Abstract: Objective: The objective of this study was to evaluate in vitro antioxidant, antimicrobial activity and characterize the chemical constituents of leaves and seeds of Abutilon indicum by using gas chromatography-mass spectrometry (GC-MS). In addition total phenolics and flavanoids were also estimated. Materials and Methods: The GC-MS analysis of plant and seed extracts was performed using a GC-Brucker 436 system and interpretation on mass spectrum GC-MS was conducted using the database of National Institute Standard and Technology (NIST). Antioxidant activity was determined in the 1,1-diphenyl-2-picrylhydrazyl (DPPH) method. The total flavonoids assay was conducted by aluminium chloride colorimetric method and otal phenolics content was determined according to the Folin-Ciocalteu method. Antibacterial activity and minimum inhibitory concentrations were calculated by the agar diffusion method. Results: Twenty five chemical compounds are identified from the plant extract and 29 compounds identified from seed extract by GC-MS analysis. The leaf and seed extracts showed significant DPPH scavenging activity (IC50 values are 48.94±3.54 and 49.89±3.29 μg mL–1) compared with the values obtained for ascorbic acid standard (IC50 value 39.22±5.50). The leaf and seed extracts prevented the growth of both Gram-positive and Gram-negative bacteria. The antimicrobial and antioxidant activities of the extracts were positively associated with the total phenolic and flavonoid contents of the extract. Conclusion:The presence of various bioactive compounds justifies the use of Abutilon indicum for various ailments by traditional practitioners.
INTRODUCTION
Reactive oxygen species, such as singlet oxygen, superoxide ion, hydroxyl ion and hydrogen peroxide are highly reactive, toxic molecules which are generated normally in cells during metabolism. They cause severe oxidative damage to proteins, lipids, enzymes and DNA by covalent binding and lipid per oxidation with subsequent tissue injury. Natural antioxidant agents have attracted much interest because of their ability to scavenge free radicals1. Free radicals have been implicated in the development of a number of disorders, including cancer, neurodegeneration and inflammation2, giving rise to studies of antioxidants for the prevention and treatment of diseases. The presence of antioxidants such as phenolics, flavonoids, tannins and proanthocyanidins in plants may provide protection against a number of diseases for example, ingestion of natural antioxidants has been inversely associated with morbidity and mortality from degenerative disorders3. Medicinal plants are therefore being investigated for their antioxidant properties and the demand for natural antioxidants and food preservatives is increasing4.
Abutilon indicum belongs to the family Malvaceae and distribute in all parts of tropical and sub tropical region of India. All parts of the plant have been recognized to have medicinal properties. The plant is commonly called as ‘Thuthi’ in Tamil Nadu. The traditional method of medicine, the plant used as anthelmintic, anti-inflammatory and is useful in urinary and uterine discharges, piles and lumbago5, jaundice, ulcer and leprosy. Abutilon indicum leaves are used in the treatment of toothache, lumbago, piles, anti-fertility and liver disorders. Although, the plant is widely used in traditional medicine, few studies have been conducted of the pharmacological activities of the plant6. Phenolic and flavonoid compounds are widespread in plant kingdom where they act as antioxidants and free radical scavengers7. The objective of this study was to determine the total phenolic and flavonoid content and the antioxidant and antimicrobial activity of a methanol extract of leaves and seeds of Abutilon indicum.
MATERIALS AND METHODS
Chemicals: Quercetin, 1,1-diphenyl-2-picrylhydrazyl (DPPH) and gallic acid were purchased from Sigma-Aldrich, USA. Folin-Ciocalteu reagent was obtained from Merck, Germany. All other chemicals and solvents used in the study were of analytical grade procured locally.
