Zingiber officinale (ginger): A systematic review and meta-analysis on antimicrobial activities

The available synthetic drugs to treat infectious diseases have many side effects on the consumer. Zingiber officinale which is known as ginger or “halia” in Malaysia has a good prospect as an alternative for safer treatment and has a low risk of side effects. It is because this herb is used as a traditional medicine in the community to treat several ailments, including infectious diseases. Several studies have shown that crude extracts and bioactive components of Z. officinale possessed diverse pharmacological properties such as anticancer, anti-inflammatory, antimicrobial, antioxidant, and immunomodulatory. The goal of this research is to find out the effects of Z. officinale on the antimicrobial activities from the selected previous studies (years 2000–2020). Briefly, this study involves 10 randomized controlled trials (RCTs) that determined the antimicrobial activities of Z. officinale . The results of the systematic analysis showed that Z. officinale exhibits antimicrobial activities for both in vitro and in vivo evaluations. The meta-analysis of appropriate data from four sources presented a substantial distinction between this plant and controls. The results present no significant difference between Z. officinale and positive controls for the antimicrobial analysis related to the overall outcome and inhibition zone [overall outcome standardized mean difference (SMD): −0.6003 (95% CI; −0.7092 to −0.4913), I 2 = 100%, inhibition zone SMD: 0.8771 (CI; −8.1288 to 9.8829), I 2 = 99%]. In conclusion, the results presented the antimicrobial activities of Z. officinale to be similar to the activity of the positive control. However, one should be aware of some limitations with the detailed reporting on the controls used in the included studies. Future well-designed RCTs with detailed reporting on the controls are required to provide additional data to prove the consequences of Z. officinale on the antimicrobial activities as well as safety data of consuming this plant.


