Author: - K.N. Wijesekara - W.S de Silva Department of Biotechnology, Horizon Campus, GARI Publisher | Medicinal | Volume: 04 | Issue: 07 Article ID: IN/GARI/ICATMMP/2018/115 | Pages: 33-42 (09) ISSN 2424-6492 | ISBN 978-955-7153-00-1 Edit: GARI Editorial Team | Received: 16.12.2018 | Publish: 20.01.2019

PRELIMINARY SCREENING OF TELEGRAPH ( MOTORIUS) EXTRACT FOR SKIN WHITENING PROPERTY AND CYTOTOXICITY ACTIVITY

K.N. Wijesekara, 1W.S de Silva Department of Biotechnology, Faculty of Science, Horizon Campus, Sri Lanka [email protected]

ABSTRACT

A perfect skin can be remained as a control was 478.800757±3.1567 µg/ml. dream if it does not maintain properly, Value of inhibition of tyrosinase was therefore most of the young women are significantly higher than to positive tempting to skin whitening products that control. The IC50 value of cytotoxicity can be composed of harmful chemicals activity for methanolic extract of leaves that cause dullness, uneven skin tone or of Telegraph plant was 1516.0538± acne breakout instead of making skin 2.407µg/ml. This analysis was revealed healthy and blooming. Natural products IC50 value of methanolic extract is are safe for consumption and will work nontoxic toxic to brine shrimps. on skin naturally and effectively by Therefore, it can be concluded that balancing skin tone and eliminating leaves possess harmful effects. This study was carried highly active antityrosinase substances out to determine skin whitening property which can be consumed for remedy of and cytotoxicity activity of Codariocalyx healthy and brighten skin. motorius. C. motorius is commonly known as “Pranajeewa” in Sinhala or Keywords: Telegraph (Codariocalyx “Telegraph Plant”. It is widely consumed motorius), Antityrosinase activity, as an antidote, cardiac-tonic and for Cytotoxicity activity, wound healing. The antityrosinase activity and cytotoxicity activity of the Mushroom tyrosinase methanolic extract of leaves of Telegraph plant have been studied on in-vitro INTRODUCTION models. The antityrosinase activity was evaluated based on inhibition of Skin is an important component of mushroom tyrosinase and cytotoxicity body image and has immense activity was evaluated based on brine physiological importance for both women shrimp lethality bioassay. The IC50 value and men. Skin pigmentation can be a of antityrosinase activity for methanolic source of significant emotional distress in extract of leaves of Telegraph plant was individuals (Stratigos and Katsambas, 282.2907299 ±4.521 µg/ml and SASAKI 2004). Both men and women have been Whitening Body Skin Serum as positive struggling with skin pigmentation Author: - K.N. Wijesekara - W.S de Silva Department of Biotechnology, Horizon Campus, Sri Lanka GARI Publisher | Medicinal Plants | Volume: 04 | Issue: 07 Article ID: IN/GARI/ICATMMP/2018/115 | Pages: 33-42 (09) ISSN 2424-6492 | ISBN 978-955-7153-00-1 Edit: GARI Editorial Team | Received: 16.12.2018 | Publish: 20.01.2019 (hyper/hypo-pigmentation). Therefore from the harmful effects of ultraviolet most of the young women are tempting to (UV) radiations by absorbing UV use skin whitening products that can be sunlight, removing reactive oxygen composed of harmful chemicals that (ROS) and scavenging toxic cause dullness, uneven skin tone or acne drugs and chemicals. Melanin also breakout instead of making skin healthy induces the expression and synthesis of a and blooming and also in long term variety of cytokines, primarily of usage, these chemicals might lead to keratinocyte origin, that act in a paracrine tumor and other unbearable diseases fashion to further induce melanogenesis instead of making skin healthy and (Yaar et al., 2006). The type and amount glowing. Even someone has a naturally of melanin synthesized by the light-skinned, external factors can melanocytes and its distribution in the generate melanin production. Some of surrounding keratinocytes determine the external factors are pollution, harmful actual color of the skin (Kim and Uyama, UV rays from the sun, stress, and 2005).If there’s formation of more unhealthy diet, hectic lifestyles, and many melanin in the skin, the darker the skin others. Considering these circumstances, tone will be arisen. Therefore, tyrosinase the best solution is to consume natural inhibition is the most common approach ingredients that are safe to use. And some to achieve skin hypopigmentation as this of benefits of consuming natural enzyme catalyses’ the rate-limiting step ingredients are skin brightening, of pigmentation (Solano et al., 2006). balancing skin tone, reducing blemishes Arbutin, a naturally occurring and marks, photo-protection, and beta-d-glucopyranoside of hydroquinone eliminating harmful bacteria. used traditionally for depigmentation, Tyrosinase (E: C: 1.14.18.1) is an involves inhibition of melanosomal essential enzyme, which contributes tyrosinase activity. Some other known towards pigment formation in natural compounds such as kojic acid, mammalian's body as well as in plants, kojic acid octanoates, salicylhydroxamic microorganisms and fungi (Momtaz