COMPARATIVE ANALYSIS OF PHYTOCHEMICAL CONSTITUENTS, FREE RADICAL SCAVENGING ACTIVITY AND GC-MS ANALYSIS OF LEAF AND FLOWER EXTRACT OF TITHONIA DIVERSIFOLIA (HEMSL.) A. GRAYHTML Full Text
COMPARATIVE ANALYSIS OF PHYTOCHEMICAL CONSTITUENTS, FREE RADICAL SCAVENGING ACTIVITY AND GC-MS ANALYSIS OF LEAF AND FLOWER EXTRACT OF TITHONIA DIVERSIFOLIA (HEMSL.) A. GRAY
M. S. Roopa *, R. Shubharani, Thejanuo Rhetso and V. Sivaram
Department of Botany, Bangalore University, Bengaluru - 560056, Karnataka, India.
ABSTRACT: The secondary plant metabolites possess therapeutic value, thus play a significant role in human health and general wellbeing. The present study aimed to estimate the secondary metabolite concentration and to evaluate the antioxidant potential of Tithonia diversifolia leaf and flower extract in methanol. The phytochemical compounds in the extracts were characterized through Gas Chromatography-Mass Spectrometry (GC-MS) analysis. The leaf extract exhibited sixteen bioactive compounds, and flower extract revealed thirteen compounds with valuable activity. The major phytocompounds in leaf extract were pentacosane (22.01%), gamma- sitosterol (15.80%) and pentatriacontane (11.95%) whereas in flower extract showed methyl linolelaidate (19.55%), methyl palmitate (18.73%), 1- dotriacontanol (15.35%), 5- eicosene, (E)- (11.90%). The compounds were found to be dissimilar in both leaf and flower. Quantitative phytochemical analysis of leaf extract was found to contain a high concentration of phenols, flavonoids and saponins, whereas total alkaloids were maximum in flower extract. In in-vitro, the antioxidant activity was evaluated using 1, 1-diphenyl-2- picrylhydrazyl (DPPH) assay. Both leaf and flower extract showed remarkable antioxidant scavenging activity with an IC50 value of 120.264 µg/ml and 121.7µg/ml, respectively. The findings of this study provide an insight into the phytochemistry and antioxidant property of methanol extract of leaf and flower of T. diversifolia. Such properties may be of great importance in alleviating the chronic effect of oxidative stress, and it can be recommended as a plant of pharmaceutical importance.
Phytochemical, GC-MS, Tithonia diversifolia, leaf extract, Flower extract, DPPH
INTRODUCTION: Tithonia diversifolia (Hemsl.) a. gray is commonly called the Mexican sunflower, tree marigold, or the Japanese sunflower. It is a subtropical plant belonging to the Asteraceae family. It is native to Mexico and Central America1 and was subsequently introduced in Africa, Australia, and Asia 2.
It is an invasive, woody shrub, reaching to heights of about 2-3 m, and the mature stem bears bright yellow to orange colored flowers and is aromatic. In Mexico and Nigeria, the plant was used by many ethnic groups in their folk medicine, especially stem and leaf extracts of T. diversifolia are taken orally for Malaria 3.
It is also used in the treatment of diabetes, diarrhea, liver disease, and stomach ache in Indonesia. In India, dried leaf powder is used in skin infection and for healing wounds 4. The main active compound isolated from aerial parts of T. diversifolia showed sesquiterpene lactones i.e. tagininins (Tagitinins A, C, E and F), titho-folinolide 5, 6 diversifolin 7 and diversifolide found in roots 8. The predominant constituents in the volatile oil of leaf and flower found to contain α-pinene, β-pinene, β-caryophyllene, germacrene D, (Z)-β-ocimene and limonene 9.
Gas Chromatography-Mass Spectrometry (GC-MS), an analytical technique, is gaining more importance in the screening of phytochemicals. It is the first step to understand the chemical profile of the plant sample. GC-MS has become a highly recommended advanced technology to identify and quantify the secondary metabolites, even at low concentrations 10. As there is no report about the GC-MS analysis of T. diversifolia plant from India, an attempt has been made to quantify the secondary metabolites, antioxidant assay, and identification of chemical constituents of methanol extract of T. diversifolia leaf and flower using GC-MS.
