Lawsonia inermis, or known as henna (English name), has local names such as hennastrauch (Germany), hena/mendhi (Pakistan, India), and inai/pacar kuku (Indonesia, Malaysia). It is one of the familiar plants widely found in Asia, including in Indonesia1. Generally, the leaves are used by the community as a natural reddish brown dye for coloring nails, hair, and skin. The community often uses L. inermis leaves to treat wounds and skin inflammation2,3.

Lawsonia inermis leaves contain large amounts of chemical compounds such as lawsone, flavonoids, tannins, coumarins, sterols, and terpenoids4. According to the phytochemical analysis5, all of the extracts contained naphthoquinones, saponins, flavonoids, and steroids. Lawsone (2-hydroxy-1,4-naphthoquinone), a kind of naphthoquinone, has been identified as the major component in L. inermis6,7.

Many studies have investigated the antimicrobial activity of L. inermis leaves extract in various solvents. Usman and Rabiu5 reported that the aqueous extract of L. inermis leaves inhibited Staphylococcus aureus and Epidermophyton floccosum. The L. inermis extract inhibited some microbial isolates at 1000 μg/mL concentrations. The most significant antimicrobial activity of methanol, ethanol, and aqueous L. inermis extract against some human pathogenic bacteria, and some fungi were possessed by methanol and ethanolic extracts8,9. However, many investigations reported that methanol extract exhibited promising antibacterial activity against some pathogenic bacteria from clinical isolated10-12. Leaves extract of L. inermis has also been reported to possess good biofilm inhibition and antibacterial activity, which can be explored to develop new drugs for MDR pathogens13.

Although L. inermis was reported to have acted as an antibacterial agent, the information on which compound is responsible for the antibacterial activity is still unclear. In this study, the methanol extract of L. inermis leaves was fractionated with n-hexane and ethyl acetate to obtain n-hexane, ethyl acetate, and methanol fractions. The purpose of the fractionation is to separate the compounds in the extract into the solvent according to their polarity. Non-polar compounds were screened out with n-hexane, semi-polar compounds were sorted out with ethyl acetate, and polar compounds were taken with methanol14. The three fractions were then tested against S. aureus and continued with TLC-bioautography. This study's objective was to comprehend and determine the antibacterial activity of the active fraction of L. inermis leaves and the class of active compounds as an antibacterial agent from the most active fraction against S. aureus.

Materials

Plant materials were obtained from Merapi Farma Herbal Yogyakarta, and the identification of the plant was carried out by the Biology Laboratory, Faculty of Science and Applied Technology, Universitas Ahmad Dahlan, Yogyakarta, with the voucher specimen number 087/Lab.bio/B/VI/2019. Staphylococcus aureus ATCC 25923, Mueller Hinton Agar (Oxoid), Brain Heart Infusion medium (Oxoid), 1% BaCl2 (Merck), 1% H2SO4 (Merck), NaCl 0.9% (Merck), 1% Dimethylsulfoxide (Merck), Vancomycin 1% (Vancep), and silica plate GF254 (Merck). The instruments used were digital balance (Ohaus), oven (Binder), micropipettes (Soccorex), biosafety cabinet (Monmouth Scientific), incubator (Binder), autoclave (Shenan), TLC chamber (Camag), UV lamps, and glassware (Pyrex).

Methods

Preparation of methanol extract

A total of 2 kg of L. inermis leaves were washed and dried in the oven. The dried L. inermis leaves were then ground with a blender and sieved with a 50-mesh sieve. An amount of 250 g of L. inermis leaves powder was macerated with 1000 mL of methanol. The maceration process was carried out at room temperature for the first six hours while shaking, then allowed to stand for 18 hours. Remaceration was done in the same manner. Afterward, the macerate was filtrated using a Buchner funnel. The filtrate was then evaporated with a rotary evaporator until a thick extract was obtained15.