et al., acid, catechins, hydroquinone, and more 2008). In humans and other mammals, recently resveratrol and oxyresveratrol the biosynthesis of melanin takes place in were also described for their tyrosinase a lineage of cells known as melanocytes, inhibition properties (Bourin et al., 2002). which contain the enzyme tyrosinase And some existing natural derivatives (Robb, 1984). Tyrosinase (phenol have got limitations in term of high oxidase) is known to be a key enzyme for toxicity. Therefore, in this study, melanin biosynthesis. This enzyme is Codariocalyx motorius (Telegraph Plant) mainly involved in the initial steps of the was investigated to determine its potential pathway which consist of the of decreasing skin pigmentation and hydroxylation of the p-monophenolic cytotoxicity by carrying out shrimp amino acid l-tyrosine (monophenolase lethality assay. The shrimp lethality assay activity of tyrosinase) and the oxidation was proposed by Michael et al., and later of the product of this reaction, the o- developed by Vanhaecke et al., and Sleet diphenolic amino acid L-DOPA and Brendel. It is based on the ability to (diphenolase activity), to give rise to o- kill laboratory-cultured Artemia nauplii dopaquinone (Bourin et al.,2002) . This brine shrimp. The assay is considered a o-quinone is transformed into melanins in useful tool for preliminary assessment of a series of non-enzymatic reactions toxicity, and it has been used for the (Prota, 1988, 1992). Melanin plays an detection of fungal toxins, plant extract important role in protecting human skin toxicity, heavy metals, cyanobacteria Author: - K.N. Wijesekara - W.S de Silva Department of Biotechnology, Horizon Campus, Sri Lanka GARI Publisher | Medicinal Plants | Volume: 04 | Issue: 07 Article ID: IN/GARI/ICATMMP/2018/115 | Pages: 33-42 (09) ISSN 2424-6492 | ISBN 978-955-7153-00-1 Edit: GARI Editorial Team | Received: 16.12.2018 | Publish: 20.01.2019 toxins, pesticides, and cytotoxicity testing This plant is famous for its rapid of dental materials (Carballo, 2002). movement of leaflets and it exists within a period of about three to five minutes. LITERATURE REVIEW The leaves move up and down rhythmically, as if the plant is dancing or Codariocalyx motorius (= D. gyrans sending out telegraphic messages. (Unnisa et al., 1994; Antkowiak & Therefore, the necessity of a time lapse Engelmann, 1995; Lev-Yadun, 2013; camera to see the movements can be Morse, 2018) = gyrans (Lev- avoided, as the rapid movements are Yadun, 2013; Morse, 2018) = observable to the naked eye. The leaf motorium (Unnisa et al., system of Codariocalyx motorius consists 1994; Antkowiak & Engelmann, 1995; of a terminal leaflet and a maximum of Lev-Yadun, 2013; Morse, 2018)), is two lateral leaflets, all on the same stalk. known as the telegraph plant (" A set of one larger and two smaller - GRIN-Global Web v 1.10.3.6", 2018; leaflets are connected by a “hinge,” Ileperuma,2015), dancing plant which allows the leaflets to lift and rotate (Ileperuma,2015), or semaphore plant themselves. Moreover, the plant also (Ileperuma,2015;"Taxonomy - GRIN- produces small, purple colored flowers Global Web v 1.10.3.6", 2018). It belongs (Ileperuma, 2015). to the Codariocalyx ("Taxonomy - GRIN-Global Web v 1.10.3.6", 2018), In Siddha medicine, it is used as antidote, (alt.Leguminosae) (Kim cardiac-tonic and wound healing et al., 2014; "Taxonomy - GRIN-Global ointment (Trout, 2002; Chidambaram et Web v 1.10.3.6", 2018), subfamily al., 2013; Kim et al., 2014; Uma et al., ("Taxonomy - GRIN-Global 2014). It has also been used in curing Web v 1.10.3.6", 2018). snake bite poisons (Chidambaram et al., 2013). The roots are used as emollient, C. motorius is widely distributed laxative, antidysentric and to treat cough, throughout -Temperate: asthma and fever. The leaves exhibit (Fujian, Jiangxi, Guangdong, Guizhou, tonic, diuretic, febrifuge and aphrodisiac Sichuan, Yunnan, and Guangxi), Eastern properties. In Sikkim, Bengal, Bihar and Asia: , Asia-Tropical :Indian Orissa, the root extract is used to cure Subcontinent[, , rheumatism (Jain 1991; Devi & (Andhra Pradesh, Arunachal Pradesh, Narmathabai, 2011). Its roots possess the Assam, Bihar, Delhi, Goa, Haryana, highly active antioxidant substance which Himachal Pradesh, Jammu and Kashmir, can be used for the treatment of oxidative Karnataka, Kerala, Madhya Pradesh, stress-related diseases (Chidambaram et Maharashtra, Meghalaya, Mizoram, al., 2013; Ileperuma, 2015). In Ayurveda, Nagaland, Orissa, Pondicherry, Punjab, In Jammu, the paste of leaves and fruits Rajasthan, Sikkim, Tamil Nadu, Tripura, are applied on wounds (Bakshi et al. Uttar Pradesh, West Bengal), , 2001; Devi & Narmathabai, 2011). Tribes , Sri Lanka],Indo-China of West Bengal, Orissa and Assam use (, , , , the juice of the flower to treat menstrual ),Malesia(, Philippines), disorder, tuberculosis, sexual disability, Australasia :Australia (tropical) boils and headache (Barua et al. 2003; ("Taxonomy - GRIN-Global Web v Das and Bhadoria 2006; Girach et al. 1.10.3.6", 2018). 2007; Devi & Narmathabai, 2011).

