Andrographis paniculata (Burm. f.) Wall. ex Nees: An Updated Review of Phytochemistry, Antimicrobial Pharmacology, and Clinical Safety and Efficacy

Infectious disease (ID) is one of the top-most serious threats to human health globally, further aggravated by antimicrobial resistance and lack of novel immunization options. Andrographis paniculata (Burm. f.) Wall. ex Nees and its metabolites have been long used to treat IDs. Andrographolide, derived from A. paniculata, can inhibit invasive microbes virulence factors and regulate the host immunity. Controlled clinical trials revealed that A. paniculata treatment is safe and efficacious for acute respiratory tract infections like common cold and sinusitis. Hence, A. paniculata, mainly andrographolide, could be considered as an excellent candidate for antimicrobial drug development. Considering the importance, medicinal values, and significant role as antimicrobial agents, this study critically evaluated the antimicrobial therapeutic potency of A. paniculata and its metabolites, focusing on the mechanism of action in inhibiting invasive microbes and biofilm formation. A critical evaluation of the secondary metabolites with the aim of identifying pure compounds that possess antimicrobial functions has further added significant values to this study. Notwithstanding that A. paniculata is a promising source of antimicrobial agents and safe treatment for IDs, further empirical research is warranted.


Introduction
Infectious disease (ID) is a serious global health problem that leads to a high mortality rate worldwide every year [1]. The world has recently witnessed a most formidable threat in recent human history, COVID-19, in the modern era of highest advancement of medical sciences. Infectious agents (i.e., invading microbes or pathogens) evolved a variety of strategies, such as modulating their cell surfaces, releasing proteins to inhibit or degrade host immune factors, or even mimicking host molecules to evade the host

Recent Progress in Publication of A. paniculata
The measurement of scientific interest in a particular topic can be revealed from its trend of publications. Due to having tremendous medicinal importance, the relentless interest in this plant and its versatile molecules have resulted in overwhelming publications over the past ten years. The publication numbers amounted to 3279 (as of 15 February 2021). In other words, we can say, daily, almost one publication (Figure 2), of which about 14% publications were about the antimicrobial study of either A. paniculata extracts or its metabolites, especially andrographolide. This bibliometric data was extracted from the Scopus database using the query of the term "Andrographis paniculata" OR "andrographolide" in titles, abstracts, and keywords. This number might be increased if data can be combined from different databases like PubMed, Web of Science and so on.

Invasive Microbes Used in the Antimicrobial Study of A. paniculata
There are about 1400 known species of human pathogens. Although this seems like a large number, they are less than 1% of the total number of microbial species on the planet earth [81]. Even though this is less than 1% of total microorganisms, a harmless microbe can sometimes be harmful under a specific condition like an immunocompromised patient. Exploring proper medications for these spontaneous behavioural changing microbes is a continuous effort of the scientific community. A. paniculata extracts and their bioactive molecules were investigated against a wide variety of pathogens, including several antibiotic-resistant species, for example, Staphylococcus aureus, Pseudomonas aeruginosa, Shigella spp., Salmonella spp., Candida spp., Streptococcus pneumoniae. We have identified a total of 59 invasive microbes that have been used to investigate the antimicrobial efficacy of A. paniculata extracts and/or their isolated pure compounds. The categorized microbes included 33 bacterial, four viral, 12 fungal and ten parasite species. The details antimicrobial effectiveness of different extracts has been discussed in the later section. The list of tested microbes, their types, mode of transmission, a disease caused, and infecting organisms are presented in the Supplementary Table S1.

Antimicrobial Secondary Metabolites of A. paniculata
A. Paniculata contains therapeutically active secondary metabolites that include lactones, diterpenes, flavonoids, quinic acid, xanthones, noriridoids, and other compounds. In our previous study [39], we reported more than 55 ent-labdane diterpenoids, 30 flavonoids, eight quinic acid derivatives, four xanthones, and five rare noriridoids in A. paniculata; however, in this study, our extensive review revealed at least 142 secondary metabolites that already isolated from A. paniculata using different plant parts and fractionations of organic solvents (i.e., acetone, butanol, chloroform, ethanol, methanol, and hexane) or water and chromatographic analysis like thin layer chromatography (TLC), high-performance thin-layer chromatography (HPTLC), liquid chromatography, micellar electrokinetic capillary chromatography (MECC), high-speed counter-current chromatography (HSCCC), high-performance liquid chromatography (HPLC), ultra-performance liquid chromatography (UPLC), Silica Gel Chromatography (SGC), and flow injection spectrophotometry (FIS) [65,80,. Among the chromatographic methods, HPLC and TLC are more commonly used. This might be due to easy accessibility and accuracy. The UPLC is good for its selectivity, linearity, precision, accuracy, stability, robustness, the limit of detection and quantification. This method showed good linearity, accuracy and satisfactory precision with a run time of less than 3 min [165].

Antibacterial Effects
For centuries, the traditional use of A. paniculata in treating several IDs caused by bacteria encouraged researchers to study its anti-bacterial properties and how it fights against invasive microbes. Our review found about 33 different types of bacteria that were inhibited by different types of extracts (Table 2 and Supplementary Table S1).

