Isolation and Antimalarial Activity of a New Flavonol from Tithonia diversifolia Leaf Extract

An antiplasmodial activity-guided isolation was carried out on the dichloromethane extract of Tithonia diversifolia dried leaves. A total of five germacranolide type sesquiterpene lactones and a new flavonol, 3,6-dihydroxy-2-(4′-hydroxyphenyl)-7-methoxy-4H-chromen-4-one, were isolated. The flavonol reported an IC50 above 6.00 µM against the chloroquine sensitive strain, NF54. The antimalarial activity of the Tithonia diversifolia dichloromethane leaf extract was attributed to orizabin and tagitinin C.


Introduction
An ethnobotanical survey indicated that Tithonia diversifolia is used as one of the traditional antimalarial remedies in Zimbabwe [1]. Tithonia is one of the genera of the Asteraceae, comprising about 11 species [2] and 13 taxa [3], and it originated from Mexico, Central America and Cuba [2]. In many African countries, such as the Democratic Republic of Congo [4], Kenya [5,6], Nigeria and Uganda [7], the use of the rotundifolia species in traditional medicines, livestock fodder, poultry feed, green manure, and field and storage pest management are widely acknowledged.
Flavonoids consist of a large group of polyphenolic compounds having a benzo-γpyrone structure, most commonly known as the C 6 -C 3 -C 6 skeleton. They are synthesized by the phenyl propanoid pathway [17]. Based on their core structure, flavonoids can be grouped into different flavonoid classes, such as flavonols, flavones, flavanones, flavanonols, anthocyanidins, isoflavones and chalcones. Flavonoids are often hydroxylated on positions 3, 5, 7, 3 , 4 , and/or 5 , and some of the hydroxyl groups are methylated, acetylated, and even with sulphate conjugation [18]. Flavonoids are usually attached to sugar moieties through the O-or C-atom in plants [18].
Plants synthesize flavonoids as a response to microbial infection [17] or to environmental changes [19]. In plants systems, flavonoids help in combating oxidative stress and act as growth regulators [17]. Hydroxyl groups in flavonoids mediate their antioxidant effects Chemistry 2021, 3 855 by scavenging free radicals and/or by chelating metal ions [17]. Fruits and vegetables are the main dietary sources of flavonoids for humans, along with tea and wine.
Many flavonoids are reported to have antioxidative activity, free radical scavenging capacity, coronary heart disease prevention, hepatoprotective, anti-inflammatory, and anticancer activities, while some of them exhibit potential antiviral activities [17]. Kavitha et al. [20] report that flavonoids play an important role in maintaining the homeostasis of the central nervous system by modulating neuronal oxidative metabolism because they are strong inhibitors of enzymes that cause neuron degeneration. There is growing evidence that long-term ingestion of diets rich in plant polyphenols offer protection against the development of cancers, cardiovascular diseases, diabetes, osteoporosis and neurodegenerative diseases [21]. It is suggested that phenolic groups can act as electron sinks in living systems, forming relatively stable phenoxyl radicals, thereby disrupting chain oxidation reactions in cellular components [21]. However, their bioavailability, metabolism, and biological activity depend upon the configuration, total number of hydroxyl groups, and substitution of functional groups about their nuclear structure.

