Development of deodorizing agent from natural plant extracts
Taek Kyu Jung, Hyun-Chul Park, Kyung-Sup Yoon Saimdang Cosmetics Co., Ltd., R&D Center, 143, Yangcheongsongdae-gil, Ochang-eup, Cheongwon-gu, cheongju-si, Chungcheongbuk-do, Korea
Keywords: Deodorizing, Plant extract, Isovaleric acid, Anti-microbial effect, Leusine dehydrogenase
Short Summary There are many kinds of unpleasant odor in our daily life, such as body odor caused by metabolism and aging. According to recent studies, it is known that there are various kinds of odor-causing substances such as steroids, fatty acids, sulfur compounds, aldehydes, ester, ketones, and nitric compounds. Also, one of the causes of odor generation is thought to be microbiologically produced from precursors contained in the apocrine secretions. The aim of this study was to investigate the antiperspirant and deodorant of natural plant extracts for the representative odor substances such as isovaleric acid, ammonia and nonenal. Also, we were to investigate the antimicrobial effect for skin microbes and the leucine dehydrogenase inhibition effect of plant extracts.
Introduction The generation of odor on various sites on the human body, e.g. foot, mouth, or axilla, is mainly caused by microbial transformation of odorless natural skin secretions into volatile odorous molecules. Thus, identification of the primary components in each body odor and clarification of the mechanisms of their generation are important for the development of methods to effectively control such odors [1]. The factor influencing human body odors were skin bacteria, life style, environmental factor (heat and humidity), physiological factors (age, gender), genetics and ethnicity, emotional factor and clinical disorders. Also, a wide range of volatile odorous substances has been implicated in axillary odor and is thought to be microbiological produced from precursors contained in the apocrine secretions [2-4]. The three major compounds of odor substances were steroids, fatty acids and sulfur compound. Other volatiles such as aldehydes, esters, ammonia, ketones, and alcohols may act as odor modifiers. Isovaleric acid was a very famous typical volatiles fatty acid with human body odor substance. Recently, extensive studies have been carried out on the deodorant using natural plant extract [5]. The aim of this study has been to test the plant extract to verify its effectiveness as a deodorant active. Especially, this study was investigated for the scavenging effect of isovaleric acid, the antimicrobial activity and the inhibition effect of leucine dehydrogenase which is involved in the generation of isovaleric acid.
Methodology Preparation of plant extract In this study, the deodorizing effect was evaluated for the plant extract of 27 kinds (samples number: DO-1 to DO-27). The plant extract of 27 kinds was prepared as follows. 50 g of dry slice of plants were extracted with 500 mL of 75% ethanol aqueous solution and then evaporated to crude extracts. Also, 75% ethanol extract of DO-4, DO-5, DO-17, DO-18, DO- 20 and DO-25 were evaporated under reduced pressure, and then fractionated by methylene chloride (MC), ethyl acetate (EA), butyl alcohol (BuOH) and H2O. List of the extracts were indicated in Table 1.
Table 1. List of plant extracts Number Scientific name Number Scientific name DO-1 Lysimachia foenum-graecum DO-15 Imperata cylindrical root stem DO-2 Nardostachys chinensis root DO-16 Artemisia princeps DO-3 Agastache rugosa DO-17 Sanguisorba hakusanensis root DO-4 Syzygium aromaticum bud DO-18 Psoralea corylifolia seed DO-5 Pterocarpus santalius stem DO-19 Carthamus tinctorius seed DO-6 Santalum album stem DO-20 Rheum palmatum root DO-7 Thymus quinquecostatus DO-21 Pulsatilla koreana root DO-8 Elsholtzia splendens DO-22 Humulus japonicus DO-9 Plantago asiatica seed DO-23 Portulaca oleracea DO-10 Typha orientalis pollen DO-24 Duchesnea indica DO-11 Lonicera japonica DO-25 Rhus semialata gall DO-12 Cynanchum paniculatum root DO-26 Zanthoxylum schinifolium fruit bark DO-13 Scrophularia buergeriana root DO-27 Betula alba bark DO-14 Scutellaria baicalensis root
