Evaluation of the Antioxidant properties of Tribulus rajasthanensis L.- A case study

Raju N. Devkar ¹ and Dr. Vishal N. Shinde ²

1. Assistant Professor in Botany, VVM’s S.G. Patil ASC College Sakri Tal. Sakri Dist. Dhule-424304 (MS) India.

Mail ID – rajudevkar094@gmail.com

• Associate Professor in Botany, ADMSP’s Late Annasaheb R D Deore Art’s and Science College, Mhasadi Tal.Sakri, Dist. Dhule- 424304 (MS) India

Mail ID – vishalshinde1001@gmail.com

ABSTRACT: Free radicals are continuously generated in the body during normal metabolic processes and though exposure to environmental factors such as infectious agents, pollution, UV light and radiations. When these harmful free radicals are not neutralized by primary and secondary defence mechanism of body, oxidative stress occurs, which is the reasons for development of various diseases. Plants have many phytoconstituents including saponin, flavonoids and polyphenol with high antioxidants properties. To determination of antioxidant properties of Tribulus spp. extracts (methanol and aqueous) DPPH (1,1- diphenyl 2- picryl hydrazyl) method was used. Whereas DPPH free radical scavenging activity of methanol extracts revealed the strongest as compared to aqueous extracts.

KEYWORDS: Antioxidants, DPPH, Phenolic compounds, Flavonoids, Tribulus rajasthanensis L.

INTRODUCTION:

          Since ancient times, the medicinal properties of plants have been investigated in the recent scientific developments throughout the world, due to their potent antioxidant activities. As antioxidants have been reported to prevent oxidative damage caused by free radicals, it can interfere with the oxidation process by reacting with free radicals, cheating, catalytic metals and also acting as oxygen scavengers ^[1]. Reactive oxygen species (ROS), such as hydrogen peroxide (H₂O₂) and hypochlorous acid (HOCl), and free radicals, such as the superoxide anion (O₂^–) and hydroxyl radical (OH), are produced as normal products of cellular metabolism. Overproduction of free radicals and ROS can lead to oxidative damage to various biomolecules including proteins, lipids, lipoproteins and DNA. This oxidative damage is a critical etiological factor implicated in several chronic disorders such as Cancer, Mellitus, diabetes, inflammatory disease, asthma, cardiovascular disease, neurodegenerative disease and premature aging ^[2,3]. Antioxidants are means for the substances or group of substances that inhibit oxidative damage to a molecule. This defense system is having many modes of classification such as based on their metabolism of action (chain breaking, preventive). Many plants contain large amounts of antioxidants such as vitamin C, vitamin E, lycopene, lutein, carotenoids, polyphenols which play important roles in adsorbing and neutralizing free radicals ^[4]. Beside this, phenolic compounds and flavonoids which have been reported to exert multiple biological effects, including free radical scavenging abilities, anti-inflammatory, anticarcinogenic etc. ^[5].

          Whereas unfavorable environmental conditions for plants, including extreme temperatures, drought, heavy metal exposure, nutrient deficiencies, and high salinity, lead to the excessive production of reactive oxygen species (ROS), which can induce oxidative stress. To counteract this damage, plant cells possess an antioxidant defense system composed of both enzymatic and non-enzymatic components. Non-enzymatic antioxidants act through various mechanisms, such as enzyme inhibition, chelation of trace elements involved in free radical generation, scavenging and neutralization of reactive species, and enhancement of protection via interaction with other antioxidant systems. Among these compounds, secondary metabolites particularly phenolic compounds play a crucial role in protecting plants against oxidative stress ^[6].

          Tribulus rajasthanensis L. belongs to the family zygophyllaceae. It is an annual plant with a wide global distribution and is commonly found throughout India. The species primarily grows wild in dry and arid regions, especially in West Rajasthan, Gujarat, Maharashtra, Uttar Pradesh, and other similar areas ^{[7, 8, 9]}.

          The plant is a decumbent herb with pinnately compound leaves. The leaves typically bear 3–10 pairs of sessile leaflets with unequal, oblique, or rounded bases. Flowers are solitary and pentamerous. The number of stamens ranges from five to ten, and the ovary is five-chambered. The fruit is the most characteristic feature of this genus. At maturity, it divides into five indehiscent mericarps, each containing two to five seeds arranged in a horizontal row.

          According to Bhandari and Sharma (1977), the species is closely allied to T. terrestris L. but can be easily distinguished by its secondary spines and the complete absence of lower pair of spines. Typical specimens with mature mericarp can be easily told apart while the intermediate forms that show the characters of both Tribulus rajasthanensis and Tribulus terrestris are difficult to separate. The typical forms of T. rajasthanensis as a variety of T. terrestris ^[10]. The aim of the present study was to evaluate the antioxidant activity of Tribulus rajasthanensis L. extracts by DPPH methods.