Isolated Compounds from the DCM Crude Extract of T. diversifolia Leaves
A total of six compounds ( Figure 1) were isolated from the antiplasmodial active fraction of the DCM crude leaf extract of T. diversifolia, comprising five germacranolide type sesquiterpene lactones and 18 mg of a new flavonol ( Figure 2). The significant antiplasmodial activities of the DCM crude extract were attributed to orizabin and tagitinin C, which reported IC 50 values of 2.28 µM (0.83 µg/mL) and 1.55 µM (0.54 µg/mL), respectively [1], while the flavonol reported an IC 50 value above 6.00 µM against the chloroquine sensitive strain, NF54. The cytotoxicity assessment of the flavonol was not done because it was not considered very active.
act as growth regulators [17]. Hydroxyl groups in flavonoids mediate their antioxidan effects by scavenging free radicals and/or by chelating metal ions [17]. Fruits and vegeta bles are the main dietary sources of flavonoids for humans, along with tea and wine.
Many flavonoids are reported to have antioxidative activity, free radical scavenging capacity, coronary heart disease prevention, hepatoprotective, anti-inflammatory, and an ticancer activities, while some of them exhibit potential antiviral activities [17]. Kavitha e al. [20] report that flavonoids play an important role in maintaining the homeostasis o the central nervous system by modulating neuronal oxidative metabolism because the are strong inhibitors of enzymes that cause neuron degeneration. There is growing evi dence that long-term ingestion of diets rich in plant polyphenols offer protection agains the development of cancers, cardiovascular diseases, diabetes, osteoporosis and neuro degenerative diseases [21]. It is suggested that phenolic groups can act as electron sink in living systems, forming relatively stable phenoxyl radicals, thereby disrupting chain oxidation reactions in cellular components [21]. However, their bioavailability, metabo lism, and biological activity depend upon the configuration, total number of hydroxy groups, and substitution of functional groups about their nuclear structure.

Isolated Compounds from the DCM Crude Extract of T. diversifolia Leaves
A total of six compounds ( Figure 1) were isolated from the antiplasmodial activ fraction of the DCM crude leaf extract of T. diversifolia, comprising five germacranolid type sesquiterpene lactones and 18 mg of a new flavonol ( Figure 2). The significant an tiplasmodial activities of the DCM crude extract were attributed to orizabin and tagitinin C, which reported IC50 values of 2.28 µ M (0.83 µ g/mL) and 1.55 µ M (0.54 µ g/mL), respec tively [1], while the flavonol reported an IC50 value above 6.00 µ M against the chloroquin sensitive strain, NF54. The cytotoxicity assessment of the flavonol was not done becaus it was not considered very active.  The five sesquiterpene lactones in Figure 1 above have been reported on; th no further studies on their physical properties were conducted. However, a searc SciFinder database retrieved no match for the flavonol in Figure 2.

The Flavonol
The HRMS spectrum of the flavonol shows a molecular ion peak at m/z 301.  The FTIR spectrum of the flavonol showed characteristic absorbencies of the c group at 1755 cm −1 [18], double bond stretching from 1552 to 1650 cm −1 , vario stretches from 1000 to 1295 cm −1 , hydroxyl groups at 3347 cm −1 and methyl stre 2922 cm −1 .

The Flavonol
The HRMS spectrum of the flavonol shows a molecular ion peak at m/z 301. The five sesquiterpene lactones in Figure 1 above have been reported on; th no further studies on their physical properties were conducted. However, a searc SciFinder database retrieved no match for the flavonol in Figure 2.

