Microbial Transformation of the Sesquiterpene Lactone, Vulgarin, by Aspergillus niger

The biotransformation of vulgarin (1), an eudesmanolides-type sesquiterpene lactone obtained from Artemisia judaica, by the microorganism, Aspergillus niger, was carried out to give three more polar metabolites; 1-epi-tetrahydrovulgarin (1α,4α-dihydroxy-5αH,6,11βH-eudesman-6,12-olide (2), 20% yield, 1α,4α-dihydroxyeudesm-2-en-5αH,6,11βH-6,12-olide (3a), 10% yield, and C-1 epimeric mixture (3a, b), 4% yield, in a ratio of 4:1, 3a/3b. The structures of vulgarin and its metabolites were elucidated by 1 and 2D NMR spectroscopy in conjunction with HRESIMS. Metabolites (3a) and (3b) are epimers, and they are reported here for the first time as new metabolites obtained by biotransformation by selective reduction at C-1. Vulgarin and its metabolites were evaluated as anti-inflammatory agents using the human cyclooxygenase (COX) inhibitory assay. The obtained data showed that (1) exhibited a good preferential inhibitory activity towards COX-2 (IC50 = 07.21 ± 0.10) and had a moderate effect on COX-1 (IC50 = 11.32 ± 0.24). Meanwhile, its metabolite (3a) retained a selective inhibitory activity against COX-1 (IC50 = 15.70 ± 0.51). In conclusion, the results of this study revealed the necessity of the presence α, β unsaturated carbonyl group in (1) for better COX-2 inhibitory activity. On the other hand, the selectivity of (1) as COX-1 inhibitor may be enhanced via the reduction of C-1 carbonyl group.


Introduction
Microbial transformation is the specific modification of a definite compound to a distinct product with structural similarity with the use of biological catalysts including microorganisms, mainly fungi. It is an alternative to chemical reactions for searching for new derivatives with enhanced biological activities [1]. In recent years, using the frequency of the microbial transformation technique has increasing significantly, from there being a limited number of trials to it being highly active area in green chemistry, including the preparation of pharmaceutical products. Microbes now are being used as biological shelf reagents [2,3].
One of the main advantages of microbial transformation is the selective introduction of functional groups to the carbon skeleton of xenobiotic resulting for the production of new metabolites, which are difficult to be obtained by chemical reactions. Moreover, microbial transformation helps to study the metabolic fate of the xenobiotics, as it is suggested that microbial transformation can mimic the mammalian metabolism. Therefore, microbial transformation can be an alternative to animal models to study xenobiotic metabolism [2]. The main goal of biotransformation is the conversion of poorly excreted lipophilic molecules into more easily excreted hydrophilic metabolites [4].
Aspergillus niger is a filamentous fungus and one of the most common species of the genus Aspergillus, and it is found in soil, decaying vegetation, seeds, and grains. It is one of the most important microorganisms used in biotechnology. A. niger is used for biotransformation and waste treatment. Since the 1960s, A. niger has been a source of a variety of enzymes that are well developed as technical aids in fruit processing and baking. In addition to its industrial uses in the production of citric acid and extracellular enzymes. A. niger has the ability to produce not only proteins and enzymes at high concentrations, but also pharmaceuticals that are beneficial for humans and animals. Intense research on A. niger over the last decade has resulted in a range of new products and processes [5].
Eudesmanolides-type sesquiterpene lactone, vulgarin (1), was isolated from different Artemisia species (family Asteraceae), including Artemisia vulgaris, after which it was named [6]. Vulgarin was assigned other names such as judaicin from Artemisia judaica [7]. In addition, vulgarin can be obtained by microbial transformation [8], as well as chemical the reduction of peroxyvulgarin [9]. Vulgarin (1) is a cytotoxic agent due to the presence of α, β unsaturated ketone [10]. It was found to be a promising candidate for treatment of different illness because of its potent anti-inflammatory, hypoglycemic, anti-bacterial, and anti-tumor activities [11][12][13][14]. The multiple biological activities of vulgarin make it an attractive target for microbial transformation studies.
The prolonged use of non-selective non-steroidal anti-inflammatory drugs (NSAIDs) results in severe side effects such as gastrointestinal hemorrhage due to inhibition of cyclooxygenase-1 (COX-1) enzyme [16], while most of the COX-2 selective drugs have been found to cause cardiovascular problems [17]. Consequently, there is a strong need to look for anti-inflammatory agents of a natural origin with minimum side effects.
The objective of this work is to utilize microorganisms for reinvestigating the biotransformation of vulgarin for the production of metabolites with enhanced anti-inflammatory activity and to study the metabolism of vulgarin by liver enzymes when used as an antiinflammatory drug depending on the capability of microbial transformation to mimic mammalian metabolism. Moreover, this work aimed to study enzymatic reactions carried out by microorganisms, which have the advantage of selectivity over chemical reactions.

