ACADEMIA Letters A herbal hand sanitizer prepared with minimal alcohol and the leaf extract of a common weed, Mimosa pudica L. Tharindi Bandara, Department of Botany, Faculty of Science, University of Peradeniya, Sri Lanka Charmalie L Abayasekara, Department of Botany, Faculty of Science, University of Peradeniya, Sri Lanka Gehan J Panagoda, Department of Oral Medicine and Periodontology, Faculty of Dental Sciences, University of Peradeniya, Sri Lanka M. R. Dammantha M Senanayake, Department of Oral Medicine and Periodontology, Faculty of Dental Sciences, University of Peradeniya, Sri Lanka Buddhie S Nanayakkara, Department of Botany, Faculty of Science, University of Peradeniya, Sri Lanka Anjani M Karunaratne, Department of Botany, Faculty of Science, University of Peradeniya, Sri Lanka Abstract Hand sanitizer (HS) usage, prevents infections of transient microflora, while alcohol based HSs are currently used widely. The high level of alcohol in the WHO-recommended HS (75%) could cause dry skin. The aim of this study, was to develop a herbal HS using a leaf extract of Mimosa pudica L. with a reduced alcohol content (40%). The newly developed herbal HS was both effective against tested transient and resident bacterial and fungal flora on hands and was consumer acceptable although its colour may be improved. The alcohol content of this herbal HS should be sufficient to reduce spread of COVID-19, as SARS-CoV-2 is recorded to be inhibited by a minimum of 30% alcohol. Academia Letters, August 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0 Corresponding Author: Anjani M Karunaratne, amkarunaratne21@gmail.com Citation: Bandara, T., Abayasekara, C.L., Panagoda, G.J., Senanayake, M.R.D.M., Nanayakkara, B.S., Karunaratne, A.M. (2021). A herbal hand sanitizer prepared with minimal alcohol and the leaf extract of a common weed, Mimosa pudica L. Academia Letters, Article 3288. https://doi.org/10.20935/AL3288. 1 Introduction Hand sanitizer (HS) usage, a practice that was mainly confined to hospitals in the pre-covid era has now become indispensable and has trickled down to all levels of society worldwide, with the onset of the COVID-19 pandemic. An important component of HSs is alcohol (Balkrishna et al., 2020) and the World Health Organization (WHO) recommends 60–80% alcohol for hand hygiene products (WHO, 2009). A higher percentage of alcohol (at least 60%) effectively kills germs (FDA, 2020) and they dry quickly leaving no sticky residues (WHO, 2006). However, a high alcohol content excessively dries the skin, and it is reported that in children below 12 years, exposure to alcohol based HSs has caused toxicity problems even resulting in a few deaths (Alghamdi, 2021). Yet, decreasing the alcohol content below 60% has not been recommended because of the reduction of its germicidal activity against Gram-positive and Gram-negative vegetative bacteria including multidrug-resistant pathogens (WHO, 2009). However, recently it has been demonstrated that alcohol (either ethyl-or isopropyl-) at a concentration of >30% (v/v) efficiently inactivated SARS-CoV-2, the virus responsible for COVID-19 (Kratzel et al., 2020). A herbal HS that is complemented with a plant component that has natural antimicrobial activity is a possible alternative to alcohol-based sanitizers currently in use. The plant to be incorporated needs to be readily available, while possessing antimicrobial properties. Mimosa pudica L. is a common garden weed which is native to South America, and now has become pantropical in its distribution (GISD, 2021). In Sri Lanka M. pudica is a well known garden weed (Dassanayake et al., 1980) and is a medicinal plant used traditionally for treating skin infections (Rajalakshmi & Banu, 2016) with antibacterial and antifungal flavonoids distributed in the whole plant (Le Thoa et al, 2015). In a previous investigation by Grace et al. (2015) on herbal sanitizer formulation, the glycerol content has been set at 5%. The objective of the present study was to use the guidelines given for the preparation of a HS by the WHO, and develop a herbal HS with a crude alcoholic leaf extract of M. pudica and a reduced level (40%) of isopropyl alcohol, and increased level of glycerol (5%) to test its activity against transient bacterial and fungal microflora of the skin. Academia Letters, August 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0 Corresponding Author: Anjani M Karunaratne, amkarunaratne21@gmail.com Citation: Bandara, T., Abayasekara, C.L., Panagoda, G.J., Senanayake, M.R.D.M., Nanayakkara, B.S., Karunaratne, A.M. (2021). A herbal hand sanitizer prepared with minimal alcohol and the leaf extract of a common weed, Mimosa pudica L. Academia Letters, Article 3288. https://doi.org/10.20935/AL3288. 2 Materials and Methods Leaf samples: Healthy young leaves of M. pudica without petioles were collected from home gardens in Kandy, Sri Lanka (Coordinates- 7.28, 80.58). They were washed thoroughly with water, then with 70% ethanol (3 min), thereafter with 5% sodium hypochlorite (5 min) and finally with distilled water. Antimicrobial compound extraction: Fresh leaves were cut into pieces (5 mm length). Cut leaves (40 g) were subjected to vacuum infiltration (2 h) in ethanol (400 ml), filtered (F1001-CHMLAB), evaporated (40 oC, 12 h) and the crude extract was collected. Microbial cultures: Pure cultures of bacteria Escherichia coli (ATCC25922), Pseudomonas aeruginosa (ATCC27853), Staphylococcus aureus (ATCC25923) and yeasts Candida albicans (ATCC 10231), Candida tropicalis (ATCC 13803) were obtained from the Division of Microbiology, Department of Oral Medicine and Periodontology, Faculty of Dental Sciences, University of Peradeniya.While Blood agar was used for sub-culturing bacteria, Sabouraud dextrose agar was used for yeasts (37 oC, 24 h). Samples for minimum inhibitory concentration: The leaf extract at five concentrations (100,000 mg/L, 50,000 mg/L, 10,000 mg/L, 5000 mg/L, and 1000 mg/L), was incorporated to prepare herbal HSs with 40% isopropyl alcohol, 5% glycerol and distilled water. While the WHO-recommended HS (WHO, 2009) comprising (v/v) 75% isopropyl alcohol, 1.45% glycerol, 0.125% hydrogen peroxide and distilled water served as the positive control, the negative control comprised 40% isopropyl alcohol, 5% glycerol and distilled water. Agar well diffusion assay: The agar well diffusion assay described in Nanayakkara et al. (2014) was performed using the five microbial cultures mentioned above, with following modifications. Equidistant wells of 9 Academia Letters, August 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0 Corresponding Author: Anjani M Karunaratne, amkarunaratne21@gmail.com Citation: Bandara, T., Abayasekara, C.L., Panagoda, G.J., Senanayake, M.R.D.M., Nanayakkara, B.S., Karunaratne, A.M. (2021). A herbal hand sanitizer prepared with minimal alcohol and the leaf extract of a common weed, Mimosa pudica L. Academia Letters, Article 3288. https://doi.org/10.20935/AL3288. 