Synergistic-antagonistic interaction of traditional herbs Acalypha indica, Centella asiatica, and Sesbania grandiflora: In antifungal-demelanising activities and nitric oxide immunomodulatory responses

Aims: The oriental-based herbs Acalypha indica (AI), Centella asiatica (CA), and Sesbania grandiflora (SG) possess a broad range of undisclosed therapeutic activities which are edible and easily available throughout the year. To convert the herb extracts into a potential drug form, aqueous (A) and methanol (M) extracts of herbs were assessed alone and in combination for their antifungal-demelanising activity and nitric oxide (NO) immunomodulatory responses. A new bioactive synergistic and antagonistic assessments approach was made on these herbs to identify which extract combination qualifies as a natural drug candidate. Methodology and results: Via micro-dilution technique, methanol extract of A. indica (AI-M) showed the strongest antifungal activity against Aspergillus niger, with a minimum inhibitory concentration (MIC) of 50 mg/mL and a minimum fungicidal concentration (MFC) of 100 mg/mL. Sublethal (50 mg/mL) and subinhibitory (25 mg/mL) doses of AI-M produced the optimal black pigmentation reduction to demelanise A. niger. The combinations AI-M+CA-M, AI-M+SG-M, and CA-M+SG-M showed similar antifungal activities (MIC = 100 mg/mL). At 500 μg/mL, CA-A and the combination CAA+SG-A successfully induced RAW264.7 cells to produce NO at 17.85 μM and 40.84 μM, respectively. The combination of herbs extract showed synergistic interaction towards stimulation of NO production. In contrast, they demonstrated antagonism towards antifungal-demelanising properties. Compound identification of AI-M, SG-M, and SG-A were performed using a UHPLC-QTrap-MS/MS system, which detected phenolic compounds from various groups (cinnamic acids, benzoic acids, and flavonoids). Conclusion, significance and impact of study: The combination of herb extracts showed better stimulation of NO production while the single herb extracts demonstrated good antifungal-demelanising activity. These findings help in the selection of herbs combination for potential natural drug discovery. A good combination of herbs demonstrated synergism to execute better bioactivities compared to individual herb extracts.


INTRODUCTION
In recent years, cost-effective and accessible alternatives have been increasingly investigated for the treatment of various ailments especially from non-animal functional materials (Wan-Mohtar et al., 2018;Kozarski et al., 2019;Ring et al., 2019;Vunduk et al., 2019). Locally available edible plant materials can provide nutritional and pharmaceutical benefits, and many Asian populations regularly consume healthy and medically important plants such as curry leaf, spinach, mint, and coriander, which are inexpensive and available throughout the year (Raju et al., 2007). Vegetables eaten cooked or raw in salads are considered part of a traditional healthy diet enriched with polyphenols, which are natural sources of antioxidants, vitamins, minerals, and fibre (Ong, 2004;Abas et al., 2006;Sulaiman et al., 2011).
A plant may exhibit unique phytochemical compounds according to their specific species or genera, and traditional practitioners thus combine different plants to obtain maximum therapeutic effectiveness in a process known as polyherbal formulation (Wink, 2003;Parasuraman et al., 2014;Baharon et al., 2019;Koh et al., 2019). For example, Triphala is a well-known Ayurvedic polyherbal compound used in skincare and consisting of three myrobylan fruits, Embelica officinalis, Terminalia chebula, and Terminala belerica. Triphala polyherbal is rich in phenolics such as ellagic acid, gallic acid, and chebulinic acid, and these compounds exhibit bioactive properties such as antioxidant, antipyretic, antiinflammatory, antibacterial, wound-healing, and anticancer activities (Varma et al., 2016).
There is considerable interest in the use of oriental herbs in the development of microbial drugs, particularly as pathogens continue to develop resistance towards conventional drugs. Liu et al. (2016) reported that MCR-1 gene mutation in Escherichia coli showed resistance towards synthetic drug peptidic colistins (the drug used as a last resort to kill the pathogen). There is, therefore, a need to identify alternative treatments from medicinal herbs since natural products are one of the important pools for the discovery of new drugs to overcome the antimicrobial resistance and treatment of various human disease (Taemchuay et al., 2009;Atanasov et al., 2015;Newman and Cragg, 2016).
According to Chou (2006), a combination of herbs might be the alternative treatment for single target or disease with multiple modes of action and decrease the development of antimicrobial drug resistance. A single extract of A. indica, C. asiatica, and S. grandiflora has been extensively studied for various bioactivities compared to combined herb extracts. Because of that, we investigated and compared the bioactivities of the single and combined extracts of the selected herbs.
A previous study reported that standard antifungal therapy using voriconazole failed to treat aspergillosis in a kidney transplant patient (Pilmis et al., 2018). In this case, Aspergillus fumigatus isolated from the wound acquired azole resistance, thus explaining the treatment failure. Aspergillosis caused by fungi from Aspergillus sp. is typically observed in immunocompromised patients including recipients of organ transplants, patients undergoing chemotherapy, and patients with human immunodeficiency virus (HIV) (Lutz et al., 2003;Prasanna et al., 2016). In rare cases, immunocompetent patients are also at risk of developing aspergillosis. One example is the infection of skin lesions with Aspergillus niger in an immunocompetent 45-year-old male from Kancheepuram district, India (Prasanna et al., 2016).
Nitric oxide (NO) is a vital factor in mammalian immune systems. It is produced by the conversion of the amino acid L-arginine into NO, catalysed by the NO synthase family of enzymes (Wilson and Garthwaite, 2009). NO serves as a critical signalling molecule in immune cells, specifically macrophages, which play an essential role in the defence mechanism against pathogens (Schairer et al., 2012). By stimulating NO production, the defense system of immunocompetent patients may be improved, thus reducing the risk of pathogen infection. According to Tapsell et al. (2006), the consumption of herbs is regarded as necessary for a healthy diet and can boost the immune system, thus the aim of the present study was to determine whether single and combined extracts derived from A. indica, C. asiatica, and S. grandiflora could significantly catalyse the production of NO by RAW264.7. The RAW264.7 cell line is derived from a mouse tumor induced with Abelson leukemia virus and has been widely utilised in the investigation of macrophage cellular physiology to understand the mechanism of inflammation in the human immune system (Maurya et al., 2013). The present study investigated the antifungal-demelanising activity of the extracts, their capacity for NO stimulation, as well as their possible synergy interaction, and sought to identify the compounds responsible for these bioactivities. To the best of our knowledge, this is the first study in which combined extracts from the three species were investigated for their synergism and polyherbal properties.

