Abstract: Background and Objective: Free radicals accumulation in the body causes many oxidative stress related diseases like arthritis, diabetes, inflammation, cancer and genotoxicity. The objectives of present study were to assess total phenolic and flavonoid content and to evaluate in vitro antioxidant activity of extracts of Hamelia patens stems. Methodology: Total phenolic and flavonoid content of the H. patens Jacq. stem extracts were determined by using the Folin-Ciocalteu method and aluminum chloride method, respectively. Antioxidant activities were performed by DPPH free radical, nitric oxide, hydrogen peroxide scavenging and metal chelating assay. Results: The total phenolic content of the petroleum ether extract (PHP), chloroform extract (CHP) and methanol extract (MHP) was 19.083±1.12, 30.58±1.28 and 99.25±1.39 mg gallic acid equivalent per gram of dried extract, respectively. The total flavonoid PHP, CHP and MHP was 8.47±0.67, 15.09±1.21 and 43.42±1.41 mg rutin equivalent per gram of dried extract, respectively. All extracts exhibited scavenging effect in concentration dependent manner. Chloroform extract showed good in vitro antioxidant activity as compared to other extracts. Antioxidant activities of the extracts were associated due to the total phenolic and flavonoid contents of the extract of H. patens. Conclusion: The present study showed that H. patens stems possess rich amounts of natural antioxidants and can be further explored for their possible use as a natural additive in food or in pharmaceutical products. These extracts can be regarded as a promising candidate for a plant derived antioxidant agent.
INTRODUCTION
The enduring increasing discovery of curative plants is owing to numerous reasons, including rising reliance in herbal drug. Allopathic medicine may alleviate a broad range of diseases although, it’s soaring prices and side-effects are causing community to come back to herbal medicines which have less side effects1.
For the majority of the developing countries, the major concern of community health is still the keen demand for crucial health care, which is miserably missing yet at the majority elementary level. This is factual in both the rapidly emerging cities and in the countryside areas. As per World Health Organization (WHO) 50% world’s population do not have access to satisfactory health care services2. This is due to the authenticity that poor people neither have access to nor can afford the current health care services. Therefore, pioneering alternative approaches are needed to address this problem. Traditional medicinal plants offer alternative medicines with incredible opportunities. They not only provide access and affordable medicine to poor people but also they generate income, employment and foreign trade for developing countries. Many traditional medicinal herbs have been shown to have medicinal value, especially in the rural areas and that these can be used to prevent, alleviate or cure several human diseases. As per WHO more than 80% of the world’s population rely either solely or largely on traditional remedies for health care3.
Molecular oxygen is an vital component for all living organisms, where it facilitate in the process of oxidation which is a basic component of aerobic life and of our metabolism4. A part of the oxygen taken into living cells may be converted to several harmful Reactive Oxygen Species (ROS) and free radicals. The formed free radicals can start a chain reaction, leading to the formation of more free radicals5. The ROS which consist of free radicals such as superoxide anion (
Plants contains free radical scavenging molecules, such as phenolic, terpenoids, vitamins, lignins, stilbenes, tannins, flavonoids, quinones, betalains, alkaloids, coumarins and other secondary metabolites which shows good antioxidant activity9.
Hamelia patens Jacq. (Rubiaceae) is commonly known as firebush or scarlet bush. It is a large perennial shrub or small tree generally grown as ornamental plant. Also, the plants are used in folk medicine against a range of ailments. Scarlet bush is used in herbal medicine to treat athlete’s foot, skin lesions and insect bites, nervous shock, rheumatism, inflammation, headache, dysentery and asthma10. Aerial parts of H. patens contain oxindole alkaloids11, flavanone glycoside12,13 while, leaves contain ephedrine14. Leaves shows anti-inflammatory activity15, anti-bacterial activity16, antioxidant activity17, cytotoxic activity18, antinociceptive effects19 and aerial parts shows wound healing activity20. Leaf, stem and root of H. patens shows anthelmintic21 while, bark has antibacterial and antifungal properties21,22.
From literature review it reveals that, antioxidant activity of H. patens is not reported. Hence, the recent trend to find naturally occurring antioxidants, this study was designed to investigate antioxidant potential and to determine total phenolic and flavonoid contents of extracts of H. patens.
MATERIALS AND METHODS
Plant material: Hamelia patens stems were collected from Nashik district in May, 2014, identified by Dr. S.G. Kotwal HOD, Department of Botany, KTHM College, Nashik and authentified by A. Benniamin, Scientist and HOD, Botanical Survey of India, Pune and herbarium specimen deposited as vou. no. FSC-1.