A. paniculata Extracts as Antibacterial Agents
Researchers used different types of extracts of A. paniculata to explore their potentiality against numerous invasive microbes, including antibiotic-resistant strains, such as methicillin-resistant S. aureus (MRSA), vancomycin-resistant E. faecalis (VRE), carbapenemresistant Actinobacillus baumannii, β-Lactamase-negative, ampicillin-resistant (BLNAR) Haemophilus influenzae, P. aeruginosa. A summary of the antibacterial activity of the different types of A. paniculata extracts is shown in Table 2.
The aqueous extract of A. paniculata showed significant antibacterial activity, which was further linked to the presence of andrographolides and arabinogalactan proteins [195]. The role of andrographolide and neoandrographolide in treating bacillary dysentery caused by Shigella sp. was reported in several studies as well [196,197]. In an experiment, A. paniculata was used to treat 1,611 cases of bacterial dysentery and 955 cases of diarrhoea [198]. The efficacy of A. paniculata extracts for dysentery was proved from laboratory stool test with 82.5% and 91.3% [198,199]. However, the crude water extract of A. paniculata leaves exhibited no effect on E. coli and K. pneumoniae. A significant activity against the Grampositive S. aureus, MRSA, and Gram-negative P. aeruginosa was reported by crude water extracts of leaves sample [200].
In contrast, methanol extract of leaves showed significant activity against E. coli along with P. aeruginosa, K. pneumonia, S. aureus, B. subtilis and Streptococcus epidemidis [201]. Furthermore, no antimicrobial effect of the aqueous extracts of the whole plant and isolated andrographolide on tested common pathogenic bacteria [170]. However, ethanol extracts were found effective against Legionella pneumophila and Bordetella pertussis only [170]. These findings indicated that the extraction process and solvent have a significant role in the efficacy of A. paniculata as the number and yield of pure metabolites greatly differ depending on the types of fractions.
Significant antibacterial activity of subsequent hexane, chloroform, n-butanol, and aqueous fractions of 50% ethyl alcohol-treated extracts of A. paniculata exhibited against E. coli [202]. Similarly, the potent inhibitory effect of ethanol extract of aerial parts on the growth of both gram-positive and gram-negative bacteria, namely, Salmonella typhi, V. cholera, V. alginolyteus, S. aureus, Shigella boydii, Shigella sonnei, E. coli, B. licheniformis, and Salmonella typhimurium. Another study [173] reported that the co-presence of andrographolide and arabinogalactan proteins in the ethanol extract was further acknowledged for its enhanced antibacterial activity compared to andrographolide and arabinogalactan proteins alone. This outcome has been derived maybe because of their synergistic effect. The ethanol extract was efficient [173]

Isolated Compound as Antibacterial Agent: Mechanisms of Action
A substantial number of evaluations proved the efficacy of different extracts of A. paniculata against many severe pathogenic microbes. Besides this, 13 pure secondary metabolites of A. paniculata were also reported to have significant antibacterial effects (Table 1). These are compounds 1, 3-5, and 7-16. These compounds have been used to evaluate antibacterial potency against a wide range of bacteria. Overall, Gram-positive bacteria were more susceptible to compound 1 than Gram-negative bacteria due to the presence of the outer membrane and the polarity nature of the compound [119,139]. Depending on the bacterial species, the mode of actions of compound 1 differs by a large extent. S. aureus was largely susceptible (MIC is 0.1 mg/mL) to compound 1 among the tested microbes [139]. Healthcare-associated infections are prevalently (10.7%) caused by S. aureus, a major bacterial human pathogen that can form biofilm [209]. It causes a wide variety of clinical manifestations, including pneumonia, mastitis, osteomyelitis, endocarditis, skin infections, abscesses, food poisoning, toxic shock syndrome, and sepsis, and treatment remains challenging due to the emergence of multi-drug resistant strains such as MRSA [210].
Compound 1 acts on bacteria themselves as well as plays an important role in the regulation of host immunity by regulating macrophage phenotypic polarization and Ag-specific antibody production [211]. When S. aureus infected the lungs, it significantly promotes NF-κB p65 phosphorylation and increases TNF-α and IL-6 production ( Figure 5). Compound 1 can downregulate them sufficiently but retain the immune cells at the level that can kill bacteria without serious immune damage. In comparison to penicillin, compound 1 showed better management of bacterial infection and persistent host immunity [212]. Since compound 1 works on immune regulation, there are fewer chances of drug resistance. Therefore, compound 1 would further reduce the problems associated with antibiotic resistance, one of the current severe health crises.
In another study, Banerjee et al. [139] reported that compound 1 could strongly inhibit DNA synthesis (approximately 31%) and consequently RNA (about 26% inhibition) and protein (around 36% inhibition) synthesis in S. aureus. This result was similar to that of antibiotic ciprofloxacin (25% incorporation). However, cell wall biosynthesis was not hampered [212]. Secondary metabolites showing antimicrobial can work on a specific target site. Compound 1 can affect the quorum sensing system (QSS), a communication system between bacteria; thereby, it is an effective antibacterial target. This system enhances the production of biofilm by bacteria, such as P. aeruginosa. The antibacterial drug effects in this system resulting in regulates the production of bacterial efflux pumps and virulence factors [213]. Compound 1 effects on the QSS, especially Las and Rhl systems, resulting in reduced production of compositions of extracellular polymeric substance (EPS), such as carbohydrate, nucleotide, and amino acid polymers, as well as inhibiting virulence factors ( Figure 5) [214,215]. In addition, compound 1 could restore the antibiotic sensitivity in P. aeruginosa by reducing expression of mexAB-oprM efflux pump [216] and inhibit bacterial adhesion, such as E. coli and S. epidermidis, to the epithelial cells of lungs; therefore, significantly reduced respiratory colonization and level of fimA, papC, and tsh ( Figure 5) [217].

