Ever since the beginning of COVID 19 pandemic the frequent hand disinfection preferably with alcohol-based hand sanitizers and washing with soap and lots of water continued as preferred option to prevent the spread of this infectious disease (Golin et al. 2020). Since excessive and repeated hand washing with soap and water is slight unpractical and non-environment friendly, as more soap-leather water drives into waste streams containing numerous chemical additives and contaminants which doesn’t degrade easily (Daverey and Dutta 2020). Therefore, use of hand sanitizers especially alcohol based are preferred due to their instantaneous disinfectant property and ease of application unless otherwise hand washing with soap is extremely required. Although wide range of hand sanitizers are available in the market with diverse composition as varied from region to region. The alcohol based hand sanitizers contains different emollients, fragrances and colours, whereas non-alcoholic hand sanitizers contains antiseptic chemicals such as triclosan, ammonium compounds and chlorohexidine etc along with synthetic fragrances and colours which may cause acute to severe dermal ailments on continuous usage (Jing et al. 2020). Moreover, the frequent use of these formulations that leaves toxic residues may also have serious damaging effects on human health and environment over long-term usage.
The phytochemicals (volatile and non-volatile) have long history of usage as effective antimicrobial agents against diverse array of infectious agents along with skin protecting properties (Minami et al. 2003; Clarke et al. 2016; Lee et al. 2018). Both tea and lemon grass are rich source of bioactive constituents with antimicrobial and skin emollient properties (Minami et al. 2003; Shin et al. 2012). The tea constituents have shown to exhibit potent antiviral property against several viruses and are extremely efficient compared to various synthetic chemical (Lee et al. 2018; Mhatre et al. 2020). We also reported in our recent studies the SARS-CoV-2 main protease inhibitor potential of tea bioactives (Bhardwaj et al. 2020). Studies have also reported the effect of ethanol on skin and cases of irritation and allergic reactions due to persistent exposure that results into dryness and skin cracking (Mahmood et al. 2020), hence alcohol needs to be supplemented with natural and sustainable phytoconstituents.
The green tea polyphenols enriched extract was prepared from secondary grade of made tea followed by quality evaluation using high-pressure liquid chromatography analysis (Figure S1). The extract showed presence of all major tea constituents such as; epigallocatechin, epicatechin, epigallocatehin, epicatechin gallate, catechin and caffeine. The quality of lemon grass oil was evaluated by gas chromatography analysis. The results have showed occurrence of limonene, 2-beta-pinene and citral as major constituents in the sample (Table 1). The content of natural ingredients tea extract (0.1%) and lemongrass oil (0.1 %) were also selected based on earlier studied results of ours and Minami et al. 2003.
Table 1
Gas chromatography mass spectral data showing major volatile constituents in lemon grass oil used in hand sanitizer formulation
Sr. no. | Name | R. Time | % Area |
1 | Camphene | 5.8 | 2.13 |
2 | 6-Methyl-5-hepten-2-one | 6.6 | 3.73 |
3 | l-Limonene | 8.0 | 0.34 |
4 | 4-Naonanone (CAS) Propyl amyl ketone | 9.3 | 2.18 |
5 | Linalool l | 10.4 | 1.63 |
6 | 1,4-Hexadiene,3,3,5-trimethyl | 11.8 | 0.26 |
7 | 2,2-Dimethyl-ota-3,4-dienal | 12.1 | 0.43 |
8 | Citronella | 12.2 | 0.32 |
9 | Bicyclo[3.1.0]hept-3-en-2-ol, 4,6,6-trimethyl- | 12.5 | 0.58 |
10 | 2-(2',3'-Epoxy-3'-methylbutyl)-3-methylfuran | 12.8 | 0.59 |
11 | Verbenol | 13.2 | 1.21 |
12 | Z-Citral | 15.3 | 33.7 |
13 | 2-Cyclohexen-1-one, 2-methyl-5-(1-methylethenyl)-(CAS) Piperitone | 15.9 | 0.66 |
14 | E-Citral | 16.4 | 43.9 |
15 | Geranyl acetate | 20.1 | 1.94 |
16 | trans-Caryophyllene | 21.6 | 0.92 |
17 | gamma-Cadinene | 24.7 | 2.98 |
18 | delta-Cadinene | 24.8 | 0.48 |
19 | (-) Caryophyllene oxide | 26.9 | 1.56 |
The formulation was optimized by using varying alcohol (ethanol) content such as 60%, 70 % and 80% ethanol v/v. These results showed 80 % alcohol formulation exhibts higher effectiveness against different microorganisms (Fig. 1). These results are in correspondence to earlier study by Suchomel et al. 2012, which also showed better results with 80 % alcohol as compared to 60 %. Further, the antimicrobial potential of the selected formulation was evaluated and compared with World Health Organization recommended formulation and commercial sample as well. Well diffusion assay revealed that our hand sanitizer formulation was effective against all the tested Gram-positive and Gram-negative bacteria (Table 2). This formulation also showed enhanced antibacterial activity than the World Health Organization recommended formulation. S. aureus MTCC 96 was the most susceptible to the effect of hand sanitizer formulation, while B. subtilis MTCC 121 was the most resistant. It was observed that this sanitizer exhibited almost equal antibacterial activity towards all the Gram-negative bacteria tested, with highest activity against S. typhi MTCC 733 (Table 2).
Table 2
Comparative inhibition study of IHBT hand sanitizer with WHO Formulation and commercial sample against three Gram-positive and three Gram-negative bacterial pathogens
| GRAM-POSITIVE | GRAM-NEGATIVE |
Samples | Bacillus subtilis MTCC 121 | Staphylococcus aureus MTCC 96 | Micrococus luteus MTCC 2470 | Salmonella typhi MTCC 733 | Escherichia coli MTCC 43 | Klebsiella pneumoniae MTCC 109 |
IHBT Hand Sanitizer | 4 ± 0 | 21.66 ± 0.5 | 9 ± 0 | 13.33 ± 0.5 | 11 ± 0 | 9.66 ± 0.5 |
WHO Formulation | 3 ± 0 | 19.33 ± 0.5 | 5 ± 0 | 10.33 ± 0.5 | 8.66 ± 1.5 | 7.66 ± 0.5 |
Commercial sample | 6 ± 0 | 9.33 ± 0.5 | 10.33 ± .05 | 4 ± 0 | 7.66 ± 0.5 | 5.33 ± 0.5 |
Positive Control | 22.33 ± 0.5 | 25 ± 1 | 28.66 ± 0.5 | 13.33 ± 0.5 | 15.66 ± 1.15 | 8.66 ± 0.5 |
Positive Control = Ampicillin 10 mcg/disc in case of Gram-positive bacteria and Streptomycin 10 mcg/disc in case of Gram-negative bacteria |
MTCC - Microbial Type Culture Collection |
The time-kill test of hand sanitizer formulation was found to cause reduction of all the three tested bacteria by more than 5 log10-steps within an exposure time of 15 seconds. The results demonstrated a log10 reduction factor (RF) above 5 against all the tested bacteria (Table 3). The efficacy of the sanitizer in a short application time provides benefits for prevention of many kinds of infections.
Table 3
Activity of IHBT hand sanitizer against one Gram-positive and two Gram-negative opportunistic bacterial pathogens (15 seconds exposure time) by presentation of the minimum log 10 reduction factor (RF)
Bacterial species | Minimum RF |
Pseudomonas aeruginosa MTCC 2453 | 5.90 |
Salmonella typhi MTCC 733 | 5.86 |
Staphylococcus aureus MTCC 96 | 5.92 |
MTCC - Microbial Type Culture Collection, RF- Reduction Factor |