Author: - K.N. Wijesekara - W.S de Silva Department of Biotechnology, Horizon Campus, Sri Lanka GARI Publisher | Medicinal Plants | Volume: 04 | Issue: 07 Article ID: IN/GARI/ICATMMP/2018/115 | Pages: 33-42 (09) ISSN 2424-6492 | ISBN 978-955-7153-00-1 Edit: GARI Editorial Team | Received: 16.12.2018 | Publish: 20.01.2019 A juicy extract from leaves and flowers phenylpropanoids (Ma et al., 2011;Kim et of this plant utilized as a home remedy to al., 2014), glycosides (Ma et al., 2011; treat wounds (Ileperuma, 2015). In China, Kim et al., 2014), and a number of it has been widely used in Ayurvedic volatile oils (Ma et al., 2011; Kim et al., medicine as ‘Codariocalyx motorius 2014), anthraquinones ( Gopalakrishnan ohashi’ and the main constituent of herbal and Rajameena, 2014), saponins tea (Sharma et al., 2003; Chidambaram et (Gopalakrishnan and Rajameena, 2014), al., 2013;Uma et al., 2014).And also it tannins (Gopalakrishnan and Rajameena, has traditionally been used in Chinese 2014), reducing sugars (Gopalakrishnan Medicine to treat various ailments such as and Rajameena, 2014), xantho proteins rheumatism, cough, malaria, pyrexia, (Gopalakrishnan and Rajameena, 2014), dysentery, hepatitis, haemoptysis, etc. amino acid (Gopalakrishnan and (Sharma et al.,2003;Ma et al.,2011;Kim Rajameena, 2014) et al., 2014;Uma et al., 2014; Ileperuma, C. motorius was categorized under 2015) . Extract from D. motorium Fabaceae family. Glycyrrhiza glabra (known as ‘‘anti-aging tea’’) was used in (liquorice) (Nerya et al., 2003), Robinia some parts of ancient Thailand to prevent pseudoacacia (black locust) (Chung et al., several diseases such as cancer of the 1999), Glycine max (soybean) (Lai et al., stomach and intestines, in curing physical 2013) are some plant of Fabaceae family. nerve damage and strengthening the These plants have been reported to immune system. (Devi & Narmathabai, possess skin whitening properties. 2011) Considering previous evidence of skin C.motorius has been reported to contain whitening properties of plant of Fabaceae antioxidant properties (Alam et family, in this study was carried out to be al.,2013;Chidambaram et al., discovered that it has been possessed skin 2013;Gopalakrishnan and Rajameena, whitening properties. 2013), anti-inflammatory properties (Lalramnghinglova 2003; Devi & MATERIALS AND Narmathabai, 2011; Kim et al., 2014) , METHODOLOGY antihyperglycemic properties (Pullaiah, 2002; Devi & Narmathabai, 2011; Uma et