Gas detection tube test Scavenging effect of the plant extracts on the odor substances were evaluated by using colorimetric gas detection tube test [6]. The plant extracts were used 75% EtOH extract. Isovaleric acid (99%, Aldrich, USA) and ammonia (2.0 M in EtOH solution, SIGMA, USA) were used by odor substances. Sampling pump of gas was used as hand pump (GASTEC, GV-100S, JAPAN), and gas test tube was used as acetic acid (as a substitute for isovaleric acid) and ammonia gas test tube. For the measurement of scavenging rate of odor gas, place 0.1 mL of Isovaleric acid (1% in 75% EtOH) into 250 mL bottle, and was added to a reaction mixture that contained 1 mL of plant extract (1% in EtOH), and control group was used 1 mL of 75% EtOH in place of plant extract. Ammonia solution (2.0 M in EtOH) was used 0.01 mL. Mixed samples were then allowed to stand for 30 minutes and then measuring the concentration of gas remaining in the bottle using gas detection system. Scavenging rate of gas was calculated by the following formula:
The value of sample (ppm) Scavenging rate of gas (%) = 100 - x 100(%) The value of control (ppm)
Agar diffusion assay The antimicrobial effect of the plant extracts on the skin microbes were evaluated by using paper disk agar diffusion method. The plant extracts were used 75% EtOH extract. Stenotrophomonas maltophilia, Staphylococcus epidermidis, Brevibacterium epidermidis, Corynebacterium xerosis, and Staphylococcus aureus were used by skin microbes. Antimicrobial tests were carried out by agar diffusion method using 100 μL of suspension containing 108 CFU/mL spread on nutrient agar plates. The discs (10 mm in diameter) were impregnated with 50 μL of extracts and placed on the inoculated agar (Difco, Becton Dickinson, NJ, USA). The diluted solution impregnated disc was used as negative control. The inoculated plates were incubated at 37℃ for 18-24 hours. Anti-microbial activity was evaluated by measuring the zone of inhibition against the test organism
Search for agents that suppress the isovaleric acid production The abilities of extracts to inhibit isovaleric acid production were evaluated by adding each of the extracts to a reaction mixture that contained 179 mM glycine (SIGMA, USA), 179 mM potassium chloride (SIGMA, USA), 18 mM L-leucine (SIGMA, USA), 1.1 mM β-NAD (SIGMA, USA), 0.37 mM potassium phosphate (SIGMA, USA) and 0.05 unit leucine dehydrogenase (TOYOBO, JAPAN). The absorbance was measured at 340 nm and 37 °C, and the decrease in the NADH production was compared with that of the control. The plant extracts were used 75% EtOH extract and solvent fractions.
Results The scavenging effect of the plant extracts on the odor substances The DO-1, DO-2, DO-5, DO-15, and DO-20 samples were relatively good scavenging effect (more than 70%) on the isovaleric acid. The DO-4, DO-5, DO-14, DO-20, and DO-26 samples were relatively good scavenging effect (more than 80%) on the ammonia (Table 2).
Table 2. The scavenging effect of the plant extracts on the odor substances Scavenging rate (%) Numbers Isovaleric acid Ammonia DO-1 75.85 2.05 60.00 0.00 DO-2 82.25 7.00 62.65 15.06 DO-3 66.85 4.60 49.00 1.41 DO-4 68.65 4.45 85.65 3.32 DO-5 75.25 2.33 83.35 4.74 DO-6 65.55 2.27 52.00 11.31 DO-7 68.25 1.34 36.65 4.74 DO-8 64.15 7.14 50.35 8.98 DO-9 44.95 0.78 59.35 10.39 DO-10 52.05 3.46 69.35 3.75 DO-11 44.80 5.52 66.65 9.40 DO-12 52.35 3.32 52.00 11.31 DO-13 65.55 2.76 50.75 3.89 DO-14 63.30 5.94 92.65 0.92 DO-15 70.95 2.47 54.35 3.32 DO-16 58.95 4.88 55.00 7.07 DO-17 61.70 2.69 56.65 4.74 DO-18 60.30 1.70 75.65 10.82 DO-19 56.75 3.18 58.65 20.72 DO-20 70.95 2.47 83.35 4.74 DO-21 60.80 1.41 77.65 7.99 DO-22 63.85 0.35 81.65 2.33 DO-23 70.05 1.20 60.00 0.00 DO-24 67.85 0.49 66.65 9.40 DO-25 59.05 0.07 56.00 5.66 DO-26 64.85 4.74 85.65 3.32 DO-27 54.25 3.04 52.65 0.92
The antimicrobial effect of the plant extracts on the skin microbes The antimicrobial effect of the plant extracts on the skin microbes, which were known as oral cavity inducer, was investigated using the paper disk agar diffusion method. The DO-4, DO-5, DO-17, DO-20, and DO-25 samples were relatively good antimicrobial effect on the microbes such as Stenotrophomonas maltophilia, Staphylococcus epidermidis, Brevibacterium epidermidis, Corynebacterium xerosis, and Staphylococcus aureus. In particular, DO-4 and DO-25 showed the best effect. (Table 3, Figure 1).