Structure Elucidation of Vulgarin Metabolites
The preparative incubation of vulgarin with Aspergillus niger (ATCC 10549) was carried out. A. niger metabolized vulgarin (1) after 12 days incubation into three metabolites (2), (3a), and (3a, b) (Figure 1), which are more polar than (1) is. After the chromatographic isolation and purification of three vulgarin metabolites, the identification of isolated pure metabolites was achieved using different spectroscopic techniques, including 1D and 2D NMR and MS analyses ( Figures S1-S25).

Structural Elucidation of Metabolite (2)
NMR spectra for vulgarin and the three metabolites showed a close resemblance with small differences, as shown in Tables 1 and 2. Metabolite (2) showed the absence of α, β unsaturated carbonyl; instead it showed one more oxygenated methine at ⸹C 73.3 ppm and two extra methylenes at 33.0 and 36.4 ppm. HRESIMS showed a quasi-molecular ion peak at 291.1564 (M+Na) + calculated for C15H24O4 (291.1572) with four degrees of unsaturation. As per Table 1, 1 HNMR revealed the presence of three methyls at ⸹H 1.00 (S), 1.23(d, J = 6.9Hz), and 1.36 (S), assigned to methyls 14, 13, and 15, respectively. A downfield double of doublet lactonic proton resonating at ⸹H 4.07 (dd, J = 10.7, 10.6 Hz) assigned to H6 and a broad singlet resonating at ⸹H 3.41 assigned to H1 are the last protons connecting directly to their corresponding carbon via one bond length in HSQC experiment at ⸹c 81.4 and 73.3, respectively. The above data, together with 13 CNMR, confirmed the structure of (2) to be vulgarin metabolite with the replacement of carbonyl at C1 by oxygenated methine group and the saturation of the olefenic double bond between carbons 2 and 3. Moreover, an APT experiment showed a total of 15 carbon atoms distinguished into three methyles, 4 methylene, 5 methins, and three quaternaries. The three quaternaries resonated at 41.6, 71.5, and 178.7, assigned to C10, C4 and C12, respectively. The key connectivity between protons 1 and 2; 2 and 3; 5 and 6; 7 and 6, 8,11; 8 and 7,9 were proved by the observed cross peaks correlation between these protons in the COSY experiment.