INTRODUCTION
Antimicrobial activity is the process of killing or preventing the growth of the microbes that cause infectious diseases . Nowadays, there are various antimicrobial agents available to treat these diseases. These antimicrobial agents are divided into groups based on the mechanisms of the antimicrobial activity such as agents that prevent cell wall synthesis, those that depolarize the cell membrane, and those that inhibit protein and nucleic acid synthesis as well as metabolic pathways in bacteria (Reygaert, 2018). There are many available synthetic drugs that are used to treat infectious diseases but also have many side effects on the consumer. For example, chloramphenicol, an antimicrobial agent, is used to treat meningitis. This drug works by passing through the blood-brain barrier and is able to cause aplastic anemia (Mohsen et al., 2020). Besides, treatment with ribavirin has been shown to reduce the ribonucleic acid of the virus but is able to cause hemolytic anemia in the patient (McFee, 2020).
This medicinal plant has been used by humans for a long time to treat many ailments as well as other essential roles. Also, medicinal plants have been important medicines in all cultures and are a source of many traditional medicines that also contribute to modern medicines (Dar et al., 2017). In pursuit of new drug candidates, plant extracts and natural molecules from plants are being extensively analyzed (Harun et al., 2018(Harun et al., , 2019(Harun et al., , 2021. Plants have been used for more than 5,000 years as agents of vaccines, antimicrobials, analgesics, cardioprotective agents, and other medicines. Human beings have used natural substances in ancient history to combat pathogens. Currently, almost 70% to 90% peoples in developing countries applied herbs to treat various diseases. The strongest and most promising components of plants are secondary metabolites. More than half of the drugs authorized by the Food and Drug Administration use natural products and their derivatives (Anand et al., 2019). As an alternative for safer treatment and low risk of side effects, Zingiber officinale or ginger is famous in the community and is traditionally used to treat many diseases including infectious diseases and fever and also was used to boost immunity.
Zingiber officinale, a member of the family Zingiberaceae and a species of the genus Zingiber, has been well known as a medicinal herbal and spice product for a long time. This herb is abundantly cultivated for commercial purposes in India, Indochina, West Indies, Mexico, Southeast Asia, and other countries as well (Banerjee et al., 2011). Ginger has been traditionally used to reduce the symptoms of headaches, colds, nausea, pain, and emesis (Mao et al., 2019;Mohamad et al., 2019). In India, the preparation of fresh ginger juice mixed with fresh garlic juice and honey is a common practice for cough and asthma (Awang, 1992). In Southeast Asian countries such as Malaysia and Indonesia, women consume ginger soup after birth to make them hot and sweat (Mohammad and Hamed, 2012). Also, Z. officinale is a common component of traditional Chinese treatments for respiratory infections (Chang et al., 2013) and also remedies for atonic dyspepsia and colic (Keys, 1985). Based on a study by Safa et al. (2020), Z. officinale-based tablets increased the recovery rate of clinical symptoms as well as the improvement of clinical and preclinical features in patients admitted with severe acute respiratory syndrome due to COVID-19 infection. Zingiber officinale and its bioactive compounds showed a variety of biological activities including antimicrobial (Aaisha et al., 2020;Elmowalid et al., 2019), antioxidant, antiarthritic (Murugesan et al., 2020), antitumor (Liao et al., 2020), anti-inflammatory, antithrombotic (Thomson et al., 2002, and hypoglycemic (Ojewole, 2006) effects. Zingiber officinale also contains many natural organic materials such as 6-gingerol, 6-shogoal, and 6-paradol that promote its biological activities.
There are numerous previous studies related to the structure-activity relationship of bioactive compounds who isolated from Z. officinale and their effects on biological activity. The study by Yamauchi et al. (2019) who isolated 13 bioactive compounds from the methanol extract of the Z. officinale rhizomes and further assessed their effects on the extracellular melanogenesis inhibitory activity. The findings showed that gingerols promoted the highest inhibitory activity of extracellular melanogenesis as compared to other vanilloid compounds. They suggest that elongation of the carbon chain as well as the carbonyl and hydroxyl groups on the carbon chain played an important role in this effect. Another study by Masuda et al. (2004) who investigated the antioxidant properties of the gingerol-related compounds and diarylheptanoid isolated from the rhizomes of ginger. The results suggested that the alkyl chain substitution of dehydrogingerdiones is able to contribute to the radical scavenging effects of autoxidation of oils as compared with gingerol-related compounds.
The pharmacological validation of the antimicrobial effect of Z. officinale is quite restricted, and several existing review publications on this plant have not been focused on this activity. Therefore, this study aimed to conduct a systematic assessment of all available data (years 2000-2020) to determine the effects of Z. officinale on antimicrobial activities. Therefore, it is crucial to prove the community's belief in traditionally consuming this herb as a treatment for infectious diseases by conducting a systematic review and meta-analysis on Z. officinale's antimicrobial activities.

METHODOLOGY
This systematic review was carried out in accordance with the principles of the Cochrane Collaboration framework and was described following the guideline by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses declaration.

Search strategies and study selection
From 2000 until 2020, an electronic search for original articles was done using two selected electronic databases which were PubMed and Science Direct. The selection of these two databases was based on the ability to get full access to the related articles from our institution. The strategic search terms were Z. officinale or ginger plus antimicrobial activities or effect of Z. officinale or ginger on antimicrobial activities. The papers that were included in the study are research that involved the use of the extract or bioactive compound of Z. officinale which contained the antimicrobial outcomes (in vivo, in vitro, or clinical studies). The papers that did not include the above criteria were excluded (Moher et al., 2009). The study selection was performed by following the steps in Figure 1.

Data extraction and quality assessment
The data were extracted using standard data extraction from the two selected databases (PubMed and Science Direct) from 2000 to 2020. The extracted data included the tested substances, antimicrobial properties, method used to test the antimicrobial properties, tested microorganism, model used, tested dose, results (using tested substances), and comparison with positive controls.
The quality of the included studies was assessed using the Cochrane risk of bias tool. Sequence generation, allocation concealment, incomplete outcome data, selective reporting, other sources of bias, and overall risk of bias were evaluated. The risk of bias assessment using the Cochrane risk of bias assessment is displayed in Table 1.

Statistical analysis
The analysis of the antimicrobial effects of Z. officinale was conducted by comparing the data for the individual function test (treated with Z. officinale) with its comparator (positive control) [standardized mean difference (SMD) with a confidence interval (CI) at 95%]. Meanwhile, the I 2 -statistic was used to assess the heterogeneity value. The direction of effects, amount, and power of heterogeneity evidence affect the value of the threshold of I 2 . Substantial heterogeneity means an I 2 value of more than 50%. All the statistical analyses were done using the RevMan software (edition 5.4).

