Identification of Antioxidative Ingredients from Feverfew (Tanacetum Parthenium) Extract Substantially free of Parthenolide and other Alpha-Unsaturated Gamma-Lactones

Extract of feverfew (Tanacetum parthenium) has anti-infl ammatory effect with various therapeutic benefi ts. Alpha-unsaturated gamma-lactones including parthenolide were recognized as part of the major active ingredients but with undesirable allergic reactions. In this research paper, feverfew extract substantially free of parthenolide and other alpha-unsaturated gamma-lactones was investigated. We are reporting the identifi cation of more than twenty ingredients from this feverfew extract. The ingredients mainly include caffeoyl derivatives and fl avonoids. Four ingredients, caffeic acid methyl ester (2), cynarin (13), 4-methoxyl caffeic acid (22) and 3,4-dimethoxyl caffeic acid (27), were discovered in feverfew for the fi rst time. The identifi cation works were performed primarily using HPLC-UV and HPLC-APCI-MS analyses and comparing with reference compounds. Ingredients caffeic acid (1), caffeic acid methyl ester (2), quercetagetin 3, 6-dimethyl ether (3), apigenin (4), and santin (6), were isolated by semi-preparative HPLC; and their structures were further confi rmed by NMR analyses. The application of APPI-MS on the analyses of 1 and 2 enabled successful molecular weight determination and eliminated lingering ambiguity caused by weak signals from APCI-MS detection. The extract of feverfew also demonstrated considerably high antioxidant capacity using a DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging model likely due to its caffeoyl derivatives and fl avonoid ingredients with phenolic moieties to contribute to its pharmacological benefi ts. Research Article Identifi cation of Antioxidative Ingredients from Feverfew (Tanacetum Parthenium) Extract Substantially free of Parthenolide and other AlphaUnsaturated Gamma-Lactones Fa Zhang*, Jianyun Zhou, Yiqun Shi and Ken Karaisz Analytical R&D, Johnson & Johnson Consumer Inc., Skillman, NJ, 08558, USA Received: 13 November, 2019 Accepted: 12 December, 2019 Published: 13 December, 2019 *Corresponding author: Fa Zhang, Analytical R&D, Johnson & Johnson Consumer Inc., Skillman, NJ, 08558, USA, E-mail:


Introduction
Feverfew (Tanacetum parthenium) is a medicinal plant that can be found in old gardens or hedgerows. The name of the herb was derived from the Latin word febrifugia, meaning "fever reducer". It has a long history of usages in traditional and alternative medicines. It was believed to have antiinfl ammatory effect and widely used to treat diseases such as fevers, arthritis, digestive problems, migraines and various other conditions. Feverfew is rich in sesquiterpene lactones, essential oils, fl avonoids and other minor chemicals [1][2][3][4][5].
Williams, et al., found several fl avonoids in feverfew such as tanetin and other fl avanol methyl esters, apigenin, luteolin, chrysoeriol as well as their glucuronides and glycosides [6][7][8]. Other research groups reported the identifi cation of jaceidin, centaureidin, sudachitin, aceronin, nevadensin. and polyphenolic acids [9][10][11][12]. The focus initially was on parthenolide which is the predominant sesquiterpene lactone presented in feverfew and has been considered as the compound mainly responsible for the anti-infl ammatory effect [1][2][3][4][5]13]. The mechanism of action is presumably through the covalent bonding by Michael addition reaction of alpha-methylenebutyrolactone moiety with cysteine residue of relevant enzymes or proteins [13]. It was also known that allergic reaction to the extract may be caused by sesquiterpene lactones such as alpha-unsaturated-gamma-lactones including parthenolide. Therefore, the therapeutic applications of feverfew extract containing less sesquiterpene lactones were widely sought after [14,15]. Considering the interest generated in feverfew, we initiated a study to profi le the structures of the active ingredients in feverfew extract that are substantially free of parthenolide and other alpha-unsaturated gamma-lactones.
Twenty-eight ingredients    structural identifi cation and isolation of caffeic acid methyl ester (2) and discovery of additional caffeoyl derivatives (13,22,27) in feverfew extract have not been reported in literature.

Experiments
Chemicals: HPLC grade acetonitrile and water were obtained from J.T. Baker. Trifl uoroacetic acid (certifi ed) and HPLC grade dimethyl sulfoxide were obtained from Fisher Scientifi c. The methods for the preparation of feverfew extracts that are substantially free of parthenolide and other alpha-unsaturated gamma-lactones were disclosed in US Patent 6224875B1 [20]. The reference compounds for the confi rmation of feverfew ingredients were obtained from multiple sources.