Mechanisms of Action Influence on Biofilm Production by Pure Compounds
Among the selected metabolites, compound 1, 5 and 15 exhibited significant antibiofilm effects against P. aeruginosa [88,176] and S. aureus [139]. Biofilm is a critical part of bacterial pathogenesis in the host. The multilayer structure of biofilm acts as a potential barrier against the host defense and antibiotics action and serves as a sign of antibiotic resistance as well. Biofilm-embedded bacteria are helped to break free from the drugs and weaken as a result [218]. Biofilm forming infections are getting severe health issues daily and significantly enhancing antibiotic resistance; some are relevant to implant-associated infections [219,220]. Exploring efficient drugs are crucial as conventional antibiotics are not sufficient to inhibit the production of biofilm. A. paniculata brought an excellent opportunity to treat biofilm infections as some of its metabolites, for example, compound 5 and 15, possess significant potentiality in inhibiting biofilm formation. Interestingly, A. paniculata metabolites showed synergistic effects with standard antibiotics (i.e., gentamicin and azithromycin), which were unable to reduce biofilm growth alone [88,176]. Compound 5 and 15 showed efficacies in a dose-dependent manner. Compound 15 at 0.125 mM concentration prevented about 54% biofilm formation. Inhibition of biofilm production was further augmented up to 60% while the concentration was used at 0.15 mM [176]. Similarly, compound 5 showed a significant reduction of biofilm growth (about 56%) by P. aeruginosa, whereas compound 1 exhibited only 40% inhibition of biofilm production [88]. Neither gentamicin nor azithromycin was incapable of inhibiting biofilm production of P. aeruginosa. However, the outcome was dramatically changed to about 90% inhibition (p < 0.0001) when the bacteria were treated in a combination of compound 15 and gentamicin [119]. In compound 5, this inhibition becomes about 92% while combined with gentamicin or azithromycin. However, compound 1 showed the least efficacy in inhibiting bacterial biofilm production (about 60% in combination) than the other two compounds [88].
EPS consists of carbohydrate, nucleotide, and amino acid polymers are considered as major components of the biofilm matrix produced by P. aeruginosa [221]. Both compound 5 and 15 significantly reduced the level of biofilm carbohydrate, extracellular DNA, and proteins up to 90% while combined with gentamicin or azithromycin. Unfortunately, compound 1 was not adequate to reduce EPS compositions. They were also efficient to reduce essential virulence factors, the exoprotease activity (up to 93% inhibition), including LasB, rhamnolipid, and pyocyanin in combined treatment as well as inhibit motility movement compared to the antibiotic gentamicin or azithromycin alone [88,176]. This synergism was achieved by inhibiting the process of biofilm development, not by killing microbes. For S. aureus biofilm formation, compound 1 at a concentration of 50 µg/mL diminished biofilm thickness by about 45% on the polystyrene surface compared to the control set after 24 h exposure [139]. Reduction of biofilm thickness was dose-dependent manner, and the outcomes demonstrated that compound 1 has an important role in the inhibition of biofilm production in S. aureus.

