© 2021 Journal of Pharmacy & Pharmacognosy Research, 9 (6), 813-823, 2021 ISSN 0719-4250

http://jppres.com/jppres

Original Article

Time-kill kinetics and mechanism of action of Caesalpinia sappan L. and Ochna integerrima (Lour.) Merr. water extracts against pathogenic bacteria [Cinética de tiempo de muerte y mecanismo de acción de extractos acuosos de Caesalpinia sappan L. y Ochna integerrima (Lour.) Merr. contra bacterias patógenas] Prapairat Seephonkai1*, Sutthira Sedlak2, Komgrit Wongpakam2, Kusavadee Sangdee3, Aphidech Sangdee3

1Nanotechnology Research Unit, Department of Chemistry, Faculty of Science, Mahasarakham University, Khamriang, Kantarawichai, Maha Sarakham, 44150, Thailand and Center of Excellence for Innovation in Chemistry (PERCH-CIC). 2Walairukhavej Botanical Research Institute, Mahasarakham University, Thailand. 3Microbiology Group, Biomedical Sciences Research Unit, Faculty of Medicine, Mahasarakham University, Thailand. 4Microbiology and Applied Microbiology Research Unit, Department of Biology, Faculty of Science, Mahasarakham University, Thailand. *E-mail: [email protected]

Abstract Resumen Context: Caesalpinia sappan and Ochna integerrima are two native plants of Contexto: Caesalpinia sappan y Ochna integerrima son dos plantas nativas Thailand and distributed widely throughout the country. Recently, de Tailandia y se distribuyen ampliamente por todo el país. water extracts from the stem bark of C. sappan and O. integerrima were Recientemente, se descubrió que los extractos de agua de la corteza del found to exhibit potently antibacterial property but the antibacterial tallo de C. sappan y O. integerrima exhiben propiedades antibacterianas mechanism of action toward target bacteria has not been investigated potentes, pero aún no se ha investigado el mecanismo de acción yet. antibacteriano hacia las bacterias diana. Aims: To study time-kill kinetic and the mechanism of action of C. Objetivos: Estudiar la cinética de tiempo de muerte y el mecanismo de sappan and O. integerrima water extracts toward target bacteria. acción de los extractos de agua de C. sappan y O. integerrima sobre las Methods: The time-kill kinetic study of bacteriostatic or bactericidal bacterias objetivo. activity against Gram-positive and Gram-negative pathogenic bacteria Métodos: Se realizó el estudio cinético de tiempo de muerte de la within 24 h experiment was conducted and the mechanism of action on actividad bacteriostática o bactericida contra bacterias patógenas Gram- cell morphology of the target bacteria by using scanning electron positivas y Gram-negativas dentro de las 24 horas del experimento y se microscopy (SEM) were investigated. investigó el mecanismo de acción sobre la morfología celular de las Results: Both of the C. sappan and O. integerrima water extracts possessed bacterias diana mediante el uso de microscopio electrónico de barrido bacteriostatic or bactericidal activity toward six tested pathogenic (SEM). bacteria. The bacteriostatic or bactericidal activity depending on Resultados: Los extractos acuosos de C. sappan y O. integerrima exhibieron bacterial strains and the test concentrations (0.5, 1 and 2 × MIC) of these actividad bacteriostática o bactericida hacia seis bacterias patógenas two extracts to the tested bacteria were observed. The bactericidal probadas. Se observó la actividad bacteriostática o bactericida activity against all of the tested bacterial was evinced at 2 × MIC. The dependiendo de las cepas bacterianas y las concentraciones de prueba morphological alterations of the target bacterial cells of changing the cell (0,5; 1 y 2 × MIC) de estos dos extractos en las bacterias probadas, size (decreased and increased), cell lysis and having cell cavity were mientras que la actividad bactericida contra todas las bacterias probadas detected by SEM after the treatment with C. sappan and O. integerrima se evidenció a 2 × MIC. Las alteraciones morfológicas de las células water extracts. bacterianas diana de cambio de tamaño celular (disminución y Conclusions: The water extracts from the stem bark of C. sappan and O. aumento), lisis celular y cavidad celular fueron detectadas por SEM integerrima exhibited bactericidal activity by changing the cell size, cell después del tratamiento con extractos de agua de C. sappan y O. lysis and cell cavity of the tested bacteria cell morphology. integerrima. Conclusiones: Los extractos acuosos de la corteza del tallo de C. sappan y O. integerrima exhibieron actividad bactericida al cambiar el tamaño celular, la lisis celular y la cavidad celular de la morfología celular de las bacterias probadas. Keywords: antibacterial activity; Caesalpinia sappan; mechanism of action; Palabras Clave: actividad antibacteriana; Caesalpinia sappan; cinética de Ochna integerrima; time-kill kinetic. tiempo de muerte; mecanismo de acción; Ochna integerrima.