HPLC-UV analyses:
The analytical HPLC method (HPLC Method I) used a C 18 column (Zorba×SB-C 18 , 5μm, 25×0.46cm,) at 40°C. A binary gradient elution using mobile phase A and B was conducted (0-20min, linear gradient from 10 to 30% B; 20-25min, linear gradient to 40% B; 25-35min, linear gradient to 60% B; 35-50min, linear gradient to 80% B) at a fl ow rate of 0.85mL/min. Mobile phase A contained water with 0.01% of trifl uoroacetic acid while mobile phase B contained acetonitrile with 0.01% of trifl uoroacetic acid. The injection volume was 10μL. An UV PDA detector was used for online UV spectral collection with wavelength at 340nm for chromatographic detection.

Semi preparative HPLC-UV isolation:
The semipreparative HPLC method (HPLC Method II) used a C18 column (Zorba×SB-C18, 5μm, 250×0.94cm,) at 40 o C. A binary linear gradient elution using the same mobile phase A and B as HPLC Method I was conducted (0-20min, linear gradient from 10 to 30% B; 20-25min, linear gradient to 40% B; 25-35min, linear gradient to 60% B; 35-50min, linear gradient to 80% B) at a fl ow rate of 4.0mL/min. The detection wavelength was at 340nm. Injection volume was 0.8mL from a solution of 2grams of extract in 20mL of dimethyl sulfoxide. The collected fractions were transferred to individual fl asks and evaporated to dryness using a Brinkmann Evaporator at 45-50°C under vacuum.

HPLC-MS and NMR analyses: HPLC-MS was performed
on a Thermo Scientifi c LXQ HPLC-MS system in APCI or APPI positive ion mode under optimized conditions. HPLC condition was the same as HPLC method I. A Bruker AVII400 FT-NMR Spectrometer was used for NMR analyses with acetone-d 6 as the solvent.

HPLC-UV screening
The feverfew extract was dissolved in a mixture of dimethyl sulfoxide and water (1:1) at 1% concentration and analyzed by HPLC-Method I. The resulted chromatogram is shown in

Identifi cation of caffeoyl derivatives
It was found that peaks 1, 2, 8, 20, 21, 22, 23, 27 and 28 all have very similar UV spectra, which suggests that they may be analogues structurally related to each other. Typical UV spectrum of 1 is presented in Figure 3. 1 and 2 were isolated as pure compounds using semipreparative HPLC Method II as described in section of Experiments. They were analyzed by direct infusion APPI-MS in positive ion mode and NMR. The structures of 1 and 2 were determined as caffeic acid and caffeic acid methyl ester. Their APPI-MS spectra are shown in Figure 4 and their 1 H and 13 C NMR data are presented in Table 1.    [11,12]. However, the detection of ingredients 2, 13, 22 and 27 in feverfew extract were reported for the fi rst time to the best of our knowledge.
The observed ingredients of feverfew extract and HPLC-APCI-MS screening results are summarized in Table 2 along with their relative levels per HPLC-UV chromatographic peak area%.

Identifi cation of fl avonoids and parthenolide
Ingredients 3, 4, and 6 exhibited protonated molecular ions at m/z 347, 271, and 345. They were isolated by semipreparative HPLC as described in the experimental section and analyzed by 1 H NMR. The NMR data are presented in Table 3.
Ingredients 3, 4, and 6 were determined as quercetagetin 3, 6-dimethyl ether, apigenin, and santin, respectively, which are all fl avonoid analogues with similar UV spectral features. As an example, the UV spectrum of apigenin (4) is presented in Figure 5.
The NMR data obtained for 3 and 6 in Table 3 matched well with what published for quercetagetin 3,6-dimethyl ether and santin [7,9]. The structures of 4 and 6 were further confi rmed by comparing their exact same HPLC-UV features with that of corresponding reference compounds.        Table 4. It indicates that the feverfew extract exhibited considerably high antioxidant activity.