Antiviral Effects
Since the last three decades, researchers have extensively studied the antiviral properties of A. paniculata. Although antiviral activity against a limited number of viruses viz.  [180], HIV [54,[177][178][179], hepatitis B [186] and Hepatitis C [194] has been reported, their findings are very encouraging and significant considering the role of these viruses on human morbidity and mortality worldwide.
It is noteworthy that the formation of syncytia in co-culture of HIV-1 infected MOLT cell lines was significantly inhibited by the methanol extracts of A. paniculata [222]. The aqueous bark extracts of A. paniculata was investigated for HIV-1 protease inhibition activity, and this result supports an earlier report by Yao, et al. [223]. They reported positive results, but the extracts were less effective (29.6% and 26.3% inhibition at 250 µg/mL and 25 µg/mL, respectively) against HIV-1 protease [179]. Methanol extracts of A. paniculata showed antiviral effects against dengue virus (DENV) serotype-1 in vitro assay. After treating with the extracts, the viability of DENV-1 infected Vero E6 cells was 113 ± 4.65% with maximum non-toxic dose (0.050 mg/mL), and the percentage of inhibition was 75% [182]. Panraksa, et al. [93] evaluated andrographolide's anti-viral activity against DENV serotype-2 in HepG2 and HeLa cell lines and DENV serotype-4 in one HepG2 cell line. They found a significant reduction of cellular infection and virus output levels in both cell lines, HepG2 (EC 50 = 21.304 µM) and HeLa (EC 50 = 22.739 µM) for DENV 2. The anti-viral activity of andrographolide was confined to a post-infection stage [93]. Andrographolide was more potent to inhibit DENV compared to the chikungunya virus (CHIKV). The CHICKV EC 50 (77 µM) was about 3.5 fold higher than the DENV, comparable to two different DENV serotypes. In addition, andrographolide affected CHIKV replication [91]. Both cases in HepG2 and HeLa cell lines did not show any toxicity sign after treating with andrographolide at a maximum concentration of 100 µM for 24 h [91,93].
Reddy, et al. [177] have investigated several pure metabolites of A. paniculata, including bis-andrographolide ether, andrographolide, 14-deoxy-11,12-didehydroandrographolide, andrograpanin, 14-deoxyandrographolide, (±)-5-hydroxy-7,8-dimethoxyflavanone and 5-Hydroxy-7,8-dimethoxyflavone against HIV. Among these compounds, only andrographolide and 14-deoxy-11,12-didehydroandrographolide have demonstrated significant anti-HIV properties with (EC 50 = 49.0 mg/mL) and (EC 50 = 56.8 mg/mL). However, these outcomes were comparatively very less than the standard HIV treatment, azidothymidine (EC 50 = 20 ng/mL) used for HIV. They used MT2 cells for the anti-HIV test and found a significant reduction of p24 antigen levels (doses used 5 to 100 mg/mL). The level of p24 antigen is one of the determinants of the anti-HIV effect since it is a viral protein that makes the viral capsid or core, and its expression is highest during the early phase of infection [224]. Andrographolide was also significantly effective to increase CD4 + lymphocytes in HIV patients. HIV RNA copy number was also decreased, but it was not significant [54].
Neoandrographolide, another most potent anti-HIV agent, have a unique C3-Oglucosyl moiety which plays a vital role in the inhibition of furin (IC 50 = 53.5 µM). This activity is around 20-fold higher than the andrographolide (IC 50 = 1.0 mM and K i = 200 µM). Moreover, furin is a protease involved in the proteolysis of HIV envelop polyprotein gp120 prior to viral assembly. Gp120 helps viruses to attach to the specific cell surface receptor [225]. A derivative of andrographolide called dehydroandrographolide succinic acid monoester (DASM) was also experimented with in H9 (T-helper-cell line) cells and human peripheral blood mononuclear cells (PBMCs) and reported to have potential anti-HIV activity [178]. DAMS showed immense improved in vitro anti-HIV activity at the concentration of 50-200 (average, 108) µg/mL that was nontoxic to the H9 cells. It also demonstrated the inhibitory effect against HIV-1 and HIV-2 strains. They reported that the subtoxic concentration of DASM (200-400 µg/mL) partially interfered with HIV-induced cell fusion and binding of HIV virions to H9 cells. Probably, DASM might also be interfered with HIV replication by inhibiting HIV infected cell proliferation at another unidentified step(s).
Time-and dose-dependent hepatitis C virus (HCV) replication suppressive effect of andrographolide also reported earlier [194]. The researchers observed the synergistic effect of andrographolide in combination with IFN-α, an inhibitor targeting HCV NS3/4A protease or NS5B polymerase. The andrographolide's effect was further linked to the up-regulation of heme oxygenase-1, which led to increased amounts of its metabolite (biliverdin) production that promoted the antiviral IFN responses inhibited NS3/4A protease activity which eventually suppressed HCV replication and showed anti-HCV activity [194]. In another study, dehydroandrographolide and andrographolide isolated from A. paniculata reported having hepatitis C virus (HBV) DNA replication suppressive activity with IC50 values of 22.58 54.07 µM and low SI values of 8.7 and 3.7 [186].
Chen, et al. [180] reported considerable inhibitory activity (both in vitro and in vivo) of 14-ά-lipoyl andrographolide (AL-1), a synthetic derivative of andrographolide, against influenza A viruses H5N1, H9N2, and H1N1. It successfully prevents mortality in mice against H1N1 infection at the dose of 200 mg/kg/d, and 80% of mice survived at both dosages of 100 gm/kg/d and 200 gm/kg/d against H9N2 and H5N1 infections. It also demonstrated the most effective inhibition of viral adsorption onto red blood cells at the concentration of 5.3 to 16.8 mM, thereby inhibiting virus transmission to the uninfected cells. The similar result was observed by Aromdee, et al. [181] for 14-acetyl analogues of an-drographolides (14-acetyl-3,9-isopropylideneandrographolide, 14-acetylandrographolide, 3,14,19-triacetylandrographolide) against HSV-1 in vitro. These three analogues were good for blocking viral entry in the pre-infection step. A cyclic dioxane analogue (3,9-isopropylideneandrographolide) was good for inhibiting viral replication at the postinfection level. However, andrographolide exhibited less effective inhibition activity against influenza A and HSV-1.
Prevention and management of coronavirus disease (COVID- 19) have not yet successful since no specific preventive measurement and treatment available. Therefore, searching for potential bioactive compounds from natural sources is an ongoing investigation as medicinal plants possess a tremendous antiviral compound. In recent in silico studies, andrographolide [227], neoandrographolide [228], glycosides 5,4 -dihydroxy-7-O-β-D-pyran-glycuronate butyl ester [229] and glycoside 3-O-β-D-glucopyranosyl-andrographolide [229] have been reported to have a potential role in inhibiting the main protease of SARS-CoV-2, including NSP9, RNA-dependent RNA polymerase, and 6LU7. Andrographolide was docked successfully in the binding site of SARS-CoV-2 Mpro. Their findings revealed that the andrographolide molecule has good solubility, pharmacodynamics property and target accuracy [230]. In another very recent report also stated that andrographolide and its derivative-14-deoxy-11,12-didehydroandrographolide have strong binding affinities with targets. They can modulate the immune system by regulating chemokine signaling, Rap1 signaling, cytokine-cytokine receptor interaction, MAPK signaling, NF-kappa B signaling, RAS signaling, p53 signaling, HIF-1 signaling, and natural killer cell-mediated cytotoxicity [231]. A couple of in silico studies suggest strong interaction of andrographolide and its derivatives against COVID-19 associated target proteins and exhibited different immunoregulatory pathways; thus, these metabolites could be potential candidates for COVID-19 treatment, and further evaluation in vitro and in vivo would be worthy.

