A free radical is a molecule containing oxygen and one or more unpaired electrons which making it highly reactive with other molecules. Antioxidants are molecules which present in the cells that prevent these reactions by donating an electron to free radicals without becoming destabilized themselves.
Free radicals initiate the oxidative stress and damage the healthy cells and DNA along with lipid peroxidation. This damages can contribute to ageing, atherosclerosis, cancer, cardiovascular diseases, and inflammatory diseases.1,2 Consumption of certain plant materials can promote health benefits and reduce the risk of stress related chronic diseases.3 Phytochemicals such as flavonoids,4 phenolic acids, alkaloids, lignins, stilbenes, and tannins are well known free radical scavengers and possessing multiple biological activities including anti-oxidant activity.5–7 Supplementation of antioxidants can reduces the oxidative stress in experimental diabetes which include vitamin E, vitamin C, non nutrient antioxidant and plant derived natural antioxidants.8
Leucas aspera belongs to Lamiaceae family and commonly found throughout India and Philippines as well as the plains of Mauritius and Java. It is a very common weed in India and it is an annual plant grows up to 15-60 cm height.9 The juice of Leucas aspera leaves are used as remedy for chronic skin eruptions, chronic rheumatism and psoriasis.10 In villages L.aspera leaves are used as insecticides and mosquito repellent.
In our present study, we attempted to evaluate the phytochemicals present in various solvent extracts obtained from leaves of Leucas aspera (Willd.) Link (L. aspera) and its antioxidant properties using different in vitro models include ABTS (2,2,-azinobis (3-ethylbenzoline-6-sulfonic acid) radical scavenging activity, DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging capacity, Nitric oxide radical inhibition assay and total antioxidant capacity in order to ensure the pharmacological effects of the plant.
MATERIALS AND METHOD
Plant collection and authentication
Well grown plants of L. aspera had been collected from Kandappakottai Village (Dindigul, Tamilnadu, India) between Nov-2014 and Jan-2015. The plant was authenticated (PARC/2014/2272) by Prof. P Jayaraman, Director, Institute of Herbal Botany and Anatomy Research Centre, Tambaram, Chennai. Then leaves were isolated, cleaned and dried at sun shadows. Dried leaves were crushed to powder and passed through pharmaceutical sieve no 40 and 80. Any materials which passed through sieve-40 and retained at sieve-80 were collected and used for extraction.
Preparation of crude extracts
Five different solvents were selected with increasing polarity, petroleum ether (60-80°C), ethyl acetate, chloroform, ethanol (70%) and isopropyl alcohol for the extraction process. Soxhlet apparatus was used for petroleum ether extraction. Hundred grams of powdered leaf was extracted with 1200 mL of petroleum ether (60-80°C). Excess solvent was the removed by distillation method. Crude extract was dried at vaccum desiccators and stored in a glass bottle at -20°C for further studies. All other solvent extracts were prepared by cold maceration technique. Weighed powder was soaked in solvents ethyl acetate (100 g + 900 mL), chloroform (100 g + 900 mL) isopropyl alcohol (100 g + 900 mL) and ethanol 70% (100 g + 1000 mL) in a separate round bottom flask for 72 hours. After soaking the mixture was heated at 30-40° C for 60 minutes. Heated solution was cooled to room temperature and filtered through muslin cloth and whatman filter paper. Excess solvent was removed by distillation method and allowed to evaporate at room temperature. Dried crude extracts were collected and stored in an individual glass bottles at -20°C for further studies.