ARTICLE INFO Received: May 3, 2021. Received in revised form: June 11, 2021. Accepted: June 12, 2021. Available Online: June 17, 2021.

______

Seephonkai et al. Caesalpinia sappan and Ochna integerrima: Time-kill kinetic and mechanism of action

INTRODUCTION diarrhea, dysentery, epilepsy, menorrhagia, leu- corrhoea, diabetes) according to Ayurveda of India Phenolic compounds are groups of phytochem- (Kirtikar and Basu, 1987; Warrier et al., 1993), to ical secondary metabolites. It contains numerous cure rheumatism and inflammatory diseases and groups of compounds such as phenols, phenolic use as an emmenagogue, and homeostatic agents acids, or polyphenols (simple flavonoids, flavones, in Vietnam (Do, 2001), and to treat tuberculosis, flavonols, tannins, coumarins, quinones, complex diarrhea, dysentery, skin infections and anemia in flavonoids and colored anthocyanins). These com- Thailand (Sireeratawong et al., 2010). pounds show antimicrobial effect and serves as plant defense mechanisms against pathogenic mi- Ochna integerrima (Lour.) Merr. (Ochnaceae) be- croorganisms (Das et al., 2010; Babbar et al., 2014). longs to the genus Ochna with more than 80 spe- They have been studied for their potential proper- cies distributed in tropical Asia, Africa and Ameri- ties and many of them exhibited antibacterial ac- ca (Rendle, 1952). There are only two species of the tivity (Bouarab-Chibane et al., 2019a; 2019b). The genus Ochna which are O. integerrima and O. kirkii antimicrobial capacity of polyphenols and flavo- Oliv. found in Thailand (Smitinand and Larsen, noids from plants has been investigated against a 1970; Smitinand, 2001) and the species O. inte- wide range of microorganisms (Ahn et al., 1991; gerrima distributed widely in Thailand (Smitinand Diker et al., 1991; Sakanaka et al., 1992; Borris, and Larsen, 1970). It is a small tree with average 1996; Cushnie and Lamb, 2005; Cŏté et al. 2010). height over 1 m for five year old tree and grown as Therefore, plant products or molecules are consid- an ornamental garden plant due to its typically ered to be good natural antimicrobial or antibacte- beautiful flower (Trang et al., 2018). In Vietnam, O. rial sources that can be used to treat microbial or integerrima has been ranked as the rare and endan- bacterial infections (Pawar, 2008; Fernebro, 2011; gered species due to its high demand trade of the Srivastava et al., 2013). beautiful species (Trang et al., 2018). In Thai folk medicine the bark of this plant has been used as a Caesalpinia sappan (L.) (Leguminosae) is distribut- digestive tonic and the root as an anthelmintic ed widely in Southeast Asia and is commonly whereas in Indonesia an infusion of the root and known as ‘sappan wood’ or ‘Brazil wood’. It is a leaf is used as an antidysenteric and an antipyretic small thorny tree of 6-9 m height and 15-25 cm in (Perry, 1980). There is none of the record of tradi- diameter with a few prickly branches. Its wood is tional use of O. integerrima to treat bacterial infec- orange-red, hard, very heavy and straight-grained tion or related symptoms. with a fine texture (Pawar, 2008). The heartwood of this plant has been traditionally used as a natu- In our continue search on antibacterial activity ral coloring agent in beverage, food, cosmetic and of local medicinal plants, the water extracts from garment for long history (Badami et al. 2004; the stem barks of C. sappan and O. integerrima was Wetwitayaklung et al., 2005; Siva, 2007; Karap- recently found to exhibit potent antibacterial anagiotis et al., 2008; Wu Toegel et al., 2012; Nir- properties against three Gram-positive; Staphylo- mal and Panichayupakaranant, 2015). In folk med- coccus aureus (MSSA) DMST 2933, Staphylococcus icine, the heartwood of C. sappan is used for varie- aureus (MRSA) DMST 20651 and Bacillus cereus ty of medicinal purposes, for examples analgesic, ATCC11778, and three Gram-negative; Escherichia thrombosis and tumor treatments (Soka, 1985), to coli ATCC 25922, Salmonella enterica serovar Typhi activate blood circulation and remove stasis, and DMST 22842 and Shigella fleneri DMST 4423, food- to use as an emmenagogue, analgesic or anti- borne pathogenic bacteria (Seephonkai et al., inflammatory agent in China (Xie et al., 2000; The 2021). It indicates that stem barks of these two Chinese Pharmacopoeia Commission, 2005), an plant extracts can be potentially used for the ingredient in vitiated conditions of Pitta (burning treatment of microbial infection or related applica- sensation, wounds, ulcers, leprosy, skin diseases, tions. However, details on antibacterial mecha- http://jppres.com/jppres J Pharm Pharmacogn Res (2021) 9(6): 814

