Part2: Anti-inflammatory And Antioxidant Properties Of Finger Millet (Eleusine Coracana (L.) Gaertn.) Varieties Cultivated in Sri Lanka

For more info. contact tina.xiang@wecistanche.com

Inflammation is an immune response of our body to fight infection and repair damaged tissue; it is a self-healing and healing process triggered by a certain disease in our body.

Many diseases of the human body are related to inflammation. For example, studies have shown that the root cause of heart disease is arterial inflammation, such as various osteoarthritis, gynecological inflammation, gastroenteritis, chronic hepatitis, cholecystitis, nephritis, pneumonia, and even rhinitis. , keratitis, otitis media...even cancer, it can be said that almost all chronic diseases are related to inflammation.

When a harmful bacteria or virus attacks us, our body's validation system kicks in and fights back; or if we sprain our ankle, our inflammatory system kicks in to repair the damaged tissue. Without the inflammatory system, we would quickly die.

Experimental studies have shown that echinacea-rich Cistanche deserticola extract has a good improvement effect on colitis mice. The phenethylgly compounds isolated from Cistanche have a protective effect on inflammatory cells caused by carbon monoxide. The total glucosides of Cistanche have a good inhibitory effect on the secretion of carbon monoxide in inflammatory cells, and the results show that it has an anti-inflammatory effect. Moreover, Cistanche has been a good tonic since ancient times, and there is no record of toxicity, which fully proves its safety.

3. Results and Discussion

3.1.Antioxidant Properties of Finger Millet Varieties 

3.1.1.Total Phenolic and Total Flavonoid Contents. TPC and TFC of ethanolic and methanolic extracts of Ravi, Rawana, and Oshadha finger millet varieties are presented in Table 1. There were significant differences(P<0.05)among the varieties and between the extracts. Methanolic extracts had significantly high(P<0.05)TPCs and TFCs compared to ethanolic extracts in all finger millet varieties. Among the three varieties, Oshadha had a significantly high (P<0.05)TPC, and Ravi had a significantly high(P<0.05 )TFC. There were no significant differences(P≥0.05)between the TFCs of Rawana and Chadha.

Significant negative(P<0.05) correlations were observed between TPCs and ICs。 values of DPPH (r=-0.602) and ABTS cation(r=-0.836)radical scavenging activities of all extracts.In addition,a significant positive (P<0.05) correlation was observed between TPCs and ORAC values(r=0.787) of the extracts. These correlations indicated the involvement of finger millet phenols in radical scavenging activities. TPCs of the extracts positively correlated with FRAP values(r=0.876, P<0.05)and FIC activities(r=0.473, P<0.05)of the extracts indicating the ability of finger millet phenols to reduce ferric ions and chelate ferrous ions. There were significant negative(P<0.05)correlations between TFCs and IC5 values of DPPH(r=-0.802)and ABTS cation(r=-0.471)radical scavenging activities of the extracts. Besides, TFCs positively correlated with FIC activities(r=0.689, P<0.05)of the extracts, suggesting the involvement of finger millet flavonoids in scavenging radicals and chelating ferrous ions.

TPCs of common cereals were reported by Abeysekera et al.[30].According to their findings, TPCs of methanolic extracts of white and red rice varieties ranged from 29.34 to 139.79mg GAEs per 100g of flour on a dry weight basis, while TPCs of ethanolic extracts ranged from 59.78 to 113.56mg GAEs per 100g of flour on a dry weight basis. TPCs of methanolic extracts of barley, corn, wheat, and oats were 93.61,75.10,71.22, and 17.24mg GAEs per 100g of flour, respectively, on a dry weight basis. TPCs of ethanolic extracts of barley, corn, wheat, and oats were 68.69,52.10, 78.05, and 15.72mg GAEs per 100g of flour, respectively, on a dry weight basis. Therefore, when compared with the aforementioned findings, Ravi, Rawana, and Oshadha finger millet varieties possess high phenolic contents than commonly consumed cereals such as rice, wheat, corn, barley, and oats. Phenolic compounds are known to be strong anti-oxidants, and therefore, any plant tissue possessing these compounds can be potentially used as effective natural anti-oxidants [2]. Consequently, the high phenolic contents indicated the antioxidant potential of the Ravi, Rawana, and Oshadha varieties.

