**2. Discussion**

#### **2.1 Determination of combination activities**

The mode of interaction between plant extracts or pure compounds or standard approved drugs is determined using fractional inhibition or interaction factor. Fractional inhibition is used to determine the pharmacological activities of extractdrug combinations, and drug-drug combinations [19]. Fractional inhibition (FI) or combination index (CI) is the inhibition that is attributable to each of the extracts, compounds, or drugs in the combination. Pharmacological activity for each combination is calculated to give a combination index (CI) or 50% fractional inhibition concentration (FIC50) using 50% inhibition concertation (IC50). These CI values are grouped into synergism (CI < 1), additivity (CI = 1), and antagonism (CI > 1), see **Figure 1**.

The Sum CI values are used to generate scatter plots (isobolographs) that explain the potency of a given combination by plotting at least all the triplicate assays on one plane. These isobole curves are used to express the activities based on the doseresponse of single components when used alone and in combination at different concentrations [20]. Sum FIC50 for each extract-drug, and drug-drug combination ratios are also determined using the equation below [4]:

*Pharmacological Properties of Ginger Combinations DOI: http://dx.doi.org/10.5772/intechopen.107214*

$$\text{LSUM CI} = \frac{\text{CI}\_{\text{90}} \text{ of A in combination}}{\text{CI}\_{\text{90}} \text{ of A alone}} + \frac{\text{CI}\_{\text{90}} \text{ of B in combination}}{\text{CI}\_{\text{90}} \text{ of B alone}} \tag{1}$$

Interaction factor (IF) also explains mode of interactions between components in extract-drug and drug-drug combinations. It is also based on isobole graphs and grouped just like fractional inhibition concentration; synergism (IF < 1), additivity (IF = 1), and antagonism (IF > 1) [14, 20].

The IF is calculated as shown below:

$$\text{IF} = \text{AM} \mid \text{AT} \tag{2}$$

AM = am measure of activity of a mixture of samples

AT = theoretically calculated activity of the mixture that is based on the doseresponse of single components in the combination at different concentrations.

#### **2.2 Antimicrobial activities of ginger combinations**

Ginger has been used widely against microbial activities [21]. When used alone, extracts of ginger showed antimicrobial activity with a mean inhibition zone, a 50% microbial inhibition (MIC50) value of 11.72 ± 0.62 mm and lemon (*Citrus limon* L.) showed MIC50 value of 27.89 ± 1.12 mm against *Streptococcus mutans*. The activity of ginger when used in combination with lemon was revealed to have a mean inhibition zone with an MIC50 value of 17.94 ± 0.46 mm [22]. This study reveals that the antimicrobial activities of ginger are enhanced when used in combination with ginger *C. limon*. Therefore *C. limon* potentiates ginger against *S. mutans*.

Synergism was observed when ginger was combined with barley (*Hordeum vulgare* L.) against *Staphylococcus aureus* and *Aeromonas hydrophila* [23]. Similar synergism was also

**Figure 1.** *CI values are grouped into; synergism (CI < 1), additivity (CI = 1), and antagonism (CI > 1).*

observed when ginger was combined with pomegranate (*Punica granatum* L.) against *A. hydrophila*. The synergism resulted in complete inhibition of *A. hydrophila.* Combinations of ginger and licorice (*Glycyrrihza glabra* L.), and ginger and sage (*Salvia officinalis* L) tested against *A. hydrophila* and *Escherichia coli* both showed synergism [23]. Combined activity of a plethora of phytochemicals in ginger extracts combined with other plant extracts is responsible for the observed synergism when tested for antimicrobial activities. These studies indicate the potential use of ginger in combination with other plant extracts against microbial diseases.