Seephonkai et al. Caesalpinia sappan and Ochna integerrima: Time-kill kinetic and mechanism of action

nisms of these two plant extracts on their target were cultured on Mueller Hinton Agar (MHA) at bacteria have not been investigated yet. Therefore, 37°C for 16–18 h. Then, single colony of bacteria the aim of our study was to investigate antibacte- pathogens was inoculated into Mueller Hinton rial activity in terms of time-kill kinetic and mode Broth (MHB) at 37°C for 4 h with shaking at 250 of action of the C. sappan and O. integerrima water rpm. After that, bacteria culture was adjusted to 4– extracts toward six strains of the foodborne tar- 5 × 106 CFU/mL and stored at 4C before use for get’s pathogenic bacteria. tested experiments.

MATERIAL AND METHODS Time-kill assay The experiment of time-kill was done as de- Chemicals and reagents scribed in the literatures (White et al., 1996; Ai- Tetracycline (Sigma, USA), Mueller Hinton yegoro et al., 2009). The concentrations of 0.5, 1 Agar (Difco, USA), Mueller Hinton Broth (Difco, and 2 × MIC of the extracts was used to test USA), glutaraldehyde (Sigma-Aldrich, Germany) against target bacteria along with the test tube of and sucrose (Ajax FineChem Laboratory Chemi- MHB without extract samples and MHB with tet- cals, New Zealand) were used for the experiments. racycline (250 μg/mL), a standard antibiotic com- pound, in the assay. After incubation (37°C), bac- Plant material and extraction terial suspension in a small volume was taken at 0, The stem barks of C. sappan (L.) and O. inte- 2, 4, and 24 h to dilute and spread onto the MHA gerrima (Lour.) Merr. were collected from Maha plate in order to count the viable colony number. Sarakham province, northeastern Thailand in May, The experiment was done in three replicates and 2018 (15°41′52″N, 103°13′36″E). The plant samples mean was calculated using Microsoft® Excel® were collected by K. Wongpakam and identified 2016 (Microsoft Corporation, Redmond, USA). A by S. Sedlak, Walai Rukhavej Botanical Research graph between viable bacteria number (mean val- Institute (WRBRI), Mahasarakham University ue) and time taken was plotted to evaluate the (MSU) where the voucher specimens of killing rate. Results with bacterial count reduction Wongpakam 19-01 for C. sappan and Wongpakam of 3 log 10 were considered to have bactericidal 19-02 for O. integerrima were deposited. Dried activity while the bacterial count reduction less plants (50 g) were extracted in distilled water (500 than 3 log 10 were defined as bacteriostatic activi- mL) by refluxing for 24 h. An aqueous solution ty. was collected after filtration, and 100 mL of the Scanning electron microscope experiment aqueous solution was subjected to freeze dry (He- to Power Dry PL3000, Thermo Fisher Scientific, The mechanism of action of the extracts on tar- USA) to obtain the water extracts from the stem get bacterial cell morphology was investigated barks of C. sappan and O. integerrima. using scanning electron microscope (SEM) exper- iment as described in literature (Sangdee et al., Microbial strains and cultivation 2018). The pathogen cells were treated with the Six pathogenic bacteria targets; three Gram- extracts at 2 × MIC level of the concentration, and positive bacteria, Staphylococcus aureus (MSSA) were then incubated (37°C, 16−18 h). Next, the DMST 2933, Staphylococcus aureus (MRSA) DMST bacterial cells were harvested and washed, and 20651 and Bacillus cereus ATCC1178, and three fixed with 2.5% glutaraldehyde in 5% sucrose. Gram-negative bacteria, Escherichia coli ATCC After that, the cell pellets were dehydrated and 25922, Salmonella enterica serovar Typhi DMST then applied to a membrane. Gold was used to 22842 and Shigella fleneri DMST 4423, were sourced sputter-coat onto a dried extract powder surface from Medical Microorganisms Department of for preparing samples which were taken to mor- Medical Sciences Thailand. The selected strains phologically examine under a SEM (JOEL Tsm-700 FSHL, Japan). The cell alterations caused by the http://jppres.com/jppres J Pharm Pharmacogn Res (2021) 9(6): 815