3.1.2.DPPH and ABTS Cation Radical Scavenging Activities. Significant differences(P<0.05) were observed in both DPPH and ABTS cation radical scavenging activities of Ravi, Rawana, and Oshadha varieties and between the extracts (Table 2). Methanolic extracts of the three varieties showed significantly high(P<0.05)DPPH and ABTS cation radical scavenging activities when compared to ethanolic extracts. Ravi and Oshadha varieties showed significantly high (P<0.05)DPPH and ABTS cation radical scavenging activities when compared to Rawana. All extracts showed dose-dependent DPPH and ABTS cation radical scavenging activities. In DPPH radical scavenging assay, the methanolic extract of Oshadha had the lowest ICs value indicating the highest activity, and in ABTS cation radical scavenging assay, the methanolic extract of Ravi had the lowest ICs value indicating the highest activity.

Significant positive(P<0.05)correlations were observed between IC values of DPPH and ABTS cation radical scavenging activities of the extracts(r=0.750)indicating that the finger millet extracts had comparable activities in the two assays. The compounds present in finger millet extracts that can scavenge DPPH radicals may be capable of scavenging ABTS cation radicals as well. Almeida et al.[31] have observed a similar correlation when studying the antioxidant activities of Brazilian fruit extracts. Suriano et al. [32] also have reported a similar correlation in barley varieties, and they have attributed it to the same reaction mechanism on which both assays rely. A good antioxidant should be capable of quenching free radicals [33,34]. Therefore, the ability to scavenge DPPH and ABTS cation radicals indicated the antioxidant potential of Ravi, Rawana, and Oshadha finger millet varieties.

3.1.3.Oxygen Radical Absorbance Capacity. There were significant differences(P<0.05) in scavenging oxygen radicals among the Ravi, Rawana, and Oshadha varieties(Table 3), and the ORAC value of Oshadha was significantly higher (P<0.05)than that of the Ravi and Rawana varieties. In Raw-ana, the ethanolic extract showed a significantly high (P<0.05) ORAC value when compared to the methanolic extract. There were no significant differences (P≥0.05)between the ORAC values of ethanolic and methanolic extracts in the Ravi and Oshadha varieties.

ORAC values of common cereals were reported in previous studies [30]. ORAC values of methanolic extracts of white and red rice varieties ranged from 150.61 to 439.64mg Trolox equivalents per 100g of flour on a dry weight basis, while ORAC values of ethanolic extracts ranged from 108.32 to 272.08mg Trolox equivalents per 100g of flour on a dry weight basis.ORAC values of methanolic extracts of barley, wheat, corn, and oats were 357.51, 322.00,249.91, and 50.71mg Trolox equivalents per 100g of flour, respectively, on a dry weight basis. ORAC values of ethanolic extracts of barley, wheat, corn, and oats were 244.33,263.90,84.04, and 73.20mg Trolox equivalents per 100g of flour, respectively, on a dry weight basis. Therefore, when compared with the aforementioned findings, Ravi, Raw-ana, and Oshadha finger millet varieties possess a higher ability to scavenge peroxyl radicals than the other commonly consumed cereals such as rice, wheat, and corn, barley, and oats. Since the ability of an extract to scavenge physiological radicals such as peroxyl radicals is an important indicator of its antioxidant capacity [33,34], these findings indicated the antioxidant potential of the Ravi, Rawana, and Oshadha varieties. 

3.1.4. Ferrous Ion Chelating Activity. There were significant differences(P<0.05) in FIC activities of Ravi, Rawana, and Oshadha varieties and between the extracts (Table 3). Methanolic extracts showed significantly high (P<0.05)activities when compared to ethanolic extracts of all finger millet varieties. Therefore, methanol extraction may be more efficient compared to ethanol extraction when extracting com-pounds, with FICability, from finger millet varieties. Ferrous ions can initiate and spread many radical generating reactions, and consequently, chelating ferrous ions are important to prevent redox-active metal catalysis-associated ROS generating reactions[35]. Therefore, the ability to chelate ferrous ions reflected the antioxidant potential of the Ravi, Rawana, and Oshadha varieties.