The Flavonol
The HRMS spectrum of the flavonol shows a molecular ion peak at m/z 301  The FTIR spectrum of the flavonol showed characteristic absorbencies of the group at 1755 cm −1 [18], double bond stretching from 1552 to 1650 cm −1 , vario stretches from 1000 to 1295 cm −1 , hydroxyl groups at 3347 cm −1 and methyl stre 2922 cm −1 .
The 13 C NMR spectrum shows a system with 14 carbons, while the APT experiment indicates 9 carbons in positive mode (8 quaternary carbons and one C=O) and 5 carbons in negative mode (1 methoxy group and 4 methine groups (CH)).
The 2D HSQC spectrum indicates that H-8 (δ H 6.59) correlates with the carbon atom at δ C 102.8, H-5 (δ H 6.77) with the carbon atom at δ C 94.7, H-3 /H-5 with the carbon at δ C 116.4 and H-2 /H-6 with the carbon at δ C 128.9.  The 13 C NMR spectrum shows a system with 14 carbons, while the APT experiment indicates 9 carbons in positive mode (8 quaternary carbons and one C=O) and 5 carbons in negative mode (1 methoxy group and 4 methine groups (CH)).
The 2D HSQC spectrum indicates that H-8 (δH 6.59) correlates with the carbon atom at δC 102.    Figure 5 shows information deduced from the 2D HMBC spectrum, indicating that H-5 (δ H 6.77) strongly correlates with the carbonyl carbon assigned as C-4, at δ C 182.5, while H-8 (δ H 6.59) slightly correlates with the same carbonyl carbon atom. The methoxy protons and H-8 correlate with the carbon at δ C 131.8, assigned as C-7. Protons 3 /5 (δ H 6.92) strongly correlate with C-4 at δ C 121.6 and weakly correlate with C-1 at δ C 161.6, while H-2 /H-6 (δ H 7.92) strongly correlate with C-1 at δ C 161.6 and C-2 at δ C 164.2, and weakly correlate with the carbons of H-3 /H-5 at δ C 116.4. The carbon at δ C 153.2 does not correlate with any proton, indicating that it is C-3. The arrows indicate correlating atoms according to the HMBC spectrum. More Supplementary Material that was used to characterize the compound is available on MDPI website 6.92) strongly correlate with C-4′ at δC 121.6 and weakly correlate with C-1′ at δC 161.6 while H-2′/H-6′ (δH 7.92) strongly correlate with C-1′ at δC 161.6 and C-2 at δC 164.2, and weakly correlate with the carbons of H-3′/H-5′ at δC 116.4. The carbon at δC 153.2 does no correlate with any proton, indicating that it is C-3. The arrows indicate correlating atom according to the HMBC spectrum. More Supplementary Material that was used to char acterize the compound is available on MDPI website A web-based search retrieved no matching responses for the flavonol. Its scientifi name could be deduced as 7-methoxy-3,4′,6-trihydroxyflavone or 3,6-dihydroxy-2-(4-hy droxyphenyl)-7-methoxy-4H-chromen-4-one.

Characterisation of Isolates
A 600 MHz Bruker Avance spectrometer (Fallanden, Switzerland) was used to record the 1 H NMR, COSY, HMBC, HMQC (600 MHz) and 13 C, APT (150 MHz) experiments in DMSO-6d (δH = 2.50; δC = 39.51) with TMS as the internal standard. Chemical shifts were expressed as parts per million (ppm) on the delta (δ) scale, and coupling constants (J) are accurate to 0.01 Hz. High-resolution mass spectral data (HRMS) were collected using a Waters Micromass LCT Premier TOF-MS (Milford, USA), while low-resolution mass spec tra (LRMS) were recorded on a Sciex 4000QTRAP hybrid triple quadrupole ion trap mas spectrometer (California, USA), and the infra-red (FTIR) spectra were recorded on a Per kin Elmer Spectrum One FT-IR (Shelton, USA).

Crude Extracts
Fleshy leaves of Tithonia diversifolia were air-dried in the shade, and then finely grounded. A sample of the powdered leaves was sequentially soaked in hexane, DCM and then in a mixture of DCM and ethyl acetate (9:1 v/v) on a shaker. The sample wa repeatedly soaked in each solvent over several nights until there was a significant loss o color and the major eluents were diminishing on the TLC. The three fractions were con centrated on a Rotavapor at 40 °C and then transferred into separate, labeled vials, which were left to dry in a fume hood.

Antiplasmodial Activity Assessment
The chloroquine-sensitive NF54 strain of the malaria parasite Plasmodium falciparum was cultured in vitro [23]. The antiplasmodial activity of the various extracts was deter mined using the tritiated hypoxanthine incorporation assay [24]), where chloroquine and quinine were used as the reference antiplasmodial agents. At least three independent ex periments were performed, from which the mean and standard deviation were deter mined. A web-based search retrieved no matching responses for the flavonol. Its scientific name could be deduced as 7-methoxy-3,4 ,6-trihydroxyflavone or 3,6-dihydroxy-2-(4hydroxyphenyl)-7-methoxy-4H-chromen-4-one.