Structural Elucidation of Metabolite (2)
NMR spectra for vulgarin and the three metabolites showed a close resemblance with small differences, as shown in Tables 1 and 2. Metabolite (2) showed the absence of α, β unsaturated carbonyl; instead it showed one more oxygenated methine at NMR spectra for vulgarin and the three metabolites showed a close rese small differences, as shown in Tables 1 and 2. Metabolite (2) showed the ab unsaturated carbonyl; instead it showed one more oxygenated methine at and two extra methylenes at 33.0 and 36.4 ppm. HRESIMS showed a quasipeak at 291.1564 (M+Na) + calculated for C15H24O4 (291.1572) with four degre ration. As per Table 1, 1 HNMR revealed the presence of three methyls a 1.23(d, J = 6.9Hz), and 1.36 (S), assigned to methyls 14, 13, and 15, respectiv field double of doublet lactonic proton resonating at ⸹H 4.07 (dd, J = 10.7 signed to H6 and a broad singlet resonating at ⸹H 3.41 assigned to H1 are th connecting directly to their corresponding carbon via one bond length in H ment at ⸹c 81.4 and 73.3, respectively. The above data, together with 13 CNM the structure of (2) to be vulgarin metabolite with the replacement of carb oxygenated methine group and the saturation of the olefenic double bond bons 2 and 3. Moreover, an APT experiment showed a total of 15 carbon guished into three methyles, 4 methylene, 5 methins, and three quaternar quaternaries resonated at 41.6, 71.5, and 178.7, assigned to C10, C4 and C12 The key connectivity between protons 1 and 2; 2 and 3; 5 and 6; 7 and 6, 8 were proved by the observed cross peaks correlation between these protons experiment.  NMR spectra for vulgarin and the three metabolites showed a close re small differences, as shown in Tables 1 and 2. Metabolite (2) showed the unsaturated carbonyl; instead it showed one more oxygenated methine and two extra methylenes at 33.0 and 36.4 ppm. HRESIMS showed a quas peak at 291.1564 (M+Na) + calculated for C15H24O4 (291.1572) with four deg ration. As per Table 1, 1 HNMR revealed the presence of three methyls 1.23(d, J = 6.9Hz), and 1.36 (S), assigned to methyls 14, 13, and 15, respect field double of doublet lactonic proton resonating at ⸹H 4.07 (dd, J = 10 signed to H6 and a broad singlet resonating at ⸹H 3.41 assigned to H1 are connecting directly to their corresponding carbon via one bond length in ment at ⸹c 81.4 and 73.3, respectively. The above data, together with 13 CN the structure of (2) to be vulgarin metabolite with the replacement of car oxygenated methine group and the saturation of the olefenic double bon bons 2 and 3. Moreover, an APT experiment showed a total of 15 carbo guished into three methyles, 4 methylene, 5 methins, and three quatern quaternaries resonated at 41.6, 71.5, and 178.7, assigned to C10, C4 and C1 The key connectivity between protons 1 and 2; 2 and 3; 5 and 6; 7 and 6 were proved by the observed cross peaks correlation between these proto experiment.

Structural Elucidation of Metabolite (2)
NMR spectra for vulgarin and the three metabolites showed a close resemblance with small differences, as shown in Tables 1 and 2. Metabolite (2) showed the absence of α, β unsaturated carbonyl; instead it showed one more oxygenated methine at ⸹C 73.3 ppm and two extra methylenes at 33.0 and 36.4 ppm. HRESIMS showed a quasi-molecular ion peak at 291.1564 (M+Na) + calculated for C15H24O4 (291.1572) with four degrees of unsaturation. As per Table 1, 1 HNMR revealed the presence of three methyls at ⸹H 1.00 (S), 1.23(d, J = 6.9Hz), and 1.36 (S), assigned to methyls 14, 13, and 15, respectively. A downfield double of doublet lactonic proton resonating at ⸹H 4.07 (dd, J = 10.7, 10.6 Hz) assigned to H6 and a broad singlet resonating at ⸹H 3.41 assigned to H1 are the last protons connecting directly to their corresponding carbon via one bond length in HSQC experiment at ⸹c 81.4 and 73.3, respectively. The above data, together with 13 CNMR, confirmed the structure of (2) to be vulgarin metabolite with the replacement of carbonyl at C1 by oxygenated methine group and the saturation of the olefenic double bond between carbons 2 and 3. Moreover, an APT experiment showed a total of 15 carbon atoms distinguished into three methyles, 4 methylene, 5 methins, and three quaternaries. The three quaternaries resonated at 41.6, 71.5, and 178.7, assigned to C10, C4 and C12, respectively. The key connectivity between protons 1 and 2; 2 and 3; 5 and 6; 7 and 6, 8,11; 8 and 7,9 were proved by the observed cross peaks correlation between these protons in the COSY experiment.