3.1.5. Ferric Reducing Antioxidant Power. There were significant differences(P<0.05) in FRAP values of Ravi, Rawana, and Oshadha varieties and between the extracts(Table 3). Methanolic extracts showed significantly high (P<0.05)FRAP values when compared to ethanolic extracts. Among the three varieties, Oshadha showed a significantly high (P<0.05)FRAP value when compared to Ravi and Rawana. Since the reducing capacity of an extract is an important indicator of its antioxidant capacity [2], these results provided evidence for the antioxidant potential of Ravi, Raw-ana, and Oshadha varieties.

FRAP values of methanolic extracts of white and red rice varieties ranged from 61.79 to 743.18mg Trolox equivalents per 100g of flour on a dry weight basis, while FRAP values of ethanolic extracts ranged from 139.36 to 255.34mg Trolox equivalents per 100g of flour on a dry weight basis. FRAP values of methanolic extracts of barley, wheat, corn, and oats were 199.45,177.95,81.06, and 61.74mg Trolox equivalents per 100g of flour, respectively, on a dry weight basis. FRAP values of ethanolic extracts of barley, wheat, corn, and oats were 107.21,84.27,49.05, and 26.60mg Trolox equivalents per 100g of flour, respectively, on a dry weight basis [30]. Therefore, when compared with the aforementioned findings, Ravi, Rawana, and Oshadha finger millet varieties possess higher abilities to reduce ferric ions when compared to rice, wheat, corn, barley, and oats. Since reducing the capacity of an extract is an important indicator of its antioxidant capacity [2], the ability to reduce ferric ions indicated the antioxidant potential of the Ravi, Rawana, and Oshadha varieties. 

3.2.Anti-inflammatory Properties of Finger Millet Varieties 

3.2.1.A5-LOX Enzyme Inhibitory Activity.A5-LOX enzyme inhibitory activities of the ethanolic and methanolic extracts of Ravi, Rawana, and Oshadha varieties are given in Table 4. Both extracts of the three-finger millet varieties are capable of inhibiting the catalytic action of the A5-LOX enzyme in a dose-dependent manner. Methanolic extracts of the three varieties exhibited significantly (P<0.05) high A5-LOX enzyme inhibitory activities when compared to ethanolic extracts. Among the six finger millet extracts, the methanolic extract of Oshadha had the lowest ICso value indicating the highest A5-LOX enzyme inhibitory activity.

ICC values of A5-LOX enzyme inhibitory activities of the extracts were negatively correlated with TPCs (r=-0.788, P<0.05)and TFCs(r=-0.399, P<0.05)of the extracts, indicating the involvement of finger millet phenols, including flavonoids, in inhibiting the catalytic action of A5-LOX enzyme. Perera et al. [2] have observed a similar correlation in Sri Lankan medicinal plants, and they have reported that anti-A5-LOX activity is proportional to the polyphenol and flavonoid contents. This is supported by the fact that most of the A5-LOX enzyme inhibitors are phenols, and furthermore, the most potent A5-LOX enzyme inhibitors, including baicalein, are flavonoids[36].

There were strong positive correlations between ICs values of A5-LOX enzyme inhibitory activities and ICs0 values of DPPH(r=0.840, P<0.05)and ABTS cation (r=0.660, P<0.05)radical scavenging activities of all extracts. These correlations indicated the involvement of antioxidants, which are present in finger millet extracts, in inhibiting the A5-LOX enzyme by scavenging the radical intermediates which are generated during the catalytic action of the A5-LOXenzyme. A nonheme iron atom is present in the active site of lipoxygenases. The catalytic reaction of lipoxygenases involves single-electron oxidation at the active site iron atom which switches between Fe2+ and Fe3+redox states. Therefore, molecules with iron-chelating and ferric ion reducing functionalities can act as potent A5-LOX inhibitors[2,37].In the present study, also strong negative correlations were observed between the ICsvalues of A5-LOX enzyme inhibitory activities and FRAP values (r=-0.873, P<0.05)as well as FIC activities(r=-0.752, P<0.05)of all extracts suggesting that reducing the ferric ions and chelating the ferrous ions as potential mechanisms of finger millet extracts in inhibiting the catalytic action of the A5-LOX enzyme.