Fractionation of methanol extract of L. inermis

A total of 15 g of L. inermis extract was subsequently dissolved in methanol and fractionated with n-hexane and ethyl acetate. Each n-hexane fraction and ethyl acetate fraction were evaporated with a rotary evaporator to get the n-hexane, ethyl acetate, and methanol fraction. Every fraction was weighed to give the yield of fractionation.

Antibacterial activity test against S. aureus

The well diffusion method was used to conduct the antibacterial activity test. A sterile cotton swab was used to apply the S. aureus bacterium suspension with a 1 x 108 CFU/mL density to the Mueller Hinton agar surface. Then the surface of the agar is perforated and dripped with test and control samples. Next, the plate was incubated at 37°C for 24 hours. After incubation, the diameter of the inhibition zones was measured.

Phytochemical compound testing of the ethyl acetate fraction by reaction test16

The test solution was made in a concentration of 1% w/v by dissolving 250 mg of the ethyl acetate fraction of L. inermis in 25 mL of distilled water. The test was carried out to determine the presence of naphthoquinone, flavonoid, tannin, and saponin compounds.

Naphthoquinone test

As much as 1 mL of the test solution was added to a few drops of 1 N NaOH, a positive solution containing naphthoquinone will show a red color.

Flavonoid test

The test solution was dropped on filter paper, then treated with ammonia vapor. If it causes a yellow color, it indicates the presence of flavonoids.

Tannin test

A positive solution containing naphthoquinone will show a red color when one mL of the test solution is added to a few drops of 1 N NaOH.

Saponin test

A total of 1 mL of the test solution was shaken vigorously for 10 seconds. If the foam is formed for not less than 10 minutes as high as 3-10 cm and by the addition of 2 N HCl the foam does not disappear, it is positive for saponins.

TLC-Bioautography

As the mobile phase, the ethyl acetate fraction of L. inermis leaves was separated using TLC with chloroform : acetone : formic acid (6 : 1.5 : 0.5). The surface medium of MH agar was sprayed with a bacterial suspension evenly. After that, the silica gel plate was placed on the surface of the MHA agar medium in an inverted position and left for 30 minutes to allow diffusion based on the reference with minor modifications17. Then the plate was removed, and the petri dish was incubated at 37°C for 24 hours. After incubation, the inhibition zone was observed. The inhibition zone that appears was measured by the Rf value and compared with the chromatogram detected with spraying reagents to determine the group of active compounds.

Yield of extraction and fractionation of L. inermis leaves

The maceration of L. inermis leaves obtained 47.96 g of methanol extract with a yield of 19.18%. In the study by Sharma and Goel18, the yield of methanol extract of L. inermis nails was 17%. The extraction yield we obtained is greater than that of the previous study due to several factors, such as geographical conditions, sampling time, or other factors.

The fractionation using n-hexane obtained a n-hexane fraction of 1.4610 g with a yield of 9.74%. The n-hexane fraction had a greenish color due to the presence of chlorophyll. With a yield of 25.55%, the ethyl acetate fraction displayed a reddish-brown color of 3.8333 g. The residue in the form of methanol fraction is 8.0585 g with a yield of 53.72%.

Antibacterial activity of the fractions of L. inermis against S. aureus

The antibacterial activity test was performed on MHA media seeded with S. aureus using the well diffusion method. Table I reveals that the inhibitory zone diameter of the methanol extract from L. inermis leaves is 11.33 mm. The n-hexane, ethyl acetate, and methanol fractions result in 8.33 mm, 9.50 mm, and 0 mm, respectively. Remarkably, the methanol extract had the most significant inhibitory zone, though the methanol fraction had none. According to Nwodo et al.19, fractionation occasionally led to increased activity but occasionally led to decreased activity. This represents a situation where fractionation leads to loss of activity, suggesting that components of the extract may have acted synergistically or additively to produce the activity observed in the extract. Another study found that some fractions of Tamarindus indica showed no activity against type P. aeruginosa and E. coli strains, unlike the extract.