MATERIALS AND METHODS:
Collection of Plant Material and Extraction: Leaves and flowers of T. diversifolia were collected from HMT quarters, Bangalore, Karnataka, India. The plant was authenticated by Dr. V. Rama Rao, Taxonomist, Regional Ayurveda Research Institute for metabolic disorders (Central Council for Research in Ayurvedic Sciences, Ministry of AYUSH, and Government of India), Bengaluru and Karnataka with the authentication number RRCBI- mus 203.
The collected leaves and flowers were shade dried, powdered and extracted separately with absolute methanol using Soxhlet apparatus for 16 h at a temperature not exceeding the boiling point of the solvent. The extracts were filtered and concentrated under reduced pressure at 40 °C using a rotary flash evaporator and stored at 4 °C until further use.
Quantitative Phytochemical Analysis: Quantification of phytochemical analysis was carried out with a focus on comparing secondary metabolites present in leaf and flower extract of T. diversifolia
Determination of Total Phenols by Folin-Ciocalteu Reagent Method: Total phenolic content (TPC) was determined by the folin- ciocalteu reagent method 11. To 1 ml of each extract, 5 ml of (1:10) folin-ciocalteu reagent and 4 ml of Na2CO3 (7.5%) were added. The above solution was incubated for 30 min at 20 °C and absorbance were read at 765 nm. Gallic acid was used as a reference standard (20-100 µg/ml). The TCPs were determined using a linear regression equation obtained from the standard plot of gallic acid. The TPC was calculated as mean SD (n=3) and expressed as mg/g gallic acid equivalent (GAE) of extract.
Estimation of Total Flavonoid Content (TFC) by Aluminium Chloride Method: Quercetin was used as a standard to construct the calibration curve. 0.5 ml of different aliquots of a standard solution of quercetin (20, 40, 60, 80 and 100 µg/ml) was mixed with 2 ml of distilled water, 0.15 ml of 5% sodium nitrite and allowed to stand for 6 min at room temperature later, 0.15 ml of 10% aluminum chloride solution was added. After 6 min of incubation, 2 ml of 4% w/v sodium hydroxide was added, and volume was made up to 5 ml with distilled water. The absorbance of the reaction mixture was measured at 510 nm using a UV-visible spectrophotometer against blank. The amount of flavonoid was calculated from the linear regression equation obtained from the quercetin calibration curve. The flavonoid content was calculated as mean SD (n=3) and expressed as mg/g of quercetin equivalent (QE) of extract12.
Estimation of Alkaloid: Atropine was used as the standard alkaloid to construct the calibration curve. Briefly, 10 mg of atropine was dissolved in methanol and then diluted to 200, 400, 600, 800, and 1000 µg/ml. The diluted standard solution of atropine or plant extracts (1 ml) of different concentrations was separately mixed with 2 ml of 2 N hydrochloric acid and 5 ml of chloroform. Vortex the above solution vigorously and take out the chloroform layer using a micropipette. To the separated chloroform layer, add 5 ml of bro-mocresol green (BCG) solution (7 mg of BCG was dissolved in 3 ml of 2 N sodium hydroxide then the volume was made up to 100 ml with distilled water) and 5 ml of sodium phosphate buffer (Ph = 4.7). The mixture was vortexes for 5 min, and the yellow color complex was formed at the bottom of the test tube. Pipette out the yellow complex, and the absorbance of the reaction mixture was measured at 470 nm in the UV-Vis spectrophotometer against blank. The total alkaloid concentration was calculated from the linear regression equation obtained from the atropine calibration curve 13.
The alkaloid content was calculated as mean SD (n=3) and expressed as mg/g of atropine equivalent (AE) of extract.
Quantitative Determination of Saponins: Different aliquots of standard saponin quillaja (1 mg/ml) were taken in different test tubes, and the volume was made up to 1 mL with absolute methanol in all the test tubes later, 500 µL of 8% vanillin, 500 µL of 72% sulphuric acid was added and incubated at 60 °C for 10 min. After incubation, the absorbance was read at 544 nm using a UV-Vis spectrophotometer. The samples were also processed similarly by taking 1 mL of each sample. The standard graph was plotted, and the amount of saponin in each sample was calculated using the linear regression equation. The total saponin content (TSC) was calculated as mean SD (n=3) and expressed as mg/g of quillaja equivalent (QJE) of extract 14.