al., 2014), antimicrobial properties (Alam et al.,2013) , antidysenteric property 3.1. Materials (Ghosal et al., 1972;Uma et al., 2014), secondary metabolites of high medicinal L-DOPA (L-3, 4- value (Ghosal et al. 1972; Trout 2004; dihydroxyphenylalanine), mushroom Panda 2004;Devi & Narmathabai, 2011), tyrosinase, DMSO (dimethyl sulphoxide) few alkaloids including were obtained from Sigma-Aldrich. hypaphorine, phenethylamines and SASAKI whitening body skin serum was 5-Methoxy-N,N- purchased from beauty parlor (Borella, dimethyltryptamine Sri Lanaka). (Trout,2002;Chidambaram et 3.2. Plant material and preparation of al.,2013;Kim et al., 2014), 5-methoxy- the extract N,N-dimethyltryptamine (Ileperuma, Fresh leaves of Telegraph plant, 2015), flavonoids(Ma etal.,2011;Kim et Codariocalyx motorius, Pranajeewa (local al., 2014), terpenoids (Ma et al., name) were collected on 6th January 2011;Kim et al., 2014), steroids (Ma et 2018 from Nilgala forest is located in al., 2011;Kim et al., 2014), phenols (Ma Monaralagala district, Uva Province of et al., 2011;Kim et al., 2014), Sri Lanaka. The collected plant part of C. Author: - K.N. Wijesekara - W.S de Silva Department of Biotechnology, Horizon Campus, Sri Lanka GARI Publisher | Medicinal Plants | Volume: 04 | Issue: 07 Article ID: IN/GARI/ICATMMP/2018/115 | Pages: 33-42 (09) ISSN 2424-6492 | ISBN 978-955-7153-00-1 Edit: GARI Editorial Team | Received: 16.12.2018 | Publish: 20.01.2019 motorius was identified and studied Where A=absorbance at 492 nm according to their families at the without test sample, and B=absorbance at Bandaranayake Memorial Ayurvedic 492 nm with test sample. Research Institute, Nawinna, Colombo, Sri Lanka 3.5. Cytotoxicity Assay The leaves were air-dried in shade and the dried material was made to a fine a) powder using an electric grinder. The Hatching brine shrimp resulting residue was dried and extracted 1. 30 ml of sea water were further methanol. The methanolic extract measured using measuring cylinder and (C. motorius) was filtered and poured into a petri dish which was half concentrated in a chamber for 3-5 days to covered by aluminum foil give a 95% methanolic extract of C. 2. About 1g of brine shrimp eggs motorius .Inhibition of tyrosinase activity was added at the top water level of the and cytotoxicity activity were observed in petri dish the methanolic fraction 3. A light (60-100 Watt bulb) was switched on and placed a few inches 3.4. Inhibition of Tyrosinase Activity away from the petri dish Leaves of C.motorius extracts and 4. After 12-24 hours, the nauplii SASAKI whitening body skin serum were hatched were dissolved in DMSO (dimethyl 5. The eggs and nauplii were sulphoxide) to a final concentration of 20 observed mg/ml separately. These stock solutions 6. The nauplii were collected after were then diluted to 600 μg/ml in 50 mM the next 24 hours potassium phosphate buffer (pH 6.5). In 96-well plate, 70 μl of each extract 7. Hatched nauplii were separated dilution was combined with 30 μl of from the empty egg. It was done by tyrosinase (333 Units/ml in phosphate switching off the lamp. The empty eggs buffer) in triplicate. After incubation at floated while the brine shrimp room temperature for 5 min, 110 μl of concentrated in the water column. substrates (12 mM L-DOPA) were added 8. 10 nauplii were transferred to a to each well. Incubation commenced for test tube using a Pasteur pipette 30 min at room temperature. SASAKI Toxicity testing whitening body skin serum used as 9. The nauplii were exposed to reference. Optical densities of the wells different concentrations of the plant were then determined at 492 nm with the extract BIO-TEK Power Wave XS multi-well 10. The number of survivors were plate reader. The concentration of plant counted and calculated the percentage of extract and SASAKI whitening body skin death after 24 hours serum solution at which half the original For each tube, count the number of tyrosinase activity is inhibited (IC50), dead and number of live nauplii, and were determined for. The percentage of determine the % death, inhibition of tyrosinase activity was Percentage of Death (%): (Total naupii calculated as follows: – Alive naupii) x100%/Total naupii