Collection and processing of plant material: The leaves and seeds of the plant Abutilon indicum collected from Siluvinippatti village, Sivagangai district, Tamil Nadu, India in the month of July, 2016 and authenticated by Dr. John Britto, Rapinat Herbarium, St., Joseph’s College, Tiruchirappalli, Tamil Nadu, India. The leaves were cleaned and shade dried for a week and grounded into uniform powder. One gram of plant material was added to 20 mL of aqueous methanol (20%, v/v) for 18 h at room temperature. The extracts were filtered and used for the estimation of total phenols, total flavanoids contents and antioxidant activity.
Total phenols determination: The amounts of total phenolic contents of plant were determined by the spectrophotometric method of Kim et al.8 with slight modification. A diluted plant sample (1 mL) was added to a 25 mL volumetric flask, containing 9 mL of distilled water. One milliliter of Folin-Ciocalteu’s phenol reagent was added to the mixture and shaken. After 5 min, 10 mL of 7% Na2CO3 solution was mixed in to the test sample solution was diluted to 25 mL distilled water and mixed thoroughly. The mixture was kept in the dark for 90 min at 23°C, after which the absorbance was read at 750 nm. Total phenol content was determined from extrapolation of calibration curve which was made by preparing gallic acid solution (20-100 μg mL–1). The estimation of the phenolic compounds was carried out in triplicate. The total phenolic content was expressed as milligrams of gallic acid (GAE) equivalents per gram dried sample.
Total flavonoids determination: The total flavonoids assay was conducted according to Damodar et al.9. Total flavonoids content was determined by using aluminium chloride colorimetric method. Plant sample (0.5 mL) was mixed with 1.5 mL of methanol, 0.1 mL of 10% aluminum chloride, 0.1 mL of 1 M potassium acetate and 2.8 mL of distilled water. It retained at room temperature for 30 min. The absorbance of the reaction mixture was measured at 510 nm using UV-visible spectrophotometer. The calibration curve was prepared by preparing quercetin solutions at concentrations 20-100 μg mL–1 in methanol. The total flavonoids content was expressed as milligrams of quercetin equivalents per gram of dried sample.
GC-MS analysis
Preparation of extract for GC-MS analysis: Leaves and seeds of Abutilon indicum were shade dried. Twenty grams of the powdered samples were soaked in 95% ethanol for 12 h. The extracts were then filtered through Whatmann filter paper No. 41 along with 2 g of anhydrous sodium sulphate to remove the sediments and traces of water in the filtrate. Before filtering, the filter paper along with anhydrous sodium sulphate was wetted with 95% ethanol. The filtrate was then concentrated by bubbling nitrogen gas into the solution. The extract contained both polar and non-polar phytocomponents of the plant material used. Two microliter of these solutions were employed for GC-MS analysis.
GC-MS specification
GC programme: Column BR-5MS (5% diphenyl/95% dimethyl poly siloxane), 30 m×0.25 mm, ID×0.25 μm df, equipment scion 436-GC bruker, carrier gas 1 mL min–1, split 10:1, detector TQ quadrupole mass spectrometer, software MS work station 8, sample injected 2 μL, oven temperature programme: 110°C hold for 3.50 min, upto 200°C at the rate of 10°C min–1, no hold, upto 280°C at the rate of 5°C min–1, 12 min hold, injector temperature 280°C, total GC running time: 40.50 min MS programme: Library used NIST version-11, inlet line temperature 290°C, source temperature 250°C, electron energy 70 eV, mass scan (m/z) 50-500 amu. Solvent delay 0-3.5 min, total MS running time: 40.50 min.
Identification of components: Interpretation on mass spectrum GC-MS was conducted using the database of National Institute Standard and Technology (NIST) having more than 62,000 patterns. The spectrum of the unknown component was compared with the spectrum of the known components stored in the NIST library. The name, molecular weight and structure of the components of the test materials were ascertained.