Discussion
Like most botanicals, feverfew is chemically very complex, containing sesquiterpene lactones, fl avonoid glycosides, pinenes and other compounds. The specifi c role that each of these component compounds plays in the biological activity of feverfew, however, is not to date fully understood. However, parthenolide has been thought to be the most active chemical component in feverfew and its anti-infl ammatory activities were proposed to be associated with specifi cally binding to and inhibiting IκB kinase complex (IKK) which plays an important role in pro-infl ammatory cytokine-mediated signaling [1,21].
Chloroform leaf extracts, rich in sesquiterpene lactones including parthenolide, inhibit production of infl ammatory prostaglandins in rat and human leukocytes. Inhibition was irreversible and the effect was not caused by cytotoxicity.
Studies have shown that lipophilic compounds other than parthenolide may be associated with anti-infl ammatory activity, particularly with reducing human neutrophil oxidative burst activity [1,[22][23][24]. Inhibition of prostaglandin synthetase also has been documented for parthenolide [1,25,26]. The anti-infl ammatory effects of feverfew could also be caused by a cytotoxic effect. Feverfew extracts were found to inhibit mitogen-induced tritiated thymidine uptake by human peripheral blood mononuclear cells, interleukin-2-induced tritiated thymidine uptake by lymphoblasts, and prostaglandin release by interleukin-1-stimulated synovial cells. Parthenolide was demonstrated to block tritiated thymidine uptake by mitogen-induced human peripheral blood mononuclear cells [1,27]. Parthenolide has been widely used as an active marker for standardization and quality control. Feverfew products are required to contain no less than 0.1% parthenolide in France and 0.2% parthenolide in the US, UK and Canada [28]. Many solvent systems have been reported to extract feverfew for high recovery of parthenolide. It was observed that methanol and 50% ethanol are the two best candidates for highest percentage of parthenolide from feverfew, 50% ethanol is a little better for feverfew extract, while methanol works better for feverfew crude material [28].
However, it is believed that alpha-unsaturated gammalactones such as parthenolide could cause many allergic reactions [14,[29][30][31][32][33], which arose the interest of using feverfew extract with reduced levels of alpha-unsaturated gamma-lactones including parthenolide for therapeutic applications since feverfew also contain other ingredients such as fl avonoids which exhibit anti infl ammatory activities [34]. Bombardelli and Morazzoni [20], disclosed a procedure to prepare extracts of feverfew with a reduced content of alphaunsaturated gamma-lactones, particularly of parthenolide by multistep extraction and elution on basic resins.
This study investigated the ingredients of the aforementioned feverfew extract substantially free of parthenolide and other alpha-unsaturated gamma-lactones. It was confi rmed that the level of parthenolide (19) is pretty low (<0.07%, Table 2) with no other alpha-unsaturated gamma-lactones detected. More than twenty ingredients were characterized to belong mainly to two categories, caffeoyl derivatives and fl avonoids. Four caffeoyl derivatives, i.e. caffeic acid methyl ester (2), cynarin (13), 4-methoxyl caffeic acid (22), and 3,4-dimethoxyl caffeic acid (27), were observed for the fi rst time from feverfew extract to the best of our knowledge. The extract was especially enriched in caffeoyl derivatives including chlorogenic acid (7) (12.00%), 3,4-di-O-caffeoyl quinic acid (14) (23.33%), and 3,5-di-O-caffeoyl quinic acid (16) (31.78% together with coeluted apigenin-7-O-glucuronide (11)). It is worthwhile to point out that caffeoyl derivatives appears to be overlooked ingredients in feverfew as refl ected in almost all the review articles on feverfew with less attention. Both caffeoyl derivatives and fl avonoids are well known antioxidants. Caffeoyl derivatives may even have higher antioxidative potency due to their polyphenolic features, which is consistent with the measured antioxidative activity by DPPH free radical scavenging assay ( Table 4) to indicate that the feverfew extract exhibited considerably high antioxidant capacity. Antioxidation is closely associated with anti-infl ammatory and other therapeutic benefi ts. The feverfew extract investigated is substantially free of parthenolide and other alpha-unsaturated gamma-lactones. It is reasonable to expect that the extract still has favorable pharmacological properties together with reduced risks of allergic reactions.

Conclusion
The feverfew extract that was substantially free of parthenolide (<0.07%) and other alpha-unsaturated gammalactones was analyzed with twenty-eight ingredients observed. Th structural identities of the ingredients were elucidated mainly using HPLC-UV, HPLC-MS and comparison with reference compounds. Five ingredients were isolated and characterized further using NMR analyses to determine their structures. Two major groups of ingredients, c affeoyl derivatives and fl avonoids as well as parthenolide at trace level, were observed. Four ingredients, i.e. caffeic acid methyl ester (2), cynarin (13), 4-methoxyl caffeic acid (22), and 3,4-dimethoxyl caffeic acid (27), were new fi ndings in feverfew extract to the best of our knowledge. The feverfew extract displayed considerably high antioxidant capacity in a DPPH free radical scavenging assay, which is likely due to the existence of phenolic moieties in c affeoyl derivative and fl avonoid ingredients. The extract was especially enriched in caffeoyl derivatives including chlorogenic acid (7) (12.00%), 3,4-di-O-caffeoyl quinic acid (14) (23.33%), and 3,5-di-O-caffeoyl quinic acid (16) (31.78% together with coeluted apigenin-7-O-glucuronide (11)). The antioxidant capacity may potentially contribute to its pharmacological benefi ts such as anti-infl ammatory, inhibition of UV induced matrix metalloproteinase-1 (MMP-1), prevention of smokeinduced loss of thiols, etc. [14,15]. The feverfew extract investigated was substantially free of parthenolide and other alpha-unsaturated gamma-lactones to reduce undesirable allergic reactions.