RAW264
.7 mouse macrophage cells were purchased from the American Type Culture Collection (ATCC) (Virginia, USA) and cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Sigma-Aldrich, USA) containing 10% foetal bovine serum (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin, and 200 µg/mL amphotericin B (PAA Lab, Austria) in a humidified incubator at 37 °C and 5% CO2. Cell culture 96-well plates and flasks were purchased from Nunc (Denmark). All chemicals and solvents used for analysis were of analytical grade. A. niger was obtained from the Institute of Biological Sciences, University of Malaya, Kuala Lumpur, Malaysia and used for screening studies. Potato dextrose agar (PDA) (Sigma-Aldrich, Dorset UK) was used for maintenance of the A. niger culture at 4 °C (Supramani et al., 2019).

Aqueous extraction
Fresh leaves (20 g) of A. indica (AI-A), C. asiatica (CA-A), and S. grandiflora (SG-A) were cut into small pieces by using a sharp blade (<3 mm 2 ) and were heated separately at 80 °C in 500 mL of distilled water for 1 h. The solutions were filtered through filter paper (Whatman ISSN (print): 1823-8262, ISSN (online):  No. 1) and the resulting aqueous filtrates were collected, freeze-dried, and stored in the dark at 4 °C until further use (Krishnan, 2005).

Methanol extraction
The leaves of A. indica (AI-M), C. asiatica (CA-M), and S. grandiflora (SG-M) were dried in an oven at 35 °C for 3-5 days. The dried leaves were ground into a powder and soaked separately in methanol (MeOH) at a ratio of 1:10 (w/v) for 72 h at room temperature. The MeOH filtrates were collected and excess solvent was evaporated to dryness under reduced pressure using a rotary evaporator at 40 °C, resulting in dark-greenish MeOH extracts. The herb extracts were stored at −20 °C until further use (Azis et al., 2017).

Preparation of single and combination herb extracts
Stock solutions of herb aqueous extracts were dissolved in sterile distilled water, filtered, and sterilised through a 0.2 µm filter cap (Orange Scientific, Belgium). Stock solutions of methanol extracts were dissolved in sterile distilled water and filtered via filter paper (Whatman No.1). Combinations of herb extracts were prepared in a 1:1 ratio (v/v) of the stock solution.