Antifungal Effects
A. paniculata crude extracts have been used for the treatment of fungal infections in folk medicines for centuries. The ethanol crude extract of the whole plant was reported to possess moderate antifungal activity against A. oryzae (60% inhibition) as well as A. niger (<60% inhibition) and Penicillium sp. (<40% inhibition) at 3% (v/v) concentration [232]. The hexane and methanol root extracts were evaluated for their antifungal activity against A. niger and Penicillium chrysogenum. Two concentrations (100 gm/mL and 200 mg/mL) of each extract were studied, which involved determining the inhibition zone diameter for a specific time. It was found that both extracts exhibited significant inhibition, 13 mm and 12 mm at 200 mg/mL concentration against A. niger and Penicillium chrysogenum, respectively. However, these inhibitions were less than the standard fluconazole, 17 mm and 16 mm at 100 µg/mL concentration, respectively [206].
Sule, et al. [183] first reported on the isolation of antifungal bioactive compounds from dichloromethane (DCM) and methanol extracts of A. paniculata whole plant. All the isolated bioactive, 3-O-β-D-glucosyl-14-deoxyandrographolide, 14-deoxyandrographolide and 14deoxy-11,12-didehydroandrographolide, showed significant antifungal activity against Microsporum canis, A. niger, and C. albicans. MIC values for all antifungal compounds ranged from 50 to 150 µg/mL, and minimum fungicidal concentration (MFC) values ranged from 50 to 200 µg/mL. Among the isolated antifungal substances, 14-deoxyandrographolide exerted the lowest MIC (50 µg/mL) and MFC (50 µg/mL) against M. canis, which indicates the most potent antifungal activity. It is noted that no anti-fungal activity was reported against T. mentagrophytes and T. rubrum at 250 µg/mL. Even though different extracts of A. paniculata reported to have potential anti-fungal activity, the mode of actions yet underreport.

Anti-Parasitic Effects
A. paniculata and its bioactive compounds have been tested and found extensive anti-parasitic activity against various parasites, such as Ascaris lumbricoidis, Plasmodium falciparum, P. berghei, Trypanosoma cruzi, etc. The extract and fractions reduced parasitaemia level in Mastomys natalensis while used in a dose-dependent manner [184]. Misra, et al. [184] have also studied the anti-malarial activity of the four diterpenes-andrographolide, neoandrographolide, deoxyandrographolide and andrographolide-isolated from A. paniculata and revealed that neoandrographolide (2.5 mg/kg BW) exhibit the highest activity when administered by gastric lavage than other diterpenes.
The fractions of A. paniculata also possessed significant anti-malarial activity [185]. The methanol extract was found to have complete inhibition at a concentration of 2.5 mg/mL by 48 h. Chloroform extract also achieved the same effect by 24 h at only 0.05 mg/mL concentration [233]. The isolated andrographolide also exhibited substantial anti-malarial activity against the MRC-pf-303 strain of P. falciparum and particularly inhibited the parasites at the ring stage [234]. Consequently, the methanol fractions soluble in chloroform were evaluated as significant inhibition of parasitaemia (74%) at the concentration of 1 mg/mL. Additionally, andrographolide showed the highest (53.9%) inhibition of parasitaemia level [235].
It has been found that the alcohol rhizome extract possessed significant in vitro activity against A. lumbricoides [236]. Two reviews were also reported A. paniculata rhizome exhibited extensive activity against A. lumbricoides [35,36]. Dutta and Sukul [237] have studied in vitro anti-filarial activity of leaves decoctions of A. paniculata against Dipetalonema reconditum and found the decoctions kill microfilaria in 40 min. In vivo study revealed that more than 85% of microfilaria in the blood reduced after three subcutaneous injections of the extract into infected dogs at 0.06 mL/kg BW.