Seephonkai et al. Caesalpinia sappan and Ochna integerrima: Time-kill kinetic and mechanism of action

extract treatment were compared with those mined by 24 h, respectively. No activity of the ex- caused by tetracycline (250 μg/mL) and the con- tract against the remaining strains was found. At trol treatments. higher concentration of 1 × MIC, the bacteriostatic activity against both stains of Gram-positive Statistical analysis Staphylococci were observed by 24 h, but it did not The data from three replicates were presented show antibacterial effect toward S. flexneri DMST as mean using Microsoft® Excel® 2016 (Microsoft 4423. Complete eliminations of bactericidal activi- Corporation, Redmond, USA). Then, the time-kill ty of three tested strains including B. cereus ATCC kinetics were plot using Microsoft® Excel® 2016. 11778, E. coli ATCC 25922 and S. Typhi DMST 22842 (Fig. 2C, D, F) were achieved by 24 h treat- ments with 1 × MIC the extract concentration. In-

RESULTS creasing the concentration up to 2 × MIC resulted in bactericidal activity against all bacterial strains Time-kill assay by 24 h (Fig. 2A-F). These results indicated the The time-kill assay of C. sappan water extract bacteriostatic or bactericidal activity of the O. inte- was shown in Fig. 1. The C. sappan extract at the gerrima extract is confirmed within 24 h experi- concentration of 0.5 × MIC had the bactericidal ment. activity against S. aureus (MRSA) DMST 20651 The time-kill study of the Gram-negative S. (Fig. 1B), and bacteriostatic activity against S. aure- flexneri DMST 4423 when treated with the C. sap- us (MSSA) DMST 2933 (Fig. 1A) and B. cereus pan and O. integerrima extracts indicated inactive ATCC 11778 (Fig. 1C). This extract did not show bacteriostatic and bactericidal activity results at antibacterial property against E. coli ATCC 25922, the concentrations of 0.5 × MIC and 1 × MIC. The S. flexneri DMST 4423 and S. Typhi DMST 22842 bactericidal effect was only observed at the highest (Fig. 1D-F) at same level of the concentration. tested concentration of 2 x MIC which were 1.56 When increasing the concentration of the extract and 6.24 mg/mL against C. sappan and O. inte- up to 1 x MIC, the bactericidal activity against all gerrima, respectively (Fig. 1E and 2E). Therefore, of the tested Gram-positive bacterial strains (Fig. both of the extracts had not much impact toward 1A-C), and the bacteriostatic property against two S. flexneri DMST 4423. strains of Gram-negative bacterial pathogen E. coli ATCC 25922 (Fig. 1D) and S. flexneri DMST 4423 Scanning electron microscope experiment (Fig. 1E) were observed by 24 h. However, there was no activity of the extract on S. Typhi DMST The water extracts of C. sappan and O. integerri- 22842 (Fig. 1F). Also, increasing the concentration ma treated with five pathogenic bacteria were ex- of the extract up to 2 × MIC resulted in bactericidal amined by SEM. The results showed similar altera- ability toward S. aureus (MSSA) DMST 2933, S. tion effects of the C. sappan and O. integerrima ex- aureus (MRSA) DMST 20651, B. cereus ATCC tracts on cell morphology of the tested bacteria. 11778, E. coli ATCC 25922 by 24 h (Fig. 1A-D), SEM micrographs of both S. aureus MSSA and whereas the bacteriostatic activity against the re- MRSA strains after 24 h treatment with the ex- maining bacterial strains was not cleared (Fig. 1E tracts revealed decreased or increased in cell size and 1F). These results suggested that the C. sappan (Fig. 3C-D and Fig. 4C-D). Cell shape of the B. ce- extract had bacteriostatic or bactericidal activity at reus ATCC 11778 (Fig. 5C-D) was abnormal when the tested concentration of 0.5-2 × MIC against the compared with their original size, showing both tested bacterial strains. cases of bacterial cells size increased or decreased. Moreover, some other cells appearing bacterial cell

For the O. integerrima water extract, at 0.5 × cavities were also observed. Cell shapes of the E. MIC concentration, the bactericidal or bacteriostat- coli ATCC 25922 (Fig. 6C-D) and S. Typhi DMST ic potency against B. cereus ATCC 11778 (Fig. 2C) 24822 (Fig. 7C-D) were induced several alterations and S. Typhi DMST 22842 (Fig. 2F) was deter- causing cell size changing (increased or de- http://jppres.com/jppres J Pharm Pharmacogn Res (2021) 9(6): 816

Seephonkai et al. Caesalpinia sappan and Ochna integerrima: Time-kill kinetic and mechanism of action

creased), cell lysis and cell cavity. However, it is mic membrane permeability and led to cell wall clear from the micrographs that the number of leakage. Untreated control of all bacterial cells ap- damaged cells greatly outnumbers those of normal peared undamaged (Fig. 3-7A) whilst the treat- cells. These results indicate that the active com- ment of all bacterial strains with standard antibi- pound in the extracts had some effects on the cy- otic tetracycline induced several alterations as sim- toplasmic membrane and cell wall of the bacteria. ilar as treated with both C. sappan and O. inte- These effects resulted in an alteration in cytoplas- gerrima extracts (Fig. 3-7B).