Antifungal-demelanising activity of herb extracts
The antifungal-demelanising activity of single and combined herb extracts was evaluated using the microdilution technique based on Heleno et al. (2013) and Wan-Mohtar et al. (2017) with slight modification. Single and combined herb extracts were dissolved in sterile distilled water and added to potato-dextrose broth (Oxoid, UK) containing fungal spore suspensions. Samples were visualised by light microscopy (Leica DM1000), and the lowest extract concentration that resulted in no visible growth (no formation of mycelium) was defined as the MIC.
On the other hand, minimum fungicidal concentrations (MFCs) were identified using a serial sub-cultivation of 2 µL of herb extracts dissolved in medium for 72 h of cultivation and subsequently transferred into 96-well plates containing 100 µL of broth per well. The plates were incubated for 72 h at 28 °C and the lowest concentration of herb extract that resulted in no visible growth was defined as the MFC, representing 99.5% killing of the inoculum. Fluconazole (2 mg/mL) was used as a positive control and 5% of DMSO was used as a negative control in these assessments. The demelanising activity of single and combined herb extracts against A. niger was further evaluated in 6-well plates to determine the minimum demelanising concentration (MDC), as according to Wan-Mohtar et al. (2017) including the sublethal and subinhibitory concentration necessary to provoke demelanisation in fungus during 72 h. Treated and untreated A. niger cultivations were observed by light microscopy (Leica DM1000). 5% of DMSO was used as a negative control. The lowest concentration that resulted in demelanisation of conidia and fungal hyphae was considered the MDC.

Fractional Inhibitory Concentration Indices (FIC) for combination herbs study
The fractional inhibitory concentration indices (FIC) calculated is limited to combinations with two extracts only. FIC index expresses the interaction of two or more agents in which the concentration of each test agent in combination is expressed as a fraction of the concentration that would produce the same effect when used independently. The FIC was calculated as the MIC of the combination (MICAB) divided by the MIC of each component extract, as shown by this formula:

Measurement of nitric oxide production
RAW264.7 cells were detached from the culture flask using a cell scraper and centrifuged at 100 × g for 10 min at 4 °C. The cells were then seeded at approximately 5×10 5 cells/mL into a 96-well plate (200 µL of cell suspension in each well) and incubated at 37 °C for 24 h at 5% CO2. The culture media was replaced with fresh DMEM supplemented with 10% FBS and cells were stimulated with extracts, with 1 µg/mL of lipopolysaccharide (LPS) from E. coli 0111:B4 used as a positive control. Cells were subsequently incubated for 24 h at 37 °C and 5% CO2. Nitric oxide production in the culture medium was measured by the Griess reaction using sodium nitrite as the standard (0-100 µM), and 100 µL of Griess reagent (1% sulphanilamide in 5% phosphoric acid and 0.1% N-(1-naphtyl) ethylenediamide dihydrochloride) was mixed with 100 µL of cell supernatant. A microplate reader was used to measure the absorbance of samples at 540 nm. Nitrite concentration was expressed as µmol/mL by using the predetermined sodium nitrite calibration curve (Wan-Mohtar et al., 2016).

Identification of compounds via UHPLC-QTrap-MS/MS analysis
Compounds present in the herb extracts were identified using a liquid chromatography UHPLC-QTrap-MS/MS system equipped with a 3200-hybrid trap (triple quad tandem mass spectrometer) (Agilent 1100 Series, California, USA). Herb extracts (AI-M, SG-M, and SG-M) were filtered using a 0.45 µM nylon filter prior to injection, and 20 µL of the sample was injected using an autosampler (Agilent 1100 G1313A, California, USA). The samples were run through a C18 column (100 mm × 3 µM × 2.0 mm) (Phenomenex Synergi, California, USA) and elution system (mobile phase) composed of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). Chromatographic separation (in the negative mode) was performed with a gradient system as follows: 10% of solvent B to 90% solvent B (0.01 min to 8.0 min), hold for 3 min, and return to original conditions (10% of solvent B) for 1 min and column reequilibration for 5 min. Compound identities were confirmed by comparison the mass of molecular ions (m/z) to the standard and correlation with the reported literature.

Statistical analysis
Each measurement was carried out in triplicate and results shown as mean ± standard deviation using GraphPad Prism 5 software (Version 5.04). Statistical differences were analysed using one-way analysis of variance (ANOVA) followed by Tukey test.