3.2.2. XO Enzyme Inhibitory Activity. Both ethanolic and methanolic extracts of Ravi, Rawana, and Oshadha finger millet varieties were capable of inhibiting the catalytic action of the xanthine oxidase enzyme in a dose-dependent manner indicating the anti-inflammatory potential of the three varieties(Table 4). Methanolic extracts exhibited significantly (P<0.05)high XO enzyme inhibitory activities when compared to ethanolic extracts. Among the six finger millet extracts, the methanolic extract of Oshadha had the lowest ICso value indicating the highest XO enzyme inhibitory activity.

ICC values of XO enzyme inhibitory activities of all extracts showed significantly (P<0.05) negative correlations with TPCs(r=-0.744), TFCs(r=-0.489), FRAP values (r=-0.793), and FIC activities(r=-0.906)indicating the involvement of antioxidants, which are present in finger mil-let extracts, in inhibiting the catalytic action of the enzyme. During the catalytic action of XO, superoxide anions and hydrogen peroxide are formed [38]. The significant (P<0.05)positive correlations between IC values of XO enzyme inhibitory activities and ICs。values of DPPH (r=0.826) and ABTS cation (r=0.790)radical scavenging activities of all extracts suggested, scavenging radical intermediates including ROS, which are generated during the catalytic action of the enzyme, as a potential mechanism of XO enzyme inhibition.

3.2.3.Hyaluronidase Enzyme Inhibitory Activity. Both ethanolic and methanolic extracts of Ravi, Rawana, and Oshadha finger millet varieties were capable of inhibiting the catalytic action of the hyaluronidase enzyme(Figure 1). Among the three varieties, Oshadha showed the highest hyaluronidase enzyme inhibitory activity although it was statistically not significant(P≥0.05) when compared to the other two varieties. There were no significant differences (P≥0.05)between the hyaluronidase enzyme inhibitory activities of the six extracts, and they were significantly (P<0.05)lower than the hyaluronidase enzyme inhibitory activity of the reference standard, tannic acid which showed 99.16% inhibition at 0.5mg/ml concentration. Girish et al. [7] and Sahasrabudhe and Deodhar [27] have reported that polyphenols are good hyaluronidase inhibitors. Furthermore, the reference standard used in this assay, tannic acid, is also a type of polyphenol [27]. In the present study also, hyaluronidase enzyme inhibitory activities of the extracts showed a positive correlation with TPCs(r=0.387), confirming the involvement of finger millet phenols in inhibiting the catalytic action of the hyaluronidase enzyme. 

3.2.4.Oxidative Burst Inhibitory Activity. The abilities of the ethanolic and methanolic extracts of Ravi, Rawana, and Oshadha finger millet varieties to inhibit the zymosan-induced oxidative burst in whole human blood was evaluated, and the results indicated that both extracts of the three-finger millet varieties are capable of inhibiting oxidative burst in whole human blood in a dose-dependent manner (Table 4). Methanolic extracts exhibited significantly (P<0.05) high inhibition when compared to ethanolic extracts. When compared with ibuprofen, methanolic extracts had a moderate oxidative burst inhibitory potential in whole human blood. All extracts were further evaluated for their oxidative burst inhibitory activity in isolated polymorphonuclear neutrophils(Table 4). Similar to the results of the study with whole human blood, all extracts showed dose-dependent oxidative burst inhibitory activities in poly-morphonuclear neutrophils, and methanolic extracts exhibited significantly (P<0.05)high inhibition when compared to ethanolic extracts. According to the results of the present study, both ethanolic and methanolic extracts of finger millet varieties exhibited a high oxidative burst inhibition in isolated polymorphonuclear neutrophils when compared with ibuprofen. Methanolic extracts of Ravi, Rawana, and Oshadha exhibited 8.5,5.2, and 5.6 times higher activities, respectively, for oxidative burst inhibition in human polymorphonuclear neutrophils when compared to ibuprofen. This may be attributed to the significantly high (P<0.05) TPCs and TFCs(Table 1) of methanolic extracts of the three-finger millet varieties.