The antibacterial activity of the methanol extract was greater compared to each fraction. The highest antibacterial activity was confirmed in crude methanol extract, possibly due to all the antibacterial compounds in its fractions20. These results indicate that the active chemical compounds as antibacterial agents were spread into these fractions and were not collected in one certain fraction. Previous studies showed that the chemical compounds contained in plants provide a synergistic or additive effect in causing pharmacological effects21. If these compounds are separated, it will cause a decrease in their pharmacological activity. However, the ethyl acetate fraction had the greatest antibacterial activity compared to the other fractions. The positive control in the antibacterial activity test was vancomycin because it is sensitive to S. aureus. As a negative control, 1% DMSO was utilized to dissolve practically all polar and non-polar substances. There was no bactericidal action in 1% DMSO. Table I shows the diameter of the inhibitory zones of the methanol extract and the fractions.

TableI. Diameter of inhibition zones of methanol extract and various fractions against S. aureus using well diffusion method

Furthermore, the antibacterial activity of the ethyl acetate fraction of L. inermis leaves was tested at different concentrations to determine the concentration that could inhibit the growth of bacteria. The concentrations of ethyl acetate fraction tested were 5, 15, and 20 %w/v. Table II shows that the diameter of the inhibition zone increased as the concentration of the ethyl acetate fraction was raised. The 20% ethyl acetate fraction produced the largest inhibition diameter of 10.67 mm. Statistical analysis with the Kruskal-Wallis test showed that there were differences in the antibacterial activity of each concentration tested.

TableII. Diameter of inhibition zones of the ethyl acetate fraction against S. aureus using well diffusion method

The phytochemical content of ethyl acetate fraction of L. inermis leaves

The results of the phytochemical screening test (Table III) show that the ethyl acetate fraction of L. inermis leaves contains naphthoquinones, flavonoids, and tannins. In the naphthoquinone test, when the ethyl acetate fraction of L. inermis leaves was dripped with 1 N NaOH solution, the color changed to brownish red due to the presence of a chromophore group in the ethyl acetate fraction of L. inermis leaves so that the addition of a hydroxyl group from NaOH will give a red color6,15. When testing for flavonoids, a more intense yellow color appears on filter paper that has been treated with ammonia vapor, indicating the presence of flavonoid22.

The test on tannin compounds, when added to the gelatin solution in the ethyl acetate fraction of L. inermis leaves, forms a precipitate due to the nature of the tannins, which can precipitate protein so that the tannin test with the addition of gelatin solution, which is a protein will be precipitated by the tannins23. In the saponin test, our finding showed that within 10 minutes, the foam slowly disappeared when HCl was added, indicating that the ethyl acetate fraction of L. inermis leaves did not contain saponin16.

TableIII. The phytochemical screening of ethyl acetate fraction of L. inermis

TLC-Bioautograahy of ethyl acetate fraction of L. inermis

The results of the TLC-bioautography of the ethyl acetate fraction of L. inermis leaves can be seen in Figures 1 and 2. Figure 1 shows two inhibition zones formed on MHA inoculated S. aureus with ​​Rf values of 0.25 and 0.53. After the TLC plate was sprayed with 10% KOH, a spot with the Rf value of 0.25 appeared to be a positive red-brown color, indicating the presence of naphthoquinone compounds (Figure 2 and Table IV). The appearance of a reddish-brown color is due to the addition of a hydroxyl group from KOH15. After the plate was sprayed with FeCl3, a spot with the Rf value of 0.53 (Figure 2 and Table IV) showed a blue-black color, indicating the presence of phenolic compounds16. The reaction forms a blue-black color due to the formation of complex compounds between metal atoms of iron (Fe) and non-metal atoms. The presence of phenolic compounds is in line with previous research, which stated that L. inermis contains phenolics11,24. Husni et al.25 reported that the ethanol extract of L. inermis leaves has a total phenolic content of 16.02 g/100 g.

