Evaluation of Antioxidant Activity of Some Medicinal plants and their Combination

Oxidative damage and antioxidants are assuming great interest in today’s modern world. Cancer, cardiovascular diseases and other types of diseases may be due to the result of free radicals.1 With newer insights into the fact that oxidative species can lead to a number of health problems and complications, finding treatment of oxidative damage and oxidative stress is acquiring greater significance. Phytochemicals obtained from plants are gaining popularity day by day in countering harmful effects of oxidative species and diseases associated with oxidative damage. Various fruits, vegetables and medicinal plants contain numerous phytochemicals.1,2 Many phytochemicals have been isolated and tested for their therapeutic potentials but many phytochemicals are still there which need to be discovered in future. Phytochemicals have a wide variety of beneficial effects. They possess antioxidant activity, enhance immunity, reduce aggregation of platelets, influence the activity of hormones and have anticancer effects. Besides the above mentioned pharmacological activities, they produce numerous other actions as well.3 Various phytochemicals have various pharmacological activities. Cellulose, lignins, gums and pectins bind toxins. Alkaloids, phenolic compounds and terpenoids have antimicrobial potential. Ascorbic acid, carotenoids, phenols and flavonoids have potent antioxidant capabilities. Antitumor activities have been shown by flavonoids, phenols etc. Carotenoids, tocopherols, phenols, flavonoids etc function as detoxifying agents.2 Phenols and flavonoids are some plant constituents that have gained popularity over the years for treatment of oxidative stress. Many medicinal plants contain different phenolic compounds and flavonoids that help in a variety of diseases. In many experiments flavonoids and phenols have proved their potential in protecting the body against harmful effects of free radicals.1,2 They protect proteins, lipids and deoxyribonucleic acid (DNA) against harmful effect of the free radicals. Also phenolic compounds and flavonoids may assist the natural antioxidant defence system of the body and help to fight oxidative damage within the body.4 In the present study three medicinal plants Ocimum kilimandscharicum (OCM), Thymus serpyllum (THY), Spilanthes acmella (SPL) and their combination in 1:1:1 ratio (COMB) were assessed for their phytochemical constituents and antioxidant activity.


INTRODUCTION
Oxidative damage and antioxidants are assuming great interest in today's modern world. Cancer, cardiovascular diseases and other types of diseases may be due to the result of free radicals. 1 With newer insights into the fact that oxidative species can lead to a number of health problems and complications, finding treatment of oxidative damage and oxidative stress is acquiring greater significance. Phytochemicals obtained from plants are gaining popularity day by day in countering harmful effects of oxidative species and diseases associated with oxidative damage. Various fruits, vegetables and medicinal plants contain numerous phytochemicals. 1,2 Many phytochemicals have been isolated and tested for their therapeutic potentials but many phytochemicals are still there which need to be discovered in future. Phytochemicals have a wide variety of beneficial effects. They possess antioxidant activity, enhance immunity, reduce aggregation of platelets, influence the activity of hormones and have anticancer effects. Besides the above mentioned pharmacological activities, they produce numerous other actions as well. 3 Various phytochemicals have various pharmacological activities. Cellulose, lignins, gums and pectins bind toxins. Alkaloids, phenolic compounds and terpenoids have antimicrobial potential. Ascorbic acid, carotenoids, phenols and flavonoids have potent antioxidant capabilities. Antitumor activities have been shown by flavonoids, phenols etc. Carotenoids, tocopherols, phenols, flavonoids etc function as detoxifying agents. 2 Phenols and flavonoids are some plant constituents that have gained popularity over the years for treatment of oxidative stress. Many medicinal plants contain different phenolic compounds and flavonoids that help in a variety of diseases. In many experiments flavonoids and phenols have proved their potential in protecting the body against harmful effects of free radicals. 1,2 They protect proteins, lipids and deoxyribonucleic acid (DNA) against harmful effect of the free radicals. Also phenolic compounds and flavonoids may assist the natural antioxidant defence system of the body and help to fight oxidative damage within the body. 4 In the present study three medicinal plants Ocimum kilimandscharicum (OCM), Thymus serpyllum (THY), Spilanthes acmella (SPL) and their combination in 1:1:1 ratio (COMB) were assessed for their phytochemical constituents and antioxidant activity.

PLANT COLLECTION AND IDENTI-FICATION
Aerial parts of Ocimum kilimandscharicum, Thymus serpyllum and Spilanthes acmella were collected from the herbal gardens of Defence Institute of Bioenergy Research, Pithoragarh. The plant samples were preliminarily identified by ICAR-National Bureau of Plant Genetic Resources, Regional station, Niglat, Bhowali, Uttarakhand. The plant samples were further sent to Botanical Survey of India, Northern Botanical Survey of India for final authentication.

Preparation of the extract
After collection, the aerial parts of the above mentioned plants were dried. After drying the aerial parts were powdered and extracted with absolute ethanol (99.9%). Soxhlet's assembly was used for the extraction. The extract so obtained was then dried using rotatory vaccum flash evaporator. 5 Experimental procedure

Phytochemical screening of the extracts
The individual extracts as well as the combined extracts were subjected to preliminary phytochemical screening tests using various methods. The results are given in the Table 1.

Reducing power assay
Individual extracts, combined extract and ascorbic acid showed different absorbance at different concentrations. A higher absorbance indicates higher reducing power due to formation of reduced intermediate ( Table 2).

DISCUSSION
In the present study it was seen that at the lowest concentration (0.0001μg/ml) the highest absorbance was shown by ascorbic acid and lowest absorbance was shown by SPL. At the highest concentration (50 μg/ml) highest absorbance was shown by ascorbic acid and lowest absorbance was shown by SPL. Highest absorbance was shown by THY in lowest concentration (0.0001μg/ml) and highest absorbance was shown by OCM at the highest concentration (50 μg/ml) among the individual extracts. Among all the extracts COMB showed better effect than the individual extracts in the reducing power assay at both the lowest and highest concentrations ( Table 2). In reducing power assay antioxidants provide a beneficial effect by breaking the free radical chain by donating a hydrogen atom. 11 In this assay antioxidants act as reductants and reduce Fe 3+ /ferricyanide complex to ferrous form. The absorbance of ferrous ions can be measured at 700 nm. In this assay increase in absorbance is an indicator of increasing reductive ability. 12 . Phytochemical screening has revealed the presence of antioxidant phytochemicals like, phenols and flavonoids in the extracts of the OCM, THY, SPL and COMB ( Table 1). As has been discussed in the introduction that phenols and flavonoids have potent antioxidant activity, thus the presence of these phytochemicals might be responsible for the antioxidant activity of these extracts.

CONCLUSION
Diseases due to oxidative stress are posing a significant burden on humans. Newer research is being carried out by scientists to study the impact of free radicals in causing diseases. Medicinal plants have the potential to become frontline medicines in the treatment of oxidative stress. Also, these plants have the advantage that they are economical and a safe alternative to allopathic drugs and synthetic antioxidant formulations. Newer medicinal plants should be explored in detail so that we can come up with potent antioxidants. In the present study ascorbic acid, individual extracts of the three plants used in the study and the combination of these individual extracts were studied for antioxidant activity using reducing power assay. The results showed that even at very low concentrations the individual extracts and their combination demonstrated decent reductive ability in comparison to the standard antioxidant ascorbic acid. Also, the combination of the plant extracts used in the study showed the highest antioxidant potential in the present study when compared to the individual extracts. Thus the extracts of the plants used in the present study and their combination can prove to be good antioxidants if properly formulated and marketed.