Structural Elucidation of Metabolite (2)
NMR spectra for vulgarin and the three metabolites showed a close resemblance with small differences, as shown in Tables 1 and 2. Metabolite (2) showed the absence of α, β unsaturated carbonyl; instead it showed one more oxygenated methine at ⸹C 73.3 ppm and two extra methylenes at 33.0 and 36.4 ppm. HRESIMS showed a quasi-molecular ion peak at 291.1564 (M+Na) + calculated for C15H24O4 (291.1572) with four degrees of unsaturation. As per Table 1, 1 HNMR revealed the presence of three methyls at ⸹H 1.00 (S), 1.23(d, J = 6.9Hz), and 1.36 (S), assigned to methyls 14, 13, and 15, respectively. A downfield double of doublet lactonic proton resonating at ⸹H 4.07 (dd, J = 10.7, 10.6 Hz) assigned to H6 and a broad singlet resonating at ⸹H 3.41 assigned to H1 are the last protons connecting directly to their corresponding carbon via one bond length in HSQC experiment at ⸹c 81.4 and 73.3, respectively. The above data, together with 13 CNMR, confirmed the structure of (2) to be vulgarin metabolite with the replacement of carbonyl at C1 by oxygenated methine group and the saturation of the olefenic double bond between carbons 2 and 3. Moreover, an APT experiment showed a total of 15 carbon atoms distinguished into three methyles, 4 methylene, 5 methins, and three quaternaries. The three quaternaries resonated at 41.6, 71.5, and 178.7, assigned to C10, C4 and C12, respectively. The key connectivity between protons 1 and 2; 2 and 3; 5 and 6; 7 and 6, 8,11; 8 and 7,9 were proved by the observed cross peaks correlation between these protons in the COSY experiment.  NMR spectra for vulgarin and the three metabolites showed a clo small differences, as shown in Tables 1 and 2. Metabolite (2) showed unsaturated carbonyl; instead it showed one more oxygenated meth and two extra methylenes at 33.0 and 36.4 ppm. HRESIMS showed a peak at 291.1564 (M+Na) + calculated for C15H24O4 (291.1572) with four ration. As per Table 1, 1 HNMR revealed the presence of three met 1.23(d, J = 6.9Hz), and 1.36 (S), assigned to methyls 14, 13, and 15, res field double of doublet lactonic proton resonating at ⸹H 4.07 (dd, J signed to H6 and a broad singlet resonating at ⸹H 3.41 assigned to H1 connecting directly to their corresponding carbon via one bond leng ment at ⸹c 81.4 and 73.3, respectively. The above data, together with the structure of (2) to be vulgarin metabolite with the replacement o oxygenated methine group and the saturation of the olefenic double bons 2 and 3. Moreover, an APT experiment showed a total of 15 c guished into three methyles, 4 methylene, 5 methins, and three qua quaternaries resonated at 41.6, 71.5, and 178.7, assigned to C10, C4 an The key connectivity between protons 1 and 2; 2 and 3; 5 and 6; 7 an were proved by the observed cross peaks correlation between these p experiment. c 81.4 and 73.3, respectively. The above data, together with 13 CNMR, confirmed the structure of (2) to be vulgarin metabolite with the replacement of carbonyl at C1 by oxygenated methine group and the saturation of the olefenic double bond between carbons 2 and 3. Moreover, an APT experiment showed a total of 15 carbon atoms distinguished into three methyles, 4 methylene, 5 methins, and three quaternaries. The three quaternaries resonated at 41.6, 71.5, and 178.7, assigned to C10, C4 and C12, respectively. The key connectivity between protons 1 and 2; 2 and 3; 5 and 6; 7 and 6, 8,11; 8 and 7,9 were proved by the observed cross peaks correlation between these protons in the COSY experiment.
An NOESY experiment was important to determine the orientation of the hydroxyl group at C1 as α oriented by observation of NOESY cross-speak correlations between H1 and Me14 and Me15 ( Figure 2). On the other hand, the α orientation of H5 was proved by the lack of NOE correlation with Me15, while the orientations at C6, C7, and C11 were proved to be as the same of those of vulgarin. The structure of compound (2) was verified as 1-epi tetrahydrovulgarin (20% yield without optimization), previously isolated from Artemisia canariensis [18] and obtained as metabolite by fungus Beauveria bassiana transformation [15]. Such as (2), compound (3a) showed similarity to vulgarin, except for the lack of carbonyl at C1 ( 13 CNMR), and instead, an extra 2ry alcoholic group appeared in 1 HNMR at 3 of 11 tic presentation of microbial transformation of vulgarin by Aspergillus niger.