MATERIALS AND METHODS:

Plant materials: The healthy infection free mature plants parts (Fruits, stem, leaves and roots) were collected from the Gomai bank of river, Shahada taluka, Nandurbar District and then they were shade dried and powdered separately in laboratory and kept safely for further research.

Preparation of crude extracts in water: 10 g of dry plant powder was taken in a beaker, 100 ml of distilled water was added, and the mixture was stirred by a magnetic stirrer for 24 h. After that it is filtered by Whatman’s filter paper No.1 and filtrate were centrifuged at 3000 rpm for 15 min. The supernatant was evaporated by rotary evaporator, to get dried form. It was weighed and kept in a refrigerator in sterilized and dark glass containers ^[11].

Preparation of crude extracts in methanol: Solvent extracts were prepared in methanol at room temperature. 10g of dry plant powder was mixed in sufficient quantity of methanol in conical flask. The conical flasks were plugged tightly with cork. Shaken the conical flask properly to mix the content then kept the conical flask for about 30 minutes for the extraction. After 30 minutes it was filtered and filtrate were collected in china dish. These dishes kept on a water bath for some time to evaporate the solvent, after that the methanolic extract were completely dried.

 Antioxidant Activity (DPPH free radical scavenging activity):

          Free radical scavenging activity was determined using the stable 1,1- diphenyl -2-picryl hydrazyl radical (DPPH) according to the method described by Shimada et al. (1992). Butylated hydroxytoluene (BHT) were used as standard control. Various concentrations of the extracts were added to 4 ml of a 0.004% methanol solution of DPPH. The mixture was shaken and left for 30 minutes at room temperature (25 ± 5⁰C) in the dark, and the absorbance was then measured with a spectrophotometer at 517 nm. All determinations were performed in triplicate ^[12,13,14]. antioxidant activity was calculated as the percent inhibition caused by the hydrogen donor activity of each sample according to the following:

Inhibition (%) = [(Absorbance _control – Absorbance _sample)/ (Absorbance _control) ×100

Where: absorbance control is the absorbance of DPPH radical plus methanol; absorbance sample is the absorbance of DPPH radical plus sample extract or standard.

RESULTS:

           Many plants exhibits in vitro and in vivo antioxidant properties owing to their phenolics, vitamins, proteins and pectins contents. In the different literatures, it has been revealed that the antioxidant activity of plant extracts is responsible for their therapeutic effect against cancer and many more disorders. Hence, Tribulus rajasthanensis L. plant extracts were evaluated for in vitro antioxidant activities. DPPH (1,1-diphenyl, 2- picryl hydrazyl) method were used for evaluation of in vitro antioxidant activity.

                  In the present study several biochemical constituents and free radical scavenging activity of Tribulus were evaluated. Free radicals are involved in many disorders like neurodegenerative diseases and cancers. Scavenging activity of antioxidants are useful for the control of these diseases. DPPH stable free radical method is a sensitive method to evaluate the antioxidant activity of plant extracts. DPPH radical scavenging activity of methanolic extracts of Tribulus showed strongest while some parts of plants revealed moderate antioxidant properties in aqueous extracts.

DISCUSSION:

           medicinal plants have been used to treat a wide range of disorders since ancient times. From simple cold to complex diseases these plants have served as effective therapeutic agents ^[15]. Tribulus rajasthanensis L. as a well- known medicinal plant, was selected for this study primarily because of it’s antioxidants potential. Plant extracts were evaluated for in vitro antioxidant activities. DPPH Method provides a good assessment for evaluation of in vitro antioxidant activity. It is based on reaction between antioxidant with nitrogen centered free radical i. e. DPPH (1,1 diphenyl, 2- picryl hydrazyl). That’s why in this experiment; we evaluated the in vitro antioxidant and radical scavenging activities of Tribulus spp. methanol extract using DPPH Method.

           Oxidative stress is a deep-rooted cause of various disorders, including rheumatoid, arthritis and inflammation, neurodegenerative disease, diabetes, cancer, aging etc. Preventing the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) during cellular metabolism is critically important. The widespread use of medicinal plants across different therapeutic contexts encouraged us to investigate Tribulus spp. to assess its antioxidant and free radical scavenging properties. Our result revealed the tremendous potential of this plant in reducing free radical through DPPH, possibly due to its high polyphenol content. However, more investigations should be carried out to clarify the specific correlations between the plant bioactive and the observed biological activities.

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