3 mm diameter were bored on each Mueller Hinton Agar (MHA) plate and the wells were sealed using molten MHA. The following (50 µl) were loaded to the wells separately; HS samples having M. pudica leaf extract at five different concentrations, negative control and positive control. The plates were incubated at 37 oC for 24 h. The radius of the zone of inhibition around each well was measured. The experiment was done in two trials in quadruplicate for each organism. Means and standard deviations for the average radii of the inhibition zones were subjected to a t-test to determine significant differences at P=0.05. Sensory evaluation test A sensory evaluation test was carried out as a discrimination test to determine differences among the three samples; WHO recommended HS, a widely available commercial HS and the developed herbal HS. The analysis included 30 untrained respondents (male-14 & female16) randomly selected in the age range of 20-70 y. The respondents were asked to apply the sample (about 1.5 ml) on to their palms, and blindly score for each organoleptic parameter (viscosity, colour, feel and aroma) and overall acceptability on a hedonic scale of 1 (Extremely disliked) to 9 (Extremely liked). Data were analyzed non-parametrically. Results & Discussion As test microorganisms, common transient and resident microflora (WHO, 2009) were chosen. The Minimum Inhibitory Concentration (MIC), of the leaf extract was detected at 10,000 mg/L. The MIC and higher concentrations, as well as the positive control gave inhibition zones against all five microorganisms (Table 1), whereas the two lower concentrations did not show any inhibition. The inhibition zones at MIC were significantly lower (P=0.05) compared with that of WHO HS, for the bacterial species, while that of both Candida spp. did not show a significant difference. Academia Letters, August 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0 Corresponding Author: Anjani M Karunaratne, amkarunaratne21@gmail.com Citation: Bandara, T., Abayasekara, C.L., Panagoda, G.J., Senanayake, M.R.D.M., Nanayakkara, B.S., Karunaratne, A.M. (2021). A herbal hand sanitizer prepared with minimal alcohol and the leaf extract of a common weed, Mimosa pudica L. Academia Letters, Article 3288. https://doi.org/10.20935/AL3288. 4 Academia Letters, August 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0 Corresponding Author: Anjani M Karunaratne, amkarunaratne21@gmail.com Citation: Bandara, T., Abayasekara, C.L., Panagoda, G.J., Senanayake, M.R.D.M., Nanayakkara, B.S., Karunaratne, A.M. (2021). A herbal hand sanitizer prepared with minimal alcohol and the leaf extract of a common weed, Mimosa pudica L. Academia Letters, Article 3288. https://doi.org/10.20935/AL3288. 5 Scores of sensory evaluation showed that the herbal HS had an overall acceptability in between WHO HS and Commercial HS (Table 2).Among the three HSs, there was no significant difference (P=0.05) for the organoleptic parameter of viscosity, while significant differences were observed for colour, feel, and aroma. While the feel and aroma of the herbal HS were preferred, its colour (moss green) was least preferred compared to that of WHO (colourless) and commercial HSs (sapphire blue), the latter being most preferred (P=0.05). The overall acceptability of the herbal HS compared well with those of the WHO and commercial HS. It is envisaged that the use of HSs has come to stay even in the post-covid era. Therefore, making HSs that are pleasing to the consumer is important in the competitive world. Academia Letters, August 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0 Corresponding Author: Anjani M Karunaratne, amkarunaratne21@gmail.com Citation: Bandara, T., Abayasekara, C.L., Panagoda, G.J., Senanayake, M.R.D.M., Nanayakkara, B.S., Karunaratne, A.M. (2021). A herbal hand sanitizer prepared with minimal alcohol and the leaf extract of a common weed, Mimosa pudica L. Academia Letters, Article 3288. https://doi.org/10.20935/AL3288. 6 While the newly developed HS inhibited major clinically important microorganisms present in the hand flora, it showed good acceptability among users, except for its colour. In the precovid era, much emphasis has been on controlling microorganisms of surgical site infections and nosocomial infections and the reference alcohol-based hand rub has been specified to contain isopropyl alcohol at 60% (v/v) (WHO, 2009). In the future it is important to test all HSs for their efficacy against viral pathogens as well. Kretzel et al. (2020) demonstrated efficient virus inactivation by the WHO-HS and they discovered that a minimum concentration of 30% (v/v) alcohol was needed to combat SARS-CoV-2.We demonstrated that the herbal HS developed using the recipe of WHO-HS with reduced alcohol is capable of inhibiting five key human pathogens (bacteria and yeasts) and based on the results of Kretzel et al. (2020) its alcohol content is sufficient to control SARS-CoV-2. Also, this formulation with reduced alcohol would be ideal to avoid adverse effects of alcohol, while also minimizing the cost of alcohol and in turn the cost of production, as the herbal extract would not cost as much, while being ecofriendly. References Alghamdi, H. A. (2021). A need to combat COVID-19; herbal disinfection techniques, formulations and preparations of human health friendly hand sanitizers. Saudi Journal of Biological Sciences, 28(2021),3943–3947.https://doi.org/10.1016/j.sjbs.2021.03.077 Balkrishna, A., Singh, K., Singh, H., Haldar, S., and Varshney, A. (2020). GermiX: A skin friendly hand sanitizer with prolonged effectivity against pathogenic bacteria. AMB Express, 10(1), 1-8. https://doi.org/10.1186/s13568-020-01151-y Dassanayake, M. D., Fosberg, F. R., and Clayton, W. D. (1980). Flora of Ceylon. Amerind publishing Co.Pvt. Ltd., New Delhi, 1, Pp. 463-464. FDA (2020). U.S. Foodand Drug Association, Coronavirus Disease 2019 (COVID-19), 2020 Performance Report – 2021. Washington, DC, Retrieved from https://www.fda.gov accessed on 1st of May, 2021. GISD (2021). Global Invasive Species Database, Mimosa pudica. Retrieved from http:// www.iucngisd.org/gisd/search.php on 11th of August 2021. Grace, X., Sowmya, K., Darsika, C., Jothy, A., & Shanmuganathan, S. (2015). Polyherbal hand sanitizer-formulation and evaluation. Indian Journal of Pharmacy and Pharmacology, 2 (2), 143-144. https://www.ijpp.org.in/media/journals/Article143-144.pdf Academia Letters, August 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0 Corresponding Author: Anjani M Karunaratne, amkarunaratne21@gmail.com Citation: Bandara, T., Abayasekara, C.L., Panagoda, G.J., Senanayake, M.R.D.M., Nanayakkara, B.S., Karunaratne, A.M. (2021). A herbal hand sanitizer prepared with minimal alcohol and the leaf extract of a common weed, Mimosa pudica L. Academia Letters, Article 3288. https://doi.org/10.20935/AL3288. 7 Kratzel, A., Todt, D., V’kovski, et al (2020). Inactivation of severe acute respiratory syndrome coronavirus 2 by WHO-recommended hand rub formulations and alcohols. Emerging infectious diseases, 26 (7), 1592.doi: 10.3201/eid2607.200915 Le Thoa, N. T., Nam, P. C., and Nhat, D. M. (2015). Antibacterial activities of the extracts of Mimosa pudica L. An in-vitro Study. International Journal on Advanced Science Engineering and InformationTechnology, 5 (5), 358-361. https://core.ac.uk/download/pdf/ 296921824.pdf Nanayakkara, B.S.,Abayasekara, C.L., Panagoda, G.J., Kanatiwela, H.M.D.K. and Senanayake, M.R.D.M. (2014). Anti-candidal activity of Piper betle (L.), Vitex negundo (L.) and Jasminum grandiflorum (L.). African Journal of Microbiology Research. 8 (23), 2307-2314. https://doi.org/10.5897/AJMR2014.6713 Rajalakshmi, K., and Banu, N. (2016). Antimicrobial activity of natural chlorophyllin from endangered medicinal plant Mimosa pudica L. International Journal of Pharmacy and Pharmaceutical Science, 8 (4), 387-389. World Health Organization & WHO Patient Safety. (2009). WHO guidelines on hand hygiene in health care : a summary. World Health Organization https://apps.who.int/iris/handle/ 10665/70126 Academia Letters, August 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0 Corresponding Author: Anjani M Karunaratne, amkarunaratne21@gmail.com Citation: Bandara, T., Abayasekara, C.L., Panagoda, G.J., Senanayake, M.R.D.M., Nanayakkara, B.S., Karunaratne, A.M. (2021). A herbal hand sanitizer prepared with minimal alcohol and the leaf extract of a common weed, Mimosa pudica L. Academia Letters, Article 3288. https://doi.org/10.20935/AL3288. 8 