TPCs of the extracts were found to be negatively correlated with IC5 values of oxidative burst inhibition in whole human blood(r=-0.431) and human polymorphonuclear neutrophils(r=-0.528, P<0.05). Similarly, TFCs of the extracts were found to be negatively correlated with IC5so values of oxidative burst inhibition in whole human blood (r=-0.715, P<0.05)and human polymorphonuclear neutrophils(r=-0.619, P<0.05). These correlations indicated the involvement of finger millet phenols, including flavonoids, in oxidative burst inhibition. The significant (P<0.05)negative correlations between FRAP values of the extracts and oxidative burst inhibitory activity in whole human blood (r=-0.541) and human polymorphonuclear neutrophils (r=-0.602)as well as FIC activities of the extracts and ICs。values of oxidative burst inhibition in whole human blood(r=-0.985)and human polymorphonuclear neutrophils(r=-0.959)suggested that antioxidant compounds which are present in the finger millet extracts are responsible for inhibiting the oxidative burst. There were significant(P<0.05) positive correlations between ICs values of ABTS cation radical scavenging activities and ICsovalues of oxidative burst inhibitory activities in whole human blood (r=0.556) and human polymorphonuclear neutrophils(r=0.652).In addition, there were strong positive correlations between IC values of DPPH radical scavenging activities and ICsvalues of oxidative burst inhibitory activities in whole human blood(r=0.889, P<0.05)and human polymorphonuclear neutrophils(r=0.815, P<0.05 ). These correlations indicated scavenging radicals as the prominent mechanism in oxidative burst inhibition. Antioxidants, which have the potential to inhibit ROS-induced oxidative burst, can serve as effective anti-inflammatory agents [1, 8]. Therefore, these results provided evidence for the anti-inflammatory potential of Ravi, Rawana, and Oshadha varieties. Oxidative burst inhibitory activities provided evidence for the anti-inflammatory potential of Ravi Rawana, and Oshadha varieties while indicating the potential application of methanolic and ethanolic extracts of Ravi, Rawana, and Oshadha finger millet varieties in the prevention and management of ROS-induced inflammatory conditions as natural sources of anti-inflammatory drug candidates.

4. Conclusions

This is the first study revealing in vitro anti-inflammatory properties of any extract of any finger millet variety cultivated in Sri Lanka using A5-LOX, XO, hyaluronidase, and oxidative burst inhibitory assays.A5-LOX and xanthine oxidase enzyme inhibitory activities of Ravi, Rawana, and Oshadha finger millet varieties reflected the potential application in preventing various inflammatory disorders accompanied by the catalytic actions of A5-LOX and XO enzymes. Promising oxidative burst inhibitory activities in whole human blood and isolated polymorphonuclear neutrophils demonstrated that both ethanolic and methanolic extracts of Ravi, Rawana, and Oshadha finger millet varieties could be rich sources of oxidative burst inhibitory bioactive compounds and consequently reflected the potential application of Ravi, Rawana, and Oshadha finger millet varieties in prevention and management of ROS-induced inflammatory conditions. Owing to the findings on anti-inflammatory properties, Ravi, Rawana, and Oshadha finger millet varieties could be effectively used as natural sources of anti-inflammatory drug candidates, fulfilling the demand for novel anti-inflammatory agents which are derived from natural sources. Further cell-based studies will be conducted to verify these findings.

This study also revealed the antioxidant properties of Ravi, Rawana, and Oshadha finger millet varieties. According to the findings, Ravi, Rawana, and Oshadha finger millet varieties contain significantly high amounts of phenolic compounds including flavonoids. All three finger millet varieties have the potential to scavenge free radicals including DPPH, ABTS cation, and oxygen radicals, to reduce metal ions and chelate metal ions terminating radical generating reactions. These findings indicated that Ravi, Raw-ana, and Oshadha finger millet varieties could be good sources of antioxidants. Consequently, the consumption of Ravi, Rawana, and Oshadha finger millet varieties on a regular basis may play an important role in the prevention and dietary management of oxidative stress-associated diseases. When compared with the reported antioxidant properties of commonly consumed cereals including rice, wheat, corn, barley, and oats, Ravi, Rawana, and Oshadha finger millet varieties possess high antioxidant properties. These findings act as a useful guide in selecting foods for daily consumption and are helpful in decision-making for the food industry to take the advantage of finger millet flour as an alternative or supplement to other cereal flours.

Collectively, the findings of the present study expanded the current knowledge of the bioactive properties of the finger millet varieties which are currently cultivated and consumed in Sri Lanka, and highlighted their therapeutic properties. Further studies will be conducted to identify the specific phenolic compounds which are responsible for each activity.