Controlled Clinical Trials of A. paniculata Treatment: A Systematic Evaluation
A. paniculata and/or its bioactive compounds have been used to treat patients with uncomplicated upper respiratory tract infections (URTIs), including common cold, rhinitis, nasopharyngitis, pharyngitis, and pharyngotonsillitis. The surrounding bacteria and viruses are the usual source of infection for the URTIs. In the treatment of URTIs, pills (made by whole powdered plant and water) and tablets (water extract of the plant) have been used, and the cure rates are 88% and 61%, respectively. The therapeutic effectiveness differed mainly due to the preparation method and duration of treatment [36]. Several randomized, double-blind, placebo-controlled trials proved the efficacy of A. paniculata standardized extracts and/or andrographolide. Some other bioactive compounds treat various infectious diseases associated with cold symptoms and infections caused by viruslike influenza [55][56][57][58][59][60][238][239][240][241][242].
Our findings depicted that about 52% of controlled clinical trials were conducted to investigate the A. paniculata extracts and their bioactive compounds effects on the disappearance of common cold, uncomplicated UTRIs symptoms, as well as recovery from viral infections including influenza and HIV (Figure 7). Among these studies, eight were randomized, double-blind, placebo-controlled studies. The characteristics of these studies and treatment outcomes are shown in Table 3. The details of A. paniculata treatment outcomes on the other health conditions and physiological effects on the healthy volunteer (HV) are given in Supplementary Table S3.

Evaluation of A. paniculata Efficacy Against Infections
Eleven randomized, and 1 non-randomized clinical trials (n = 2008) were met inclusion criteria. The compliance rate of this study was 95.97%. Of these, nine were double-blind, of which eight were placebo-controlled. Others were either simple randomized control or randomized parallel-group or randomized controlled open-label. Melchior, et al. [240] and Kulichenko, et al. [57] included one pilot trial and one main trial in one article. Therefore, we split their findings and presented them separately in Table 3. A comparative, randomized, double-blind study [60] had been done on 152 Thai patients with pharyngotonsillitis to investigate the efficacy of A. paniculata extracts using either paracetamol (3.9 g/day) or encapsulated A. paniculata dried leaves for low dose group (LDG) (3 g/day) or high dose group (HDG) (6 g/day) for seven days. There was no significant difference in efficacy of relieving fever (p = 0.16) and sore throat (p = 0.49) among three groups on day 7. Most of Paracetamol and HDG stopped taking medication on day three because their symptoms had disappeared. However, LDG patients discontinued taking medications due to persistent adverse side effects or worsening symptoms. The incident of mild or self-limiting side effects (i.e., nausea, vomiting, abdominal discomfort, dizziness, drowsiness, and malaise) was not statistically significant among the three groups (p = 0.8), and patients were almost equally satisfied with paracetamol and a high dose of A. paniculata treatment. The findings indicated that daily 6 g of A. paniculata dried leaves extracts that contain at least 6% of andrographolide can be replaced by paracetamol treatment as a standard treatment for the patients with symptoms of pharyngotonsillitis.
In a placebo-controlled study conducted by Caceres, Hancke, Burgos and Wikman [241], Kan Jang tablets, a standardized A. paniculata extract, had been administrated to 107 healthy students in a rural school at a dose of two tablets (200 mg) per day for three months to evaluate the efficacy of Kan Jang to prevent common colds. The common colds were successfully prevented; 2.1-fold higher prevention in the Kan Jang group compared to the placebo group. The incidence of common colds was 30% (16 out of 54) and 62% (33 out of 53) in the Kan Jang group and placebo group, respectively. Melchior, et al. [58] conducted another similar study on 50 volunteers suffering from common colds and sinusitis using Kan Jang tablets for five days. Both subjective symptoms and duration of the symptoms were significantly reduced; 68% of patients recovered entirely in the Kan Jang group. In contrast, only 36% of patients recovered in the placebo group.
Another randomized, double-blind placebo-controlled study on 158 adult patients of both sexes used A. paniculata dried extract SHA-10 for five days to investigate the efficacy in plummeting the prevalence and intensity of sign and symptoms of common colds. The extract (1.2 g/day) showed the ability to reduce the intensity of the symptoms of tiredness, sleeplessness, sore throat, and nasal secretion compared to the placebo group. The intensity of symptoms started to decrease from the second day. All symptoms disappeared on day four without showing any adverse effect [242]. In similar two other Swedenian studies, the Kan Jang, a standardized extract of A. paniculata in a fixed combination with Eleutherococcus senticosus, has been used three times daily for 3-8 days. This study evaluated the efficacy in the treatment of uncomplicated URTIs on 46 and 179 patients, respectively. The treatment group showed highly significant improvement compared to the placebo group in terms of both the total symptom score (p < 0.0006) and total diagnosis score (p < 0.003). In addition, relief of throat symptom was highly significant in both studies [240].
A three-arm clinical study has been carried out on 130 children (ages 4-11 years) over ten days to evaluate the effects of Kan Jang on uncomplicated respiratory diseasecommon colds. In the three groups, the control group treated with standard common cold treatment and the other two groups adjuvant and adjuvant control group treated with Kan Jang tablets and Immunal (a preparation of Echinacea purpurea L. extract) concomitant to standard treatment, respectively. The Kan Jang showed more significant effects by the rapid recovery process. Using a less standard medication, resulting in the symptoms, particularly nasal secretion and congestion, was less severe at an early stage of uncomplicated common colds in the Kan Jang treatment group. However, Immunal did not exhibit the same efficacy.
The use of Kan Jang as an alternative medication was found to be safe and efficacious in treating uncomplicated upper respiratory tract diseases [238].
A randomized, controlled clinical study has also been conducted to investigate Kan Jang's effects in treating influenza viral infection on 540 patients. In (same as before) group, Kan Jang treated patients recovered very quickly from infections, and Kan Jang reduced the risk of post-influenza complications. Moreover, patients tolerated the Kan Jang very nicely [57]. In addition, the addition of A. paniculata to paracetamol in influenza patients could further reduce the severity of influenza symptoms compared to the control group who was given paracetamol alone [55].
Existing clinical studies showed that A paniculata has a clinical effect on infectious diseases and influences several other diseases. A. paniculata can decrease fasting and postprandial glucose and decrease body mass index in diabetes mellitus patients. A. paniculata also influences mild to moderately active ulcerative patient. Administration of 1800 mg showed a significant clinical response (rectal bleeding) compared to placebo (p = 0.0183) [61].