ACADEMIA Letters A herbal hand sanitizer prepared with minimal alcohol and the leaf extract of a common weed, Mimosa pudica L. Tharindi Bandara, Department of Botany, Faculty of Science, University of Peradeniya, Sri Lanka Charmalie L Abayasekara, Department of Botany, Faculty of Science, University of Peradeniya, Sri Lanka Gehan J Panagoda, Department of Oral Medicine and Periodontology, Faculty of Dental Sciences, University of Peradeniya, Sri Lanka M. R. Dammantha M Senanayake, Department of Oral Medicine and Periodontology, Faculty of Dental Sciences, University of Peradeniya, Sri Lanka Buddhie S Nanayakkara, Department of Botany, Faculty of Science, University of Peradeniya, Sri Lanka Anjani M Karunaratne, Department of Botany, Faculty of Science, University of Peradeniya, Sri Lanka Abstract Hand sanitizer (HS) usage, prevents infections of transient microflora, while alcohol based HSs are currently used widely. The high level of alcohol in the WHO-recommended HS (75%) could cause dry skin. The aim of this study, was to develop a herbal HS using a leaf extract of Mimosa pudica L. with a reduced alcohol content (40%). The newly developed herbal HS was both effective against tested transient and resident bacterial and fungal flora on hands and was consumer acceptable although its colour may be improved. The alcohol content of this herbal HS should be sufficient to reduce spread of COVID-19, as SARS-CoV-2 is recorded to be inhibited by a minimum of 30% alcohol. Academia Letters, August 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0 Corresponding Author: Anjani M Karunaratne, amkarunaratne21@gmail.com Citation: Bandara, T., Abayasekara, C.L., Panagoda, G.J., Senanayake, M.R.D.M., Nanayakkara, B.S., Karunaratne, A.M. (2021). A herbal hand sanitizer prepared with minimal alcohol and the leaf extract of a common weed, Mimosa pudica L. Academia Letters, Article 3288. https://doi.org/10.20935/AL3288. 1 Introduction Hand sanitizer (HS) usage, a practice that was mainly confined to hospitals in the pre-covid era has now become indispensable and has trickled down to all levels of society worldwide, with the onset of the COVID-19 pandemic. An important component of HSs is alcohol (Balkrishna et al., 2020) and the World Health Organization (WHO) recommends 60–80% alcohol for hand hygiene products (WHO, 2009). A higher percentage of alcohol (at least 60%) effectively kills germs (FDA, 2020) and they dry quickly leaving no sticky residues (WHO, 2006). However, a high alcohol content excessively dries the skin, and it is reported that in children below 12 years, exposure to alcohol based HSs has caused toxicity problems even resulting in a few deaths (Alghamdi, 2021). Yet, decreasing the alcohol content below 60% has not been recommended because of the reduction of its germicidal activity against Gram-positive and Gram-negative vegetative bacteria including multidrug-resistant pathogens (WHO, 2009). However, recently it has been demonstrated that alcohol (either ethyl-or isopropyl-) at a concentration of >30% (v/v) efficiently inactivated SARS-CoV-2, the virus responsible for COVID-19 (Kratzel et al., 2020). A herbal HS that is complemented with a plant component that has natural antimicrobial activity is a possible alternative to alcohol-based sanitizers currently in use. The plant to be incorporated needs to be readily available, while possessing antimicrobial properties. Mimosa pudica L. is a common garden weed which is native to South America, and now has become pantropical in its distribution (GISD, 2021). In Sri Lanka M. pudica is a well known garden weed (Dassanayake et al., 1980) and is a medicinal plant used traditionally for treating skin infections (Rajalakshmi & Banu, 2016) with antibacterial and antifungal flavonoids distributed in the whole plant (Le Thoa et al, 2015). In a previous investigation by Grace et al. (2015) on herbal sanitizer formulation, the glycerol content has been set at 5%. The objective of the present study was to use the guidelines given for the preparation of a HS by the WHO, and develop a herbal HS with a crude alcoholic leaf extract of M. pudica and a reduced level (40%) of isopropyl alcohol, and increased level of glycerol (5%) to test its activity against transient bacterial and fungal microflora of the skin. Academia Letters, August 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0 Corresponding Author: Anjani M Karunaratne, amkarunaratne21@gmail.com Citation: Bandara, T., Abayasekara, C.L., Panagoda, G.J., Senanayake, M.R.D.M., Nanayakkara, B.S., Karunaratne, A.M. (2021). A herbal hand sanitizer prepared with minimal alcohol and the leaf extract of a common weed, Mimosa pudica L. Academia Letters, Article 3288. https://doi.org/10.20935/AL3288. 2 Materials and Methods Leaf samples: Healthy young leaves of M. pudica without petioles were collected from home gardens in Kandy, Sri Lanka (Coordinates- 7.28, 80.58). They were washed thoroughly with water, then with 70% ethanol (3 min), thereafter with 5% sodium hypochlorite (5 min) and finally with distilled water. Antimicrobial compound extraction: Fresh leaves were cut into pieces (5 mm length). Cut leaves (40 g) were subjected to vacuum infiltration (2 h) in ethanol (400 ml), filtered (F1001-CHMLAB), evaporated (40 oC, 12 h) and the crude extract was collected. Microbial cultures: Pure cultures of bacteria Escherichia coli (ATCC25922), Pseudomonas aeruginosa (ATCC27853), Staphylococcus aureus (ATCC25923) and yeasts Candida albicans (ATCC 10231), Candida tropicalis (ATCC 13803) were obtained from the Division of Microbiology, Department of Oral Medicine and Periodontology, Faculty of Dental Sciences, University of Peradeniya.While Blood agar was used for sub-culturing bacteria, Sabouraud dextrose agar was used for yeasts (37 oC, 24 h). Samples for minimum inhibitory concentration: The leaf extract at five concentrations (100,000 mg/L, 50,000 mg/L, 10,000 mg/L, 5000 mg/L, and 1000 mg/L), was incorporated to prepare herbal HSs with 40% isopropyl alcohol, 5% glycerol and distilled water. While the WHO-recommended HS (WHO, 2009) comprising (v/v) 75% isopropyl alcohol, 1.45% glycerol, 0.125% hydrogen peroxide and distilled water served as the positive control, the negative control comprised 40% isopropyl alcohol, 5% glycerol and distilled water. Agar well diffusion assay: The agar well diffusion assay described in Nanayakkara et al. (2014) was performed using the five microbial cultures mentioned above, with following modifications. Equidistant wells of 9 Academia Letters, August 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0 Corresponding Author: Anjani M Karunaratne, amkarunaratne21@gmail.com Citation: Bandara, T., Abayasekara, C.L., Panagoda, G.J., Senanayake, M.R.D.M., Nanayakkara, B.S., Karunaratne, A.M. (2021). A herbal hand sanitizer prepared with minimal alcohol and the leaf extract of a common weed, Mimosa pudica L. Academia Letters, Article 3288. https://doi.org/10.20935/AL3288. 