Assay of free Radical Scavenging Activity by DPPH Method: The free radical scavenging activity of different concentrations of plant samples of T. diversifolia and standard ascorbic acid was estimated by Vasundhara et al., 2017 method 15. One milliliter of various concentrations (1, 10, 25, 50, 75, 100 µg/ml) of the sample or standard ascorbic acid was taken in a separate test tube. Three milliliters of 1 mmol/L DPPH solution prepared in absolute methanol was added to each test tube. The solvents were mixed and kept in the dark at 37 °C for 15 min to complete the reaction. The blank was prepared without sample or ascorbic acid. The absorbance was read at 517 nm using a UV-Vis spectrophotometer. The percentage of free radical inhibition activity of sample and positive control ascorbic acid was calculated by using the following formula.
Free radical inhibition activity (%) = Ac - As × 100 / Ac
Where Ac - Absorbance of control and As - Absorbance of the sample at 517 nm.
The concentration of the sample required to scavenge 50% of the DPPH free radical (IC50) was determined from the curve of percent inhibitions plotted against the respective concentration.
GC-MS Analysis: The methanolic leaf and flower fraction of T. diversifolia were investigated using Gas Chromatography and Mass Spectrometry (GC-MS) 9. The Shimadzu GC-MS, of model number QP2010S, was used with a silica column rxi-5 sil MS, of length: 30 m; internal diameter: 0.25 mm; thickness: 0.25 μm. Helium gas (99.99%) was used as carrier gas with split-less injection mode having column flow: 1.0 ml/min, pressure; 65.2 kPa, linear velocity: 36.8 cm/sec; purge flow: 3.0 ml/min and split ratio: 50.0 the injector was maintained at a temperature of 260.0 °C and column oven temperature, at 80.0 °C. The GC instrument was operated at an ion source temperature of 200 °C, with an interface temperature of 280.0 °C and a solvent cut time of 6.50 min. The mass spectrometer was operated from 7.00 min to 50.00 min with an event time of 0.05 sec, and fragments from m/z 50.00 to 500.00 were programmed. The area percentage of each chemical compound was determined by comparing its average peak area to the total area. The software used to run mass spectra, and the chromatogram was GC-MS solution. The spectrum of unknown components was compared with the known components, stored in the database of the National Institute of Standards and Technology (NIST) 11 and WILEY 8. The identification of the phytochemical compounds was based on the peak area, retention time, and molecular formula.
Data Analysis: Analysis of the experimental data was performed in triplicate and expressed as mean ± sem. Statistical one-way ANOVA was calculated using the software tool graph pad prism 5.01. Differences were considered statistically significant P<0.05.
RESULTS AND DISCUSSION: Since the dawn of civilization, plants are used as the main source of medicine in curing a wide range of ailments in humans and animals 16. Today, many pharmaceutical industries depended directly or indirectly on the floral kingdom to produce an effective drug. Thus, extraction and analysis of plant material play an important role in the development of qualitative herbal formulation 17.
In the aforementioned study, quantification of the phytochemical compounds in the leaf and flower extract of Tithonia diversifolia in methanol was carried out Table 1 illustrates that leaf extract contained a higher concentration of phenols, flavonoids, alkaloids, and saponins compared to flower extract. A significant difference in the total phenols, total flavonoids, total alkaloids, and total saponins was observed by ANOVA one-way test.
