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Cite this: DOI: 10.1039/c3an01085f

Kaajal ghts against eye pathogens and is safe for eye make-up: a reinvestigation of an ancient practice
Santi M. Mandal,*a Suman Saha,c Jayangshu Senguptac and Sanjay Pratihar*b

Received 30th May 2013 Accepted 13th July 2013 DOI: 10.1039/c3an01085f www.rsc.org/analyst

The present report includes a scientic view of the traditional use of Kaajal from the leaf of Euphorbia neriifolia, an Indian spurge tree locally called the Monosha plant. A thorough analysis of the material demonstrates that it contains carbon nanoparticles having two uniform sizes of 23 nm and 3545 nm. Further study revealed that Kaajal has antimicrobial properties against eye pathogens and is biocompatible for cosmetic use.

In the past few decades, nanomaterials have been the focus of much interest in the pharmaceutical and cosmetics industries. It is evident that nanomaterials have been consumed by humans in the form of food caramels from ancient times when humans rst started eating bread.13 Few nanoparticles (NPs) have been entered into clinical trials and marketed, but the idea of the consumption of NPs from topical application or some natural process, either for curing a disease or for having nutritional or avouring benets, creates public alarm and concern for public health.13 While NPs are generally considered a discovery of modern science, some examples, like the unique Lycurgus Cup from the fourth century A.D,4 and dyes used for coloring hair black during the Greco-Roman period,5 are evidence that the technology based on nanomaterials was known to dierent civilizations, although the nanoscale nature of their functional constituents has only been revealed recently. The use and presence of nanoparticles in any substances, and their characterization through sophisticated microscopic and analytical techniques, is a fervently challenging area, owing to their short and long term eects on human health and the environment.13 Since the Protodynastic period of Egypt, Kohl has been used as an eye cosmetic, traditionally made by
a

grinding galena (lead sulde) and other ingredients. For decades, various conicting reports have been found relating to whether Kohl application to the eyes is responsible for causing a higher lead concentration in the blood or not.6 Finally, in January 2010, French researchers reported that, at low levels, the specially made lead compounds from the heavy eye make-up of ancient Egyptians actually helped the immune system by stimulating the production of nitric oxide.7 In some rural parts of India, Kaajal is made from the Monosha plant. In a special process, the leaf of the Monosha plant is covered with oil (generally mustard oil) and kept over a burning mud lamp. Within a few minutes, the leaf is covered with creamy so black soot, which is used in the rural area of West Bengal, India, on infants to protect the eye from common infections (Fig. 1). Until now, no reports have been available on the physico-chemical characteristics of Kaajal, nor have there been any assessments of its biocompatibility. The present study is focused on the novel preparation protocol of Kaajal using the ancient approach (see video clip in the ESI) and its characterization with the help of dierent analytical instruments. Moreover, its biological compatibility was also assessed following MTT and haemolytic assay. Transmission electron microscopic (TEM) and X-ray diraction (XRD) analysis revealed that Kaajal contains carbon nanoparticles of dierent sizes ranging from 23 nm and 3545 nm. TEM images of the sample demonstrated the presence of well dispersed and spherical carbon NPs (Fig. 2). Interestingly, two types of carbon nanoparticles were observed with average size of 23 nm (Fig. 2b) and 3545 nm (Fig. 2c). The wide-angle region of the X-ray diraction (XRD) patterns show a

Central Research Facility, Indian Institute of Technology, Kharagpur 721302, India. E-mail: mandalsm@gmail.com; Fax: +91-3222-282481; Tel: +91-3222-282486 Department of Chemical Sciences, Tezpur University, Napaam, 784028, Asam, India

b c

Priyamvada Birla Aravind Eye Hospital, 10, Loudon Street, Kolkata-700017, WB, India Electronic supplementary information (ESI) available: Video clip of ancient synthesis procedure. See DOI: 10.1039/c3an01085f Fig. 1 Preparation (a), use (b), and TEM image (c) of Kaajal.

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Fig. 2 Analytical images of Kaajal prepared from the Monosha leaf. The XRD pattern of the prepared Kaajal (a); TEM images of Kaajal (b & c).

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high-intensity diraction peak at 2q 24.40 and one additional peak at 2q 43.70 that are ascribed to the (002) and (101) diractions of graphitic carbon respectively. Next, we had attempted to check the biocompatibility of the prepared Kaajal to understand the eects of the material in biological systems. For this, we have used the non-carcinoma mouse embryo broblast (3T3) cell lines following MTT assay.8 Interestingly, we did not observe any morphological changes in the 3T3 cells supplemented with Kaajal (up to 1.0 mg mL1) over 24 h of incubation (data not shown). Finally, the results obtained from the MTT assay are found to be in good agreement with their very low toxicity. In this study, haemolytic assay was also performed to examine the interactions of Kaajal with red blood cell membranes. No signicant dierence in haemolysis could be detected between the sample and a negative control aer 1 h of incubation (data not shown). These results are also in good agreement with the morphological and cell cycle analysis studies, which further conrm the biocompatibility of Kaajal. In addition to this, we have performed an in vivo assay on rabbit (Lepus curpaeums) eyes aer creating an infection by putting drops of C. albicans into the eyes four times daily for three days and using the prepared Kaajal to control the infection. Kaajal was used four times daily for 15 days, and the observed infection was not spread further and healed slowly (Fig. 3). This study revealed that Kaajal has no adverse eect on mammalian cells up to a concentration of at least 1 mg mL1 and can be used for eye make-up. So, besides its cosmetic use, is there any benet we can get from the use of Kaajal? For this we have tested the antibacterial and antifungal activity of Kaajal against Pseudomonas aeruginosa ATCC27853, and Candida albicans (keratitis-associated eye pathogens) strains following CLSI guidelines.9 In order to evaluate the antibacterial activity of Kaajal, minimum inhibitory concentration (MIC) values were determined when no visible growth was observed.10,11 The MIC value of Kaajal against all tested fungal and bacterial pathogens was 1.0 g L1. The carbon nanoparticles of Kaajal adhered to the bacterial cells as shown by eld emission scanning electron