Figure1. The bioautography result of the ethyl acetate fraction of L. inermis leaves on Mueller Hinton Agar inoculated by S. aureus. The inhibition zones are depicted with black circles.

a b

Figure2. The chomatogram of ethyl acetate fraction of L. inermis leaves after sprayed with FeCl3 (a), and with KOH 10% (b).

TableIV. Result of TLC-bioautography of the ethyl acetate fraction of L. inermis leaves

The mechanism of phenolic compounds in inhibiting bacterial growth is to irreversibly bind to nucleophilic amino acids from proteins, causing protein inactivation and becoming non-functional while also inactivating adhesins and enzymes on microbial membranes. The presence of phenolic groups with a high protein binding affinity can inhibit microbial enzymes while also boosting membrane affinity, resulting in increased antibacterial action26. Luis et al.27 investigated the mechanism of action of a phenolic compound and hypothesized that it was linked to polyphenol-membrane contact. The presence of a phenolic compound was connected to increased permeability and depolarization of the cell layer, as well as a decrease in respiratory action in the S. aureus ATCC 25923 strain. The component of activity of a phenolic compound is connected to cell layer damage and changes in the vigorous metabolism of S. aureus cells8,27. A phenolic compound suppresses a hemolysin secretion in S. aureus; a membrane-dependent activity further supports the initial findings28. Simple phenols' activities are thought to be mediated through contact with sulfhydryl groups in microbial enzymes, inhibiting those enzymes or nonspecific protein interactions29.

Naphthoquinones are found naturally in various plants and are considered promising antibacterial agents. Increased ROS production, followed by apoptotic cell death, is the mechanism of action for this antibacterial agent. Various naphthoquinone compounds have pharmacological effects, including antibacterial, anticancer, antitubercular, antimalarial, and trypanocidal properties. Naphthoquinone analogs are highly lethal to infected cells due to their capacity to create reactive oxygen species (ROS) and can inhibit cellular enzymes involved in apoptosis and cell proliferation. Consequently, these compounds serve as models for developing clinical antibacterial drugs30. Another study confirmed 2-hydroxy-1,4-naphthoquinone found in L. inermis as the main compound that may be an antibacterial agent31. However, its specific mechanism of action needs further research. Although the class of compounds with antibacterial action was identified in this investigation, the actual name of the active chemical cannot be determined. Therefore, additional investigation is required to identify and isolate the active substance.

These results indicate that the ethyl acetate fraction of L. inermis leaves contains naphthoquinones, flavonoids, and phenolic compounds that can inhibit bacterial growth. The results also suggest that other phytochemical compounds may contribute to the antibacterial activity of L. inermis leaves, and further study needs to be done to explore them.

We want to thank Nurma Harfita Sari for assisting with this research. The authors received no financial support for the study or publication of this article.

Sri Mulyaningsih: designed, directed, and managed the study; drafted manuscript preparation; edited and reviewed article. Febriyati Adji Rachmadani: collected data.

None.

The authors declare no conflict of interest.

1. Charoensup R, Duangyod T, Palanuvej C, Ruangrungsi N. Pharmacognostic Specifications and Lawsone Content of Lawsonia inermis Leaves. Pharmacognosy Res. 2017;9(1):60-4. doi:10.4103/0974-8490.199775

2. Sharma RK, Goel A, Bhatia AK. Lawsonia Inermis a Plant with Cosmetic and Medical Benefits. Int J Appl Sci Biotechnol. 2016;4:15-20. doi:10.3126/ijasbt.v4i1.14728

3. Leela K, Singh ARJ. Bioactive Compound Studies of Lawsonia inermis L. (Henna) –Its Ethnomedicinal and Pharmacological Applications: A Review. Int J Mod Trends Sci Technol. 2020;6(9):187-200. doi:10.46501/IJMTST060929

4. Semwal RB, Semwal DK, Combrinck S, Cartwright-Jones C, Viljoen A. Lawsonia inermis L. (henna): ethnobotanical, phytochemical and pharmacological aspects. J Ethnopharmacol. 2014;155(1):80-103. doi:10.1016/j.jep.2014.05.042