Elucidation of Metabolite (2)
ra for vulgarin and the three metabolites showed a close resemblance with s, as shown in Tables 1 and 2. Metabolite (2) showed the absence of α, β bonyl; instead it showed one more oxygenated methine at ⸹C 73.3 ppm ethylenes at 33.0 and 36.4 ppm. HRESIMS showed a quasi-molecular ion (M+Na) + calculated for C15H24O4 (291.1572) with four degrees of unsatuable 1, 1 HNMR revealed the presence of three methyls at ⸹H 1.00 (S), ), and 1.36 (S), assigned to methyls 14, 13, and 15, respectively. A downdoublet lactonic proton resonating at ⸹H 4.07 (dd, J = 10.7, 10.6 Hz) asd a broad singlet resonating at ⸹H 3.41 assigned to H1 are the last protons ctly to their corresponding carbon via one bond length in HSQC experiand 73.3, respectively. The above data, together with 13 CNMR, confirmed (2) to be vulgarin metabolite with the replacement of carbonyl at C1 by thine group and the saturation of the olefenic double bond between caroreover, an APT experiment showed a total of 15 carbon atoms distinree methyles, 4 methylene, 5 methins, and three quaternaries. The three onated at 41.6, 71.5, and 178.7, assigned to C10, C4 and C12, respectively. tivity between protons 1 and 2; 2 and 3; 5 and 6; 7 and 6, 8,11; 8 and 7,9 the observed cross peaks correlation between these protons in the COSY ity (J in parentheses in Hz) of vulgarin and its metabolites as per 1 H-NMR spectral

Structural Elucidation of Metabolite (2)
NMR spectra for vulgarin and the three metabolites showed a close resemblance w small differences, as shown in Tables 1 and 2. Metabolite (2) showed the absence of unsaturated carbonyl; instead it showed one more oxygenated methine at ⸹C 73.3 p and two extra methylenes at 33.0 and 36.4 ppm. HRESIMS showed a quasi-molecular peak at 291.1564 (M+Na) + calculated for C15H24O4 (291.1572) with four degrees of uns ration. As per Table 1, 1 HNMR revealed the presence of three methyls at ⸹H 1.00 1.23(d, J = 6.9Hz), and 1.36 (S), assigned to methyls 14, 13, and 15, respectively. A do field double of doublet lactonic proton resonating at ⸹H 4.07 (dd, J = 10.7, 10.6 Hz) signed to H6 and a broad singlet resonating at ⸹H 3.41 assigned to H1 are the last prot connecting directly to their corresponding carbon via one bond length in HSQC exp ment at ⸹c 81.4 and 73.3, respectively. The above data, together with 13 CNMR, confirm the structure of (2) to be vulgarin metabolite with the replacement of carbonyl at C1 oxygenated methine group and the saturation of the olefenic double bond between bons 2 and 3. Moreover, an APT experiment showed a total of 15 carbon atoms dis guished into three methyles, 4 methylene, 5 methins, and three quaternaries. The th quaternaries resonated at 41.6, 71.5, and 178.7, assigned to C10, C4 and C12, respectiv The key connectivity between protons 1 and 2; 2 and 3; 5 and 6; 7 and 6, 8,11; 8 and were proved by the observed cross peaks correlation between these protons in the CO experiment.  The key connectivity between protons 1 and 2; 2 and 3; 5 and 6; 7 an were proved by the observed cross peaks correlation between these pr experiment.

