Evaluation of Antioxidant Interactions in Combined Standardized Extracts of Tinospora Cordifolia (Willd), Terminalia Arjuna (Roxb) and Ocimum Sanctum (LINN)

Introduction

Free radicals or reactive oxygen species (ROS) are synthesized in our body due to biological oxidation and participate in different cellular functions including gene expression and apoptosis.¹ The excess production of free radicals such as super oxide anion radical, hydroxyl radical, hydrogen peroxide causes damage to different organs and contributes to oxidative stress. Oxidation of lipid, DNA and proteins causes chronic degenerative diseases including hypertension, cancer, diabetes, coronary artery disorders etc. ^2,3

Endogenous mechanism protects the organisms from free radicals along with the dietary antioxidants.⁴ However, synthetic antioxidants like butylated hydroxyanisole and butylated hydroxytoluene have restricted use in foods due to their specific limitations. Hence, the search for safe, natural antioxidants has greatly increased in the recent years. The researchers have now focused on natural antioxidants such as standardized plant extracts and their compounds. These have been recognized to have beneficial effects against ROS in biological systems as antioxidants.^5,6

Medicinal plants containing antioxidant constituents such as ascorbic acid, polyphenols, carotenoids, vitamins, and hydrolysable tannins play a vital role in alleviating human diseases.⁷ Antioxidants are the chemicals which destroy the free radicals. Major plant antioxidants like flavonoid, tannins, phenolics, coumarins, chalcone are mainly biosynthesized via shikimic acid pathway and phenyl-propanoid metabolism^8-11.

Tinospora cordifolia (T. cordifolia) (menispermaceae) is a shrub which is extensively used in ayurvedic system and folk medicine as tonic, vitalizer. It is reported to have anti-diabetic, anti-inflammatory, antiallergic, spasmodic, neuroprotective and antioxidant properties.¹² Tinosporone, tinosporaside, tinosporic acid, cordifolisides A to E, syringen, giloin, giloinin and, picrotene, bergenin, and arabinogalactan are the major constituents of this plant. Recent studies on standardized extract of T. cordifolia revealed metabolism mediated interaction potential through rat and human liver microsomes, modulation alcohol induced steroid synthesis.^13,14

Terminalia arjuna is also known as ‘Arjuna’ and different parts of this plant such as leaves, fruits and bark are extensively used in traditional system of medicine. The stem bark possesses laxative antidysentric, and purgative properties. It is prescribed in the treatment of ulcers, inflammation, tumors, and diarrhea associated with blood as well as documented to have anti-nociceptive and immunomodulatory activities. Moreover, antioxidant activity and cytotoxic in hepatocellular carcinoma cells has been reported with bark extract. The stem bark possesses a substantial amount of secondary metabolites, which may act as resource of pharmacologically active agents and natural antioxidants. Phytochemicals such as gallic acid, ellagic acid, quercetin, which have been reported to be present in T. arjuna bark hold varying degree of antioxidant, protective and intestinal anti-inflammatory effects in rat models of colitis.^15,16

Ocimum sanctum (O. sanctum) (the holy basil) belongs to the family Lamiaceae and is reported to possess antioxidant properties. The plant branches out from its base, with angle stems and open foliage. The plant shows aromatic flavor, is pungent and is commonly used for culinary purposes. Within Ayurveda, Ocimum sanctum is said to prevent diseases, promote general health and wellbeing. Previous studies have shown the multitude potential of tulsi including anti-stress, anticonvulsant, anxiolytic and antioxidant activities. Majority of constituents of hydroalcoholic extract of O. sanctum leaves include flavonoids (oleanolic acid, ursolic acid, orientin, and vicenin).¹⁷

The anticonvulsant potential of tulsi extracts has been delineated by few previous in vivo studies, conducted in acute seizure models such as maximal electroshock seizure (MES) and pentylenetetrazole (PTZ) model. In one of previous studies, O. sanctum extract has shown 50% protection from MES and PTZ induced seizures.¹⁸

Since antioxidant mechanism plays vital role in the treatment of different disorders, the present investigation aims to check the synergistic/antagonistic antioxidant effects of standardised extracts of Tinospora cordifolia (Willd) (TCE), Terminalia arjuna (Roxb) (TAE), Ocimum sanctum (Linn) (OSE) in combinations.

Materials and methods

Chemicals

Standardized extracts of TCS, TAE and OCE were obtained from Sami Labs Limited (19/1, 19/2, I Main, II phase, Peenya Industrial Area, Bangalore, Karnataka-560058) as gift samples. Other chemicals are purchased from SD Fine Chemicals Ltd. (Mumbai, India). All chemicals used in the experiment were of analytical grade.