5. Usman R, Rabiu U. Antimicrobial activity of Lawsonia inermis (henna) extracts. Bayero J Pure Appl Sci. 2019;11(1):167-71. doi:10.4314/bajopas.v11i1.27S

6. Garg R, Tripathi R, Batav N, Singh R. Phytochemical High Performance Thin Layer Chromatography based Estimation of Lawsone in Lawsonia inermis (Henna) obtained from Two Natural Habitats and Dye Products Collected from Local Market. Med Aromat Plants. 2017;6:3. doi:10.4172/2167-0412.1000290

7. Xavier MR, Santos MMS, Queiroz MG, Silva MSdL, Goes AJS, De Morais Jr MA. Lawsone, a 2-hydroxy-1,4-naphthoquinone from Lawsonia inermis (henna), produces mitochondrial dysfunctions and triggers mitophagy in Saccharomyces cerevisiae. Mol Biol Rep. 2020;47(2):1173-85. doi:10.1007/s11033-019-05218-3

8. Miklasińska-Majdanik M, Kępa M, Wojtyczka RD, Idzik D, Wąsik TJ. Phenolic Compounds Diminish Antibiotic Resistance of Staphylococcus Aureus Clinical Strains. Int J Environ Res Public Health. 2018;15(10):2321. doi:10.3390/ijerph15102321

9. Al-Snafi AE. A Review on Lawsonia Inermis: A Potential Medicinal Plant. Int J Curr Pharm Res. 2019;11(5):1-13. doi:10.22159/ijcpr.2019v11i5.35695

10. Shahabinejad S, Kariminik A. Antibacterial activity of methanol extract of Lawsonia inermis against uropathogenic bacteria. MicroMedicine. 2019;7(2):31-6. doi:10.5281/zenodo.3473381

11. Akhtar J, Bashir F, Bi S. Scientific Basis for the Innovative Uses of Henna (Lawsonia inermis L.) mentioned by Unani Scholars in different ailments. J Complement Altern Med Res. 2021;14(1):1-21. doi:10.9734/jocamr/2021/v14i130234

12. Nigussie D, Davey G, Legesse BA, Fekadu A, Makonnen E. Antibacterial activity of methanol extracts of the leaves of three medicinal plants against selected bacteria isolated from wounds of lymphoedema patients. BMC Complement Med Ther. 2021;21(1):2. doi:10.1186/s12906-020-03183-0

13. Ag T, Kumar MS, Shivannavar CT Gaddad SM. Antibacterial and anti-biofilm activities of crude extracts of Lawsonia inermis against Methicillin Resistant Staphylococcus aureus. Asian J Pharm Clin Res. 2016;9(6):263-5. doi:10.22159/ajpcr.2016.v9i6.14362

14. Abubakar AR, Haque M. Preparation of Medicinal Plants: Basic Extraction and Fractionation Procedures for Experimental Purposes. J Pharm Bioallied Sci. 2020;12(1):1-10. doi:10.4103/jpbs.jpbs_175_19

15. Zainab Z, Muthoharoh A. Penapisan Fitokimia, Penetapan Kadar Naftokuinon total, dan Aktivitas Antifungi Fraksi Tidak Larut Etil Asetat Ekstrak Etanol Daun Pacar Kuku (Lawsonia inermis L.) TERHADAP Candida albicans ATCC. Pharmaciana. 2015;5(2):199-208. doi:10.12928/pharmaciana.v5i2.2371

16. Shaikh JR, Patil M. Qualitative tests for preliminary phytochemical screening: An overview. Int J Chem Stud. 2020;8(2):603-8. doi:10.22271/chemi.2020.v8.i2i.8834

17. Kusumaningtyas E, Astuti E, Darmono D. Sensitivitas Metode Bioautografi Kontak dan Agar Overlay dalam Penentuan Senyawa Antikapang. J Ilmu Kefarmasian Indones. 2008;6(2):75-80.

