Formulation and Biochemical Analysis of Herbal Soap Prepared Using Leaf Extracts of Mentha and Eucalyptus
Microorganisms, primarily bacteria, are responsible for various infections in humans. We are living in a world full of bacteria and it is impacting us more than we had thought . These bacteria come in contact with our body either directly from the environment or by touch. This can lead to several dermatological infections and ingestion can lead to various types of infectious diseases such as typhoid, dysentery, etc. Due to the pathogenic effects that bacteria have on humans, there is a need of antibiotic formulations and skin being the most exposed part of the body has to be protected. In recent decades, the research on plants has intensified due to the fact that these plants contain some pharmacologically active compounds. These active compounds can become new antibiotics after the evaluation of pure constituents and extracts of plants.
In the present study, extracts of Mentha Arvensi and Eucalyptus obliqua leaves were prepared and analyzed for their anti-oxidative properties. These extracts demonstrating high anti-oxidant activity were used for formulating herbal soap that targets skin infecting bacteria and other microorganisms. Promising results were obtained after in vitro anti-microbial analysis and anti-oxidant activity analysis of prepared plant extracts. Further evaluation and fractionation of extracts would help to formulate a potent herbal soap composition that directly acts on skin problems as acne, rashes and inflammation.
Keywords: Herbal soap, Mentha Arvensi, Eucalyptus obliqua, Anti-microbial, Mint
Eucalyptus plant was traditionally used as antiseptic and the benefits of eucalyptus extract on skin health and integrity was also reported. Essential oils of Eucalyptus globulus have moderate anti-microbial activity on Gram-negative (Salmonella enteritidis, Escherichia coli and Pseudomonas aeruginosa) and Gram-positive bacteria (Staphylococcus aureus, Enterococcus faecium). This effect on bacteria may be due to the dominant presence of eucalyptol, which has a strong antimicrobial activity against many important pathogens.
Mentha, commonly known as mint, has biologically active compounds against infectious diseases. Preparations of peppermint is done by leafs, leaf extracts and water. The plant is cultivated mainly for its essential oil, which is obtained by distillation from freshly grounded leaves and is known to have antibacterial properties. Peppermint extracts have significant antiviral activities. Peppermint oil and extracts showed a good antimicrobial activity against: 1) Escherichia coli, 2) Salmonella pullorum, 3) Streptococcus faecalis, 4) Lactobacillus bulgaricus, 5) Staphylococcus pyogenes, 6) Staphylococcus aureus, 7) Streptococcus pyogenes, 8) Serratia marcescens, 9) Salmonella typhi, 10) Shigella dysenteriae and some others. Some studies have showed that the antibacterial activity of peppermint leaves extract was higher than of its stem extract against Gram negative bacilli.
With the growing concern of people for the use of synthetic chemicals used in cosmetics, the demand of herbal products has increased rapidly. Thus, they prefer more natural products free from harmful chemicals. Herbal products for skin care such as soap are safe to use, budget friendly, compatible with all skin types and have no side effects.
This work aims to evaluate the anti-microbial potential of the extracts of Mentha Arvensi and Eucalyptus obliqua against some common microorganisms. Further, the aim is to formulate an anti-bacterial herbal soap and to evaluate its physiochemical properties and stability.
Materials And Methods
Collection of samples
The plants Mentha Arvensi and Eucalyptus obliqua were collected from the Herbal Garden of IMS Engineering College and the market areas of Ghaziabad in the year 2018.
Washing of samples: All the sample leaves were firstly washed with normal water and then thrice with distilled water to eliminate the dirt completely. Further, washing was done by ethanol to kill the microorganisms present on the sample.
Sample drying: All samples were dried in the laboratory for 15-20 days till the moisture content was removed completely.
Grinding of samples: The dried sample leaves were grinded to powder form by the mixer grinder in the laboratory.
Storage of samples: The sample was then stored in an air tight jar to maintain the aroma and avoid the entry of moisture and microorganisms. The jar was stored in a cool and dry place to maintain the quality of powder.
Preparation of extracts
2 gm of grinded powder was taken and mixed in 40 ml of distilled water. The solution was kept in a conical flask and placed on shaking condition in BOD incubator at 30°C for 24 hours. After 24 hours, extract was filtered using a filter paper or blotting paper and the filtered extract was stored in a cool and dry place.
