Antimicrobial and anti-inflammatory activities of selected medicinal plants against pathogens causing sexually transmitted infections

Introduction Sexually transmitted infections (STIs) among young adults in many rural communities have a profound impact on sexual and reproductive health and remain a major public health concern with more than one million STIs acquired daily, worldwide (1-3). The World Health Organization (WHO) identified Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum and Trichomonas vaginalis as the pathogens that cause STIs. N. gonorrhoeae and T. pallidum, on the other hand, have been considered the most common causes of STIs in developing countries (4,5). The STIs caused by N. gonorrhoeae result in women infertility and pelvic inflammatory disease, while T. pallidum is considered the causative agent of syphilis that increases the risk of HIV acquisition and numerous hemolysin (6-10). Syphilis leads to approximately 305 000 fatal neonatal deaths in pregnant women every year, http://www.herbmedpharmacol.com doi: 10.34172/jhp.2020.17


Introduction
Sexually transmitted infections (STIs) among young adults in many rural communities have a profound impact on sexual and reproductive health and remain a major public health concern with more than one million STIs acquired daily, worldwide (1)(2)(3). The World Health Organization (WHO) identified Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum and Trichomonas vaginalis as the pathogens that cause STIs. N. gonorrhoeae and T. pallidum, on the other hand, have been considered the most common causes of STIs in developing countries (4,5).
The STIs caused by N. gonorrhoeae result in women infertility and pelvic inflammatory disease, while T. pallidum is considered the causative agent of syphilis that increases the risk of HIV acquisition and numerous hemolysin (6)(7)(8)(9)(10). Syphilis leads to approximately 305 000 fatal neonatal deaths in pregnant women every year, thus leaving around 200 000 infants with increased risk of prematurity, low-birth-weight and congenital disease (3,7).
Furthermore, infectious diseases caused by antimicrobial-resistant microbes are becoming a serious problem all over the world (11,12). Inappropriate use of antibiotics has been attributed to the development of antibiotic resistance and the global emergence of multidrug resistant bacteria that gradually limit the efficacy of existing drugs resulting in treatment failure (13). Molecular mechanisms responsible for the development of drug resistance are a subject of great interest and have also been highlighted in various reports revealing clinical resistance to antifungal agents (14).
Medicinal plants have been touted as the possible sources of new classes of antimicrobial agents with novel modes of action and represent a viable alternative to treat and/or cure the infections that result from the resistant microbes. Medicinal plants have been used as remedies for the treatment of STIs since ancient times (15). The WHO reported that about 80% of the population in developing countries are dependent on medicinal plants as the primary source of health care (16). Accordingly, medicinal plants have been used to treat different ailments and they are also regarded as remedies with several advantages such as fewer side effects, low cost and are readily available (17)(18)(19). Plant extracts constituents of bioactive secondary metabolites such as flavonoid, alkaloids, tannins, terpenoids and phenolics act synergistically to combat the microbial growth (20). The validation and approval of medicinal plants for the treatment of diseases caused by pathogens could be of great value to most people in rural communities who have limited access to vital, life-saving and often costly modern medicine (21)(22)(23). Therefore, in this study, we aimed to evaluate the antimicrobial and anti-inflammatory activities of selected medicinal plants against bacteria or pathogens that cause STIs. Firstly, we evaluated the antimicrobial activity using different solvents. We, then determined the anti-inflammatory activities using the plant extracts on 15-lipoxygenase (15-LOX) enzyme. Lastly, we determined the total phenolic content and the inhibitory activity of plant extracts on HIV-1 reverse transcriptase (RT) enzyme.

Plants materials
Medicinal plants were selected based on their ethnomedicinal uses in the treatment of STIs as previously described (24)(25)(26)(27)(28)(29)(30)(31). Plant materials were collected from the Walter Sisulu National Botanical Garden in Roodepoort, Gauteng province (South Africa). A voucher specimen of each plant were also prepared ( Table S1).
Preparation of plant extracts Plant materials were air-dried and ground into a fine powder. Different powdered plant materials were weighed and dissolved in dichloromethane (DCM), ethyl acetate and methanol, respectively. The honey jars were vigorously shaken for 24 hours using a shaker at room temperature. The extracts were filtered using Whatman filter paper and the remaining plant materials were discarded. The extracts were then transferred into pre-weighed labeled bottles and placed in the fume hood to evaporate the remaining solvents.