Chemicals: All chemicals used in the study were of analytical grade. Chemicals used in this study were DPPH obtained from Sigma-Aldrich, India, NADH and sulfanilamide obtained from Himedia, Laboratory Pvt. Ltd., India, Ascorbic acid, tocopherol, ferrozine obtained from qualigens fine chemicals, N-1-naphthylethylenediamine dihydrochloride, sodium nitroprusside, sodium nitrite, ferrous chloride Sd fine chemicals Ltd., India.
Preparation of extracts: The plant materials were air dried under shade, pulverized and successively extracted with petroleum ether (60-80°C), chloroform and methanol in Soxhlet apparatus. The petroleum ether extract (PHP), chloroform extract (CHP) and methanol extract (MHP) obtained were dried in vacuum dryer at 40°C.
Preliminary phytochemical tests: The dried extracts subjected for various preliminary screening of phytochemicals such as glycosides (General test), alkaloids (Dragendroff’s test, Mayer’s reagent, Wagner’s reagent, Hager’s reagent), flavonoids (Shinoda and NaOH tests), saponins (foam and haemolysis tests), sterols (Liberman-Burchard and Salkowski tests), tannins (Ferric chloride test, matchstick test, gelatin test), carotenoids (Carr-price test), iridoidal glycosides (Trim-Hill test), coumarin glycosides (Ammonia and alkali test), cyanogenetic glycosides (Grignard’s test), naphthaquinones (Juglone test), anthraquinones (Modified Brontager’s test), proteins (Biuret test) and carbohydrates (Moilsch’s test).
Total phenolic content: The phenolic content was determined according to the method described by Spanos with slight modifications23. The assay was determined using 1 mL of each extract stock solution (1 mg mL–1) and 1 mL of each standard gallic acid solution was taken in 25 mL volumetric flask, added 10 mL of water, 1.5 mL of Folin-Ciocalteu reagent and allowed to stand for 10 min then 4 mL of sodium carbonate solution was added in each volumetric flask and volume was adjusted with distilled water. Absorbance were measured after 1 h at 765 nm by UV visible spectrophotometer against blank24. The total phenolic content was calculated from the calibration curve of gallic acid and the results were expressed in mg gallic acid equivalent per gram of dried extract.
Total flavonoid content: The flavonoids content was determined according to the method described by Kumaran and Karunakaran with slight modifications25. Total flavonoid content was determined using 0.50 mL of each extract stock solution (1 mg mL–1) and each dilution of standard rutin solution (10-100 μg mL–1) taken separately in test tubes. To each test tube 1.50 mL methanol, 0.10 mL aluminium chloride solution, 0.10 mL potassium acetate solution and 2.80 mL distilled water were added and shaked. Sample blank for all extract and all the dilution of standard rutin and were prepared in similar manner by replacing aluminium chloride solution with distilled water. All the prepared solutions were filtered through Whatmann filter paper No. 1 before measuring their absorbance. Absorbance was measured at 510 nm against the suitable blank. From a calibration curve of rutin, the total flavonoid content was calculated and the result was expressed in mg rutin equivalent per gram dry weight extract.
Antioxidant method
Nitric oxide scavenging assay: Aqueous sodium nitroprusside solution at physiological pH, instinctively produce nitric oxide. This nitric oxide reacts with oxygen to produce nitrite ions, which determined by Griess Illosvoy reaction. Griess Illosvoy reagent was slightly modified instead of 1-naphthylamine (5%) naphthylethylenediamine dihydrochloride (0.1% w/v) was used. The nitric oxide scavengers compete with oxygen and reduce the production of nitric oxide26. The reaction mixture contained 2 mL of 10 mM sodium nitroprusside, 0.5 mL of phosphate buffer in saline (pH 7.4, 0.01 M) and 0.5 mL of extract was incubated for 150 min at 25°C. After 150 min 0.5 mL of the reaction mixture was pipetted and mixed with 1 mL of sulphanilic acid reagent (0.33% in 20% glacial acetic acid) and allowed to stand for 5 min for completing diazotization reaction. Then, 1 mL of 0.1% naphthylethylenediamine dihydrochloride was added and allowed to stand for 30 min in dark place27. The absorbance of the pink coloured chromophore was measured at 540 nm by UV visible spectrophotometer against the blank solutions. Percentage inhibition was calculated by using following formula:
where, Acontrol is the absorbance of the control and Atest is the absorbance in the presence of the sample of extract and standard. Plotted the graph percentage inhibition vs concentration and calculated the IC50.
Hydrogen peroxide scavenging assay: The capacity of the extracts to scavenge H2O2 was determined according to Halliwell et al.28. A solution of 40mM hydrogen peroxide was prepared in phosphate buffer solution (pH 7.4). Extracts (25-125 μg mL–1) in methanol were added to a test tube contained 0.6 mL hydrogen peroxide solution. The concentration of hydrogen peroxide wasdetermined by absorbance taken at 230 nm using UV visible spectrophotometer after 10 min against a blank solution containing phosphate buffer without hydrogen peroxide. Percentage inhibition and IC50 calculated similar as given in nitric oxide scavenging activity29.
