An antioxidant is any substance, if present at low concentrations in combination with an oxidisable substrate, significantly delays or prevents oxidation of the substrate.  Based  on  the  historical  success  of   natural products, a number of medicinal plants have been evaluated for their antioxidant potential (Argolo et al., 2004; Burits et al., 2001; Helle et al., 2004).

 

Free radical mediated damage is connected with several diseases, and therefore its prevention can play an important role in the cure of those diseases (Kanwal et al., 2011; Kulisica et al., 2004; Sharma and Trivedi, 2002; Smith et al., 2007). For example, oxidative stress has been widely postulated to be involved in the development and progression of some chronic diseases such as cardiovascular disease, neuronal disease, cataracts, and several types of cancer (Gua et al., 2009). There is increasing search for antioxidants that remove occurring naturally in vegetables, fruits and functional herbs to replace synthetic antioxidants. It has been found out that some synthetic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) have been revealed to be potentially toxic and carcinogenic, they have been found to induce DNA damage (Helle et al., 2004).

 

Tagetes minuta (Asteraceae) is an annual  herb that belongs to the Asteraceae family. Its leaves are slightly glossy, green and pinnately dissected into 4–6 pairs of pinnae (Cerruti et al., 2010; Daizy et al., 2007). It is used in common medicine and grows in temperate regions of South America, some parts of Africa and Asia (Hamill et al., 2000; Kamatenesi-Mugisha and Oryem-Origa, 2007; Tabuti et al., 2003; Vasudevan et al., 1997a). Infusions of leaves from di?erent species of Tagetes have been used to treat stomach and intestinal diseases (Gakuya et al., 2013; Harris et al., 2002; Paul and Kasenene, 2007; Tabuti et al., 2003; Vasudevan et al., 1997a), and other species have been found to possess di?erent biological activities, such as, antimicrobial, antiin?ammatory, antioxidant and antiviral (Andreotti et al., 2013; Dharmagadda et al., 2005; Hamil et al., 2000; Paul and Kasenene, 2007).

 

T. minuta is a wild shrub in Uganda that thrives mostly in the rainy season (Tabuti et al., 2003).  Tagetes species, commonly known as marigold are also grown as ornamental plants and thrive in varied agro-climates (Vasudevan et al., 1997b). Bioactive extracts of different Tagetes parts exhibit nematocidal, fungicidal and insecticidal activity (Vasudevan et al., 1997b). T. minuta has been used by the local people in Uganda to relieve a number of ailments (Hamil et al., 2000; Paul and Kasenene, 2007).

 

Previous work on Tagetes species, Tagetes maxima reavealed strong antioxidant properties of its ethylacetate extracts (Parejo et al., 2005). T. maxima was found to exhibit strong radical scavanging and antioxidant activities (Parejo et al., 2005) .There is a great possibility of similar activity in other Tagetes species. Antioxidant activity of T. minuta from Uganda has not been determined according to literature, but since it belongs to the same family as other Tagetes species with strong antioxidant properties, it was necessary to determine its potential as an antioxidant.

 

In this research, antioxidative compounds of T. minuta were isolated both from the Ethyl acetate extract and the essential oil. It was done by determining  the  scavenging activity using 2,2-diphenyl-1-picrylhaydrazyl free radical (DPPH). The active compounds were tested quantitatively for their radical scavenging activity.

Plant material

 

Fresh  aerial parts of T. minuta were collected from Mabira Forest in the morning hours in the month of November 2012. The sample  was transported to Makerere University, Department of Chemistry Laboratory. Essential oils from T. minuta were extracted on arrival in the Laboratory. The remaining plant material was dried under  shade for 3 weeks, ground in a mortal to obtain fine powder. A voucher specimen (CK001) was deposited at Makerere University Herbarium.

 

Hydrodistillation

 

Essential oil from fresh T. minuta was extracted by hydro-distillation in a Clevenger type apparatus for 3 h with a separated extraction chamber. The resulting essential oils were dried over anhydrous-sodium sulphate to extract the water. The oil was kept in refrigerated conditions at 8°C prior to the antioxidant activity determination and GC-MS analysis (Conti et al., 2010; Polatoglu et al., 2012).

 

Chemicals

 

All chemicals and reagents used in extraction, isolation and analysis of the active compounds were obtained from Sigma-Aldrich (Germany). These chemicals and reagents were of analytical grade. The standards were also purchased from sigma-Aldrich.

 

Solvent extraction- Cold extraction

 

T. minuta dry powder (1000 g) was extracted  four times with 2000 ml of ethyl acetate at 40 to 45°C. The supernatant (extract) was separated from the residue by paper filtration (Whatman No. 1 filter, whatman paper Ltd., UK). It was  dried in vaccum using a rotary evaporator at 40°C to remove all the ethyl acetate  to give a residue. The powder  was dried and re-extracted three times with 2000 ml methanol. The extract was combined and evaporated at 40°C to dryness. Both methanolic and ethyl acetate extracts were kept in a dry place for further  testing (Gua et al., 2009).