Antifungal activity
The antifungal activity of single and combined herb extracts is shown in Table 1. Only methanol extracts (single and combined) exhibited antifungal properties, among which AI-M showed the highest and significant (p <0.001) antifungal activity with a MIC of 50 mg/mL and MFC of 100 mg/mL. Single extracts of CA-M and SG-M and combination extracts of AI-M+CA-M, AI-M+SG-M, and CA-M+SG-M had a similar MIC value of 100 mg/mL and MFC values of 200 mg/mL. The MICs for all single and combined aqueous herb extracts were greater than 300 mg/mL. Our findings showed that the MIC and MFC of the single herb extracts were lower than the combination. This explains that the combination of herb extracts exhibited weak or almost similar antifungal effect as the single herb extracts.

Demelanising activity
The demelanising activity of single ( Figure 1) and combined ( Figure 2) herb methanol extracts was evaluated against A. niger at the concentrations of 6.25-200 mg/mL, and the results showed that the demelanising activity was dose-dependent. Greater numbers of black   Figure 3C). The demelanisation and reduction of heads of A. niger were observed by light microscopy ( Figure 3D-F).
The green-colored chromoprotein or black-insoluble pigment contained in the heads of conidiophores of some Aspergillus and Penicillium species belong to the group of melanin pigments (Heleno et al., 2013). Melanin can contribute to fungal virulence and survival by functioning as a 'shield' for fungal (Gómez and Nosanchuk, 2003;Rosa et al., 2010). In the current study, methanol extracts (single and combined) displayed a sublethal and subinhibitory MDC, indicating that they may be directly involved in the suppression or modification of the demelanisation mechanism. The demelanisation results reported here are of significance because the MDC (sublethal) for the fungus was at small doses of extract compared with the inhibitory and fungicidal doses for ISSN (print): 1823-8262, ISSN (online): 2231-7538

Production of nitric oxide by stimulated-RAW264.7 cells
The single and combined herb extracts induced nitric oxide (NO) production from RAW264.7 cells in a dosedependent manner in comparison with untreated controls (Figure 4). In general, aqueous extracts stimulated more NO production compared with the methanol extracts. Only AI-A and CA-A significantly (p<0.0001) stimulated NO production. At 100 µg/mL, AI-A and CA-A produced 4.57 µM and 12.36 µM of NO, respectively. At 500 µg/mL, AI-A and CA-A produced 5.52 µM and 17.85 µM of NO production, respectively. AI-M stimulated the production of 3.11 µM NO at 100 mg/mL and 7.66 µM NO at 500 mg/mL. However, no significant NO production was observed when RAW264.7 cells were treated with SG-A, CA-M, or SG-M (1.10-3.13 µM). NO production by single herb extracts was ranked as follows: CA-A > AI-A > AI-M.
Interestingly, all combined herb extracts stimulated NO production by RAW264.7 cells. At 100 µg/mL, only AI-A+CA-A (13.65 µM) and CA-A+SG-A (13.10 µM) significantly stimulated the production of NO. At 500 µg/mL, AI-A+CA-A (38.89 µM) and CA-A+SG-A (40.84 µM) stimulated the highest production of NO followed by AI-A+SG-A (15.03 µM), AI-M+CA-M (7.28 µM), AI-M+SG-M (6.07 µM), and CA-M+SG-M (5.96 µM). RAW264.7 cells treated with the combination of AI-A+CA-A and CA-A+SG-A produced more NO compared with the positive control, LPS (1 µg/mL = 30.28 µM). NO production by combined herb extracts was ranked as follows: CA-A+SG-A > AI-A+CA-A > AI-A+SG-A > AI-M+CA-M > AI-M+SG-M > CA-M+SG-M. From our results, both aqueous and methanol herb extracts showed the different potential of NO stimulatory. Here, the methanol herb extracts displayed weak NO stimulatory compared to the aqueous herbs extracts and this might be due to the different phytochemical compounds present in each herb extract from different extraction solvent (Thouri et al., 2017). Based on Schairer et al. (2012), the function of NO is determined by its concentration. At low concentrations (<1 µM), NO serves as an intracellular signal to control the activity and growth of immune cells. At higher concentrations (>1 µM), NO acts as a 'pathogen hunter' in which it covalently binds proteins, DNA, and lipids, thus killing or inhibiting pathogens. Inflammation is a vital phase in the wound healing process in which macrophages remove pathogens regulated by NO. However, excess NO can lead to prolonged inflammation and a delayed healing process. A previous study by Punturee et al. (2004) reported both inhibitory and stimulatory activities of ethanol and aqueous extract of C. asiatica, respectively, on NO production in J774.2 mouse macrophages. These findings agree with the results of the current study, which shows increased production of NO when RAW264.7 macrophage cells were stimulated with aqueous extracts and no significant effect following treatment with methanol extracts. The RAW264.7 and J774.2 are the in vitro model that widely used to study inflammation with different mechanism of action (Cabral et al., 2018). The infected RAW264.7 cells were able to produce more than 30-fold TNF-α (pro-inflammatory cytokines) and induce higher inflammation response compared to the J774.2 cells (Lindmark et al., 2004;El Aamri et al., 2015). Further studies are required to study the mechanism of A. indica, C. asiatica, and S. grandiflora for modulation of NO production to facilitate the development of novel immunomodulatory drugs. Table 2  Our results demonstrated that the execution of antifungal properties of herb extracts was better independently, compared to the combined extracts. Meanwhile, analysis of synergistic interaction of the combined herb extracts on stimulation of NO production also shown in Table 2. As the concentration of extract increased (25, 50, and 100 µg/mL), AI-A+CA-A, CA-A+SG-A, CA-M+SG-M demonstrated synergism (CI <1) at all combination data points. AI-A+SG-A and AI-M+CA-M showed antagonism (CI >1) at 25-50 µg/mL and synergism (CI <1) at 100 µg/mL). AI-M+SG-M displayed antagonistic (CI >1) interaction at 25 µg/mL and synergism (CI <1) at 50-100 µg/mL. Nowadays, there is an increase of interest in the combination therapy studies for the treatment of diseases such as cancer, acquired immunodeficiency syndrome (AIDS), and pulmonary tuberculosis (Mitchison, 2012;Schrijvers and Debyser, 2012;Che et al., 2013;Maio et al., 2013). Synergistic behaviour analyses of medicinal herbs might lead towards a better formulation of treatment, which reduces the dosage of drugs and eliminate the need of taking more than one drugs at a time (Chou,   2010; Parasuraman et al., 2014). In this study, the herb extracts have antifungal and NO stimulatory properties hence could be utilized as a supplement to maintain healthy body immune system.