3 mm diameter were bored on each Mueller Hinton Agar (MHA) plate and the wells were sealed using molten MHA. The following (50 µl) were loaded to the wells separately; HS samples having M. pudica leaf extract at five different concentrations, negative control and positive control. The plates were incubated at 37 oC for 24 h. The radius of the zone of inhibition around each well was measured. The experiment was done in two trials in quadruplicate for each organism. Means and standard deviations for the average radii of the inhibition zones were subjected to a t-test to determine significant differences at P=0.05. Sensory evaluation test A sensory evaluation test was carried out as a discrimination test to determine differences among the three samples; WHO recommended HS, a widely available commercial HS and the developed herbal HS. The analysis included 30 untrained respondents (male-14 & female16) randomly selected in the age range of 20-70 y. The respondents were asked to apply the sample (about 1.5 ml) on to their palms, and blindly score for each organoleptic parameter (viscosity, colour, feel and aroma) and overall acceptability on a hedonic scale of 1 (Extremely disliked) to 9 (Extremely liked). Data were analyzed non-parametrically. Results & Discussion As test microorganisms, common transient and resident microflora (WHO, 2009) were chosen. The Minimum Inhibitory Concentration (MIC), of the leaf extract was detected at 10,000 mg/L. The MIC and higher concentrations, as well as the positive control gave inhibition zones against all five microorganisms (Table 1), whereas the two lower concentrations did not show any inhibition. The inhibition zones at MIC were significantly lower (P=0.05) compared with that of WHO HS, for the bacterial species, while that of both Candida spp. did not show a significant difference. Academia Letters, August 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0 Corresponding Author: Anjani M Karunaratne, amkarunaratne21@gmail.com Citation: Bandara, T., Abayasekara, C.L., Panagoda, G.J., Senanayake, M.R.D.M., Nanayakkara, B.S., Karunaratne, A.M. (2021). A herbal hand sanitizer prepared with minimal alcohol and the leaf extract of a common weed, Mimosa pudica L. Academia Letters, Article 3288. https://doi.org/10.20935/AL3288. 4 Academia Letters, August 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0 Corresponding Author: Anjani M Karunaratne, amkarunaratne21@gmail.com Citation: Bandara, T., Abayasekara, C.L., Panagoda, G.J., Senanayake, M.R.D.M., Nanayakkara, B.S., Karunaratne, A.M. (2021). A herbal hand sanitizer prepared with minimal alcohol and the leaf extract of a common weed, Mimosa pudica L. Academia Letters, Article 3288. https://doi.org/10.20935/AL3288. 5 Scores of sensory evaluation showed that the herbal HS had an overall acceptability in between WHO HS and Commercial HS (Table 2).Among the three HSs, there was no significant difference (P=0.05) for the organoleptic parameter of viscosity, while significant differences were observed for colour, feel, and aroma. While the feel and aroma of the herbal HS were preferred, its colour (moss green) was least preferred compared to that of WHO (colourless) and commercial HSs (sapphire blue), the latter being most preferred (P=0.05). The overall acceptability of the herbal HS compared well with those of the WHO and commercial HS. It is envisaged that the use of HSs has come to stay even in the post-covid era. Therefore, making HSs that are pleasing to the consumer is important in the competitive world. Academia Letters, August 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0 Corresponding Author: Anjani M Karunaratne, amkarunaratne21@gmail.com Citation: Bandara, T., Abayasekara, C.L., Panagoda, G.J., Senanayake, M.R.D.M., Nanayakkara, B.S., Karunaratne, A.M. (2021). A herbal hand sanitizer prepared with minimal alcohol and the leaf extract of a common weed, Mimosa pudica L. Academia Letters, Article 3288. https://doi.org/10.20935/AL3288. 6 While the newly developed HS inhibited major clinically important microorganisms present in the hand flora, it showed good acceptability among users, except for its colour. In the precovid era, much emphasis has been on controlling microorganisms of surgical site infections and nosocomial infections and the reference alcohol-based hand rub has been specified to contain isopropyl alcohol at 60% (v/v) (WHO, 2009). In the future it is important to test all HSs for their efficacy against viral pathogens as well. Kretzel et al. (2020) demonstrated efficient virus inactivation by the WHO-HS and they discovered that a minimum concentration of 30% (v/v) alcohol was needed to combat SARS-CoV-2.We demonstrated that the herbal HS developed using the recipe of WHO-HS with reduced alcohol is capable of inhibiting five key human pathogens (bacteria and yeasts) and based on the results of Kretzel et al. (2020) its alcohol content is sufficient to control SARS-CoV-2. Also, this formulation with reduced alcohol would be ideal to avoid adverse effects of alcohol, while also minimizing the cost of alcohol and in turn the cost of production, as the herbal extract would not cost as much, while being ecofriendly. References Alghamdi, H. A. (2021). A need to combat COVID-19; herbal disinfection techniques, formulations and preparations of human health friendly hand sanitizers. Saudi Journal of Biological Sciences, 28(2021),3943–3947.https://doi.org/10.1016/j.sjbs.2021.03.077 Balkrishna, A., Singh, K., Singh, H., Haldar, S., and Varshney, A. (2020). GermiX: A skin friendly hand sanitizer with prolonged effectivity against pathogenic bacteria. AMB Express, 10(1), 1-8. https://doi.org/10.1186/s13568-020-01151-y Dassanayake, M. D., Fosberg, F. R., and Clayton, W. D. (1980). Flora of Ceylon. Amerind publishing Co.Pvt. Ltd., New Delhi, 1, Pp. 463-464. FDA (2020). U.S. Foodand Drug Association, Coronavirus Disease 2019 (COVID-19), 2020 Performance Report – 2021. Washington, DC, Retrieved from https://www.fda.gov accessed on 1st of May, 2021. GISD (2021). Global Invasive Species Database, Mimosa pudica. Retrieved from http:// www.iucngisd.org/gisd/search.php on 11th of August 2021. Grace, X., Sowmya, K., Darsika, C., Jothy, A., & Shanmuganathan, S. (2015). Polyherbal hand sanitizer-formulation and evaluation. Indian Journal of Pharmacy and Pharmacology, 2 (2), 143-144. https://www.ijpp.org.in/media/journals/Article143-144.pdf Academia Letters, August 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0 Corresponding Author: Anjani M Karunaratne, amkarunaratne21@gmail.com Citation: Bandara, T., Abayasekara, C.L., Panagoda, G.J., Senanayake, M.R.D.M., Nanayakkara, B.S., Karunaratne, A.M. (2021). A herbal hand sanitizer prepared with minimal alcohol and the leaf extract of a common weed, Mimosa pudica L. Academia Letters, Article 3288. https://doi.org/10.20935/AL3288. 7 Kratzel, A., Todt, D., V’kovski, et al (2020). Inactivation of severe acute respiratory syndrome coronavirus 2 by WHO-recommended hand rub formulations and alcohols. Emerging infectious diseases, 26 (7), 1592.doi: 10.3201/eid2607.200915 Le Thoa, N. T., Nam, P. C., and Nhat, D. M. (2015). Antibacterial activities of the extracts of Mimosa pudica L. An in-vitro Study. International Journal on Advanced Science Engineering and InformationTechnology, 5 (5), 358-361. https://core.ac.uk/download/pdf/ 296921824.pdf Nanayakkara, B.S.,Abayasekara, C.L., Panagoda, G.J., Kanatiwela, H.M.D.K. and Senanayake, M.R.D.M. (2014). Anti-candidal activity of Piper betle (L.), Vitex negundo (L.) and Jasminum grandiflorum (L.). African Journal of Microbiology Research. 8 (23), 2307-2314. https://doi.org/10.5897/AJMR2014.6713 Rajalakshmi, K., and Banu, N. (2016). Antimicrobial activity of natural chlorophyllin from endangered medicinal plant Mimosa pudica L. International Journal of Pharmacy and Pharmaceutical Science, 8 (4), 387-389. World Health Organization & WHO Patient Safety. (2009). WHO guidelines on hand hygiene in health care : a summary. World Health Organization https://apps.who.int/iris/handle/ 10665/70126 Academia Letters, August 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0 Corresponding Author: Anjani M Karunaratne, amkarunaratne21@gmail.com Citation: Bandara, T., Abayasekara, C.L., Panagoda, G.J., Senanayake, M.R.D.M., Nanayakkara, B.S., Karunaratne, A.M. (2021). A herbal hand sanitizer prepared with minimal alcohol and the leaf extract of a common weed, Mimosa pudica L. Academia Letters, Article 3288. https://doi.org/10.20935/AL3288. 8 