DPPH free radical scavenging assay: The DPPH scavenging potential of different extracts of H. patens was measured based on scavenging ability of stable 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radicals. The method modified by Brand-Williams was employed to perform the free radical scavenging activity30. Freshly prepared 2 mL DPPH solution (33 mg L–1) was mixed with 2 mL of different dilution of H. patens extracts. The reaction mixture was stand for 15 min at room temperature in dark place. Absorbance of the resultant mixture was recorded at 517 nm using UV visible spectrophotometer26. The percentage of DPPH scavenging by the extracts and standard compounds were calculated as given in nitric oxide scavenging activity.
Metal chelating assay: The chelating activity of the H. patens extracts toward ferrous ions was studied by the method of Dinis et al.31 with minor modification. Different dilutions of each extracts in methanol (25-125 μg mL–1) were prepared from the dried extracts. Take 1 mL of each dilution of extracts, 0.1 mL of FeCl2 (2 mM) was added and the reaction was initiated by the addition 0.2 mL of ferrozine (5 mM), the mixture was shaken vigourously for 10 sec. The mixture was stand at room temperature for 10 min and the absorbance was determined at 562 nm. Methanol without test sample was used as a control and methanol without ferrozine mixture was used as a sample blank. The EDTA was used as a standard for the assay32.
Chelating activity was expressed as IC50, the concentration that chelates 50% of Fe2+ were calculated as follows:
RESULTS AND DISCUSSION
Phytochemical investigations: The preliminary phytochemical investigations was studied to explore its type of chemical component, the study reveals the presence sterols and triterpenoids in pet-ether extract and chloroform extract showed the presence of alkaloids, glycoside, sterols and triterpenoids. Methanol extract showed the presence of alkaloids, sterols, glycosides, flavonoids, tannins, proteins and carbohydrate.
Total phenolic content: The phenolic compounds are main source of antioxidants and free radical scavengers hence, there should be a close correlation between the phenolic content and antioxidant activity. It was reasonable to determine the total phenolic content in the plant extract. The total phenolic content of extracts were determined from regression equations for the calibration curves of gallic acid (y = 0.004x, R2 = 0.992). The TP content of the PHP, CHP and MHP are listed in Table 1.
Total flavonoid content: Total flavonoids content of the crude extract of H. patens was determined using the aluminium chloride colorimetric method. The aluminium chloride forms acid stable complexes with the C-4 keto group and either the C-3 or C-5 hydroxyl group of flavonols and flavones33. For building the calibration curve, rutin is used as standard materials. The TFP of extracts were determined from regression equations for the calibration curve of rutin (y = 0.007x, R2 = 0.996). The FP content of the PHP, CHP and MHP are listed in Table 1.
Antioxidant activity
Nitric oxide scavenging activity: Nitric Oxide (NO) is an important pleiotropic mediator generated by endothelial cells, macrophages, neurons, etc. Nitric oxide is involved in the regulation of various physiological processes such as smooth muscle relaxant, neuronal signaling, regulation of cell mediated toxicity and inhibition of platelet aggregation. Excess concentration of NO is associated with many diseases34,35. These compounds are responsible for changing the structural and functional behavior of several cellular components. Incubation of sodium nitroprusside solutions at 25°C for 150 min resulted in linear time dependent nitrite production which is reduced by the extracts of H. patens. In order to evaluate the antioxidant potency through NO scavenging by extracts, the concentraction NO was monitored. The NO scavenging capacity is determined by the decrease in the absorbance at 550 nm, induced by antioxidants36. Figure 1a shows the comparative NO scavenging activity of the extract. Nitric oxide inhibitors have been shown to have beneficial effects on some aspect of inflammation and tissue damage seen in inflammatory diseases37. Extracts of H. patens significantly inhibited nitric oxide in a concentration-dependent manner.
| Table 1: | Total phenolic and flavonoid content of different extracts of H. patens |
Values represent Mean±Standard Deviation (n = 3), PHP: Petroleum ether extract, CHP: Chloroform extract and MHP: Methanol extract | |
| Fig. 1(a-d): | Antioxidant activity of Hamelia patens extracts at different concentrations. Each value represents mean (n = 3), (a) Nitric oxide free radical scavenging activity, (b) Hydrogen peroxide scavenging activity, (c) DPPH free radical scavenging and (d) Ferrous ion chelating activity |
| Fig. 2: | Antioxidant profile (IC50 values) of different extract of H. patens for nitric oxide free radical scavenging activity, hydrogen peroxide scavenging activity, DPPH free radical scavenging and ferrous ion chelating activity |
Petroleum ether, chloroform, methanol extracts of Hamelia patens stems and ascorbic acid were found to be scavenger of nitric oxide free radical with an IC50 of 219.97, 61.33, 94.57 and 12.88 μg mL–1, respectively. Chloroform extract was found to be good scavenger of Nitric oxide free radical than methanol extract and Petroleum ether extract. Comparison of IC50 value of extracts and standards are given in Fig. 2. The result indicated that the extract contain compounds able to inhibit nitric oxide free radical.