In vitro antioxidant activity
DPPH radical-scavenging activity: Different concentrations of leaf and seed extracts (20, 40, 60 and 80 μg mL–1) were chosen for in vitro antioxidant activity. The L-ascorbic acid (20, 40, 60 and 80 μg mL–1) was used as the reference standard. The DPPH radical-scavenging activity was determined by the method of Shimada et al.10. Briefly, a 2 mL aliquot of DPPH methanol solution (25 μg mL–1) was added to 0.5 mL sample solution at different concentrations. The mixture was shaken vigorously and allowed to stand at room temperature in the dark for 30 min. Then the absorbance was measured at 517 nm in a spectrophotometer. Lower absorbance of the reaction mixture indicated higher free-radical scavenging activity:
where, AC is control of the absorbance and AS is sample of the absorbance of reaction mixture (in the presence of sample).
Assay of antimicrobial activity: Twenty five grams of plant powder was weighed and macerated in methanol, individually in the ratio of 1:4 (w/v). They were kept at the room temperature for 24 h. Each mixture was stirred every hours using sterile glass rod. Then it was filtered through the Whatmann No. 1 filter paper. The extracting procedure was done further twice for complete extraction of the active compounds. The obtained filtrate was combined together and concentrated using Soxhlet apparatus. The dried residue of respective solvent extract was used for evaluating the antibacterial activity. They were kept in refrigerator until use. Sterile empty antibiotic discs (6 mm diameter) were purchased from Hi-media Company, Mumbai. Twenty milligrams of dried crude extract was dissolved in 1 mL of 20% DMSO (Dimethyl sulphoxide). From this stock solution 10 μL of respective solvent extracts were added to the disc (0.2 mg disc–1) individually and aseptically. Each disc contained 0.2 mg of extract. Then the discs were allowed for drying at room temperature. After drying they were used for screening the antibacterial activity.
Bacteria causing infectious diseases both in animals and humans were used in the present study. They were both Gram positive and Gram negative. Such as Aspergillus niger, Bacillus subtilis and E. coli were used. They were collected from the Microbial Type Culture Collection (MTCC) at Chandigarh, India. Pure cultures of bacterial pathogens in nutrient agar slant was transferred to tryptone broth and incubated at 37°C for 24 h.
Antibacterial assay was carried out by agar diffusion method11. The sterile Muller-Hinton agar plates were prepared. The test organisms were spread over the Muller-Hinton agar plates by using separate sterile cotton swabs. The prepared sterile disc was placed on the surface of the medium at equidistance. Then the plates were incubated at 37°C for 24 h to determine the antibacterial activity of the respective solvent extraction. Gentamicin antibiotic discs (30 mg disc–1) were used as positive control and disc with respective solvents (10 μL) were used as negative control. Each extract was treated in triplicate for calculation of mean value.
Statistical analysis: The results were presented as Mean±SD. Data was statistically analyzed using student "t" test. For the calculation of IC50, linear regression analysis was done using GraphPad prism statistical software.
RESULTS AND DISCUSSION
Phenolic and flavonoid contents: The total phenolic content of the methanolic leaf and seed extract, calculated from the calibration curve (R2 = 0.997) were 24.60±1.68 and 30.20±2.10 mg gallic acid equivalents g–1 and the total flavonoid content (R2 = 0.997) were 148.10±10.30 and 80.9±5.60 mg quercetin equivalents g–1 (Table 1). Phenolic compounds have redox properties, which allow them to act as antioxidants12. As their free radical scavenging ability is facilitated by their hydroxyl groups, the total phenolic concentration could be used as a basis for rapid screening of antioxidant activity. Flavonoids, including flavones, flavanols and condensed tannins are plant secondary metabolites, the antioxidant activity of which depends on the presence of free OH groups, especially 3-OH. Plant flavonoids have antioxidant activity in vitro and also act as antioxidants in vivo13.