TABLE 1: LINEAR REGRESSION EQUATION OF STANDARD OF TOTAL PHENOLICS, TOTAL FLAVONOIDS, TOTAL ALKALOIDS AND TOTAL SAPONINS CONTENT IN METHANOL EXTRACT OF LEAF AND FLOWER OF T. DIVERSIFOLIA. VALUES ARE MEAN ± SEM; P < 0.05.
|Qualitative analysis||Linear regression equation of standard||Leaf extract||Flower extract|
|Total phenolics (mg/g)||Y = 0.0138 × + 0.0244, R2 = 0.999(Gallic acid)||48.304 ± 2.765||34.101 ± 1.999|
|Total flavonoids (mg/g)||Y = 0.0029 × + 0.0033, R2 = 0.999 (Quercetin)||24.78 ± 0.172||21.9 ± 1.0456|
|Total alkaloids (mg/g)||Y = 0.0003 × + 0.0008, R2 = 0.999 (Atropine)||43.33 ± 0.577||88.333 ± 0.192|
|Total saponin (mg/g)||Y = 0.0002 × + 0.0031, R2 = 0.999 (Quillaja)||694.5 ± 1.528||304.5 ± 2.082|
Where y is the absorbance and x is the concentration of the standard in µg/ml
The current findings support the study of Olutobi and Olasupo 18, which confirms the presence of phytochemicals such as alkaloids, flavonoids, phenols, saponins, tannins, and terpenoids in methanol extract of T. diversifolia leaves. However, ethanol extract of dried inflorescence collected from Brazil showed the presence of phenol, flavonoids, and tannins 19 similar results were obtained by Essiett and Akpan 20 only differing in the presence of saponins. The variation in the phytochemical composition of T. diversifolia may depend on the environmental changes, climatic factors, and geographical distribution 21. Phenolic compounds can scavenge free radicals and thus gained importance in pharmaceutical, nutraceutical, and herbal industries. Free radicals are the reactive oxygen species created in the body during normal metabolism or introduced from the environment 22. An imbalance between free radicals and antioxidants in the body leads to oxidative stress involved in the development of chronic diseases such as Parkinson's disease, Huntington's disease, dementia, heart failure, autism, cancer, atherosclerosis, aging-related diseases, etc. 23, 24. Table 2 and Fig. 1 summarize the free radical scavenging activity of leaf and flower extract of T. diversifolia compared to a standard (Ascorbic acid).
TABLE 2: PERCENTAGE INHIBITION OF METHANOL EXTRACT OF LEAF AND FLOWER OF T. DIVERSIFOLIA AND STANDARD ASCORBIC ACID AT VARIOUS CONCENTRATIONS (µG/ML) IN THE DPPH SCAVENGING MODEL VALUES ARE MEAN % INHIBITION ± SEM ; P < 0.0
|Concentration (µg/ml)||Ascorbic acid||Leaf extract||Flower extract|
|10||15.41 ± 0.203||36.96 ± 0.77||13.62 ± 0.400|
|25||29.49 ± 0.282||38.03 ± 0.776||20.27 ± 0.306|
|50||49.22 ± 0.147||40.20 ± 0.325||26.40 ± 0.573|
|75||62.26 ± 0.144||43.99 ± 0.284||38.30 ± 0.553|
|100||83.26 ± 0.144||48.20 ± 0.037||41.18 ± 0.895|
FIG. 1: DPPH SCAVENGING ACTIVITY OF STANDARD ASCORBIC ACID, METHANOL EXTRACT OF LEAF AND FLOWER OF T. DIVERSIFOLIA
The half-maximal inhibitory concentration (IC50) of DPPH was calculated under the experimental condition. The leaf extract was found to have a slightly lesser IC50 value (120.264 µg/ml) than flower extract (121.7 µg/ml) and ascorbic acid was 54.839 µg/ml. Both the extracts showed less antioxidant property when compared to standard ascorbic acid. Lesser the IC50 value stronger the scavenging activity. Scavenging activity of the standard ranged from 15.41% at 10 µg/ml concentrations to 83.26% µg/ml at 100 µg/ml concentrations. Leaf extract was able to scavenge 48.20% at 100 µg/ml concentration, whereas flower extract depicted 41.18% at 100 µg/ml concentration. Studies have shown that T. diversifolia leaves were used in folkloric medicine of Africa to treat neurodegenerative diseases, and scientific data proves that it has antioxidant and cholinesterase inhibitory activity 25. Thus, the present study signifies that both leaf and flower extract of T. diversifolia has the notable effect of free radical scavenging activity. GC-MS analysis of leaf extract of T. diversifolia showed sixteen bioactive compounds Fig. 2 and Table 3.