ACADEMIA Letters A herbal hand sanitizer prepared with minimal alcohol and the leaf extract of a common weed, Mimosa pudica L. Tharindi Bandara, Department of Botany, Faculty of Science, University of Peradeniya, Sri Lanka Charmalie L Abayasekara, Department of Botany, Faculty of Science, University of Peradeniya, Sri Lanka Gehan J Panagoda, Department of Oral Medicine and Periodontology, Faculty of Dental Sciences, University of Peradeniya, Sri Lanka M. R. Dammantha M Senanayake, Department of Oral Medicine and Periodontology, Faculty of Dental Sciences, University of Peradeniya, Sri Lanka Buddhie S Nanayakkara, Department of Botany, Faculty of Science, University of Peradeniya, Sri Lanka Anjani M Karunaratne, Department of Botany, Faculty of Science, University of Peradeniya, Sri Lanka Abstract Hand sanitizer (HS) usage, prevents infections of transient microflora, while alcohol based HSs are currently used widely. The high level of alcohol in the WHO-recommended HS (75%) could cause dry skin. The aim of this study, was to develop a herbal HS using a leaf extract of Mimosa pudica L. with a reduced alcohol content (40%). The newly developed herbal HS was both effective against tested transient and resident bacterial and fungal flora on hands and was consumer acceptable although its colour may be improved. The alcohol content of this herbal HS should be sufficient to reduce spread of COVID-19, as SARS-CoV-2 is recorded to be inhibited by a minimum of 30% alcohol. Academia Letters, August 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0 Corresponding Author: Anjani M Karunaratne, amkarunaratne21@gmail.com Citation: Bandara, T., Abayasekara, C.L., Panagoda, G.J., Senanayake, M.R.D.M., Nanayakkara, B.S., Karunaratne, A.M. (2021). A herbal hand sanitizer prepared with minimal alcohol and the leaf extract of a common weed, Mimosa pudica L. Academia Letters, Article 3288. https://doi.org/10.20935/AL3288. 1 Introduction Hand sanitizer (HS) usage, a practice that was mainly confined to hospitals in the pre-covid era has now become indispensable and has trickled down to all levels of society worldwide, with the onset of the COVID-19 pandemic. An important component of HSs is alcohol (Balkrishna et al., 2020) and the World Health Organization (WHO) recommends 60–80% alcohol for hand hygiene products (WHO, 2009). A higher percentage of alcohol (at least 60%) effectively kills germs (FDA, 2020) and they dry quickly leaving no sticky residues (WHO, 2006). However, a high alcohol content excessively dries the skin, and it is reported that in children below 12 years, exposure to alcohol based HSs has caused toxicity problems even resulting in a few deaths (Alghamdi, 2021). Yet, decreasing the alcohol content below 60% has not been recommended because of the reduction of its germicidal activity against Gram-positive and Gram-negative vegetative bacteria including multidrug-resistant pathogens (WHO, 2009). However, recently it has been demonstrated that alcohol (either ethyl-or isopropyl-) at a concentration of >30% (v/v) efficiently inactivated SARS-CoV-2, the virus responsible for COVID-19 (Kratzel et al., 2020). A herbal HS that is complemented with a plant component that has natural antimicrobial activity is a possible alternative to alcohol-based sanitizers currently in use. The plant to be incorporated needs to be readily available, while possessing antimicrobial properties. Mimosa pudica L. is a common garden weed which is native to South America, and now has become pantropical in its distribution (GISD, 2021). In Sri Lanka M. pudica is a well known garden weed (Dassanayake et al., 1980) and is a medicinal plant used traditionally for treating skin infections (Rajalakshmi & Banu, 2016) with antibacterial and antifungal flavonoids distributed in the whole plant (Le Thoa et al, 2015). In a previous investigation by Grace et al. (2015) on herbal sanitizer formulation, the glycerol content has been set at 5%. The objective of the present study was to use the guidelines given for the preparation of a HS by the WHO, and develop a herbal HS with a crude alcoholic leaf extract of M. pudica and a reduced level (40%) of isopropyl alcohol, and increased level of glycerol (5%) to test its activity against transient bacterial and fungal microflora of the skin. Academia Letters, August 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0 Corresponding Author: Anjani M Karunaratne, amkarunaratne21@gmail.com Citation: Bandara, T., Abayasekara, C.L., Panagoda, G.J., Senanayake, M.R.D.M., Nanayakkara, B.S., Karunaratne, A.M. (2021). A herbal hand sanitizer prepared with minimal alcohol and the leaf extract of a common weed, Mimosa pudica L. Academia Letters, Article 3288. https://doi.org/10.20935/AL3288. 2 Materials and Methods Leaf samples: Healthy young leaves of M. pudica without petioles were collected from home gardens in Kandy, Sri Lanka (Coordinates- 7.28, 80.58). They were washed thoroughly with water, then with 70% ethanol (3 min), thereafter with 5% sodium hypochlorite (5 min) and finally with distilled water. Antimicrobial compound extraction: Fresh leaves were cut into pieces (5 mm length). Cut leaves (40 g) were subjected to vacuum infiltration (2 h) in ethanol (400 ml), filtered (F1001-CHMLAB), evaporated (40 oC, 12 h) and the crude extract was collected. Microbial cultures: Pure cultures of bacteria Escherichia coli (ATCC25922), Pseudomonas aeruginosa (ATCC27853), Staphylococcus aureus (ATCC25923) and yeasts Candida albicans (ATCC 10231), Candida tropicalis (ATCC 13803) were obtained from the Division of Microbiology, Department of Oral Medicine and Periodontology, Faculty of Dental Sciences, University of Peradeniya.While Blood agar was used for sub-culturing bacteria, Sabouraud dextrose agar was used for yeasts (37 oC, 24 h). Samples for minimum inhibitory concentration: The leaf extract at five concentrations (100,000 mg/L, 50,000 mg/L, 10,000 mg/L, 5000 mg/L, and 1000 mg/L), was incorporated to prepare herbal HSs with 40% isopropyl alcohol, 5% glycerol and distilled water. While the WHO-recommended HS (WHO, 2009) comprising (v/v) 75% isopropyl alcohol, 1.45% glycerol, 0.125% hydrogen peroxide and distilled water served as the positive control, the negative control comprised 40% isopropyl alcohol, 5% glycerol and distilled water. Agar well diffusion assay: The agar well diffusion assay described in Nanayakkara et al. (2014) was performed using the five microbial cultures mentioned above, with following modifications. Equidistant wells of 9 Academia Letters, August 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0 Corresponding Author: Anjani M Karunaratne, amkarunaratne21@gmail.com Citation: Bandara, T., Abayasekara, C.L., Panagoda, G.J., Senanayake, M.R.D.M., Nanayakkara, B.S., Karunaratne, A.M. (2021). A herbal hand sanitizer prepared with minimal alcohol and the leaf extract of a common weed, Mimosa pudica L. Academia Letters, Article 3288. https://doi.org/10.20935/AL3288. 