Figure 1. In vitro bacteriostatic and bacteriocidal activities of C. sappan water extract against S. aureus (MSSA) DMST 2933, MIC 0.78 mg/mL (A); S. aureus (MRSA) DMST 20651, MIC 0.78 mg/mL (B); B. cereus ATCC 11778 MIC 0.39 mg/mL (C); E. coli ATCC 25922, MIC 3.12 mg/mL (D); S. flexneri DMST 4423, MIC 0.78 mg/mL (E); and S. Typhi DMST 22842, MIC 0.78 mg/mL (F) at the concentration of 0.5 x MIC (▲), 1 x MIC (×), and 2 x MIC () compared with the untreated control () and tetracycline at 250 g/mL (). Data represent mean from three replicates.

Figure 2. In vitro bacteriostatic and bacteriocidal activities of O. integerrima water extract against S. aureus (MSSA) DMST 2933, MIC 0.78 mg/mL (A); S. aureus (MRSA) DMST 20651, MIC 1.56 mg/mL (B); B. cereus ATCC 11778, MIC 0.78 mg/mL (C), E. coli ATCC 25922, MIC 6.25 mg/mL (D); S. flexneri DMST 4423, MIC 3.12 mg/mL (E); and S. Typhi DMST 22842, MIC 3.12 mg/mL (F) at the concentrations of 0.5 × MIC (▲), 1 × MIC (×) and 2 × MIC () compared with the untreated control () and tetracycline at 250 g/mL (). Data represent mean from three replicates.

http://jppres.com/jppres J Pharm Pharmacogn Res (2021) 9(6): 817

Seephonkai et al. Caesalpinia sappan and Ochna integerrima: Time-kill kinetic and mechanism of action

Figure 3. SEM micrographs of untreated control S. aureus (MSSA) DMST 2933 (A); compared with treated with tetracycline at 250 g/mL (B); C. sappan water extract (C); and O. integerrima water extract (D).

Figure 4. SEM micrographs of untreated control S. aureus (MRSA) DMST 20651 (A); compared with treated with tetracycline at 250 g/mL (B); C. sappan water extract (C); and O. integerrima water extract (D).

http://jppres.com/jppres J Pharm Pharmacogn Res (2021) 9(6): 818

Seephonkai et al. Caesalpinia sappan and Ochna integerrima: Time-kill kinetic and mechanism of action

Figure 5. SEM micrographs of untreated control B. cereus ATCC 11778 (A); compared with treated with tetracycline at 250 g/mL (B); C. sappan water extract (C); and O. integerrima water extract (D).

Figure 6. SEM micrographs of untreated control E. coli ATCC 25922 (A); compared with treated with tetracycline at 250 g/mL (B); C. sappan water extract (C); and O. integerrima water extract (D).

http://jppres.com/jppres J Pharm Pharmacogn Res (2021) 9(6): 819

Seephonkai et al. Caesalpinia sappan and Ochna integerrima: Time-kill kinetic and mechanism of action

Figure 7. SEM micrographs of untreated control S. Typhi DMST 22842 (A); compared with treated with tetracycline at 250 g/mL (B); C. sappan water extract (C); and O. integerrima water extract (D).