Preliminary screening of anti-microbial activity of extracts
The extracts derived were subjected to anti-microbial screening. LB broth was prepared, autoclaved and inoculated with Escherichia coli and pseudomonas (5 ml in 100 ml broth). It was kept in BOD incubator at 37°C for 12 hours.
Discs were prepared using whatman filter paper and punching machine and then autoclaved.
NAM media was prepared, autoclaved and then poured into pre-autoclaved petri-plates. It was allowed to solidify. The fresh culture was used to inoculate the plates. Sterilized discs were dipped into plant extracts and in water to work as control. Plates were divided into 2 sections: S for sample and C for control. Soaked discs were then placed on inoculated plates in their sections respectively and then placed in a BOD incubator at 37°C for 12-14 hours.
The plates were examined after the incubation time.
Extraction of oil
Fresh leaves of eucalyptus and mint were cut into small pieces and weighed around 250 gm with an electronic balance. Leaves were placed inside a thick thimble made of thick filter paper and loaded into the main chamber of soxhlet extractor. For mint leaves, 300 ml of ethanol and for eucalyptus leaves, 300 ml of water was added to the round bottom flask. The soxhlet extractor was then placed onto the bottom flask with the extraction solvent. Heating mantle temperature was set to 90°C. As the boiling started, temperature was set to 45°C for 4 hours. The flask was put on heating mantle. The solvent heated to reflux. The vapors of solvent travel to distillation arm, flood into the chamber housing the thimble of solid. The condenser of the soxhlet ensures that all the solvent vapors cool and drip back down into the chamber housing the solid material. The chamber slowly gets filled with warm solvent. As the chamber is almost full, it is emptied automatically by a siphon side arm with the solvent running back down to the distillation flask. This cycle is repeated several times and the desired compound is concentrated in the distillation flask. The solvent is removed after extraction by rotary vapors yielding the extracted compound.
Preparation of soap
The glycerin soap was cut into small pieces to facilitate melting by a double boiler. The aqueous extract was added to the melted soap base and the extracted oil was added to the solution and mixed properly. This final solution was poured into moulds and allowed to harden at room temperature. To make the soap firm and cool, it was kept in freezer for 20 minutes. The soaps were carefully removed from moulds and left for drying for 2 days.
Quality tests of soap
pH determination: 1 gm in 20 ml soap shavings was dissolved in a 100 cm volumetric flask and made up to prepare 10% soap solution. pH was tested.
Acidity/Alkalinity test: Drops of phenolphthalein indicator was added to 5 ml of soap and color change was observed.
Lathering test: 5 ml of soap sample was vigorously shaken in a test tube. Lather amount was observed and recorded.
Foam test: 2 gm of soap was added to 100 cm cube of distilled water and shaken vigorously for about 2 minutes. The cylinder was allowed to stand for 2 minutes and the height of foam was recorded.
Moisture content: 10 gm of scrapped soap was taken in a petridish and placed in oven for 1 hour at 100°C. Then it was allowed to cool and moisture content was calculated by the following formula:
%W = (A-B)/B * 100
%W = % of moisture, A = weight of wet sample, B = weight of dry sample
Preliminary screening of anti-microbial activity of extracts
The initial microbial susceptibility of the Mentha Arvensi and Eucalyptus obliqua plant extracts was done and the results are recorded in Table 1.
Area inhibited in Pseudomonas
Area inhibited in E.coli
Extraction of Oil
pH test of Soap
Name of soap
Acidity and Alkalinity test
Name of soap
Name of soap
Length of foam formed
Name of soap
Wt. of wet sample (gm)
Wt. of dry sample (gm)
Moisture content in soap (%)
The paper focuses on the importance of use of natural herbal products. It lays emphasis on the use of herbal soap with anti-microbial properties against skin pathogens as a replacement for the commonly used chemical based products.
The soap was formulated using the extracts and essential oils of Mentha Arvensi and Eucalyptus obliqua in aseptic conditions. The various quality tests performed on the soap were successful.
By the various quality tests performed, it can be concluded that herbal soap can be effectively used to prevent and eliminate bacterial infection and also overcome the drawbacks of chemical based products. Essential oils will also provide healthier skin. This is a small initiative to make people aware of the fact and to scale up the process.
- We are living in a bacterial world, and it’s impacting us more than previously thought. February 15, 2013 by Lisa Zyga, Phys.org
- P. A. Melendez and V. A. Capriles, “Antibacterial properties of ´ tropical plants from Puerto Rico,” Phytomedicine, vol. 13, no. 4, pp. 272–276, 2006.