3 mm diameter were bored on each Mueller Hinton Agar (MHA) plate and the wells were sealed using molten MHA. The following (50 µl) were loaded to the wells separately; HS samples having M. pudica leaf extract at five different concentrations, negative control and positive control. The plates were incubated at 37 oC for 24 h. The radius of the zone of inhibition around each well was measured. The experiment was done in two trials in quadruplicate for each organism. Means and standard deviations for the average radii of the inhibition zones were subjected to a t-test to determine significant differences at P=0.05. Sensory evaluation test A sensory evaluation test was carried out as a discrimination test to determine differences among the three samples; WHO recommended HS, a widely available commercial HS and the developed herbal HS. The analysis included 30 untrained respondents (male-14 & female16) randomly selected in the age range of 20-70 y. The respondents were asked to apply the sample (about 1.5 ml) on to their palms, and blindly score for each organoleptic parameter (viscosity, colour, feel and aroma) and overall acceptability on a hedonic scale of 1 (Extremely disliked) to 9 (Extremely liked). Data were analyzed non-parametrically. Results & Discussion As test microorganisms, common transient and resident microflora (WHO, 2009) were chosen. The Minimum Inhibitory Concentration (MIC), of the leaf extract was detected at 10,000 mg/L. The MIC and higher concentrations, as well as the positive control gave inhibition zones against all five microorganisms (Table 1), whereas the two lower concentrations did not show any inhibition. The inhibition zones at MIC were significantly lower (P=0.05) compared with that of WHO HS, for the bacterial species, while that of both Candida spp. did not show a significant difference. Academia Letters, August 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0 Corresponding Author: Anjani M Karunaratne, amkarunaratne21@gmail.com Citation: Bandara, T., Abayasekara, C.L., Panagoda, G.J., Senanayake, M.R.D.M., Nanayakkara, B.S., Karunaratne, A.M. (2021). A herbal hand sanitizer prepared with minimal alcohol and the leaf extract of a common weed, Mimosa pudica L. Academia Letters, Article 3288. https://doi.org/10.20935/AL3288. 4 Academia Letters, August 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0 Corresponding Author: Anjani M Karunaratne, amkarunaratne21@gmail.com Citation: Bandara, T., Abayasekara, C.L., Panagoda, G.J., Senanayake, M.R.D.M., Nanayakkara, B.S., Karunaratne, A.M. (2021). A herbal hand sanitizer prepared with minimal alcohol and the leaf extract of a common weed, Mimosa pudica L. Academia Letters, Article 3288. https://doi.org/10.20935/AL3288. 5 Scores of sensory evaluation showed that the herbal HS had an overall acceptability in between WHO HS and Commercial HS (Table 2).Among the three HSs, there was no significant difference (P=0.05) for the organoleptic parameter of viscosity, while significant differences were observed for colour, feel, and aroma. While the feel and aroma of the herbal HS were preferred, its colour (moss green) was least preferred compared to that of WHO (colourless) and commercial HSs (sapphire blue), the latter being most preferred (P=0.05). The overall acceptability of the herbal HS compared well with those of the WHO and commercial HS. It is envisaged that the use of HSs has come to stay even in the post-covid era. Therefore, making HSs that are pleasing to the consumer is important in the competitive world. Academia Letters, August 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0 Corresponding Author: Anjani M Karunaratne, amkarunaratne21@gmail.com Citation: Bandara, T., Abayasekara, C.L., Panagoda, G.J., Senanayake, M.R.D.M., Nanayakkara, B.S., Karunaratne, A.M. (2021). A herbal hand sanitizer prepared with minimal alcohol and the leaf extract of a common weed, Mimosa pudica L. Academia Letters, Article 3288. https://doi.org/10.20935/AL3288. 6 While the newly developed HS inhibited major clinically important microorganisms present in the hand flora, it showed good acceptability among users, except for its colour. In the precovid era, much emphasis has been on controlling microorganisms of surgical site infections and nosocomial infections and the reference alcohol-based hand rub has been specified to contain isopropyl alcohol at 60% (v/v) (WHO, 2009). In the future it is important to test all HSs for their efficacy against viral pathogens as well. Kretzel et al. (2020) demonstrated efficient virus inactivation by the WHO-HS and they discovered that a minimum concentration of 30% (v/v) alcohol was needed to combat SARS-CoV-2.We demonstrated that the herbal HS developed using the recipe of WHO-HS with reduced alcohol is capable of inhibiting five key human pathogens (bacteria and yeasts) and based on the results of Kretzel et al. (2020) its alcohol content is sufficient to control SARS-CoV-2. Also, this formulation with reduced alcohol would be ideal to avoid adverse effects of alcohol, while also minimizing the cost of alcohol and in turn the cost of production, as the herbal extract would not cost as much, while being ecofriendly. References Alghamdi, H. A. (2021). A need to combat COVID-19; herbal disinfection techniques, formulations and preparations of human health friendly hand sanitizers. Saudi Journal of Biological Sciences, 28(2021),3943–3947.https://doi.org/10.1016/j.sjbs.2021.03.077 Balkrishna, A., Singh, K., Singh, H., Haldar, S., and Varshney, A. (2020). GermiX: A skin friendly hand sanitizer with prolonged effectivity against pathogenic bacteria. AMB Express, 10(1), 1-8. https://doi.org/10.1186/s13568-020-01151-y Dassanayake, M. D., Fosberg, F. R., and Clayton, W. D. (1980). Flora of Ceylon. Amerind publishing Co.Pvt. Ltd., New Delhi, 1, Pp. 463-464. FDA (2020). U.S. Foodand Drug Association, Coronavirus Disease 2019 (COVID-19), 2020 Performance Report – 2021. Washington, DC, Retrieved from https://www.fda.gov accessed on 1st of May, 2021. GISD (2021). Global Invasive Species Database, Mimosa pudica. Retrieved from http:// www.iucngisd.org/gisd/search.php on 11th of August 2021. Grace, X., Sowmya, K., Darsika, C., Jothy, A., & Shanmuganathan, S. (2015). Polyherbal hand sanitizer-formulation and evaluation. Indian Journal of Pharmacy and Pharmacology, 2 (2), 143-144. https://www.ijpp.org.in/media/journals/Article143-144.pdf Academia Letters, August 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0 Corresponding Author: Anjani M Karunaratne, amkarunaratne21@gmail.com Citation: Bandara, T., Abayasekara, C.L., Panagoda, G.J., Senanayake, M.R.D.M., Nanayakkara, B.S., Karunaratne, A.M. (2021). A herbal hand sanitizer prepared with minimal alcohol and the leaf extract of a common weed, Mimosa pudica L. Academia Letters, Article 3288. https://doi.org/10.20935/AL3288. 7 Kratzel, A., Todt, D., V’kovski, et al (2020). Inactivation of severe acute respiratory syndrome coronavirus 2 by WHO-recommended hand rub formulations and alcohols. Emerging infectious diseases, 26 (7), 1592.doi: 10.3201/eid2607.200915 Le Thoa, N. T., Nam, P. C., and Nhat, D. M. (2015). Antibacterial activities of the extracts of Mimosa pudica L. An in-vitro Study. International Journal on Advanced Science Engineering and InformationTechnology, 5 (5), 358-361. https://core.ac.uk/download/pdf/ 296921824.pdf Nanayakkara, B.S.,Abayasekara, C.L., Panagoda, G.J., Kanatiwela, H.M.D.K. and Senanayake, M.R.D.M. (2014). Anti-candidal activity of Piper betle (L.), Vitex negundo (L.) and Jasminum grandiflorum (L.). African Journal of Microbiology Research. 8 (23), 2307-2314. https://doi.org/10.5897/AJMR2014.6713 Rajalakshmi, K., and Banu, N. (2016). Antimicrobial activity of natural chlorophyllin from endangered medicinal plant Mimosa pudica L. International Journal of Pharmacy and Pharmaceutical Science, 8 (4), 387-389. World Health Organization & WHO Patient Safety. (2009). WHO guidelines on hand hygiene in health care : a summary. World Health Organization https://apps.who.int/iris/handle/ 10665/70126 Academia Letters, August 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0 Corresponding Author: Anjani M Karunaratne, amkarunaratne21@gmail.com Citation: Bandara, T., Abayasekara, C.L., Panagoda, G.J., Senanayake, M.R.D.M., Nanayakkara, B.S., Karunaratne, A.M. (2021). A herbal hand sanitizer prepared with minimal alcohol and the leaf extract of a common weed, Mimosa pudica L. Academia Letters, Article 3288. https://doi.org/10.20935/AL3288. 8 