Qualitative screening of phytochemicals presents in L. aspera leaves
All the five crude extracts were screened for the presence of phytochemicals as described in textbook by Harborne JB (eds.).11 Freshly prepared extracts were tested for flavonoids (Shinoda’s test, Alkaline reagent test, Lead acetate test and Sulphuric acid test), alkaloids (Dragendorff’s test, Mayer’s test, Hager’s test, Wagner’s test and Tannic acid test), steroids (Libermann-Burchard’s test and Salkowsky’s test), glycosides (Legal’s test, Baljet test, Keller- killiani test and Borntrager’s test), saponins (Foam test and Lead acetate test), tannins (Gelatin test, Ferric chloride test) Vanillin hydrochloride test, Lead acetate test, Ammonia test and Potassium dichromate test.), phenolic components, fixed oils (Spot test and Saponification test.), carbohydrates (Molisch’s test, Fehling’s test, Benedict’s test, Tollen’s test, Seliwanoff’s test and Bromine water test.), proteins (Biuret test, Ninhydrin test, Xanthoproteic test and Millon’s test) and terpenoids (Knoller’s test).
Quantitative estimation of phyto-ingredients present in L. aspera leaves
Estimation of total flavonoid compounds
Total flavonoids were estimated by using spectrophotometric technique. Flavonoids give yellow colour when treated with aluminium chloride reagent (1% w/v) and this principle was used in flavonoid estimation. Rutin was used as the standard solution (100 mg/mL) with methanol. Test sample was prepared with methanol to the final concentration of 100 mg/mL. One mL of test and standard solutions were taken in a separate 10 mL volumetric flask and 1.0 mL of aluminium chloride reagent was added to both the test and standard solutions. Volumes were made up to 10 mL with ethanol and optical density was measured at 410 nm against blank solution exactly at 15 min after adding aluminium chloride reagent.
Estimation of total phenolic compounds
Total phenolic content was calculated by calibration curve method using Gallic acid, Folin Ciocalteu reagent and sodium carbonate (20%) solution reagent. 100 mg of crude extract was added to 0.5 mL distilled water and 0.25 mL of the Folin-Ciocalteu reagent. Then 1.25 mL of sodium carbonate solution was added and vortexed. The absorbance of final solution was recorded at 725 nm after 40 min. Total phenols were calculated using the Gallic acid equivalent from the calibration curve equation.
Estimation of total alkaloid compounds
Total alkaloid compounds were estimated by using the method adopted from Anjanal et.al.,.12 Small quantity of extract was dissolved in 2N hydrochloric acid and filtered. One mL of this solution washed with about 10 mL chloroform for three times in a separation funnel. The pH was adjusted to neutral with addition of 0.1N sodium hydroxide. Five mL of bromocresol green solution and 5 mL phosphate buffer were added to this solution and shaken well, then extracted with 1,2,3 and 4mL of chloroform by vigorous shaking and collected in a 10 mL volumetric flask then diluted with chloroform. The absorbance was measured at 470 nm and atropine was used as standard reference.
Estimation of total tannins
Total tannin compounds were estimated by spectrophotometric procedure using potassium ferricyanide reagent, ferric chloride reagent and tannic acid as standard. 100 mg of crude extract was refluxed with purified water in a water bath at 100°C±2°C for 60 min and cooled them volume made to 100 mL with purified water. Standard solution of tannic acid (0.01 mg/mL) was prepared with purified water and for working standard 1.0 mL of standard solution was added to 1.0 mL each potassium ferricyanide and ferric chloride in a 10 mL standard flask. The volume was made up to 10 mL with purified water. For working sample solution 0.2 mL of sample preparation was added to 1.0 mL of potassium ferricyanide and ferric chloride in a 10 mL standard flask. The volume was made up to 10 mL with purified water. Both standard and test samples optical density was measured at 720 nm exactly after 30 min from addition of reagents.
Assessment of in vitro antioxidant activity of crude extracts of L. aspera leaves
DPPH (Diphenyl-2-picrylhydrazyl) radical scavenging activity
DPPH free radical scavenging activity was measured in terms of hydrogen donating or radical scavenging ability using stable DPPH radical, method described by Blois.13,14 One mL of 0.1mM DPPH solution in ethanol was added to 3.0 mL of extract solution (or standard) and volume made up with water at different concentrations (10-100 μg/mL). Thirty minutes later absorbance was measured at 517 nm. Lower absorbance indicates the higher free radical scavenging activity. Capability to scavenge the DPPH radical was calculated using the following equation. Mean values were obtained from triplicate of experiment values.