Microbial strains
The following microorganisms were used in this study: Neisseria gonorrhoeae (ATCC 49226), Gardnerella vaginalis (ATCC14018) and Candida albicans (ATCC10231). All pathogens were cultured in a sterile fume hood. N. gonorrhoea was inoculated in Mueller-Hinton broth and incubated at 37 o C for 24 hours in a humid environment enriched CO 2 . G. vaginalis was inoculated in brain and heart infusion broth and incubated at 37 o C for 24 hours. C. albicans was inoculated in tryptone soya broth and incubated at 37 o C for 24 hours.
Antimicrobial activity Antimicrobial activity was determined using broth microdilution method in 96-well plate as previously described (32). Briefly, 50 mg of each plant extract was weighed in 2 mL microcentrifuge tubes and each extract was dissolved in 1 mL of 10% dimethyl sulfoxide (DMSO). Microorganisms were inoculated in sterile broth and prepared to a density of 1.5 × 10 8 colony forming units (CFU) per mL, corresponding with the 0.5 McFarland standard. Then, a 100 µL of broth was added to the wells of the plate and a 100 µL of each dissolved plant extract (50 mg/mL) was added to the first well in triplicate and serially diluted. Later, a 100 µL of pathogen suspension was added to each well except control wells, ciprofloxacin that was used as a control compound for all the extracts, and 10% of DMSO used as a negative control. The plates were incubated at 37 o C for 24 hours. Finally, the results were read based on colour change after adding 40 µL of 0.2 mg/mL Presto Blue dye. A pink colour change indicated microbial growth whereas clear wells indicated inhibition of growth by test plant extract. The lowest concentration that showed no visible pathogen growth was recorded as the MIC for each microorganism.

Anti-inflammatory activity
The anti-inflammatory activity of the plant extracts was performed as previously described (33). Briefly, plant extracts were prepared at 10 mg/mL in DMSO and the 15-LOX enzyme was prepared on ice up to the final concentration of 200 units/mL. A volume of 12.5 µL of each plant sample and controls were then added to 487.5 µL of 15-LOX in 96-well microtiter plate and incubated for 5 minutes at room temperature. After incubation 500 µL of substrate solution was added to 96 well microtiter plate and incubated for 5 minutes at room temperature. After incubation, the absorbance was measured at 234 nm using the microtiter plate reader. Quercetin (1 mg/mL) was used as a control compound and DMSO as a negative control. The results were expressed as mean ± standard error of mean obtained from two independent experiments.

HIV-1 reverse transcriptase inhibition studies
The activity of plant extracts on reverse transcriptase activity was determined using the HIV-1 RT colorimetric ELISA kit (Roche). Firstly, all plant materials were weighed up to 3 mg and dissolved in 1 mL DMSO to make a final concentration of 3 mg/mL stock solution. A stock solution of 10 μL was added to 90 µL of lysis buffer to make a final concentration of 0.3 mg/mL. The RT enzyme was prepared to a stock solution of 0.764 mg/mL and 0.327 µL was added to 1000 µL. Lastly, 20 µL of enzyme, diluted extracts and reaction mixture (10 µM dUTP/dTTP, template/primer hybrid) were added together into wells of the plate. Then, doxorubicin at various concentrations (25 μg/mL, 50 μg/ mL and 100 μg/mL) dissolved in 10% DMSO was used as a control compound, whilst lysis buffer with reaction mixture was used as a negative control. The plates were incubated for 1 hour at 37°C. After incubation, the wells were rinsed three times with 200 µL of washing buffer per well. After washing, 200 µL of anti-digoxigenin-peroxidase (DIG-POD) solution, prepared to a final concentration of 200 mU/mL, was added to each well, covered with foil and incubated for 1 hour at 37°C. After incubation, the solutions were removed from the plate wells and washed three times with 200 µL of washing buffer. Thereafter, 200 µL of 2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) substrate solution was added to each well and allowed to stand for 10 minutes. The absorbance was then measured on a microtiter plate reader at a wavelength of 405 nm with a reference wavelength of 490 nm. The assay was performed in triplicate and percentage inhibition calculated using the following formula: where OD is the optical density of the sample at 405 nm.
Determination of total phenolic content The amount of total phenolic content in plant samples was determined according to the Folin-Ciocalteu method as previously described but with slight modifications (34). In brief, 25 µL of the extract was treated with 250 µL of Folic-Ciocalteu reagent for 5 minutes. The reaction was stopped by adding 750 µL of 20% anhydrous sodium carbonate. The volume was made up to 5 mL with distilled water and incubated at room temperature for 2 hours. After incubation, the absorbance was read at 760 nm using the spectrophotometer. The phenolic content was determined from the standard curve of different concentrations of gallic acid and results were expressed as mg/g gallic acid equivalent (GAE).

Antimicrobial activity
The antimicrobial activity results are expressed in minimum inhibitory concentration (MIC) values shown in Ethyl acetate, DCM and methanol leave extracts of B. pilosa showed best antimicrobial activity for C. albicans with an MIC value of 0.4 mg/mL. Meanwhile, ethyl acetate, DCM and methanol leaves extracts of K. africana and T. sericea indicated some antifungal activity with the MIC value of 8.1 mg/mL and 6.3 mg/mL, respectively ( Figure 1).

Anti-inflammatory activity
The ethyl acetate and DCM extracts of K. africana and methanol extract of B. pilosa contained compounds with higher activity compared to quercetin (control compound) whereas all extracts of T. sericea showed low inhibitory activity ( Table 2).