Table 3. The antimicrobial effect of the plant extracts on the skin microbes: size of inhibition zone Skin Microbes Numbers Stenotrophomonas Staphylococcus Brevibacterium Corynebacterium Staphylococcus maltophilia epidermidis epidermidis xerosis aureus DO-1 -* - - 18.5 1.0 - DO-2 - 12.1 0.0 - 24.9 1.8 12.1 0.0 DO-3 - 10.9 0.5 - 22.0 1.3 - DO-4 17.3 0.3 15.2 0.6 14.5 0.2 39.6 1.3 18.6 0.1 DO-5 - 18.3 0.9 14.5 0.2 42.3 0.2 14.9 0.0 DO-6 - - - - - DO-7 - 12.6 0.3 - 21.1 0.6 - DO-8 - 10.5 0.2 - 20.4 0.7 - DO-9 - 12.1 0.3 - 21.4 0.6 - DO-10 - - - 21.7 0.5 - DO-11 - - - 18.8 0.9 - DO-12 - - - 18.3 1.8 - DO-13 - - - 18.8 0.2 - DO-14 - 11.6 0.6 - 36.3 0.5 - DO-15 - - - - - DO-16 - - - 22.4 0.7 - DO-17 17.2 0.0 17.2 0.4 20.1 0.5 30.5 0.8 16.1 0.0 DO-18 - 15.0 0.3 15.5 0.1 21.4 0.9 14.4 1.2 DO-19 - - - - - DO-20 - 17.3 0.4 17.6 0.6 24.2 0.4 15.7 0.0 DO-21 - - - 23.3 1.4 - DO-22 - - - 17.2 0.4 - DO-23 - - - - - DO-24 - - - 19.9 0.06 - DO-25 24.3 1.1 19.7 0.4 26.8 0.7 33.6 1.4 16.3 0.0 DO-26 - - - 26.8 1.8 - DO-27 - 12.8 0.3 - 29.9 0.1 - * -: no effect
Figure 1. Inhibition zone of DO-4, DO-5, DO-17, DO-18, DO-20 and DO-25 against Corynebacterium xerosis with a variation of solvent fraction.
Evaluation of the leucine dehydrogenase inhibition effect The 27 naturally occurring plant extracts were evaluated. Table 4 shows the leucine dehydrogenase inhibition effect of 75% EtOH extract of 27 kinds. The 75% EtOH of DO-20 showed relatively high effect for the leucine dehydrogenase inhibition effect (49.5%, 1 mg/mL). Especially, the inhibition rate of 75% EtOH Ex.-EA fraction of DO-20 was 86.36% at 1 mg/mL (Figure 2).
Table 4. The leucine dehydrogenase inhibition effect of plant extracts
Number Inhibition (%) Number Inhibition (%) DO-1 -102.1 DO-15 -88.7 DO-2 -15.5 DO-16 -102.1 DO-3 35.1 DO-17 12.4 DO-4 -1.0 DO-18 2.1 DO-5 38.5 DO-19 -75.3 DO-6 25.8 DO-20 49.5 DO-7 -29.9 DO-21 30.9 DO-8 5.2 DO-22 12.4 DO-9 28.9 DO-23 13.4 DO-10 14.4 DO-24 -38.1 DO-11 12.4 DO-25 5.2 DO-12 38.5 DO-26 11.3 DO-13 11.3 DO-27 -4.1 DO-14 10.2 Control 0.0
* *
*
* p <0.05 Figure 2. The leucine dehydrogenase inhibition effect of DO-20 with a variation of solvent fraction. g
Conclusions and Discussion In humans, the formation of body odors is mainly caused by skin glands excretions and bacterial activity. Between the different types of skin glands, the human body odor is primarily the result of the apocrine sweat glands, which secrete the majority of chemical compounds needed for the skin flora to metabolize it into odorant substances. The main components of human axillary odor are unsaturated or hydroxylated branched fatty acids with isovaleric acid. Isovaleric acid (3-methyl butanoic acid) is the other source of body odor as a result of actions of the bacteria Staphylococcus epidermidis. In this study, we targeted to prevent body odor by odor-causing substances, microbial metabolism and enzyme inhibition effect. The plant extracts of this study can be prevented to generation of odor-causing substance. Also, it is possible to reduce the generated odor-causing substance with isovaleric acid. In conclusion, these results suggest that the DO-20 samples from the extract of 27 kinds may have potential as deodorizing agents.
Acknowledgements This work was supported by Chungbuk institute for regional program evaluation (CBIRPE) promotion project of the MOTIE (Ministry of Trade, Industry and Energy) Republic of Korea.
References 1. X. Zeng, J. J. Leyden, J. G. Brand, A. I. Spielman, and K. J. Mcginley, An investigation of human apocrine gland secretion for axillary odor precursors, Journal of Chemical Ecology, 18(7) 1039-1045 (1992). 2. K. Ara, M. Hama, S. Akiba, K. Koike K. Okisaka, T. Hagura, T. Kamiya, and F. Tomita, Foot odor due to microbial metabolism and its control, Can. J. Microbiol., 50, 357-364 (2006). 3. S. Mikoshiba, H. Takenaka, T. Okumura, K. Someya, and M. Ohdera, The suppressive effect of apricot kernel extract on 5--androst-16-en-3-one generated by microbial metabolism, Int. J. Cosmet. Sci., 28(1), 45-52 (2006) 4. A. G. James, C. J. Austin, D. S. Cox, D. Taylor, and R. Calvert, Microbiological and biochemical origins of human axillary odor, FEMS Microbiol Ecol., 83(3) 527-540 (2013). 5. M. A. Shahtalebi, M. Ghanadian, A. Farzan, N. Shiri, D. Shokri, and S. A. Fatemi, Deodorant effects of a sage extract stick: antibacterial activity and sensory evaluation of axillary deodorancy, Inhibitory effect of fractionated Trapa japonica extracts on UVB- induced skin photoaging, J. Res. Med. Sci., 18(10), 833-839 (2013). 6. Haag and R. Werner, Interchangeability of gas detection tubes and hand pumps, AIHAJ : a journal for the science of occupational and environmental health and safety, 62(1), 65- 69 (2001).