In vitro free radical scavenging activity

DPPH Scavenging Activity¹⁹

The DPPH free radical scavenging activity of diverse combinations of any two standardized extracts (1:1) at different concentrations were measured from bleaching of the purple colour of 2,2 Diphenyl -1-picryl hydrazyl (DPPH). A 3 ml solution of different concentrations of extract was added to 1 ml of DPPH and kept in dark for 30 min. DPPH assay was performed in triplicates. The absorbance of the colour was measured at 517 nm and the percentage of scavenging activity was calculated by using the following equation.

Percentage inhibition (%) = (A0-A1/A0)×100

where:

A0 is the Absorbance of control

A1 Absorbance of test

The results are expressed in terms of IC50 value.

Hydroxyl radical scavenging activity²⁰

Hydroxyl radicals were generated by Fenton reaction (Fe3+-ascorbate-EDTA-H2 O2 system), and the scavenging capacity towards the hydroxyl radicals was measured by using deoxyribose method. The reaction mixture contained 2-deoxy-2-ribose (2.8 mM), phosphate buffer (0.1 mM, pH 7.4), ferric chloride (20 μM), EDTA (100 μM), hydrogen peroxide (500 μM), ascorbic acid (100 μM) and different concentrations (100-600 μg/ml) of the different herb-herb combinations (1:1) in a final volume of 1 ml. The mixture was incubated for 1 h at 37°C. After the incubation, an aliquot of the reaction mixture (0.8 ml) was added to 2.8% TCA solution (1.5 ml), followed by TBA solution (1% in 50 mM sodium hydroxide, 1 ml) and sodium dodecyl sulphate (0.2 ml). The mixture was then heated (20 min at 90°C) to develop the colour. After cooling, the absorbance was measured at 532 nm against an appropriate blank solution. This scavenging activity was performed in triplicates. The percentage of inhibition was expressed according to the following equation:

Percentage inhibition (%) = (A0-A1/A0)×100

where:

A0 is the Absorbance of control

A1 is the Absorbance of test.

The results are expressed in terms of IC50 value.

Reducing Power Assay²¹

The reducing power of the diverse combinations of any two standardized extracts (1:1) was measured by the method described by Oyaizu.21 An aliquot of extracts (1.0 ml) at different concentrations from 50-450 μg/ml was mixed with phosphate buffer (0.2 M, pH 6.6, 2.5ml) and 1% potassium ferricyanide (2.5 ml, 1%). The mixture was incubated at 50°C for 20 min. After adding 10% trichloroacetic acid (2.5 ml, 10%), the mixture was centrifuged at 6500 rpm for 10 min. The supernatant (2.5 ml) was mixed with distilled water (2.5 ml) and 0.1% iron (III) chloride (0.5 ml) and the absorbance was measured at 700 nm using phosphate buffer as blank. These were done in triplicate and the mean values were given in the results.

Statistical analysis

All the experimental results were expressed in terms of IC50 value, which is the effective concentration at which the antioxidant activity is 50%.

Results

DPPH Scavenging Activity

DPPH assay method is mainly based on the reduction of methanolic solution of blue to dark purple coloured free radical DPPH by free radical scavenger. Basically, a higher DPPH radical-scavenging activity is associated with a lower IC50 value. Reaction with DPPH radicals of combination of standardized extracts showed better scavenging activity compared to individual extracts. The IC50 values for TCE+TAE, TCE+OSE, TAE+OSE were found to be 74.12 µg/ml, 78.62 µg/ml and 71.70 µg/ml respectively whereas IC50 value for ascorbic acid was found to be 69.05 μg/ml. A linear correlation coefficient (r2 =0.809, 0.863, 0.853) was obtained (Fig.1). Whereas TCE, OSE, TAE showed IC50 values of 97.09, 98.01, 96.07 respectively with linear correlation coefficient r2 = 0.970, 0.931, 0.889.

Hydroxyl radical scavenging activity

The assay showed the capacity of the extract and standard mannitol to decrease hydroxyl radical-mediated deoxyribose degradation in an Fe3+-EDTA-Mannitol and H2 O2 reaction mixture. The IC50 values for different herb combinations viz., TCE+TAE, TCE+OSE, TAE+OSE were found to be 320.28 µg/ml, 340 µg/ml and 261 µg/ml respectively. A linear correlation coefficient (r2 =0.986, 0.992, 0.936) was obtained (Table 1). The IC50 value for standard mannitol showed 181.65µg/ml with a linear correlation coefficient (r2 =0.927) respectively. Whereas TCE, OSE, TAE showed IC50 values of 399.11, 343.37, 372.78 respectively with linear correlation coefficient r 2 =0.958, 0.997, 0.992.