ACADEMIA Letters A herbal hand sanitizer prepared with minimal alcohol and the leaf extract of a common weed, Mimosa pudica L. Tharindi Bandara, Department of Botany, Faculty of Science, University of Peradeniya, Sri Lanka Charmalie L Abayasekara, Department of Botany, Faculty of Science, University of Peradeniya, Sri Lanka Gehan J Panagoda, Department of Oral Medicine and Periodontology, Faculty of Dental Sciences, University of Peradeniya, Sri Lanka M. R. Dammantha M Senanayake, Department of Oral Medicine and Periodontology, Faculty of Dental Sciences, University of Peradeniya, Sri Lanka Buddhie S Nanayakkara, Department of Botany, Faculty of Science, University of Peradeniya, Sri Lanka Anjani M Karunaratne, Department of Botany, Faculty of Science, University of Peradeniya, Sri Lanka Abstract Hand sanitizer (HS) usage, prevents infections of transient microflora, while alcohol based HSs are currently used widely. The high level of alcohol in the WHO-recommended HS (75%) could cause dry skin. The aim of this study, was to develop a herbal HS using a leaf extract of Mimosa pudica L. with a reduced alcohol content (40%). The newly developed herbal HS was both effective against tested transient and resident bacterial and fungal flora on hands and was consumer acceptable although its colour may be improved. The alcohol content of this herbal HS should be sufficient to reduce spread of COVID-19, as SARS-CoV-2 is recorded to be inhibited by a minimum of 30% alcohol. Academia Letters, August 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0 Corresponding Author: Anjani M Karunaratne, amkarunaratne21@gmail.com Citation: Bandara, T., Abayasekara, C.L., Panagoda, G.J., Senanayake, M.R.D.M., Nanayakkara, B.S., Karunaratne, A.M. (2021). A herbal hand sanitizer prepared with minimal alcohol and the leaf extract of a common weed, Mimosa pudica L. Academia Letters, Article 3288. https://doi.org/10.20935/AL3288. 1 Introduction Hand sanitizer (HS) usage, a practice that was mainly confined to hospitals in the pre-covid era has now become indispensable and has trickled down to all levels of society worldwide, with the onset of the COVID-19 pandemic. An important component of HSs is alcohol (Balkrishna et al., 2020) and the World Health Organization (WHO) recommends 60–80% alcohol for hand hygiene products (WHO, 2009). A higher percentage of alcohol (at least 60%) effectively kills germs (FDA, 2020) and they dry quickly leaving no sticky residues (WHO, 2006). However, a high alcohol content excessively dries the skin, and it is reported that in children below 12 years, exposure to alcohol based HSs has caused toxicity problems even resulting in a few deaths (Alghamdi, 2021). Yet, decreasing the alcohol content below 60% has not been recommended because of the reduction of its germicidal activity against Gram-positive and Gram-negative vegetative bacteria including multidrug-resistant pathogens (WHO, 2009). However, recently it has been demonstrated that alcohol (either ethyl-or isopropyl-) at a concentration of >30% (v/v) efficiently inactivated SARS-CoV-2, the virus responsible for COVID-19 (Kratzel et al., 2020). A herbal HS that is complemented with a plant component that has natural antimicrobial activity is a possible alternative to alcohol-based sanitizers currently in use. The plant to be incorporated needs to be readily available, while possessing antimicrobial properties. Mimosa pudica L. is a common garden weed which is native to South America, and now has become pantropical in its distribution (GISD, 2021). In Sri Lanka M. pudica is a well known garden weed (Dassanayake et al., 1980) and is a medicinal plant used traditionally for treating skin infections (Rajalakshmi & Banu, 2016) with antibacterial and antifungal flavonoids distributed in the whole plant (Le Thoa et al, 2015). In a previous investigation by Grace et al. (2015) on herbal sanitizer formulation, the glycerol content has been set at 5%. The objective of the present study was to use the guidelines given for the preparation of a HS by the WHO, and develop a herbal HS with a crude alcoholic leaf extract of M. pudica and a reduced level (40%) of isopropyl alcohol, and increased level of glycerol (5%) to test its activity against transient bacterial and fungal microflora of the skin. Academia Letters, August 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0 Corresponding Author: Anjani M Karunaratne, amkarunaratne21@gmail.com Citation: Bandara, T., Abayasekara, C.L., Panagoda, G.J., Senanayake, M.R.D.M., Nanayakkara, B.S., Karunaratne, A.M. (2021). A herbal hand sanitizer prepared with minimal alcohol and the leaf extract of a common weed, Mimosa pudica L. Academia Letters, Article 3288. https://doi.org/10.20935/AL3288. 2 Materials and Methods Leaf samples: Healthy young leaves of M. pudica without petioles were collected from home gardens in Kandy, Sri Lanka (Coordinates- 7.28, 80.58). They were washed thoroughly with water, then with 70% ethanol (3 min), thereafter with 5% sodium hypochlorite (5 min) and finally with distilled water. Antimicrobial compound extraction: Fresh leaves were cut into pieces (5 mm length). Cut leaves (40 g) were subjected to vacuum infiltration (2 h) in ethanol (400 ml), filtered (F1001-CHMLAB), evaporated (40 oC, 12 h) and the crude extract was collected. Microbial cultures: Pure cultures of bacteria Escherichia coli (ATCC25922), Pseudomonas aeruginosa (ATCC27853), Staphylococcus aureus (ATCC25923) and yeasts Candida albicans (ATCC 10231), Candida tropicalis (ATCC 13803) were obtained from the Division of Microbiology, Department of Oral Medicine and Periodontology, Faculty of Dental Sciences, University of Peradeniya.While Blood agar was used for sub-culturing bacteria, Sabouraud dextrose agar was used for yeasts (37 oC, 24 h). Samples for minimum inhibitory concentration: The leaf extract at five concentrations (100,000 mg/L, 50,000 mg/L, 10,000 mg/L, 5000 mg/L, and 1000 mg/L), was incorporated to prepare herbal HSs with 40% isopropyl alcohol, 5% glycerol and distilled water. While the WHO-recommended HS (WHO, 2009) comprising (v/v) 75% isopropyl alcohol, 1.45% glycerol, 0.125% hydrogen peroxide and distilled water served as the positive control, the negative control comprised 40% isopropyl alcohol, 5% glycerol and distilled water. Agar well diffusion assay: The agar well diffusion assay described in Nanayakkara et al. (2014) was performed using the five microbial cultures mentioned above, with following modifications. Equidistant wells of 9 Academia Letters, August 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0 Corresponding Author: Anjani M Karunaratne, amkarunaratne21@gmail.com Citation: Bandara, T., Abayasekara, C.L., Panagoda, G.J., Senanayake, M.R.D.M., Nanayakkara, B.S., Karunaratne, A.M. (2021). A herbal hand sanitizer prepared with minimal alcohol and the leaf extract of a common weed, Mimosa pudica L. Academia Letters, Article 3288. https://doi.org/10.20935/AL3288. 