Fig. 4 SEM micrographs of bacteria and fungus interacting with Kaajal. C. albicans without treatment (a), treated with Kaajal at 1 mg mL1 (b) and P. aeruginosa without treatment (c), treated with Kaajal at 1 mg mL1 (d). The images indicate the adherence of nanoparticles to the bacterial cells at their surfaces.

microscopic (FESEM) analysis (Fig. 4). SEM images of the bacteria and fungus treated with the nanoparticles showed that the nanoparticles adhered to their cell surfaces and caused drastic damage to their cell walls and cell membranes. The overall charge of the Pseudomonas (gram-negative) cell surface is negative due to the presence of high amounts of lipopolysaccharide containing a negatively charged carboxylic group. The Candida cell wall also contains several groups of lipid moieties that inuence the interaction between the carbon nanoparticles and the cell surface occurring by adsorption or hydrophobic interactions.12 All of the above mentioned studies suggest that Kaajal made from the leaf of the Monosha plant is not toxic and can be used safely for both cosmetic and medicinal uses for the eye.

Conclusions
In summary, our study revealed the presence of carbon nanoparticles in Kaajal, which can ght against the bacterial and fungal pathogens in keratitis-associated eye infections. Biological studies revealed that Kaajal can be considered to have no or minimal risks to human health and may be used for eye make-up safely.

Acknowledgements
The author, SP is nancially supported by DST-New Delhi (to SP for INSPIRE fellowship and research grant), which is gratefully acknowledged.

Notes and references

1 P. Sanguansri and M. A. Augustin, Nanoscale materials development a food industry perspective, Trends Food Sci. Technol., 2006, 17, 547556. 2 T. V. Duncan, The communication challenges presented by nanofoods, Nat. Nanotechnol., 2011, 6, 683688. 3 Md. S. k. Palashuddin, A. Jaiswal, A. Paul, S. S. Ghosh and A. Chattopadhyay, Presence of amorphous carbon nanoparticles in food caramels, Sci. Rep., 2012, 2, 383.
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Fig. 3 Histopathological view of rabbit cornea adjacent epithelial tissue. Images are captured from tissue with a Candida infection (marked with an arrow) (a) and from healed tissue (b).

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Communication 4 D. J. Barber and I. C. Frestone, An investigation of the origin of the color of the Lycurgus cup by analytical transmission electron microscopy, Archaeometry, 1990, 32, 3345. 5 P. Walter, et al., Early use of PbS nanotechnology for an ancient hair dyeing formula, Nano Lett., 2006, 6, 22152219. 6 G. Padmanabhan, Issue Kaajal is good for the eyes, say researchers. But, choose what you wear with care, The Hindu, 18 December 2010. 7 I. Tapsoba, S. Arbault, P. Walter and C. Amatore, Finding out egyptian gods' secret using analytical chemistry: biomedical properties of egyptian black makeup revealed by amperometry at single cells, Anal. Chem., 2010, 82, 457460. 8 S. M. Mandal, L. Migliolo, S. Das, M. Mandal, O. L. Franco and T. K. Hazra, Identication and characterization of a bactericidal and proapoptotic peptide from Cycas revoluta seeds with DNA binding properties, J. Cell. Biochem., 2012, 113, 184193.

Analyst 9 Clinical and Laboratory Standards Institute Performance standards for antimicrobial susceptibility testing. Seventeenth informational supplement Document M100-S17, CLSI, Wayne, PA: CLSI, 2007. 10 J. Sengupta, S. Saha, A. Khetan, S. K. Sarkar and S. M. Mandal, Eects of lactoferricin B against keratitisassociated fungal biolms, J. Infect. Chemother., 2012, 18, 698703. 11 S. M. Mandal, L. Migliolo, O. L. Franco and A. K. Ghosh, Identication of an antifungal peptide from Trapa natans fruits with inhibitory eects on Candida tropicalis biolm formation, Peptides, 2011, 32, 17411747. 12 D. De, S. M. Mandal, S. S. Gauri, R. Bhattacharya, S. Ram and S. K. Roy, Antibacterial eect of lanthanum calcium manganate (La0.67Ca0.33MnO3) nanoparticles against Pseudomonas aeruginosa ATCC 27853, J. Biomed. Nanotechnol., 2010, 6, 138144.

Published on 30 July 2013. Downloaded by Sri Venkateswara University on 04/08/2013 11:04:22.

This journal is The Royal Society of Chemistry 2013

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