HIV-1 reverse transcriptase inhibition
The percentage inhibition activity of plant extracts against RT enzyme are shown in Figure 1. Inhibitory activities of the plant extracts against HIV-1 RT ranged from 15.79% to 80.16% inhibition at a final concentration of 100 µg/ mL. The results were compared to the control compound, doxorubicin, which has been shown and validated to inhibit the RT enzyme ( Figure 1). Ethyl acetate, DCM and methanol leaf extracts of T. sericea exhibited high inhibition activity (>70%).
Ethyl acetate and DCM extracts of T. dregeana leaf extract showed moderate inhibitory activity while methanol extract of T. dregeana indicated low inhibitory activity (Figure 1). Ethyl acetate and DCM extracts of C. papaya showed moderate inhibitory activity whilst methanol extract of C. papaya had no inhibitory activity. Ethyl acetate extract of B. pilosa displayed high inhibitory activity with an inhibition percentage of 80%. DCM and methanol extracts of B. pilosa yielded moderate inhibitory activity of 65% and 55%, respectively. Inhibitory activities of ethyl acetate and methanol extracts of K. africana were moderate at 65% and 60%, respectively whilst DCM extract of K. africana had the highest inhibitory activity of 70 % (Figure 1). The difference between the control (untreated) and the treated samples was determined by one-way ANOVA test (Bonferroni multiple comparison test). ‫٭‬P < 0.05 compared to control group. The total phenolic contents of the extracts were examined using the Folin-Ciocalteu method and reported as a gallic acid equivalent (mg/g GAE) (Figure 2). The highest number of phenolic compounds were obtained from methanol extract of T. sericea at 270 mg/g GAE followed by ethyl acetate extract of C. papaya and methanol extract of B. pilosa with the total phenolic content of 130 mg/g GAE. Ethyl acetate extract of K. africana and DCM extract of T. dregeana had the lowest amount of phenolic compounds at 20 mg/g GAE. Interestingly, no phenolic compounds were detected in the DCM extract of both B. pilosa and K. africana and methanol extract of K. africana. conditions, the season of collection and the age of the plant (37). However, van Vuuren (35) tested T. sericea against anti-microial activity and the results of aqueous extract correlated well with our findings. Furthermore, Naidoo and colleagues' study using a combination of plant extracts from C. papaya, T. dregeana, K. africana and B. pilosa demonstrated moderate antimicrobial activities that also correlated well with our findings in terms of organic extract (38). C. papaya has been shown to be efficacious in the treatment of STIs and has subsequently displayed good wound healing activities in the past (39). Steenkamp et al (40) tested the roots of T. sericea against C. albicans and showed inhibition of STIs bacteria. Similarly, Moshi and Mbwambo (41) tested the inhibition of C. albicans growth using methanol extracts of T. sericea and their results also indicated a notable inhibition of C. albicans growth that correlates with the findings of this study.

Discussion
Anti-inflammatory activity Inflammation plays an important role in the resolution of many diseases as well as the STIs (36) and the plant extracts that have anti-inflammatory activities usually inhibit the cyclooxygenase (COX) and/or LOX activity (36). Such plants usually have active therapeutic effects that promote healing and repair of tissue cells (42). T. sericea contains a phenolic anti-inflammatory compound, triterpenoid saponin, which can inhibit the COX-2 and 5-LOX enzymes (43). The observed anti-inflammatory activity may be due to the overall effects of plants constituents or compounds having similar action to the anti-inflammatory agent and non-steroidal anti-inflammatory drugs (48). However, all the tested leave extracts of T. sericea exhibited low 15-LOX inhibitory activities. As a result, T. sericea might have compounds that have anti-inflammatory activities that are lower than that of quercetin. Also, the low inhibition   (47). Due to these pharmacological properties, plant phenolic compounds have gained increasing attention in both modern and traditional medicine as a possible source of new therapeutics. In this study, methanol extract of T. sericea with lower crude extract yield of 2.19 % contained relatively high amounts of phenolics content which could be responsible for antimicrobial and anti-inflammatory activity observed. Anokwuru et al (49) indicated that methanol extract of T. sericea had the highest total phenolic content of 440 mg/g GAE. On the other hand, Singh et al (50) reported the total phenolic content of methanol extract from B. pilosa to be about 72 mg/g GAE. In addition, leaves of C. papaya were shown to have high total phenolic content that might provide the best sources of dietary antioxidants (51).

Conclusion
After evaluation of five selected medicinal plants against the pathogens causing STIs, some plants (T. sericea and B. pilosa) were shown to have good antimicrobial activity and had the potential to be used as alternative therapeutic options for the treatment of STIs. Additionally, some plants had promising activities against the propagation of inflammation. Accordingly, for a medicinal plant to be considered the candidate plant with good antimicrobial activity, it should contain the lowest MIC value in each microorganism tested. The plant extracts, which can inhibit the pro-inflammatory activities of the 15-LOX enzyme, could also be used for the discovery and development of new and novel anti-inflammatory compounds. Further characterization studies such as isolation, identification, solubility, pharmacokinetics and toxicity profiles of bioactive compounds in these plants should be conducted to explore other pharmacological properties.