RESULTS
The database search resulted in the discovery of 363 articles. Because there was no duplication, none of the articles were removed. There were 96 titles, abstracts, and keywords evaluated in all. Nineteen of the full-text research articles were reviewed from the screened titles, abstracts, and keywords, and all the papers were included in the systematic review. The flow of study selection for the antimicrobial activities is presented in Figure 2. After analyzing the 19 included studies, 10 studies (52.63%) had sufficient data for the comparison between Z. officinale and positive control while 9 other studies (47.37%) did not have sufficient data for this. All the information on the antimicrobial activities of this plant (model and method used, tested substances, tested microorganisms, results, tested dose, and comparison with control) is summarized in Table 2.
The quality assessment of the data is presented in Table 3. Fifteen studies (78.94%) showed a low possibility of bias. Four studies (21.05%) were indistinguishable. Despite the fact that all studies claimed that they were randomized controlled trials, two studies showed an unclear risk of bias for "sequence generation" due to a lack of description of the sequence generation methods. Two other studies showed an indistinguishable possibility of bias for "allocation concealment" because the explanation for the "allocation concealment" method was not present.
The antimicrobial-related outcome was categorized into overall outcomes, inhibition zone, and minimum inhibition concentration (MIC) for the meta-analysis. Qualitative analysis of heterogeneity for the "overall outcome" findings is shown in Figure 3. An analysis specifically done on the qualitative visual method of the findings suggests variability present between the studies. The individual study point evaluations of the effect of treatment (green squares) are on the same line of the upright axis, representing similar treatment amount effects between studies. The prediction of the effect on the study treatment of the population showed the difference value as presented at the horizontal lines in the figure, and the result suggests the presence of heterogeneity. The I 2 -value that presented the quantitative tests of heterogeneity was 100% and suggests there was study variability (i.e., heterogeneity).
Qualitative analysis of heterogeneity for the "inhibition zone" findings is shown in Figure 4. Observational analysis of the results suggests there is between-study variability. The individual study point evaluations of the effect of treatment (green squares) are on both sides. However, they are not located on the upright axis, representing a modification in treatment amount effect between studies. Meanwhile, the prediction of the effects of the study treatment of the population showed the difference value as presented at the horizontal lines in the figure, and the result suggests the presence of heterogeneity. The I 2 value was 99% and suggests that there was study variability (i.e., heterogeneity).
Furthermore, an observational analysis of the MIC result suggests medium between-study-group variability, as shown in Figure 5. Based on the figure, the location of the green squares  (Moher et al., 2009). "n" is the number of papers. showed similar approximation of the amount of the treatment effects for the groups. However, there was a similar prediction for the effect of population treatment groups, as illustrated by the CIs for the groups overlapping each other in the figure, suggesting there is medium heterogeneity. The I 2 -value was 34%. This quantitative result suggests medium between-study variability (i.e., heterogeneity). Based on the data of the included studies, 19 studies described the antimicrobial activities of Z. officinale, but only 4 had satisfactory data for further proceeding with meta-analysis. The four studies were considered in a specific area for the purpose of determining the antimicrobial effects of Z. officinale. The domains included: 1) overall effect outcomes, 2) inhibition zone, and 3) MIC. For the overall outcome and inhibition zone, the I 2 -values were 100% and 99%, respectively. The results presented high heterogeneity between the parameters included in this study. For the MIC, the I 2 value was 34%, which means there was medium heterogeneity. There was a significant difference between Z. officinale and positive controls on the MIC [SMD: 0.0201 (CI; 0.0166-0.0235), I 2 = 34%], while there was no significant difference between Z. officinale and positive controls for the overall outcome and inhibition zone [overall outcome SMD: −0.6003 (95% CI; −0.7092 to −0.4913), I 2 = 100%: inhibition zone SMD: 0.8771 (CI; −8.1288 to 9.8829), I 2 = 99%]. All the results are presented in Table 4.