Total antioxidant activity (Phosphomolybdic acid method)
Total antioxidant activity of the extract was evaluated by the transformation of Mo (VI) to Mo (V) to form phosphomolybdenum complex.15 An aliquot of 0.2 mL extract solution (10-100 μg/mL) was combined in a vial with 2.0 mL of reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The vials were capped and incubated in a water bath at 95°C for 30 mins. Samples were cooled to room temperature and the absorbance was measured at 695 nm against blank solution. Total antioxidant activity was expressed relative to that of ascorbic acid.
Nitric oxide radical scavenging
Nitric oxide radical scavenging activity procedure was adopted from the method reported.16 and rutin was used as standard. Nitric oxide was generated from sodium nitro prusside in aqueous solution at physiological pH, which interacts with oxygen to produce nitric ions, which may be determined by the Griess Ilosvay reaction. Two mL of 10 mM sodium nitro prusside and 0.5 mL of phosphate buffer saline (pH 7.4) was mixed with 0.5 mL of sample at different concentrations (10-100 μg/mL) and the mixture incubated at 25°C for 2.5 h. Incubated mixture (1.5 mL) was added to 1.5 mL of griess reagent (1% sulphanilamide, 2% o-phosphoric acid, 0.1% napthyl ethylene diamine di hydrochloride) and again incubated at room temperature for 5 min. The absorbance of the mixture was read at 546 nm.
ABTS (2, 2,-azinobis (3-ethylbenzoline-6-sulfonic acid) radical scavenging activity
Scavenging activity of extract was tested by using ABTS+ assay and quercetin was used as standard. This method was described with a slight modification.17 The ABTS+ radical solution was prepared by mixing 14 mM ABTS stock solution with 4 mM ammonium per sulphate and incubating for 16 h at room temperature in the dark until the reaction was stable. Absorbance of ABTS+ solution was equilibrated to 0.70±0.02 by diluting with ethanol at room temperature. To 1.0mL of ABTS+ solution various concentrations of test samples (10-100 μg/mL) was added. Six minutes later the absorbance was measured at 734 nm. Percentage inhibition of absorbance was calculated by using the following equation.
Preliminary phytochemical studies
The present study was carried out on petroleum ether, ethyl acetate, chloroform, ethanol and isopropyl alcohol extracts of L. aspera leaves. The results have confirmed that L. aspera leaf contains flavonoids, alkaloids, glycosides, carbohydrates and less extent of steroids, terphenoids, tannins, phenolic compounds. Maximum number of phytochmicals were found in petroleum ether extract and ethanol extract. Complete details of presence of phytochemicals in each crude extracts were presented in Table 1.
Our current study demonstrated the amount of total flavonoid compounds, total phenolic compounds, total tannins and total alkaloids present in various solvent extract of L. aspera leaves. The complete details of phyto-ingredients present in each crude extract were given in Table 2. values are expressed in grams per 100 grams.
In vitro antioxidant studies DPPH scavenging activity
Petroleum ether, ethanol and isopropyl alcohol extracts of L. aspera leaves were exhibited a significant dose dependent DPPH scavenging activity (Figure 1). Amongst, petroleum ether extract exhibitted 66%, 68%, 69%, 70%, 71% and 72% of scavenging activity at 10, 20, 40, 60, 80 and 100 μg/mL concentrations respectively. Whereas ethanol extract exhibited 64%, 67%, 71%, 76%, 81% and 87% with the same concentrations. On the other hand, ascorbic acid standard showed 21%, 33%, 34%, 46%, 58% and 74% of activity with the same concentrations. Percentage of scavenging activity or percentage inhibition was calculated by linear regression method. The IC50 values (Table 3) of ascorbic acid, petroleum ether extract, ethanol extract and isopropyl alcohol extract was found to be 27.05μg/mL, 18.96μg/mL, 19.90μg/mL. and 24.74 μg/mL respectively.