DISCUSSION 4423 and S. Typhi DMST 22842. These results may be due to the mode of antibacterial action of the Nowadays, increasing in the incidence of drug- extract that responsible to the different target mol- resistant bacterial pathogens infections have ecules in cell wall structure of the Gram-negative prompted renewed efforts to identify biologically and Gram-positive bacteria (Russell, 2003). The O. active molecules from natural sources that could integerrima extract at the same concentration of 2 × be useful for antibacterial therapies. Plant extracts MIC possessed bactericidal ability against all of have one of the candidate sources due to their va- the tested bacterial strains by 24 h time-kill kinetic rieties of bioactive phytochemicals and their safety experiment. The antibacterial activity as bacteri- for using on human health. According to our cide of the O. integerrima extract seems to be study, the microbial death kinetic and possible stronger than the activity of C. sappan extract. The mechanism of action of the C. sappan and O. inte- antibacterial activity of the C. sappan extract has gerrima water extracts toward six foodborne path- been studies (Xu and Lee, 2004; Kim et al., 2004; ogenic bacteria were first reported. The results Srinivasan et al., 2012; Nirmal and Panichayupa- demonstrated that the antibacterial activity de- karanant, 2015) while the antibacterial activity of pended on the extract and the strains of the tested the O. integerrima extract has been explored by our bacteria. It indicates clearly that the C. sappan and group recently. However, the potent antibacterial O. integerrima extracts exhibited concentration- property of Ochna pretoriensis species collected in and time-dependent bacteriostatic or bactericidal South Africa against E. coli and E. faecalis has been activity. documented (Makhafola and Eloff, 2012). There- Based on the results of death kinetic at the con- fore, the stem bark of O. integerrima can be alterna- centration of 2 × MIC level, the C. sappan extract tive potential plant sources of natural antibacterial exhibited greater antibacterial activity against the substances according to our present results. Gram-positive bacteria than Gram-negative bacte- Results from the SEM images clearly showed ria. The extract had bactericidal property against bacterial cell morphological alterations such as cell all of the Gram-positive bacteria while the bacteri- lysis and cell cavity observing in the B. cereus ostatic activity was observed in S. flexneri DMST ATCC 11778, E. coli ATCC 25922, S. flexneri DMST http://jppres.com/jppres J Pharm Pharmacogn Res (2021) 9(6): 820

Seephonkai et al. Caesalpinia sappan and Ochna integerrima: Time-kill kinetic and mechanism of action

4423 and S. Typhi DMST 22842 after the treatment REFERENCES with the C. sappan and O. integerrima extracts. Ahn YJ, Kawamura T, Kim M, Yamamoto T, Mitsuoka T These alterations may be due to flavonoid and (1991) Tea polyphenols: Selective growth inhibitors of phenolic compounds, which are groups of phyto- Clostridium spp. Agric Biol Chem 551: 425−1426. chemicals presented in the barks of C. sappan and Aiyegoro OA, Afolayan AJ, Okoh AI (2009) In vitro O. integerrima extracts (Kaewamatawong et al., antibacterial time kill studies of leaves extracts of 2002; Ichino et al., 2006; Reutrakul et al., 2007; Helichrysum longifolium. J Med Plant Res 3(6): 462−67. Pawar et al., 2008; Nirmal et al., 2015) that affect- Babbar N, Oberoi HS, Sandhu SK, Bhargav VK (2014) ing the cell wall and cytoplasmic membrane of Influence of different solvents in extraction of phenolic compounds from vegetable residues and their evaluation bacterial pathogens. Flavonoids represent another as natural sources of antioxidants. J Food Sci Technol 51: class of bioactive compounds that inhibit bacterial 2568−2575. growth by involving the inhibition of DNA gyrase, Badami S, Moorkoth S, Suresh B (2004) Caesalpinia sappan a disrupting the cytoplasmic membrane function medicinal and dye yielding plant. Nat Prod Rad 3(2): and inhibiting of energy metabolism (Cushnie et 75−82. al., 2016). Phenolic compounds also play an im- Borris RP (1996) Natural products research: Perspectives from portant role to inhibit bacterial cell growth by dis- a major pharmaceutical company. J Ethnopharmacol 51: 29−38. rupting the cytoplasmic membrane of bacterial cell, then causing a change in membrane permea- Bouarab-Chibane L, Degraeve P, Ferhout H, Bouajila J, Oulahal N (2019a) Plant antimicrobial polyphenols as bility and finally causing leakage of cytoplasmic potential natural food preservatives. J Sci Food Agric 99: constituents (Johnston et al., 2003). Therefore, the 1457−1474. bacterial cell alteration changes found in this study Bouarab-Chibane L, Forquet V, Lantéri P, Clément Y, may suggest that the mode of antibacterial action Léonard-Akkari L, Oulahal N, Degraeve P, Bordes C of the extract involves disruption of peptidoglycan (2019b) Antibacterial properties of polyphenols: Characterization and QSAR (Quantitative Structure- biosynthesis (Cushnie et al., 2016). Activity Relationship) models. Front Microbiol 10: 1−23. Cŏté J, Caillet S, Doyon G, Sylvain JP, Lacroix M (2010) CONCLUSIONS Bioactive compounds in cranberries and their biological − Both of the C. sappan and O. integerrima extracts properties. Crit Rev Food Sci Nutr 50: 666 679. showed time- and concentration-dependent bacte- Cushnie TPT, Lamb AJ (2005) Antimicrobial activity of flavonoids. Int J Antimicrob Agents. 2005; 26: 343−356. riostatic or bactericidal activity against all of the Cushnie TPT, O’Driscoll NH, Lamb AJ (2016) Morphological tested pathogenic bacteria. The bacteriostatic or and ultrastructural changes in bacterial cells as an bactericidal property were observed at the tested indicator of antibacterial mechanism of action. Cell Mol concentrations of 0.5  MIC and 2  MIC while the Life Sci 73: 4471−92. bactericidal activity against all of the tested bacte- Das K, Tiwari RKS, Shrivastava DK (2010) Techniques for rial was found at 2  MIC. The morphological al- evaluation of medicinal plant products as antimicrobial terations of changing the bacterial cell size, cell agent: Current methods and future trends. J Med Plant Res 4(2): 104−111. lysis and having cell cavity were observed. Diker KS, Akan M, Hascelik G, Yurdakök M (1991) The bacterial activity of tea against Campylobacter jejuni and CONFLICT OF INTEREST Campylobacter coli. Lett App Microbiol 12: 34−35. The authors declare no conflicts of interests. Do TL (2001) Vietnamese Medicinal Plants; Medicine Publisher: Hanoi, p. 50. ACKNOWLEDGMENTS Fernebro J (2011) Fighting bacterial infections - Future treatment options. Drug Resist Updates 14: 125−39. This research was financially supported by Mahasarak- Ichino C, Kiyohara H, Soonthornchareonnon N, Chuakul W, ham University (Grant year 2019). PS gratefuls the Center of Ishiyama A, Sekiguchi H, Namatame M, Otoguro K, Excellence for Innovation in Chemistry (PERCH-CIC), Minis- Omura S, Yamada H (2006) Antimalarial activity of try of Higher Education, Research and Innovation and Faculty biflavonoids from Ochna integerrima. Planta Med 72(7): of Science, Mahasarakham University. 611−614.