TABLE 3: THE PHYTOCOMPOUNDS OBSERVED IN THE METHANOL LEAF EXTRACT OF T. DIVERSIFOLIA
The major phytocompounds were pentacosane (22.01%), gamma-sitosterol (15.80%) and penta-triacontane (11.95%) and minor phytocompounds were tridecyl acrylate (4.85%), neophytadiene (4.18%), phytol, acetate (1.32%), 17-penta-riacontene (1.6%), tetracontane (5.22%)-methyldocosane (7.89%), nerolidol a (cis or trans) (4.51%), dodecyl palmitate (1.6%), penta-triacontane (1.77%), behenyl chloride (3.32%), 2-methyltetracosane (4.6%), stigmasta-5,22-dien-3-ol (6.01%) and β-sitosterol (3.36%). Phytocompounds identified from the flower extract revealed thirteen phytochemicals Fig. 3 and Table 4. The major compounds were methyl linolelaidate (19.55%), methyl palmitate (18.73%), 1-dotriacontanol (15.35%), 5-eicosene, (E)-(11.90%) and the minor compounds were squalene (8.83%), dl-alpha-tocopherol (8.58%), 9,12,15- octadecatrienoic acid, methyl ester, (Z,Z,Z)-(4.31%), octacosane (3.09%), methyl lignocerate (2.69%), methyl isostearate (2.15%), cholest- 22- ene- 21- ol, 3,5- dehydro- 6- methoxy-, pivalate (2.11%), phytol, acetate (1.36%), octadecane, 1- (ethenyloxy)- (1.34%). The literature survey reveals that GC-MS analysis of methanol leaf extract (maceration technique) of T. diversifolia from Nigeria showed 29 bioactive compounds 26 whereas amana tie 27observed only two secondary metabolites from ethyl acetate fraction of T. diversifolia leaf. However, to the best of our knowledge, there is no report of GC-MS based metabolite profile of methanol extract of T. diversifolia leaf and flower from India.
FIG. 2: MASS CHROMATOGRAM OF GC-MS ANALYSIS OF METHANOL LEAF EXTRACT OF TITHONIA DIVERSIFOLIA
FIG. 3: MASS CHROMATOGRAM OF GC-MS ANALYSIS OF METHANOL FLOWER EXTRACT OF TITHONIA DIVERSIFOLIA
TABLE 4: THE PHYTOCOMPOUNDS OBSERVED IN THE METHANOL FLOWER EXTRACT OF T. DIVERSIFOLIA
The highest phytocompounds were detected in methanol leaf extract 16 followed by flower extract 13. In this study, the greater antioxidant activity of methanol leaf extract could be correlated to the occurrence of higher quantitative of secondary metabolites Table 1. We know that most of the bioactive compounds display several pharmacological activities. Neophytadiene is a good analgesic, antipyretic, anti-inflammatory, anti-microbial and antioxidant compound 28. Phytol acetate is known to exhibit cancer-preventive property, antimicrobial, anti-inflammatory, and diuretic. Stigmasta-5, 22-dien-3-ol also referred to as stigmasterol shows anti-hepatotoxic, antiviral, antioxidant, hyper-cholesteremic, and cancer preventive 29. 17-penta-triacontendisplay anti-bacterial, antiviral, anti-oxidant and anti-inflammatory 30-32. Tetracontane has anti-inflammatory and analgesic activity, whereas pentacosane shows antibacterial property 33. Nerolidol is sesquiterpene alcohol used a food flavoring agent, anti-microbial, anti-biofilm, anti-oxidant, anti-parasitic, skin-penetration enhancer, skin-repellent, anti-nociceptive, anti-inflammatory, and anti-cancer 34. It has been reported that gamma- sitosterol have hypolipidemic, antioxidant, antibacterial, anti-diabetic, anti-angiogenic, anti-cancer, antimicrobial, anti-inflammatory, anti-diarrhoeal and antiviral properties 35, 28. 2- Methyltetracosane is a good free radical scavenger 36. β-sitosterol, a plant phytosterol having various biological activities such as anti-inflammatory activity, apoptosis inducer, chemoprotective, hypo-cholesterolemic, angiogenic, antimutagenic, anticancer, antioxidant, neuroprotector, antidiabetic 37. Methyl palmitate is used in the preparation of detergents, emulsifiers, wetting agents, stabilizers, resins, lubricants, plasticizers, and animal feeds 38.