Characterisation of Isolates
A 600 MHz Bruker Avance spectrometer (Fallanden, Switzerland) was used to record the 1 H NMR, COSY, HMBC, HMQC (600 MHz) and 13 C, APT (150 MHz) experiments in DMSO-6d (δH = 2.50; δC = 39.51) with TMS as the internal standard. Chemical shifts were expressed as parts per million (ppm) on the delta (δ) scale, and coupling constants (J) are accurate to 0.01 Hz. High-resolution mass spectral data (HRMS) were collected using a Waters Micromass LCT Premier TOF-MS (Milford, USA), while low-resolution mass spectra (LRMS) were recorded on a Sciex 4000QTRAP hybrid triple quadrupole ion trap mass spectrometer (Foster City, CA, USA), and the infra-red (FTIR) spectra were recorded on a Perkin Elmer Spectrum One FT-IR (Shelton, CT, USA).

Crude Extracts
Fleshy leaves of Tithonia diversifolia were air-dried in the shade, and then finely grounded. A sample of the powdered leaves was sequentially soaked in hexane, DCM and then in a mixture of DCM and ethyl acetate (9:1 v/v) on a shaker. The sample was repeatedly soaked in each solvent over several nights until there was a significant loss of color and the major eluents were diminishing on the TLC. The three fractions were concentrated on a Rotavapor at 40 • C and then transferred into separate, labeled vials, which were left to dry in a fume hood.

Antiplasmodial Activity Assessment
The chloroquine-sensitive NF54 strain of the malaria parasite Plasmodium falciparum was cultured in vitro [23]. The antiplasmodial activity of the various extracts was determined using the tritiated hypoxanthine incorporation assay [24]), where chloroquine and quinine were used as the reference antiplasmodial agents. At least three independent experiments were performed, from which the mean and standard deviation were determined.

Isolation of Antiplasmodial Active Fractions
The DCM fraction was further fractionated on a silica gel column with hexane-ethyl acetate (H-EA) solutions (15:1; 10:1; 5:1; 3:2 and 2:3 (v/v)). The column was followed by TLC. Sub-fractions 9 (0.59 g) and 10 (1.67 g) of the DCM from eluents H-EA 5:1 and 3:2 had very similar TLC profiles, and reported the highest antimalarial activities as reflected by IC 50 values of 0.31 ± 0.07 and 0.62 ± 0.04 µg/mL, respectively, and an average inhibition of 53.7 %, which were sustained even at a low concentration of 0.5 µg/mL [1]. The two sub-fractions were combined (1.60 g) and then further subjected to silica gel column chromatography with DCM-EA eluents (20:1; 12:1; 7:1; 5:2: 3:2, and 2:3 (v/v)). All the isolates, except the flavonol, were cleaned by re-crystallization in ethyl acetate and hexane mixtures [1] The major sub-fraction from eluent DCM-EA 7:1 was re-dissolved in ethyl acetate after rotary evaporation, and on standing, light yellowish-green amorphous particles separated, which were filtered off. On further standing, two more crops were harvested. The precipitate did not dissolve in ethyl acetate, chloroform, methanol or ethanol, but dissolved in DMSO. The isolate was dissolved in DMSO-d 6 , and an 1 H NMR was run, which indicated that it was a pure compound [1].

Conclusions
A combination of gravity chromatography and re-crystallization afforded five germacranolide type of sesquiterpene lactones and a new flavonol from the DCM fraction of the T. diversifolia leaf extract. Antiplasmodial activity of DCM crude extract of T. diversifolia leaves is mainly due to orizabin and tagitinin C.
The six isolated compounds may further be assessed for GABA A and Acetylcholinesterase inhibition effects. Additionally, the flavonol could be tested for anticancer and antidiabetic activities because flavonoids exhibit antioxidant and anticancer activities. Furthermore, flavonol could be severally functionalized and the products assessed on various diseases.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.