http://jppres.com/jppres J Pharm Pharmacogn Res (2021) 9(6): 821

Seephonkai et al. Caesalpinia sappan and Ochna integerrima: Time-kill kinetic and mechanism of action

Johnston MD, Hanlon GW, Denyer SP, Lambert RJW (2003) (2021) Antioxidant, xanthine oxidase inhibitory and Membrane damage to bacteria caused by single and antibacterial activities of selected galactogogue Thai combined biocides. J Appl Microbiol 94: 1015−1023. medicinal plant water and ethyl acetate extracts. J Res Kaewamatawong R, Likhitwitayawuid K, Ruangrungsi N, Pharm (http://www.jrespharm.com/abstract.php? Takayama H, Kitajima M, Aimi N (2002) Novel lang=en&id=908) [June 10, 2021]. biflavonoids from the stem bark of Ochna integerrima. J Sireeratawong S, Piyabhan P, Singhalak T, Wongkrajang Y, Nat Prod 65: 1027−1029. Temsiririrkkul R, Punsrirat J, Ruangwises N, Saraya S, Karapanagiotis I, Lakka A, Valianou L, Chryssoulakis Y (2008) Lerdvuthisopon N, Jaijoy K (2010) Toxicity evaluation of High-performance liquid chromatographic determination sappan wood extract in rats. J Med Assoc Thai 93: of colouring matters in historical garments from the Holy S50−S57. Mountain of Athos. MCA 160: 477−483. Siva R (2007) Status of natural dyes and dye-yielding plants in Kim KJ, Yu HH, Jeong SI, Cha JD, Kim SM, You YO (2004) India. Curr Sci 92: 916−925. Inhibitory effects of Caesalpinia sappan on growth and Smitinand T, Larsen K (1970) Flora of Thailand, Tistr Press, invasion of methicillin-resistant Staphylococcus aureus. J Bangkok. Ethnopharmacol 91(1): 81−87. Smitinand T (2001) Thai Plant Names, Funny Publishing, Kirtikar KR, Basu BD (1987) Indian Medicinal Plants. Bangkok, Thailand. International Book Distributors, Dehradun, India, pp. Soka T (1985) Dictionary of Chinese Drugs, Shanghai Science 847−848. Technology Shogaukan (Eds.), Shogakukan Press, Tokyo, Makhafola TJ, Eloff JN (2012) Five Ochna species have high pp. 1627−1628. antibacterial activity and more than ten antibacterial Srinivasan R, Selvam GG, Karthik S, Mathivanan K, Baskaran compounds. S Afr J Sci 108(1/2): 1−6. R, Karthikeyan M, Gopi M, Govindasamy C (2012) In Nirmal NP, Panichayupakaranant P (2015) Antioxidant, vitro antimicrobial activity of Caesalpinia sappan L. Asian antibacterial, and anti-inflammatory activities of Pac J. Trop Biomed 2(1): 136−139. standardized brazilin-rich Caesalpinia sappan extract. Srivastava J, Chandra H, Nautiyal AR, Kalra SJS (2013) Pharm Biol 53(9): 1339−1343. Antimicrobial resistance (AMR) and plant-derived Nirmal NP, Rajput MS, Prasad RGSV, Ahmad M (2015) antimicrobials (PDAms) as an alternative drug line to Brazillin from Caesalpinia sappan heartwood and its control infections. 