In another study, methanol extracts from ginger showed antibacterial activities against 32 *S. aureus* strains with an MIC90 value of 3.56 mg/mL when used alone. Synergism was observed with CI90 values of less than 0.9 when two standard antibacterial drugs, tetracycline and netilmicin were each combined with ginger extracts [24]. This shows the potential use of ginger extracts in combination with tetracycline and netilmicin in controlling antimicrobial diseases and reduction of antimicrobial drug resistance. In the same study, additivity and antagonism were observed when ginger was combined with chloramphenicol, erythromycin, gentamycin, vancomycin, ampicillin, cefoxitin, cotrimoxazole, and ofloxacin, all tested against *S. aureus* [24]. In another comparative study, methanol and water leaves and rhizome ginger extracts were shown to be more effective against *S. aureus* and *S. pyogenes* than three standard antibiotics: chloramphenicol, ampicillin, and tetracycline. This depicts the possible use of ginger alongside standard antibiotics in the treatment of microbial diseases [12].

One of the major burdens of disease control is the emergence of drug-resistant strains. When two drugs that have different mechanisms of action are used in combination the likelihood of the emergence of drug-resistant strains is reduced. The pace of emergence of drug resistance is also reduced since a lower amount of each drug is used in combination treatment [19]. Therefore, these studies underpin the use of ginger combinations against antimicrobial diseases. The potential use of ginger in combination with other plants used against microbial diseases and a combination of ginger with standard antibiotic drugs is also depicted in overcoming antimicrobial drug resistance.

### **2.3 Antioxidant activities of ginger combinations**

Ginger has been reported to have high phenolic content and high antioxidant activity [7]. The combination of ginger and coffee (*Coffea arabica* L.) has shown antioxidant synergistic effects. A combination of ethanol extracts of coffee and ginger showed the highest 2,2-dipheny-1-picryl-hydrazyl-hydrate (DPPH) radical scavenging activity than when used singly. Similarly, combined ginger and tea aqueous extracts showed the highest ABTS radical scavenging activity [13]. This shows that the antioxidant activities of ginger and tea are higher when used in combination than when used singly.

Juice extracts of ginger, kesum (*Polygonum minus* H.), and turmeric (*Curcuma longa* L.) without using any solvent showed were tested for antioxidant activities using DPPH radical scavenging assay and ferric-reducing antioxidant power (FRAP) assay [25]. When tested singly for FRAP ginger showed antioxidant activities of 26.2 μmol/g, kesum 46.3 μmol/g, and turmeric 23.3 μmol/g (μmol of Fe II/g of extracts). When the three plant species were combined at a ratio of 1:1:1, they had an antioxidant activity of 23.1 μmol/g which shows that kesum and turmeric juice extracts potentiate antioxidant activities of ginger. When ginger was combined separately

with kesum (1:1) and turmeric (1:1) for FRAP, antagonism was displayed with CI5o values of greater than 1. For DPPH radical scavenging activities, juice extracts of ginger, kesum, and turmeric showed percentage antioxidant activities of 79.0%, 82.6%, and 64.6%, respectively, when tested singly. When ginger was combined separately with extracts of both kesum (1:1) and turmeric (1:1) and when they were all combined (1:1:1) for DPPH radical scavenging activities, they showed additivity with CI5o values of 1 [25].

In another study, the combination of ginger, garlic (*Allium sativum* L.), and cayenne pepper (*Capsicum fructensces* L.) extract at a ratio of 1:1:1 showed synergism with CI5o values of less than 1. All extracts showed higher antioxidant activities than when these three natural spices are used alone [26] indicating that the extracts potentiate each other's antioxidant activities. This is probably because the many phytochemicals in the combination increase the superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GRed) enzymes. These enzymes catalyze the conversion of reactive oxygen species to harmless species [26].

Ethanol extracts of ginger and secang wood (*Caesalpinia sappan* L.) were tested for antioxidant activities using DPPH method both in combination and singly [27]. The concertation of antioxidant compounds was highest in a combination of scang and ginger (2:1) with an IC50 value of 90,14; absorbance was measured using UV. When tested singly, the IC50 for scang and ginger was 54,53 and 197,74, respectively [27]. In another similar study, ginger combination with eucalyptus honey (*Eucalyptus globulus* Labill.) portrayed synergistic activities with CI5o values of less than 1 in which antioxidant activities were increased where H6G5 displayed 60.0% and 90.0% using DPPH method [28].
