time, that the methanolic root extract of H. contortus has moderate sunscreen activity (in terms of SPF value) at very low concentrations: concentrations of 0.05, 0.1 and 0.2 mg/mL had SPF values of 12.85, 12.80 and 13.02, respectively. Corresponding SPF values for Dermatone®, reference agent at 0.05, 0.1 and 0.2 mg/mL were 8.21, 14.00 and 27.32, respectively. This indicates that root extract is less efficacious compared to Dermatone®. Most organizations and dermatologists recommend to apply a topical sunscreen formulation having SPF value 15 or more, preferably, year around to protect the skin and hair against harmful UV B rays^1,2. Moreover, SPF values 2-12, 12-30 and > 30 are recognized as having mild, moderate and high sun protective activity⁷. What is more, a sunscreen with SPF value of 15 is claimed to protect against 93% of harmful UV B rays and no available sunscreen is capable of protecting 100% UV B rays²⁵. These facts suggest that the root extract of H. contortus can be developed as a safe inexpensive and user-friendly topical sunscreen.

It is well-known that UV B rays provoke the production of variety of aggressive free radicals/molecules such as ^.O₂, ^.OH, HOO^. in the skin^4,6,9,26. Free radicals are closely linked with UV B induced pathogenesis of skin damages^4,6,26. Sun protective activity of herbal products is often attributed to their antioxidant activity; quenching of free radicals^19,26,27. Antioxidant activity of herbal sunscreens are usually mediated by flavonoids^19,27, tannins^19,27and alkaloids²⁸. The H. contortus root extract contained flavonoids, phenols and alkaloids. Accordingly, it is quite possible that the sunscreen activity of H. contortus root extract is also mediated by synergestic antioxidant activity of flavonoids, phenolics and alkaloids present in it.

It is concluded that the methanolic root extract of Sri Lankan grass, H. contortus possesses moderate sunscreen activity at very low concentrations. This is a novel finding. Possibility exists to develop a cheap, safe and efficacious sunscreen from roots of H. contortus.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 08.02.2018 Accepted on 24.03.2018

Asian J. Pharm. Ana. 2018; 8(2):65-68.

DOI: 10.5958/2231-5675.2018.00012.1