DISCUSSION
The results of preliminary research employing the disc diffusion approach reported that the methanol extract of Z.
officinale exhibited antibacterial potentials against pathogenic bacteria such as Escherichia coli, Pseudomonas aeruginosa, Pasteurella multocida, Staphylococcus aureus, Bacillus subtilis, Klebsiella pneumoniae, and Proteus mirabilis. In this study, the methanol extract of Z. officinale showed antibacterial activities with an inhibition zone of 10-15 mm (Chakraborty et al., 2014). In addition, the similar extract of Z. officinale presented a broad range of inhibition towards P. aeruginosa, Streptococcus mutans, and Streptococcus sobrinus. The researcher used the agar well diffusion and agar diffusion methods to figure out these antibacterial activities, and the outcome exhibited that the methanol extract at doses of 0.2 and 2 mg/ml suppressed most major inhibitory capabilities against those microorganisms (Babaeekhou and Ghane, 2020;Chakotiya et al., 2017). The ethanol extract of Z. officinale was able to inhibit the growth of many microorganisms including E. coli, P. multocida, B. subtilis, and S. aureus on the basis of the disc diffusion method. The inhibition zone by the ethanol extract ranged from 10.6 to 15.7 mm (Abdul Qadir et al., 2017;Chakraborty et al., 2014). The ethanol extract also presented significant antibacterial properties by inhibiting the growth of the enterococcal species, Enterobacter species, Proteus species, and Klebsiella species on the basis of the agar well diffusion method and serial tube dilution technique. The ethanol extract (0.025-100 mg/ml) was able to inhibit microorganism growth with inhibition zones ranging from 4 to 20 mm (Karuppiah and Rajaram, 2012;Revati et al., 2015). In addition, the acetone extract also showed promising outcomes in inhibiting the growth  Continued Table 3. The quality assessment of the data included in the study.  The characteristics of the included studies for the systematic review of the antimicrobial-related outcomes. The results were presented in mean ± standard deviation (SD). MIC: Minimum inhibition concentration; n: sample size.