Hydrogen peroxide scavenging assay: Scavenging of H2O2 by extracts may be attributed to their phenolics and flavonoids which can donate electrons to H2O2, thus neutralizing it to water38. The ability of the extracts to effectively scavenge hydrogen peroxide, determined according to the method of Gulcin, where they are compared with that of tocopherol as standard. The extracts were capable of scavenging hydrogen peroxide in a concentration-dependent manner (Fig. 1b). Petroleum ether, chloroform, methanol extracts of Hamelia patens stem and tocopherol were found to be scavenger of H2O2 with an IC50 of 172.54, 66.09, 93.51 and 11.58 mg mL–1, respectively. Chloroform extract was found to be good scavenger of H2O2 than methanol extract and petroleum ether extract. Although, hydrogen peroxide itself is not very reactive but it produces hydroxyl radicals in the cell and cause cytotoxicity. Thus, removing H2O2 is very important throughout food systems39.
DPPH radical-scavenging activity: The model of scavenging the stable DPPH radical is a widely used method to perform the free radical scavenging ability of test components40. The DPPH is a nitrogen-centered a stable free radical with a maximum absorption at 517 nm. The DPPH can readily undergo scavenging by an antioxidant by hydrogen or electron-donation. The DPPH changes from violet to yellow upon reduction. Substances which are able to perform this reaction can be considered as antioxidants41. Extracts of H. patens significantly shows radical-scavenging activities in a concentration-dependent manner (Fig. 1c). Petroleum ether, chloroform, methanol extracts of Hamelia patens stems and tocopherol were found to be scavenger of DPPH radical with an IC50 of 250.58, 46.03, 83.44 and 16.07 mg mL–1, respectively. Chloroform extract was found to be good scavenger of DPPH radical than methanol extract and petroleum ether extract.
Metal chelating activity: Ferrous ions are one of the most effective prooxidants they interact with hydrogen peroxide in biological systems can lead to formation of highly reactive hydroxyl radicals. Ferrozine is a ferroin compound and forms complex with ferrous ions. In the presence of chelating agents, complex (magenta colored) formation is interrupted and as a result, absorbance of the complex is decreased. Thus, the chelating effect of the coexisting chelator can be determined by measuring the rate of color reduction42. The formation of the ferrozine-ferrous complex is interrupted by the H. patens extract, indicating ferrous chelating activity with concentration dependent manner (Fig. 1d). Chelating agents that forms bonds with a metal are effective as secondary antioxidants because they reduce the redox potential and thereby stabilize the oxidized form of the metal ion. Petroleum ether, chloroform, methanol extracts of Hamelia patens stems and EDTA were found to be ferrous ion chelating activity with an IC50 of 294.12, 126.90, 112.36, 19.63 μg mL–1, respectively. Chloroform extract was found to be good scavenger of DPPH radical than methanol extract and petroleum ether extract.
Phytochemicals have been of enormous awareness as a supply of natural antioxidants used for health support, food safeguarding, food flavoring and cosmetics as they are safer than synthetics43.
The antioxidant activities of various extracts of Hamelia patens stems are in accordance with their amount of phenolic contents. At present there has been an improved interest globally to identify antioxidant compounds from plant sources which are pharmacologically potent and have small or no side effects for use in protective medicine and the food industry. Increasing acquaintance in antioxidant phytoconstituents and including them in daily uses and diet can give enough support to human body to fight those diseases. Phytochemical analysis reveals the presence of tannins, flavonoids, steroids and alkaloids. This study affirms the in vitro antioxidant potential of crude methanol, chloroform and petroleum ether extracts of the Hamelia patens stems, with results comparable to those of the standard compounds such as ascorbic acid. This assay is very useful and inexpensive way of assessing the bioactivity of plant extracts. These extracts can be regarded as a promising candidate for a plant derived antioxidant agent.
CONCLUSION
This study affirmed that H. patens extracts is a potential source of antioxidant and could be used in preservative in food, medicine and other non-food material as a natural antioxidant. Further phytochemical analysis is required to isolate the phytoconstitents from the H. patens that shows pharmacological activity related to diseases resulting from oxidative stress.
ACKNOWLEDGMENT
The authors are grateful to Principal, SMBT College of Pharmacy for providing necessary facilities and support.