 

DPPH assay

 

The capacities to donate hydrogen atoms/electrons by the essential oil and solvent extracts from the test samples were preliminarily detected using thin layer chromatography (TLC) and further measured spectrophotometrically.

 

TLC screening for antioxidants

 

Dilutions of volatile oils (5 µl, 1:5 in hexane), and the crude extracts were spotted on silica gel sheets and developed in ethylacetate:hexane (7:3 v/v). The plates were sprayed with 0.2% solution of the stable radical, diphenylpicrylhydrazil ( DPPH) (Brand Williams et al., 1995; Burits et al., 2001; Helle et al., 2004; Xiao et al., 2010). Active spots were detected as yellow spots  on  a  purple background. Zones where the colour changed within 30 min (after spraying) were taken as positive results (Burits et al., 2001).

 

DPPH spectrophotometric assay

 

This assay uses DPPH as a reagent (Argolo et al., 2004; Brand-Williams et al., 1995; Burits et al., 2001; Helle et al., 2004). 50 µl of various concentrations  of the volatile oils were added to 5 ml of 0.004% methanolic solution of DPPH. After 30 min of incubation period at room temperature, the absorbance was read against the blank  at  517 nm   using   a   U-1100   UV-VIS   Spectrophotometer  (Hitachi Ltd; Tokyo Japan). The tests were carried out in duplicate. DPPH solution (1.0 ml; 0.3 mM) plus methanol (2.5 ml) was used as a negative control. After 30 min the absorbance values were measured at 517 nm and converted into the percentage antioxidant activity (AA) using the following formula:

 

%AA, which was, %aa=((A_C(0) – A_A(t))/A_C(0))*100

 

Where A_C(0) is the AA for the control solution at t=o minutes, and A_A(ti) is the AA  after the given time intervals, for I = 5, 10, 15, 20, 25, and 30 min (Kulisica et al., 2004).

 

GC-MS analysis

 

GC-MS analysis was used to identify the compounds in the essential oil and solvent extracts that had antioxidant activity.The GC-MS results of T. minuta  was already determined in a previous research (Kyarimpa et al., 2014).

 

High performance liquid chromatography

 

The active fractions were purified with HPLC. A  Dionex Ultimate 3000 HPLC (Dionex) equipped with a diode array detector and operated by Chromeleon Version 6.80 SR9 software. 2.0 ml each of the active fractions was injected onto a 150 x 2.1 mm, 100 A, 2.6 µm Phenomenex Kinetex C18-column at 35°C. N-Hexane was used as a mobile phase. The flow rate was 237 µl/min. To selectively detect antioxidants, the detector recorded the signal at 520 nm (Application Note 281, Dionex Corporation, Sunnyvale, CA, USA). HPLC was carried out only samples with antioxidant activity and the solvent system was chosen based on Rf values of the TLC experiments.

 

Nuclear magnetic resonance (NMR) spectroscopy

 

Nuclear magnetic resonance (NMR) spectroscopy is (arguably) the most powerful tool available for determining the structure of organic compounds. It is used to identify and/or elucidate detailed structural information about chemical compounds. In this case it was used to determine the structure of the pure active compound  in the sample.

 

All NMR spectra were recorded on a Bruker Avance II 400 (resonance frequencies 400.13 MHz for ¹H and 100.63 MHz for 13C) equipped with a 5 mm broadband observe probe head (BBFO) with z–gradients at room temperature with standard Bruker pulse programmes. The sample was dissolved in 0.6 ml of CDCl3 (99.8% D). Chemical shifts are given in ppm, referenced to residual solvent signals (7.26 ppm for ¹H, 77.0 ppm for ¹³C). ¹H NMR data were collected with 32k complex data points and apodized with a Gaussian window function (lb = −0.3 Hz and gb = 0.3 Hz) prior to Fourier transformation. ¹³C-jmod spectra with WALTZ16 ¹H decoupling was acquired using 64k data points. Signal-to-noise enhancement was achieved  by  multiplication  of  the  FID  with  an exponential window function (lb = 1Hz). All two-dimensional experiments were performed with 1k × 256 data points, while the number of transients (2–8 scans) and the sweep widths were optimized individually. The resulting FIDs were zero-filled to a 2k × 1k data matrix and apodized with a sine function for COSY in both the ω1 and ω2 dimensions prior to Fourier transformation.