Total antioxidant activity
Total antioxidant capacity of various solvent extracts of L. aspera leaves were calculated using the standard curve of ascorbic acid (y = 0.029+0.021; R2 = 0.813) and expressed as number of equivalent of ascorbic acid per gram of extract. The IC50 values of total antioxidant capacity of ascorbic acid, petroleum ether extract, ethanol extract, isopropyl alcohol extract, chloroform extract and ethyl acetate extract was found to be 17.23 μg/mL, 17.22 μg/mL, 21.58 μg/mL, 10.40 μg/mL, 37.78 μg/mL and 23.61 μg/mL respectively (Figure 1). Amongst petroleum ether, ethanol and isopropyl alcohol exhibited significant dose dependent antioxidant activity (IC50 values are presented in Table 3).
|Antioxidant activity||Crude extract of L. aspera leaves IC50 values (μg/mL)|
|Nitric oxide scavenging||11.87*||20.75||13.47*||24.26||13.58*||-|
PET-petroleum ether / EA-ethyl acetate / Et.OH-ethanol / IPA-isopropyl alcohol / ChCl3-chloroform / Std-standard used.
$all values are expressed in grams per 100 grams. Values are obtained from average of 3 determinations.
Nitric oxide scavenging activity
Nitric oxide is a free radical and over production of nitric oxide radicals contributes to some inflammatory diseases. Ethanol extract and petroleum ether extract were exhibited the higher nitric oxide scavenging activity among the five extracts (Figure 1). The IC50 values of ethanol and petroleum ether extracts are 11.87μg/mL and 13.47μg/mL respectively (Table 3).
The scavenging activity of 2,2,-azinobis (3-ethylbenzoline-6-sulfonic acid (ABTS+) assay was measured with the comparison of quercetin standard reference. Among the extracts, petroleum ether, ethanol and chloroform extracts were exhibited better inhibition compared to quercetin standard (Figure 1). Petroleum ether extract exhibited 33%, 38%, 53, 64%, 71% and 72% of scavenging activity and ethanol extract had shown 3%, 8%, 15%, 23%, 34% and 77% at 10, 20, 40, 60, 80 and 100 μg/mL concentrations respectively. The IC50 values (Table 3) of quercetin petroleum ether, chloroform and ethanol was found to be 16.66 μg/mL, 16.0 μg/mL, 17.08 μg/mL and 11.6μg/mL respectively.
Constant generation of free radicals occurs in living system and it causes extensive damages to the cells, tissues and organs which may lead to various disease statuses, especially degenerative disorders and inflammatory diseases. Antioxidants offer resistance to cells and prevent the diseases through scavenging the free radical molecules, by inhibiting lipid peroxidations and many other mechanisms.18 Numbers of plant constituents had been proven for free radical scavenging or antioxidant activities, among phenolic compounds are very important plant constituents. Total phenolic compounds of many plants and antioxidant activity has positive relationship due to the presence of hydroxyl group which has a scavenging ability.19 By donating an electron a phenolic compound can also scavenge the hydrogen peroxide and neutralize it to water.20
Researchers continuously exploit the medicinal plants and their extracts in order to discover the potential drugs with reduced toxicity. In this context we have reported the significant in vitro antioxidant activity of various solvent extracts of L. aspera leaves. The various antioxidant activities of solvent extracts such as petroleum ether, ethyl acetate, ethanol, chloroform and isopropyl alcohol extracts from the leaves of L. aspera were tested and the results are summarized in Table 3.