Evaluation of Safety of A. paniculata Treatment
The incidents of the adverse effect of treatment with the natural product are sporadic. To our best knowledge, there was no such severe adverse event reported for A. paniculata treatment to the drug safety body. Our critical observations revealed that A. paniculata treatment showed a mild adverse event in some cases. Out of 14 reports (Table 4), six reports stated (n = 579) that they did not observe any adverse effects (or report not provided). These studies cover only 1991-2010. Our systematic investigation did not reveal any clinical studies conducted from 2010 to now. However, several clinical studies conducted using A. paniculata extracts or pure compounds on the healthy volunteer to check semen quality [243], pharmacokinetics and tolerance ability [244], ulcerative colitis [62,63], arthritis [66,245], fatigue [67], Familial Mediterranean Fever [246], hypertriglyceridemia [64], and type 2 diabetes mellitus [61] (Supplementary Table S3).
Saxena, et al. [59] (n = 223) reported mild adverse effect (2.73%): vomiting (1 case), epistaxis (1 case), Urticaria (1 case) and diarrhoea (3 case). Except for vomiting (patient in the treatment group) and urticaria, all other effects stopped spontaneously without any medication. Minimal and self-limiting side effects (n = 152) (i.e., nausea, vomiting, abdominal discomfort, dizziness, drowsiness, and malaise) were found about 20% in treatment (LDG & HDG) and paracetamol groups (9-11 cases) [60]. These are pervasive mild effects that usually recovered shortly without any medications. Three cases out of 200 subjects were also reported mild side effects: increase in nasal discharge and epigastric pain (1), nose blocked (1), and severe headache (1) for the treatment of A. paniculata fixed combination Kan Jang in URTIs and sinusitis [239]. For viral infections, the treatment group (Kan Jang) experienced dry cough, rhinitis, and pain in the throat (22 cases out of 540). Control group received antiviral agent amantadine which showed significantly (p < 0.01) higher (67.8% cases) influenza complications compared to treatment group (30.1% cases) [57]. For main trials, influenza complications were found in 31.43% of A. paniculata treated patients and 70.97% of standard medicine (amantadine) treated patients (p < 0.01). one HIV positive experience an anaphylactic reaction in phase I clinical trials [54]. All but one (92%) reported at least one adverse event during the study. About 75% reported an adverse event by the healthy volunteer. All conditions were returned to normal by week 9 [54]. A. paniculata standardized extract treatment of uncomplicated acute URTI patient experienced unpleasant sensations in the chest and intensified headache (1 case out of 180) [240], and for common cold and sinusitis, two patients out of 50 experienced urticaria [58].
Clinical studies other than respiratory infections also reported no adverse effects or mild effects. Only two patients (n = 180) with mild-to-moderate ulcerative colitis experienced severe adverse effects [63]. However, clinical response was significantly higher in the A. paniculata group than in the placebo (p = 0.0183). The best efficacy was observed with the HDG of A. paniculata. In another clinical trial with ulcerative colitis (n = 108), both A. paniculata extract-treated group and control group experiences several side effects, including aphthous ulcer (1), WBC decrease (1), abdominal pain (1), blood in the stool (1), fever (1), elevated glucose (1), rash (1) [treatment group]; blood in the urine (2), elevated CRP (1), WBC decrease (1), blood in the urine (2), fever (1), WBC decrease (1), abdominal pain (1), dry mouth (1), oedema lower extremity (1) cough (2), diarrhoea (2), dizziness and nausea (1), WBC elevated in urine (1) and other [control group], where comparatively treatment groups showed less adverse effects [62]. This indicated that the drug tolerance capacity of ulcerative patients is less regardless type of drugs, and they should be taken precaution measurement before taking any medicine, including A. paniculata.
Considering the type of adverse events and frequency and overall efficacy in treating subjective symptoms of common cold or URTIs either alone or in combination with Acanthopanax senticosus, A. paniculata might be safe for both adults and children. However, the treatment must need to follow the prescribed regime and daily recommended dose. Based on the findings of this study, 90-150 mg andrographolide daily could be recommended as a safe dose for the treatment of URTIs and other similar complications. Additionally, URTIs patients with ulcerative colitis should take extensive precaution taking any medications, and they are not recommended or encouraged for self-medication of any herbal drugs, including A. paniculata.