18. Sharma R, Goel A. Identification of Phytoconstituents in Lawsonia inermis Linn. Leaves Extract by GC-MS and their Antibacterial Potential. Pharmacogn J. 2018;10(6):1101-8. doi:10.5530/pj.2018.6.187

19. Nwodo UU, Ngene AA, Iroegbu CU, Obiiyeke G. Effects of fractionation on antibacterial activity of crude extracts of Tamarindus indica. African J Biotechnol. 2010;9(42):7108-13. doi:10.5897/AJB09.1662

20. Voukeng IK, Nganou BK, Sandjo LP, Celik I, Beng VP, Tane P, et al. Antibacterial activities of the methanol extract, fractions and compounds from Elaeophorbia drupifera (Thonn.) Stapf. (Euphorbiaceae). BMC Complement Altern Med. 2017;17(1):28. doi:10.1186/s12906-016-1509-y

21. Mundy L, Pendry B, Rahman M. Antimicrobial resistance and synergy in herbal medicine. J Herb Med. 2016;6(2):53-8. doi:10.1016/j.hermed.2016.03.001

22. Sembiring EN, Elya B, Sauriasari R. Phytochemical screening, total flavonoid and total phenolic content and antioxidant activity of different parts of Caesalpinia bonduc (L.) Roxb. Pharmacogn J. 2018;10(1):123-7. doi:10.5530/pj.2018.1.22

23. Dhanani T, Shah S, Gajbhiye NA, Kumar S. Effect of extraction methods on yield, phytochemical constituents and antioxidant activity of Withania somnifera. Arab J Chem. 2017;10(S1):1193-9. doi:10.1016/j.arabjc.2013.02.015

24. Hadef KZ, Boufeldja W. Antimicrobial activity of lawsonia inermis leaf extract collected from south of algeria touat (Adrar) and tidikelt (in salah). Asian J Plant Sci. 2020;15(1):9-16. doi:10.3923/jps.2020.9.16

25. Husni E, Suharti N, Atma APT. Karakterisasi Simplisia dan Ekstrak Daun Pacar Kuku (Lawsonia inermis Linn) serta Penentuan Kadar Fenolat Total dan Uji Aktivitas Antioksidan. J Sains Farm Klin. 2018;5(1):12-6. doi:10.25077/jsfk.5.1.12-16.2018

26. Bouarab-Chibane L, Forquet V, Lantéri P, Clément Y, Léonard-Akkari L, Oulahal N, et al. Antibacterial Properties of Polyphenols: Characterization and QSAR (Quantitative Structure-Activity Relationship) Models. Front Microbiol. 2019;10:829. doi:10.3389/fmicb.2019.00829

27. Luís Â, Silva F, Sousa S, Duarte AP, Domingues F. Antistaphylococcal and biofilm inhibitory activities of gallic, caffeic, and chlorogenic acids. Biofouling. 2014;30(1):69–79. doi:10.1080/08927014.2013.845878

28. Cowan M. Plant products as antimicrobial agents. Clin Microbiol Rev. 1999;12(4):564-82. doi:10.1128/cmr.12.4.564

29. Aldulaimi OA. General Overview of Phenolics from Plant to Laboratory, Good Antibacterials or Not. Pharmacogn Rev. 2017;11(22):123-7. doi:10.4103/phrev.phrev_43_16

30. Ravichandiran P, Sheet S, Premnath D, Kim AR, Yoo DJ. 1,4-Naphthoquinone Analogues: Potent Antibacterial Agents and Mode of Action Evaluation. Molecules. 2019;24(7):1437. doi:10.3390/molecules24071437

31. Pour AP, Farahbakhs H. Lawsonia inermis L. leaves aqueous extract as a natural antioxidant and antibacterial product. Nat Prod Res. 2020;34(23):3399-403. doi:10.1080/14786419.2019.1569006