Chemical composition by GC-MS analysis: Interpretation on mass spectrum GC-MS was conducted using the database of National Institute Standard and Technology (NIST). The spectrum of the unknown component was compared with the spectrum of the known components stored in the NIST library. In the GC-MS analysis, 25 bio-active phytochemical compounds were identified in the ethanol extract of leaves (Fig. 1, Table 2) and 29 bio-active compounds were identified in the ethanol extract of seeds (Fig. 2, Table 3). Biological activity of some identified components listed in Table 4.
| Table 1: | Quantitative analysis of total phenolic and flavonoids content of leaf and seeds |
| Fig. 1: | GC-MS chromatogram of leaves extract |
| Fig. 2: | GC-MS chromatogram of seeds extract |
| Table 2: | Compounds identified in the leaves of Abutilon indicum |
| Table 3: | Compounds identified in the seeds of Abutilon indicum |
| Table 4: | Biological activity of some identified components |
| *Source: Dr. Duke’s phytochemical and ethnobotanical databases [Online database] | |
Corresponding to the peak at RT 17.66 and peak area 53.32% is phytol. The molecular formula and molecular weight of this compound is C20H40O and 296, respectively. Phytol is known to be antimicrobial, anti-cancer, anti-inflammatory, hepatoprotective and anti-androgenic14. Guanethidine (Peak area 0.32%) is an antihypertensive drug that reduces the release of catecholamines, such as norepinephrine.
| Table 5: | DPPH radical scavenging activity of leaf and seed extract at different concentrations |
| Values were expressed as Mean±SD for triplicate | |
| Table 6: | Assay of antibacterial activity |
Guanethidine is transported across the sympathetic nerve membrane by the same mechanism that transports norepinephrine itself (NET, uptake 1) and uptake is essential for the drug’s action. Once guanethidine has entered the nerve, it is concentrated in transmitter vesicles, where it replaces norepinephrine. It may also inhibit the release of granules by decreasing norepinephrine and was often used safely during pregnancy. Eugenol (4-allyl-2-methoxyphenol), one of these phytochemicals is a biologically active phenolic component of syzigium aromaticum (cloves). Eugenol has been used traditionally in Asian countries, mainly as a medicinal antiseptic, analgesic and antibacterial agent. Eugenol has been used as a flavoring agent in cosmetics and food products and also plays a role in dentistry as cavity filling cement. Eugenol is said to possess various biological properties like antiviral, antioxidant, anti-inflammatory, etc. At low concentrations it usually acts as an antioxidant and anti-inflammatory agent, whereas at higher concentration act as a pro-oxidant causing increased generation of tissue-damaging free radicals. It has been reported to possess anti-genotoxic activity15. The β-vitamin E (RT 32.76) has many biological functions, the antioxidant function being the best known. Vitamin E has also found use as a commercial antioxidant in ultra high molecular weight polyethylene (UHMWPE) used in hip and knee implants to replace faulty joints, to help resist oxidation. Vitamin E also plays a role in neurological functions and inhibition of platelet coagulation. Vitamin E also protects lipids and prevents the oxidation of polyunsaturated fatty acids16. Sitosterol (RT 37.23) reduce carcinogen-induced cancer of the colon. It shows anti-inflammatory, anti-pyretic, anti-arthritic, anti-ulcer, insulin releasing and oestrogenic effects and inhibition of spermatogenesis stigmasterol (RT 35.54) is a plant sterol able to bind to chondrocyte membrane and possesses potential anti-inflammatory, anti-osteoarthritic and anti-catabolicproperties. It is thought to have anti-inflammatory effects. It was demonstrated that it inhibits several pro-inflammatory and matrix degradation mediators typically involved in osteoarthritis-induced cartilage degradation. The β-sitosterol is mainly known and used for its anti-inflammatory cholesterol lowering property, easing symptoms of benign prostatic enlargement, reducing risk of cancer and prevention of oxidative damage through its antioxidant activity17. Results reveals that most of the predominate compounds were identified in leaf and seed extracts are biologically active molecules. Among the identified phytochemicals, 2,3-dihydro benzofuran and guanethidine were identified first time in this plant. These identified active compounds are considered to be plant defence systems. Some of the chemical compounds were isolated and identified in different crude extracts from Abutilon indicum samples those active compounds are currently used as natural antioxidant, antimicrobial agents and in the formulation of different medicines.