Methodology
The information related to this article was systematically collected from worldwide accepted scientific databases including PubMed (http://www.ncbi.nlm.nih.gov/pubmed (accessed on 27 March 2021)), ScienceDirect (http://www.sciencedirect.com/ (accessed on 27 March 2021)), Scopus (http://www.scopus.com/ (accessed on 27 March 2021)), Web of Science (https://apps.webofknowledge.com/ (accessed on 27 March 2021)), Springer Nature (http://link.springer.com/ (accessed on 27 March 2021)), Wiley Online Library (http://onlinelibrary.wiley.com/ (accessed on 27 March 2021)), and advanced search in Google Scholar (http://scholar.google.com.my/ (accessed on 27 March 2021)), as well as recognized books, abstract, and thesis/dissertation using the keywords "Andrographis paniculata", "antimicrobial", "anti-bacterial" "antiviral" "antifungal". In the aforementioned databases, other relevant papers from the list of references of all available articles were searched. For searching the controlled clinical trials, the following keywords: "antiparasitic", "clinical trials", "controlled clinical trials", and "randomized clinical trials" were used in PubMed, Scopus, and web of science. Controlled clinical trials were systematically screened and selected for further evaluation of their outcomes in this study. The studies were selected for this review (clinical section) if they were controlled clinical trials dealing with A. paniculata to treat infections incredibly uncomplicated upper respiratory tract infection. Used of A. paniculata for other health conditions and healthy volunteers were also selected in this study. English language only restriction was imposed.

Conclusions and Recommendations
Human invasive microbes are growing resistant to the available antibiotics for many reasons. As A. paniculata works on immune regulation, there are fewer chances of drugresistance occurrence. Even though A. paniculata has a potential antimicrobial activity, the detailed study regarding the mode of actions, effects concerning the available antimicrobial agents and specific administration route as well as schedule remain to be explored. The active constituents of A. paniculata could be a potential source of antimicrobial agent, and exploring the therapeutic potentiality of them based on the clinical implications are worthwhile. We have explored substantial antimicrobial agents in A. paniculata (Table 1); however, very little is still known about their molecular mechanisms in response to microbes or host-infected cells. A majority of metabolites are not investigated to identify the molecular target to understand the drug-target-disease network. In addition, our checklist of secondary metabolites of A. paniculata (Supplementary Table S2) can be used for further exploration of their effectiveness because about 44% of metabolites are yet to be evaluated.
The outcomes from clinical effects suggest that A. paniculata offers a promising treatment for alleviating symptoms of infections caused by bacteria or virus that appeared in the URTIs. Nevertheless, a few common mild adverse events were reported from both short term and long term treatments. Therefore, self-medication using A. paniculata should be cautious to avoid potential adverse effects such as vomiting, diarrhea etc. If we consider the overall efficacy of A. paniculata treatment, it would be a worthy consideration as a natural product treatment option for acute URTIs as currently, there is a lack of compelling therapeutic opportunity for IDs.
In some cases, pure compounds from medicinal plants, such as aristolochic acids, possess severe side effects like kidney failure and urinary tract cancers [247]. Therefore, moving forward, the further requires us to take a more comprehensive approach to harness the true potential of A. paniculata for IDs fully. Different disease conditions have diverse responses to the drugs; therefore, to obtain a complete picture of the drug-target-disease network, elucidating each secondary metabolite's mechanism(s) is crucial. Andrographolide has the potential to target multiple sites since it has shown significant efficacy against different disease conditions. Therefore, this natural product could be considered as a potential candidate for polypharmacology. We would obtain the full advantage of using andrographolide for therapeutics in the near future.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/life11040348/s1, Table S1. Invasive test microbes for the evaluation of antimicrobial efficacy of different extracts of Andrographis paniculata and its isolated metabolites, Table S2. Isolated pure metabolites of Andrographis paniculata and the part used, Table S3. Study characteristics and intervention outcomes of clinical trials of Andrographis paniculata for healthy volunteers and other health complications included familial Mediterranean fever, diabetes mellitus, hypertriglycemia, ulcerative colitis, fatigue and arthritis pain.  Institutional Review Board Statement: This study was a in house study, no animal or human subject was involved; therefore, ethical approval was not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the article or Supplementary Material.