- Chevallier A., Encyclopedia of Medicinal Plants. St. Leonards, New South Wales, Australia: DK Publishing 2001.
- Ishikawa J, Shimotoyodome Y, Chen S, et al. Eucalyptus increases ceramide levels in keratinocytes and improves stratum corneum function. Int J Cosmet Sci 2012; 34(1): 17-22.
- Ait-Ouazzou A, Lorán S, Bakkali M, et al. Chemical composition and antimicrobial activity of essential oils of Thymus algeriensis, Eucalyptus globulus and Rosmarinus officinalis from Morocco. J Sci Food Agric 2011; 91(14): 2643-51.
- Bakkali F, Averbeck S, Averbeck D, Idaomar M. Biological effects of essential oils-a review. Food Chem Toxicol 2008; 46(2): 446-75.
- Mucciarelli M, Camusso W, Maffei M, Panicco P, Bicchi C (2007) Volatile terpenoids of endophyte-free and infected peppermint (mentha piperita l): Chemical partitioning of a symbiosis. Microb Ecol 54: 685-696.
- Cosentino M, Bombelli R, Conti A, Colombo ML, Azzetti A, et al. (2009) Antioxidant properties and in vitro immunomodulatory effects of peppermint (mentha x piperita l.) essential oils in human leukocytes. J Pharm Sci Res 1: 33-43.
- Mairapetyan S, Mamikonyan V, Alexanyan J, Tovmasyan A, Daryadar M(2016) Productivity, biochemical indices and antioxidant activity of peppermint (mentha piperita l.) and basil (ocimum basilicum l.) in conditions of hydroponics. J Aquac Res Development 7: 2.
- Uribe E, Marín D, Veg Gálvez A, Quispe-Fuentes I, Rodríguez A (2016) Assessment of vacuum-dried peppermint (mentha piperita l.) as a source of natural antioxidants. Food Chem 190: 559-565.
- Riachi LG, De Maria (2015) C.A.B. Peppermint antioxidants revisited. Food Chem 176: 72-81.
- Choi O, Cho SK, Kim J, Park CG, Kim J (2016) Antibacterial properties and major bioactive components of mentha piperita essential oils against bacterial fruit blotch of watermelon. Arch Phytopath Plant Protect 49: 325-334.
- Brand YM, RoaLinares VC, BetancurGalvis LA, DuránGarcía DC, Stashenko E (2016) Antiviral activity of colombian labiatae and verbenaceae family essential oils and monoterpenes on human herpes viruses. J Essent Oil Res 28: 130-137.
- Schuhmacher A, Reichling J, Schnitzler P (2003) Virucidal effect of peppermint oil on the enveloped viruses herpes simplex virus type 1 and type 2 in vitro. Phytomedicine 10: 504-510.
- Shalayel M, Asaad A, Qureshi M, Elhussein A (2016) Anti-bacterial activity of peppermint (mentha piperita) extracts against some emerging multi-drug resistant human bacterial pathogens. J Herb Med 22: 152.
- Bekhit AE, Cheng VJ, McConnell M, Zhao JH, Sedcole R et al. (2011) Antioxidant activities, sensory and anti-influenza activity of grape skin tea infusion. Food Chem 129: 837-845.
- Bohnert T, Patel A, Templeton I, Chen Y, Lu C (2016) Evaluation of a new molecular entity as a victim of metabolic drug-drug interactions-an industry perspective. Drug Metab Dispos dmd 115: 690-696.
- Rodrigues F, Dupret JM (2002) 3d model of human arylamine n-acetyltransferase 2: Structural basis of the slow acetylator phenotype of the r64q variant and analysis of the active-site loop. Biochem. Biophys. Res. Commun 291: 116-123.
- Sun Z, Wang H, Wang J, Zhou L, Yang P (2014) Chemical composition and anti-inflammatory, cytotoxic and antioxidant activities of essential oil from leaves of mentha piperita grown in china. PloS one 9: e114-767.
- Shaikh S, Yaacob HB, RahimZHA (2014) Prospective role in treatment of major illnesses and potential benefits as a safe insecticide and natural food preservative of mint (mentha spp.): A review. Asian J Biomed Pharm 4: 1.
- Escamilla M, Ferre A, Hidalgo C, Fuentes N, Kaps R, et al.(2012) Revision of European Ecolabel Criteria for Soaps, Shampoos and Hair Conditioners, Joint Research Centre European Commission 1-40.