Antioxidant activity: The effect of antioxidants on DPPH radical scavenging is thought to be due to their hydrogen-donating ability. The DPPH is a stable free radical and accepts an electron or hydrogen radical to become a stable diamagnetic molecule18. Extracts of plants are allowed to react with the stable radical, DPPH in methanol solution. The reduction capability of DPPH radicals is determined by the decrease in its absorbance at 517 nm, induced by an antioxidant (AH) after 30 min, as follows:
DPPH.+AH→DPPH-H+A
The concentration of antioxidant needed to decrease the initial DPPH concentration by 50% (IC50) is a parameter widely used to measure the antioxidant activity19. A lower IC50 value corresponds with a higher antioxidant power. The leaf and seed extracts showed significant DPPH scavenging activity (IC50 values are 48.94±3.54 and 49.89±3.29 μg mL–1) compared with the values obtained for ascorbic acid standard (IC50 value 39.22±5.50). All results are shown in Fig. 3 and Table 5.
Antimicrobial activity: The antibacterial properties of methanolic extract of the leaves and seeds of Abutilon indicum are presented in Table 6. The extracts had antibacterial activity against both Gram-positive and Gram-negative bacteria with MICs of 10-22 mm in diameter. The leaves extract had the greatest activity against Bacillus subtilis and the least against Aspergillus niger (Fig. 4). The seeds extract had the greatest activity against E. coli and the least against Bacillus subtilis. These results suggest that Abutilon indicum is a potential source of broad-spectrum antimicrobial agents. The antimicrobial activity of the extract may be attributed to the high content of flavonoids, which have been reported to be involved in inhibition of nucleic acid biosynthesis and other metabolic processes20.
The objective of this study is to identify good number of organic chemical compounds in leaf and seed extracts of Abutilon indicum might have some ecological and medicinal benefits. This study shows that the identified chemical might be used as a vital source of natural antioxidant for food and pharmaceutical industry. Results suggest that Abutilon indicum is a potential source of antioxidant and antimicrobial agents and could be used as a natural antioxidant and preservative in food and non-food systems. Further phtyochemical analysis is required to isolate the elements of the plant that show a broad spectrum of pharmacological activity.
| Fig. 3: | DPPH radical scavenging activity of leaf and seed extracts at different concentrations |
| Fig. 4(a-c): | Antimicrobial activity of Abutilon indicum leaf and seed extracts on pathogenic microorganisms, (a) A. niger, (b) B. subtilis and (c) E. coli |
CONCLUSION
The present study provided the potential antimicrobial and antioxidant properties of Abutilon indicum leaf and seed. The radical scavenging activity and other complementary assays are also in good correlation. In the GC-MS analysis, 25 phytochemicals were identified in the leaves extract and 29 phytochemicals were identified in the seeds extract. Moreover, the potency of the constituents such as trans isoeugenol and sterols could provide a chemical basis for some of the health benefits claimed for Abutilon indicum . The present results of this study do not reveal that which chemical compound is responsible for different activity. To the best of our knowledge this is the first report to investigate in vitro antioxidant activity as well as the chemical composition of leaf and seed extracts of Abutilon indicum by GC-MS. Hence, the whole plant could be of use as a good source of antioxidant. Further studies are needed for the isolation and identification of individual compounds from the plant crude extracts Abutilon indicum also in vivo studies are needed for better understanding of their mechanism of action as antioxidant.
ACKNOWLEDGMENT
The authors are grateful to Indian Institute of Crop Processing Technology (IICPT), Thanjavur, Tamil Nadu for providing laboratory facilities for GC-MS analysis.