Reducing Power Assay

Reducing power of individual standardized herbal extract and in combination increased with increase in concentration. The standardized herb combinations viz., TCE+TAE, TCE+OSE, TAE+OSE showed more effective reductive ability when compared to that of individual standardized herbal extract (Fig. 2). Here, Fe (III) reduction is frequently used as a notable indicator of electron donating activity which is a prime mechanism of phenolic antioxidant action by breaking the free radical chain by donating a hydrogen atom.

Discussion

Effect of plant extract combinations or phytochemicals leading stronger effect as compared to individual extracts/compounds at the same concentrations is termed as synergistic effect. Synergistic antioxidant effect was observed with compounds such as vitamins E and C²², vitamin E and β-carotene²³, catechin and malvidin-3- glucoside²⁴, tea polyphenols and vitamin E²⁵ and with varieties of fruits.²⁶ In this study, for the first time, we employed to assess synergistic antioxidant effects raised with the combination of standardized extracts of T. cordifolia, T. arjuna and O. sanctum.

Natural polyphenols and carotenoids are the most potent antioxidants in human food, and their free radical scavenging properties are related to substitution of hydroxyl groups in the aromatic rings of phenolics.²⁷ The plant type, geographic location, growing time, and storage condition can all affect the concentrations of polyphenols in food. Dietary polyphenols could be classified into five classes: flavonoids, coumarins, stilbenes, tannins and phenolic acids. Flavonoids are grouped as flavones, flavanols, flavanones, anthocyanidins, and isoflavonoids. Total phenolic content and total antioxidant activity in phytochemical extracts of different herbs may have a direct relationship. When the herbs contain high amount of total phenolic contents, they possess potential antioxidant activity. For example, the scavenging activity of grape seed extract against ABTS radical was strongly linked with the level of phenolic compounds.²⁸

OCE is rich in volatile oil (0.7%), phenolics, flavonoids, neolignans, terpenoids and fatty acid derivatives.²⁹ Caffeic acid, chlorogenic acid, vanillic acid, ocimumnaphthanoic acid and menthylsalicylic glucoside were also isolated from the aerial parts of O. Sanctum.³⁰ Eugenol is one of the most distributed antioxidant phenyl propanoid in the essential oil of O. sanctum leaves. Other phenyl propane derivatives such as ociglycoside or eugenyl-βD-glucoside, citrusin C, ferulaldehyde, bieugenol and dehydrodieugenol were isolated from the leaves of O. Sanctum.³¹

The major constituents of T. arjuna in stem bark are flavonoids and phenolics like arjunone, luteolin, baicalein, ethyl gallate, gallic acid, kempfero, pelargonidin, quercetin, (+)-catechin, (+)-gallocatechin and (-) -epigallocatechin, gallic acid, ellagic acid and its derivatives which are responsible for its antioxidant activity. T. arjuna also contains triterpenoids; arjunin, arjunic acid, arjungenin, terminic acid, terminoltin, arjunolic acid which further enhances its antioxidant activity.³²

T. cordifolia exhibits an antioxidant potential by scavenging the free radicals and ROS. T. cordifolia significantly decreases the regulating lipid peroxidation process and in turn reduces the level of ROS in a diabetic rat model (alloxan induced diabetes). It regulates antioxidant enzymes like glutathione and catalase indicating its antioxidant effects. A clinical research has reported that the extract shows antioxidant effect by raising the level of GSH and reducing the expression of inducible nitric oxide synthase gene. The plant derived polysaccharide compound named as ‘arabinogalactan’ shows protection against free radicals in rat model exhibiting its antioxidant action. In Ayurvedic medicine, Pepticare, a herbomineral formulation which includes T. cordifolia has also been reported to possess potent antioxidant effect in rat model.³³

Conclusion

The above discussion revealed that O. sanctum contains phenolics, flavonoids, triterpenoids; whereas, T. arjuna contains triterpenoids, alkaloids, steroids and phenolics, flavonoids, triterpenoids and tannins are present in T. Cordifolia. These potent antioxidant phyto constituents are responsible for synergistic antioxidant activity. Hence, we conclude that combinations of TCE+TAE, TCE+OSE, TAE+OSE have shown synergistic antioxidant activity compared to individual extracts.

Conflicts of interest

None

Acknowledgements

The authors are thankful to the President, Soniya Education Trust and Principal, SET’s College of Pharmacy, Dharwad for encouragement, support and for providing the necessary facilities to carry out the research work. We are also thankful to Sami Labs Limited, Bangalore, Karnataka for providing standardised plant extracts as gift samples.