Inhibition %= (A - B)/A × 100 DATA ANALYSIS AND DISCUSSION Author: - K.N. Wijesekara - W.S de Silva Department of Biotechnology, Horizon Campus, Sri Lanka GARI Publisher | Medicinal Plants | Volume: 04 | Issue: 07 Article ID: IN/GARI/ICATMMP/2018/115 | Pages: 33-42 (09) ISSN 2424-6492 | ISBN 978-955-7153-00-1 Edit: GARI Editorial Team | Received: 16.12.2018 | Publish: 20.01.2019 4.1. Antityrosinase IC50 Value

Due to the ethical issues in toxicological tests, substituting animals with alternative models is very important (Hamidi et al., 2014). The effectiveness of the C. motorius bioassay for predicting the toxicity of plant extracts was evaluated by comparing the LC50 results In here, C.motorius methanol extract for the brine shrimps. According to was screened to determine its inhibitory Meyer’s toxicity index, extracts with activities of tyrosinase which key enzyme LC50 < 1000 μg/ml are considered as in melanin synthesis with reference which toxic, while extracts with LC50 > 1000 is a commercially available SASAKI μg/ml are considered as non-toxic (Meyer Whitening Body Skin Serum. Its et al., 1982). Clarkson’s toxicity criterion composition is alpha arbutin, glutathione, for the toxicity assessment of plant vitamin C, E, B3. IC50 value of extracts classifies extracts in the C.motorius methanol extract and following order: extracts with LC50 SASAKI Whitening Body Skin Serum above 1000 μg/ml are non-toxic, LC50 of were given as follows respectively, 500- 1000 μg/ml are low toxic, extracts 282.2907299 ±4.521 µg/ml and with LC50 of 100 - 500 μg/ml are 478.800757±3.1567 µg/ml. This study medium toxic, while extracts with LC50 revealed that C.motorius methanol extract of 0 -100 μg/ml are highly toxic have higher anti tyrosinase activity when (Clarkson et al., 2004). The LC50 value compared to SASAKI Whitening Body obtained for C. motorius methanol extract Skin Serum. Methanolic extract from the using the Brine Shrimp Lethality Assay is leaves of C.motorius was verified a 1516.0538± 2.407µg/ml that indicating significant capability of inhibiting according Meyer’s toxicity index, while it mushroom tyrosinase, are described for is nontoxic. the first time for this biological property. For further studies human melanocytes CONCLUSION must be done in order to clarify antityrosinase activity. The leaf extracts of C. motorius revealed cytotoxic activity against the brine shrimp and considered as containing nontoxic components and also possess highly active antityrosinase substances which can be consumed for remedy of healthy and brighten skin. This Author: - K.N. Wijesekara - W.S de Silva Department of Biotechnology, Horizon Campus, Sri Lanka GARI Publisher | Medicinal Plants | Volume: 04 | Issue: 07 Article ID: IN/GARI/ICATMMP/2018/115 | Pages: 33-42 (09) ISSN 2424-6492 | ISBN 978-955-7153-00-1 Edit: GARI Editorial Team | Received: 16.12.2018 | Publish: 20.01.2019 preliminary screen can be leaded of Baurin, N., Arnoult, E., Scior, T., Do, Q. manufacturing skin whitening cosmetics and Bernard, P. (2002). “Preliminary and therapeutic drugs by developing out screening of some tropical plants for anti- of this research. tyrosinase activity”, Journal of Ethnopharmacology, 82(2-3), pp.155-158.

Carballo, J.L., Hernández-Inda, Z.L., ACKNOWLEDGMENT Pérez, P. and García-Grávalos, M.D. (2002) “A comparison between two brine shrimp I would like to heartiest appreciation, assays to detect in vitro cytotoxicity in marine deepest sense of respect and gratitude to natural products”, BMC biotechnology, 2(1), Dr. Lekha Wanasakera, Dean of Science p.17. Faculty of Horizon Campus, Sri Lanka Chidambaram, U., Pachamuthu, V., for giving approval for my research, then Natarajan, S., Elango, B., Suriyanarayanan my honorable supervisor Mr. Sadin De and Ramkumar, K. (2013), “In vitro evaluation of free radical scavenging activity Silva for his guidance during of Codariocalyx motorius root extract”, Asian development of my research project. Pacific Journal of Tropical Medicine, 6(3), Without his valuable assistance, this work pp.188-194. would not have been a success. It has Chung, J., Jin, G. and Park, Y. 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