3 mm diameter were bored on each Mueller Hinton Agar (MHA) plate and the wells were sealed using molten MHA. The following (50 µl) were loaded to the wells separately; HS samples having M. pudica leaf extract at five different concentrations, negative control and positive control. The plates were incubated at 37 oC for 24 h. The radius of the zone of inhibition around each well was measured. The experiment was done in two trials in quadruplicate for each organism. Means and standard deviations for the average radii of the inhibition zones were subjected to a t-test to determine significant differences at P=0.05. Sensory evaluation test A sensory evaluation test was carried out as a discrimination test to determine differences among the three samples; WHO recommended HS, a widely available commercial HS and the developed herbal HS. The analysis included 30 untrained respondents (male-14 & female16) randomly selected in the age range of 20-70 y. The respondents were asked to apply the sample (about 1.5 ml) on to their palms, and blindly score for each organoleptic parameter (viscosity, colour, feel and aroma) and overall acceptability on a hedonic scale of 1 (Extremely disliked) to 9 (Extremely liked). Data were analyzed non-parametrically. Results & Discussion As test microorganisms, common transient and resident microflora (WHO, 2009) were chosen. The Minimum Inhibitory Concentration (MIC), of the leaf extract was detected at 10,000 mg/L. The MIC and higher concentrations, as well as the positive control gave inhibition zones against all five microorganisms (Table 1), whereas the two lower concentrations did not show any inhibition. The inhibition zones at MIC were significantly lower (P=0.05) compared with that of WHO HS, for the bacterial species, while that of both Candida spp. did not show a significant difference. Academia Letters, August 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0 Corresponding Author: Anjani M Karunaratne, amkarunaratne21@gmail.com Citation: Bandara, T., Abayasekara, C.L., Panagoda, G.J., Senanayake, M.R.D.M., Nanayakkara, B.S., Karunaratne, A.M. (2021). A herbal hand sanitizer prepared with minimal alcohol and the leaf extract of a common weed, Mimosa pudica L. Academia Letters, Article 3288. https://doi.org/10.20935/AL3288. 4 Academia Letters, August 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0 Corresponding Author: Anjani M Karunaratne, amkarunaratne21@gmail.com Citation: Bandara, T., Abayasekara, C.L., Panagoda, G.J., Senanayake, M.R.D.M., Nanayakkara, B.S., Karunaratne, A.M. (2021). A herbal hand sanitizer prepared with minimal alcohol and the leaf extract of a common weed, Mimosa pudica L. Academia Letters, Article 3288. https://doi.org/10.20935/AL3288. 5 Scores of sensory evaluation showed that the herbal HS had an overall acceptability in between WHO HS and Commercial HS (Table 2).Among the three HSs, there was no significant difference (P=0.05) for the organoleptic parameter of viscosity, while significant differences were observed for colour, feel, and aroma. While the feel and aroma of the herbal HS were preferred, its colour (moss green) was least preferred compared to that of WHO (colourless) and commercial HSs (sapphire blue), the latter being most preferred (P=0.05). The overall acceptability of the herbal HS compared well with those of the WHO and commercial HS. It is envisaged that the use of HSs has come to stay even in the post-covid era. Therefore, making HSs that are pleasing to the consumer is important in the competitive world. Academia Letters, August 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0 Corresponding Author: Anjani M Karunaratne, amkarunaratne21@gmail.com Citation: Bandara, T., Abayasekara, C.L., Panagoda, G.J., Senanayake, M.R.D.M., Nanayakkara, B.S., Karunaratne, A.M. (2021). A herbal hand sanitizer prepared with minimal alcohol and the leaf extract of a common weed, Mimosa pudica L. Academia Letters, Article 3288. https://doi.org/10.20935/AL3288. 6 While the newly developed HS inhibited major clinically important microorganisms present in the hand flora, it showed good acceptability among users, except for its colour. In the precovid era, much emphasis has been on controlling microorganisms of surgical site infections and nosocomial infections and the reference alcohol-based hand rub has been specified to contain isopropyl alcohol at 60% (v/v) (WHO, 2009). In the future it is important to test all HSs for their efficacy against viral pathogens as well. Kretzel et al. (2020) demonstrated efficient virus inactivation by the WHO-HS and they discovered that a minimum concentration of 30% (v/v) alcohol was needed to combat SARS-CoV-2.We demonstrated that the herbal HS developed using the recipe of WHO-HS with reduced alcohol is capable of inhibiting five key human pathogens (bacteria and yeasts) and based on the results of Kretzel et al. (2020) its alcohol content is sufficient to control SARS-CoV-2. Also, this formulation with reduced alcohol would be ideal to avoid adverse effects of alcohol, while also minimizing the cost of alcohol and in turn the cost of production, as the herbal extract would not cost as much, while being ecofriendly. References Alghamdi, H. A. (2021). A need to combat COVID-19; herbal disinfection techniques, formulations and preparations of human health friendly hand sanitizers. 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Indian Journal of Pharmacy and Pharmacology, 2 (2), 143-144. https://www.ijpp.org.in/media/journals/Article143-144.pdf Academia Letters, August 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0 Corresponding Author: Anjani M Karunaratne, amkarunaratne21@gmail.com Citation: Bandara, T., Abayasekara, C.L., Panagoda, G.J., Senanayake, M.R.D.M., Nanayakkara, B.S., Karunaratne, A.M. (2021). A herbal hand sanitizer prepared with minimal alcohol and the leaf extract of a common weed, Mimosa pudica L. Academia Letters, Article 3288. https://doi.org/10.20935/AL3288. 7 Kratzel, A., Todt, D., V’kovski, et al (2020). Inactivation of severe acute respiratory syndrome coronavirus 2 by WHO-recommended hand rub formulations and alcohols. Emerging infectious diseases, 26 (7), 1592.doi: 10.3201/eid2607.200915 Le Thoa, N. T., Nam, P. C., and Nhat, D. M. (2015). Antibacterial activities of the extracts of Mimosa pudica L. An in-vitro Study. International Journal on Advanced Science Engineering and InformationTechnology, 5 (5), 358-361. https://core.ac.uk/download/pdf/ 296921824.pdf Nanayakkara, B.S.,Abayasekara, C.L., Panagoda, G.J., Kanatiwela, H.M.D.K. and Senanayake, M.R.D.M. (2014). Anti-candidal activity of Piper betle (L.), Vitex negundo (L.) and Jasminum grandiflorum (L.). African Journal of Microbiology Research. 8 (23), 2307-2314. https://doi.org/10.5897/AJMR2014.6713 Rajalakshmi, K., and Banu, N. (2016). Antimicrobial activity of natural chlorophyllin from endangered medicinal plant Mimosa pudica L. International Journal of Pharmacy and Pharmaceutical Science, 8 (4), 387-389. World Health Organization & WHO Patient Safety. (2009). WHO guidelines on hand hygiene in health care : a summary. World Health Organization https://apps.who.int/iris/handle/ 10665/70126 Academia Letters, August 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0 Corresponding Author: Anjani M Karunaratne, amkarunaratne21@gmail.com Citation: Bandara, T., Abayasekara, C.L., Panagoda, G.J., Senanayake, M.R.D.M., Nanayakkara, B.S., Karunaratne, A.M. (2021). A herbal hand sanitizer prepared with minimal alcohol and the leaf extract of a common weed, Mimosa pudica L. Academia Letters, Article 3288. https://doi.org/10.20935/AL3288. 8