Comparison of antifungal demelanising and NO activity of A. indica, C. asiatica, and S. grandiflora with results from previous studies
The current results indicate that methanol extracts of A. indica, C. asiatica, and S. grandiflora have potent antifungal properties in comparison with aqueous extracts, in agreement with the results of previous studies as shown in Table 3. However, comparative analyses of antifungal activity are unavailable as there are very few studies up to date have investigated the antifungal properties of these three herbs. Previous studies by Solomon et al. (2005) and Sakthi et al. (2011) have reported that ethanol and ethyl acetate extracts of A. indica did not show antifungal effects towards A. niger. In contrast, AI-M showed inhibitory effects (MIC = 50 mg/mL), suggesting that the compound may be suitable for the treatment of aspergillosis and wound infections caused by A. niger (Pasqualotto and Denning, 2006). This discrepancy might be attributable to differences in the extraction method and the variety of phenolic groups and structures responsible for the numerous therapeutic effects of each plant, including antifungal activity (Harborne, 1994). To the best of our knowledge, our study is the first to report antifungal activity for combinations of A. indica, C. asiatica, and S. grandiflora extracts. From the data, the single AI-M extract appeared to exhibit the most potent antifungal-demelanising activity among the evaluated extracts.
Among the three herbs used in this study, the immunomodulatory effects of C. asiatica have been extensively studied compared to A. indica and S. grandiflora. From literature survey, no combination and synergism studies have been done to investigate the immunomodulatory activities of these herbs. As shown in Table 3, a previous study showed that J774.2 mouse macrophages treated with water and methanol extract of C. asiatica produced 1.6 µM and <0.2 µM of NO, respectively (Punturee et al., 2004). However, in the present study, RAW264.7 mouse macrophages treated with aqueous extract of C. asiatica produced increased levels of NO. NO production may vary by cell line and investigated compound or extract. Methanol herb extracts stimulated low NO production compared with aqueous extracts in both studies, and NO production from RAW264.7 cells was increased following treatment with aqueous herb extracts.

CONCLUSION
In conclusion, single and combined herb extracts of A. indica, C. asiatica, and S. grandiflora exhibited considerable antifungal-demelanising effects against A. niger and significant immunomodulatory activities in RAW264.7 macrophage cells. These findings suggest that single and combined herb extracts may have the potential for the development of antimicrobial-synergistic and hostimmunomodulatory drugs to treat new types of infections. Further studies should be done to study the specific mechanism of these herbs for their antifungal and inflammatory effect.