It exhibits a strong acaricidal activity, anti-inflammatory property, protective effect against bleomycin-induced lung inflammation, and inhibits macrophages in rats. Also, it possesses a potent anti-fibrotic effect against carbon tetrachloride-induced liver fibrosis 39-41. 9, 12, 15- octadecatrienoic acid, methyl ester, (Z, Z, Z) is the linolenic acid compound that acts as an antiinfla-mmatory, hypocholesterolemic cancer-preventive, heaptoprotective, nematicide insectifuge, anti-histaminic antieczemic, antiacne, 5-alpha reductase inhibitor antiandrogenic, anti-arthritic, anti-coronary, insectifuge.
It also has antimicrobial, anticancer, hepatoprotective, anti-arthritic, anti-asthma and diuretic property 42, 43. 5-eicosene is a fatty acid, exhibits antimicrobial and cytotoxic properties 44. Octa-decane, 1-(ethenyloxy) is ether and reported as antisepsis 45. Squalene is a natural 30-carbon isoprenoid compound, seen both in plants and animals.
It is an intermediate metabolite in the synthesis of cholesterol having pharmacological properties such as anti-bacterial, anti-oxidant, anti-tumor, cancer preventive, immunostimulant, chemopreventive, a lipoxygenase inhibitor, pesticide, diuretic 46, 47 reported that octacosane from plant Couroupita guianensis L. flower extract showed the highest mortality against spodopteralitura.
Alpha-tocopherol is a fat-soluble vitamin, biologically active form of vitamin E, and essential for the stabilization of biological membranes. Dl-alpha-tocopherol is a potent antioxidant having peroxyl radical scavenging activity and important in protecting cells from oxidative stress 48.
However, some of the other compounds such as tridecyl acrylate, 4-methyldocosane, dodecyl palmitate, pentatriacontane, behenyl chloride, pentacosane, methyl palmitate, methyl lino-lelaidate, methyl isostearate, cholest-22-ene-21- ol, 3,5-dehydro-6-methoxy-pivalate, methyl lingo-cerate and 1-dotriacontanol are yet to be described in detail. Nonetheless, extended research is essential in the field of isolation, characterization, and assessment of bioactivity of each compound from T. diversifolia to authenticate their pharma-cological importance.
CONCLUSION: This is the first scientific data to report the phytochemical profile of leaf and flower extract of T. diversifolia from India. The findings of this research give an insight into the bioactive compounds of Tithonia diversifolia and its antioxidant properties. The results support the use of T. diversifolia in folk medicine to treat different ailments. Thus, it can be concluded that this plant has phytopharmaceutical importance and may serve as the new potential source of herbal drug.
ACKNOWLEDGEMENT: The authors gratefully acknowledge the Department of Botany, Bangalore University, Bengaluru - 560056, Karnataka, India, for providing support in carrying out phytochemical analysis, antioxidant experiments and the Kerala Forest Research Institute (KFRI), Peechi, Thrissur, Kerala, India for GC-MS instrument.
CONFLICTS OF INTEREST: The authors declare that they have no conflict of interest.
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How to cite this article:
Roopa MS, Shubharani R, Rhetso T and Sivaram V: Comparative analysis of phytochemical constituents, free radical scavenging activity and GC-MS analysis of leaf and flower extract of Tithonia diversifolia (Hemsl.) a. gray. Int J Pharm Sci & Res 2020; 11(10): 5081-90. doi: 10.13040/IJPSR.0975-8232.11(10).5081-90.
All © 2013 are reserved by the International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.
M. S. Roopa *, R. Shubharani, T. Rhetso and V. Sivaram
Department of Botany, Bangalore University, Bengaluru, Karnataka, India.
22 October 2019
31 January 2020
12 March 2020
01 October 2020