Structural Elucidation of Metabolite (2)
NMR spectra for vulgarin and the three metabolites showed a close resemblance with small differences, as shown in Tables 1 and 2  at ra for vulgarin and the three metabolites showed a close resemblance with s, as shown in Tables 1 and 2. Metabolite (2) showed the absence of α, β bonyl; instead it showed one more oxygenated methine at ⸹C 73.3 ppm ethylenes at 33.0 and 36.4 ppm. HRESIMS showed a quasi-molecular ion (M+Na) + calculated for C15H24O4 (291.1572) with four degrees of unsatuable 1, 1 HNMR revealed the presence of three methyls at ⸹H 1.00 (S), ), and 1.36 (S), assigned to methyls 14, 13, and 15, respectively. A downdoublet lactonic proton resonating at ⸹H 4.07 (dd, J = 10.7, 10.6 Hz) asd a broad singlet resonating at ⸹H 3.41 assigned to H1 are the last protons ctly to their corresponding carbon via one bond length in HSQC experiand 73.3, respectively. The above data, together with 13 CNMR, confirmed (2) to be vulgarin metabolite with the replacement of carbonyl at C1 by thine group and the saturation of the olefenic double bond between caroreover, an APT experiment showed a total of 15 carbon atoms distinree methyles, 4 methylene, 5 methins, and three quaternaries. The three onated at 41.6, 71.5, and 178.7, assigned to C10, C4 and C12, respectively. tivity between protons 1 and 2; 2 and 3; 5 and 6; 7 and 6, 8,11; 8 and 7,9 the observed cross peaks correlation between these protons in the COSY ity (J in parentheses in Hz) of vulgarin and its metabolites as per 1 H-NMR spectral c 70.4 and was assigned to C4. The final structure of metabolites (3a) was proved to be 1α,4α-dihydroxyeudesm-2-en-5α,6β,11β-6,12-olide (3a) (15% yield without optimization) based on the significant J 2 and J 3 HMBC correlations from H1 to C2, C3, C5, and C10; from H2 to C3, C5 and C10; from methyl 13 to C7; C11 and C12. The location of OH at C1 was confirmed on the lower surface of the molecule (α oriented) by noticing the NOESY cross-peaks between H1 and methyl 14 and methyl 15 (Figure 2). The aforementioned data confirmed the structure of (3) to be 1α,4α-dihydroxyeudesm-2-en-5α,6β,11β-6,12-olide (3) (10% yield without optimization) previously isolated from the aerial parts of Artemsia spicigera [19] and reported here for the first time by microbial transformation.