The DPPH scavenging activity of the extracts evaluated against the positive control ascorbic acid and the DPPH reduction is directly proportional to the antioxidant content in the extract. Higher the antioxidant contents produced higher DPPH reduction. Petroleum ether extract exhibited the maximum DPPH scavenging with the value of 92% at a concentration of 100 μg/ml with IC50 value of 18.96 μg/ml and it showed the antioxidant activity in a concentration dependent manner (20 to 92 %) in the DPPH assay. The scavenging activity of L. aspera leaf extracts are in the order of petroleum ether > ethanol > isopropyl alcohol > ethyl acetate > chloroform. L. aspera leaf extracts showed the potential scavenging of DPPH radicals similar to that of ascorbic acid, it’s likely due to the proton-donating ability of L. aspera leaf and stabilizes the free radicals in association with a number of hydroxyl groups.21 The result of this study suggests that these crude extracts of L. aspera leaves contains phytochemicals that are capable of donating hydrogen to a free radical.
The total antioxidant capacity of isopropyl alcohol extract was found to be higher (82% at 100 μg/mL) when compared to other solvents used. The total antioxidant capacity of different solvent extracts of leaves of L. aspera at various concentrations (10–100 μg/ml) was in the following order: isopropyl alcohol > petroleum ether > ethanol > ethyl acetate > chloroform.
Nitric oxide is a free radical produced from mammalian cells and it involved in various physiological processes. The excess production of nitric oxide is directly toxic to the cells and associated to septic shock, vascular collapse, arthritis and diabetes mellitus.22 Nitric oxide is generated from sodium nitroprusside and it produces nitrite when reacts with oxygen. This crude extracts of L. aspera leaves scavenge the free nitric oxides in the medium and prevent the formation of nitrite. Crude extract of petroleum ether exhibited higher nitric oxide scavenging at the concentration of 100μg/mL (IC50 = 11.87) and the order of nitric oxide scavenging activity of various solvent extracts of L. aspera leaf was found to be petroleum ether > ethyl alcohol > chloroform > ethyl acetate > isopropyl alcohol.
ABTS assay is a decolourization method, the ABTS oxidant generated from 2,2,-azinobis (3-ethylbenzoline-6-sulfonic acid) by persulfate oxidation. The antioxidant activity of extracts of leaves of L. aspera in this method is due to either inhibiting or scavenging the radicals. Because both the inhibition and scavenging activity of ABTS++ was reported earlier.23 The order of antioxidant potency of L. aspera leaf extract in our study was found to be, ethanol > petroleum ether > chloroform > ethyl acetate > isopropyl alcohol.
The data of our present study revealed that Leucas aspera (Willd.) Link leaves have significant antioxidant potential and scavenging activity. Petroleum ether, ethanol and isopropyl alcohol extracts of Leucas aspera (Willd.) Link leaves showed better DPPH scavenging activity. These could be due the presence of flavonoids, phenolic compounds, tannins, saponins and alkaloids. Similarly isopropyl alcohol (polar solvent), ethanol (polar solvent) and petroleum ether (non-polar solvent) exhibited better ABTS scavenging activity and total antioxidant activity compared to chloroform and ethyl acetate extracts. From our study results we suggest, ethyl alcohol or isopropyl alcohol (polar solvent selection) and petroleum ether (non-polar solvent selection) for the better extraction of phytochemicals from plants. This in vitro antioxidant study indicates Leucas aspera (Willd.) Link is a significant natural source of antioxidants and may be helpful in preventing the oxidative stress and damages to cells. Our present study warrants further research to isolation, characterization and in vivo experiments on animal models to assess the potency and safety before the clinical use.
Petroleum ether, ethanol and isopropyl alcohol extracts of L. aspera leaves showed better DPPH scavenging activity.
Isopropyl alcohol, ethanol and petroleum ether exhibited better ABTS scavenging activity and total antioxidant activity.
This in vitro antioxidant study indicates Leucas aspera (Willd.) Link is a significant natural source of antioxidants and may be helpful in preventing the oxidative stress and damages to cells.