3 Biotech 4: 451−60. pharmacological activities: A review. Asian Pac J Trop The Chinese Pharmacopoeia Commission (2005) The Med 8(6): 421−430. Pharmacopoeia of the People's Republic of China, (Part I), Pawar CR, Landge AD, Surana SJ (2008) Phytochemical and Chemical Industry Press, Beijing, China, p. 113. pharmacological aspects of Caesalpinia sappan. J Pharm Trang DH, Dong DV, Chung BH, Gioi DH, Khanh TD (2018) Res 1(2): 131−138. Identification of Vietnamese Ochna integerrima (Lour.) Rendle AB (1952) The Classification of Flowering Plants vol. 2, Merr species based on ribosomal DNA internal Cambridge University Press London. transcribed spacer sequence. Int Lett Nat Sci 68: 9−18. Perry LM (1980) Medicinal Plants of East and Southeast Asia, Warrier PK, Nambiar VPK, Ramankutty C (1993) Indian Boston, The MIT Press, pp. 289-290. Medicinal Plants, A Compendium of 500 Species,” Vol. 1, Reutrakul V, Ningnuek N, Pohmakotr M, Yoosook C, Orient Longman Ltd., Chennai, India, pp. 291−293. Napaswad C, Kasisit J, Santisuk T, Tuchinda P (2007) Wetwitayaklung P, Phaechawud T, Keokitichai S (2005) Anti-HIV-1 flavonoid glycosides from Ochna integerrima. Antioxidant activity of Caesalpinia sappan L. heartwood in Planta Med 73(7): 683v−688. various ages. NUJST 3(2): 43-−52. Russell AD (2003) Similarities and differences in the responses White RL, Burgess DS, Manduru M, Bosso JA (1996) of microorganisms to biocides. J Antimicrob Chemother Comparison of three different in vitro methods of 52: 750−63. detecting synergy: time-kill, checkerboard, and E test. Sakanaka S, Shimura N, Aizawa M, Kim M, Yamamoto T Antimicrob Agents Chemother 40(8): 1914−18. (1992) Preventive effect of green tea polyphenols against Wu Toegel SQ, Otero M, Goldring MB, Leelapornpisid P, dental caries in conventional rats. Biosci Biotech Bioch 56: Chiari C, Kolb A, Unger FM, Windhager R, Viernstein H 592−594. (2012) Caesalpinia sappan extract inhibits IL1β-mediate Sangdee A, Sangdee K, Buranrat B, Thammawat S (2018) overexpression of matrix metalloproteinase in human Effects of mycelial extract and crude protein of the chondrocytes. Genes Nutr 7(2): 312−318. medicinal mushroom, Ophiocordyceps sobolifera, on the Xie YW, Ming DS, Xu HX, Dong H, But PP (2000) pathogenic fungus, Candida albicans. Trop J Pharm Res Vasorelaxing effects of Caesalpinia sappan involvement of 17(12): 2449−2454. endogenous nitric oxide. Life Sci 67: 1913−1918. Seephonkai P, Mongkolsiri N, Thiabphet W, Traisathit R, Xu HX, Lee SF (2004) The antibacterial principle of Caesalpinia Sedlak S, Wongpakam K, Dhammaraj T, Sangdee A sappan. Phytother Res 18(8): 647−651. http://jppres.com/jppres J Pharm Pharmacogn Res (2021) 9(6): 822

Seephonkai et al. Caesalpinia sappan and Ochna integerrima: Time-kill kinetic and mechanism of action

______

AUTHOR CONTRIBUTION:

Contribution Seephonkai P Sedlak S Wongpakam K Sangdee K Sangdee A

Concepts or ideas x x x x x

Design x x x x x

Definition of intellectual content x x x x

Literature search x x x x

Experimental studies x x x x x

Data acquisition x x x x x

Data analysis x x x x x

Statistical analysis x x

Manuscript preparation x x x x

Manuscript editing x x x x x

Manuscript review x x x x x

Citation Format: Seephonkai P, Sedlak S, Wongpakam K, Sangdee K, Sangdee A (2021) Time-kill kinetics and mechanism of action of Caesalpinia sappan L. and Ochna integerrima (Lour.) Merr. water extracts against pathogenic bacteria. J Pharm Pharmacogn Res 9(6): 813–823.

http://jppres.com/jppres J Pharm Pharmacogn Res (2021) 9(6): 823