Random sequence generation
of some microorganisms such as E. coli, P. multocida, S. aureus, K. pneumoniae, B. subtilis, P. aeruginosa, and P. mirabilis (Abdul Qadir et al., 2017;Chakraborty et al., 2014). The Z. officinale root essential oils exhibited effective inhibitors for pathogens (Silva et al., 2018). Ginger essential oil (GEO) was effective against Gram-positive and Gram-negative bacteria. Essential oil at concentrations of 0.01 and 50 mg/ml inhibited the growth of both Gram-positive and Gram-negative bacteria with inhibition zones ranging from 6 to 22.33 mm (Imane et al., 2020;Snuossi et al., 2016). On the basis of the agar diffusion method, GEO (300 mg/ml) inhibited the growth of S. aureus, Listeria monocytogenes, Salmonella typhimurium, and P. aeruginosa. In addition, GEO also revealed antifungal activity by inhibiting Penicillium citrinum, E. coli, and Penicillium chrysogenum through broth dilution antifungal susceptibility testing (Sharifzadeh et al., 2016). Susceptibility testing of GEO against Mycobacterium tuberculosis and nontuberculous mycobacteria presented significant results at the concentration of 0.25 mg/ml with an MIC value of 0.25 mg/ml (Baldin et al., 2019).
A finding by Chang et al. (2013) showed that the hot water extract of ginger was able to reduce the infection rate of the human respiratory syncytial virus (HRSV) by 50% at a dose of 0.3 mg/ml. Ginger nanofiber (GNF) is a product from the remains after ginger oil and oleoresins are extracted from the ginger. The research conducted by Jacob et al. (2019) stated that the bacterial susceptibility of GNF by the agar diffusion assay was 0.013 mg/ ml against Bacillus cereus, 0.012 mg/ml against E. coli, 0.018 mg/ ml against S. aureus, and 0.031 mg/ml against S. typhimurium. The phenolic extract also showed a significant antibacterial property against S. aureus, K. pneumoniae, P. mirabilis, E. coli,   and Helicobacter pylori on the basis of the agar well diffusion, agar diffusion, and conventional broth dilution methods. The dose of 0.05 mg/ml on the agar well produced inhibition zones ranging from 16 to 25 mm with MICs ranging from 1.59 to 2.2 mg/ml while the dose of 0.01 mg/ml in the conventional broth dilution and agar produced a 20 mm inhibition zone and an MIC value of 0.049 mg/ml (Saleh et al., 2018;Siddaraju and Dharmesh, 2007).
The crude extract of ginger inhibits the growth of S. mutans at a dosage of 2 mg/ml with an inhibition zone of 7.65 mm. Meanwhile, the aqueous extract of ginger inhibits S. mutans at a similar concentration with an inhibition zone of 14.02 mm, which is higher than the crude extract. The finding showed that the aqueous extract of ginger is more effective than the crude extract in inhibiting the growth of S. mutans (Jain et al., 2015). The ginger compounds ([6]-dehydrogingerdione, [6]-shogaol, [10]-gingerol, and [6]-gingerol) derived from ginger display good antibacterial properties against extensively drug-resistant Acinetobacter baumannii when using the broth microdilution method (MIC value: 0.132-0.347 mg/ml) (Wang et al., 2010). A further experiment was done on the basis of the alamarBlue assay to test gingerol against the M. tuberculosis drug-sensitive and drug-resistant clinical strains. Gingerol at a dose of 0.025 mg/ml inhibits the growth of the drug-sensitive and drug-resistant clinical strains of M. tuberculosis with MIC values ranging from 0.0015 to 0.05 mg/ml (Bhaskar et al., 2020).
The concentrations of Z. officinale in previous papers included in this study were different with a range of 0.01 to 300 mg/ml. Variation in Z. officinale extract concentrations was also observed, which included four studies that used the methanol extract ranging from 0.03 to 100 mg/ml, six studies that used the ethanol extract ranging from 0.03 to 300 mg/ml, five studies that used the essential oil ranging from 0.01 to 300 mg/ml, and two studies that used acetone ranging from 0.03 to 100 mg/ml and two studies that used the crude extract ranging from 2 to 5 mg/ ml. Additionally, the concentration of Z. officinale in some of the included studies was unclear. Hence, there is insufficient evidence to support the antimicrobial activity of Z. officinale. The SMD is used to measure the antimicrobial activities of Z. officinale across the included studies. SMD converts data from different scales to a common scale which causes the original information for each measurement of the included studies to be missing. However, the value of SMD is able to provide a significant level of the effect of Z. officinale when compared to the positive controls (Higgins and Green, 2011). The result of the meta-analysis for the mean inhibitory concentration displays that the effects of the positive controls in the previous research selected in this study were more effective than the effects of the Z. officinale extract [SMD: 0.0201 (CI; 0.0166-0.0235), I 2 = 34%]. However, the previous reports showed that Z. officinale were more effective than its selective positive control. These findings included in the study that used GNF showed an MIC value higher than that of ampicillin which were 0.031 and 0.012 mg/ml, respectively (Jacob et al., 2019). Secondly, the study that used the methanol, n-hexane, and ethyl acetate extracts of ginger at a dose of 2 mg/ml presented higher inhibition zones than that of penicillin (40 mg/ml) (Babaeekhou and Ghane, 2020). The other related study was conducted by Baldin et al. (2019) who demonstrated that GEO (0.25 mg/ml) has a higher MIC value than isoniazid and ciprofloxacin. The results might be because of insufficient relevant data from these studies to be included in the meta-analysis.

CONCLUSION
In a nutshell, the overall findings revealed that the Z. officinale extracts and bioactive compounds have antimicrobial activities similar to the positive controls for antimicrobial analysis related to the overall outcome and inhibition zone [overall outcome SMD: −0.6003 (95% CI; −0.7092 to −0.4913), I 2 = 100%, inhibition zone SMD: 0.8771 (CI; −8.1288 to 9.8829), I 2 = 99%]. However, the verification of the Z. officinale as an antimicrobial agent still needs further study with more available data from several other databases.

CONFLICTS OF INTEREST
The authors declared no conflicts of interest. The results were presented in mean ± standard deviation (SD). The inhibition zone is in mm units, while the MICis in mg/ml units. "n" is the sample size.

FUNDING
There is no funding to report.