 

Heteronuclear spectra were zero-filled only in F1 to a 1k × 512 data matrix, and apodized in both dimensions with a shifted sine function. The heteronuclear single quantum coherence (HSQC) experiment was acquired using adiabatic pulse for inversion of ¹³C and GARP-sequence for broadband ¹³C-decoupling, optimized for 1J(CH) = 145 Hz.

The essential oil from tagets minuta exhibited strong antioxidant activity on TLC. Some components of the crude extract were also found to be active (Figure 2). Track 1 on the first TLC plate from the left (Figure 2) was the essential oil, the other tracks were different extracts from T. minuta and Tephrosia Vogelli. It was noted that some of the components of the two plants had strong antioxidant activity. These fractions were isolated using Column Chromatography, HPLC. The pure fractions were analysed using NMR spectroscopy. Track 1 had very strong antioxidant activity as shown by the DPPH reaction on the TLC plate (Figure 2). This oil was later used for quantitative measurement again using DPPH and Ultra Violet Spectrophotometer (Table 1). The total antioxidant capacity revealed that the essential oil from T. minuta had a high antioxidant activity. Free radical scavenging activity of  the extracts was assesed using the stable free radical DPPH. Plant extracts which reduce DPPH by donating hydrogen ions are considered as antioxidants having free radical scavenging activity.  The results  from Table 1, were used to calculate the amount of DPPH scavenged over a period of time  according to the formula {%AA, which was , %aa=((AC(0) – AA(t))/AC(0))*100}, and the LC₅₀ was determined (Appendix 1B). DPPH solution alone served as control (A0). It is evident from the study, that the investigated extracts and essential oil have the ability to quench free radicals. This indicates that T. minuta is  a potential source of natural antioxidants.

 

Nuclear magnetic resonance (NMR) Spectroscopy

 

One active  pure compound was identified using both the proton NMR and the carbon NMR as shown in Figure 1 and Table 2. The 1 H NMR spectra of this compound  revealed the presence of two isolated olefinic spin systems without any further coupling partners: on the one hand a vinyl group, indicated by its characteristic ABX-system at α 5.05 (d, J = 10.8 Hz), α 5.23 (d, J = 17.6 Hz) and α 6.37 (dd, J = 17.6 , 10.8 Hz), and on the other hand an olefinic methylene group with broad singuletts at α 4.99 and α 5.00 ppm, respectively. Besides a  triplett  at α 2.18 ppm, a bulk of aliphatic methylene and methine protons in the region between 1.60 to 1.00 ppm and signals of different methyl groups at around 0.90 ppm no more signals were found in the 1H NMR spectra. In combination with the hsqc experiment the j-modulated 13C nmr spectra showed signals of 4 olefinic carbons – one quaternary, one methine, and two methylenes – 4 methyl, 3 methine and 9 methylene carbons. In addition to the molecular mass peak at m/z = 278 these results indicated that compound is a noncyclic, nonoxidized diterpene with a molecular formula  of  C₂₀H₃₈.  Extensive analysis of heteronuclear 2D NMR led to the elucidation  of  that  structure  which   turned   out   to   be neophytadiene, a widespread component of essential oils from different plant sources. Whereas Burkhardt et al. published only NMR data of the olefinic part of the molecule, we present here to our best knowledge  for  the first time the fully assigned nmr resonances (Table 2).

 

Neophytadiene, is a fatty acid-related compound  which plays an important part in competitive inhibition of cyclooxygenase  or   lipoxygenase   in   an   inflammation reduction, resulting in decreased production of prostaglandins and leukotriene (Pillai and Nair, 2013).

 

 

DPPH is a free radical, stable at room temperature, which produces a violet solution in methanol. It is reduced in the presence of an antioxidant molecule, giving rise to uncoloured methanol solutions. The use of DPPH provides an easy and rapid way to evaluate antioxidants. According to the results obtained from data in Tables 1 and 2 as well as those of statistical analysis, we can say that extracts from T. minuta as well as its essential oil possess strong antioxidant properties with an LC₅₀ of 1.49 g/l^-1 after 30 min as compared to other antioxidants reported in Parejo et al. (2005) and Xiao et al. (2010). A well-known antioxidant, ascorbic acid, was used as positive control. DPPH scavenging patterns for T. minuta  versus time, along with IC50 values, are presented in Appendix 1 A and B.

We declare no competing interests in this research.

The authors wish to acknowledge the Robert Mc. Namara Fellowship Scheme of the World Bank for the sponsorship. The University of Natural Resources and Applied life Sciences, Vienna  for hosting the researcher and providing techinical guidance. Special thanks go to Prof. Stefan Bohmdorfer ,Prof  Thomas Rosenau and Dr. Markus Bacher for their techinical support. The authors express their gratitude to CARNEGIE Corporation of New York for the financial support through The Science Initative Group's RISE-AFNNET, Makerere University.