Structural Elucidation of Metabolite (3a, b)
Metabolite (3a, b) (4% yield without optimization) was isolated as a mixture of two compounds. It showed two molecular ion peaks, one at M+ Na at 289.2706 corresponding to (3a), and the other for (3b) appeared at 289.1405 for M-2 in HRESIMS. NMR spectra showed a little difference between (3a) and (3b), suggesting that the two compounds are isomers of each other. The main differences were noticed in 13 CNMR (Table 2) for C1, C5, C9, and C10, with downfield shifts of NMR spectra for vulgarin and the three metabolites showed a close resemblance with small differences, as shown in Tables 1 and 2. Metabolite (2) showed the absence of α, β unsaturated carbonyl; instead it showed one more oxygenated methine at ⸹C 73.3 ppm and two extra methylenes at 33.0 and 36.4 ppm. HRESIMS showed a quasi-molecular ion peak at 291.1564 (M+Na) + calculated for C15H24O4 (291.1572) with four degrees of unsaturation. As per Table 1, 1 HNMR revealed the presence of three methyls at ⸹H 1.00 (S), 1.23(d, J = 6.9Hz), and 1.36 (S), assigned to methyls 14, 13, and 15, respectively. A downfield double of doublet lactonic proton resonating at ⸹H 4.07 (dd, J = 10.7, 10.6 Hz) assigned to H6 and a broad singlet resonating at ⸹H 3.41 assigned to H1 are the last protons connecting directly to their corresponding carbon via one bond length in HSQC experiment at ⸹c 81.4 and 73.3, respectively. The above data, together with 13 CNMR, confirmed the structure of (2) to be vulgarin metabolite with the replacement of carbonyl at C1 by oxygenated methine group and the saturation of the olefenic double bond between carbons 2 and 3. Moreover, an APT experiment showed a total of 15 carbon atoms distinguished into three methyles, 4 methylene, 5 methins, and three quaternaries. The three quaternaries resonated at 41.6, 71.5, and 178.7, assigned to C10, C4 and C12, respectively. The key connectivity between protons 1 and 2; 2 and 3; 5 and 6; 7 and 6, 8,11; 8 and 7,9 were proved by the observed cross peaks correlation between these protons in the COSY experiment.    Table 1, 1 HNMR revealed the presence of three methyls at ⸹H 1.23(d, J = 6.9Hz), and 1.36 (S), assigned to methyls 14, 13, and 15, respectively. field double of doublet lactonic proton resonating at ⸹H 4.07 (dd, J = 10.7, 10.6 signed to H6 and a broad singlet resonating at ⸹H 3.41 assigned to H1 are the las connecting directly to their corresponding carbon via one bond length in HSQC ment at ⸹c 81.4 and 73.3, respectively. The above data, together with 13 CNMR, co the structure of (2) to be vulgarin metabolite with the replacement of carbonyl oxygenated methine group and the saturation of the olefenic double bond betw bons 2 and 3. Moreover, an APT experiment showed a total of 15 carbon atom guished into three methyles, 4 methylene, 5 methins, and three quaternaries. T quaternaries resonated at 41.6, 71.5, and 178.7, assigned to C10, C4 and C12, resp The key connectivity between protons 1 and 2; 2 and 3; 5 and 6; 7 and 6, 8,11; 8 were proved by the observed cross peaks correlation between these protons in t experiment.  1.36 (3b), respectively. The rest of NMR spectral data were almost the same for both metabolites, as shown in Tables 1 and 2. The NOESY experiment was valuable for confirming the orientation of OH at position 1 to be β orientation by the absence of NOESY cross-peaks between H1 and methyls 14 and 15 (Figure 2). The above-mentioned data confirmed that (3a) and (3b) are epimers of each other and present in the mixture at a ratio of 4:1 3a/3b. Metabolites (3a) and (3b) are reported here for the first time as new metabolites obtained by the biotransformation of vulgarin, and the structure of (3b) is 1β,4α-dihydroxyeudesm-2-en-5α,6β,11β-6,12-olide.
in close similarity to those of vulgarin, and this confirming the eudesamnolide basic nucleus for (3a).

Structural Elucidation of Metabolite (3a, b)
Metabolite (3a, b) (4% yield without optimization) was isolated as a mixture of two compounds. It showed two molecular ion peaks, one at M+ Na at 289.2706 corresponding to (3a), and the other for (3b) appeared at 289.1405 for M-2 in HRESIMS. NMR spectra showed a little difference between (3a) and (3b), suggesting that the two compounds are isomers of each other. The main differences were noticed in 13 CNMR (Table 2) for C1, C5, C9, and C10, with downfield shifts of ⸹H 2.1, 1.8, 1.0, and 1.4 ppm, respectively. A significant difference was observed for C1 and C5 chemical shifts. Moreover, 1 HNMR for oxygenated carbon at position 1 shifted upfield from 3.49 in (3a) to 3.41 ppm in (3b). The methyl chemical shift at position 14 was also downfield shifted from ⸹H 0.97 in (3a) to 1.00ppm in (3b), while for methyls 13 and 14, they showed a little upfield shift from 1.25 to 1.22 ppm in (3a) and from 1.39 (3a) to 1.36 (3b), respectively. The rest of NMR spectral data were almost the same for both metabolites, as shown in Tables 1 and 2. The NOESY experiment was valuable for confirming the orientation of OH at position 1 to be β orientation by the absence of NOESY cross-peaks between H1 and methyls 14 and 15 ( Figure 2). The above-mentioned data confirmed that (3a) and (3b) are epimers of each other and present in the mixture at a ratio of 4:1 3a/3b. Metabolites (3a) and (3b) are reported here for the first time as new metabolites obtained by the biotransformation of vulgarin, and the structure of (3b) is 1β,4α-dihydroxyeudesm-2-en-5α,6β,11β-6,12olide.

Proposed Mechanism of Selective Reduction
Aspergillus niger is considered as a source of oxidoreductases enzymes. NAD(P)Hdependent oxidoreductases enzymes, such as alcohol dehydrogenase (ADH), can selectively control the locus of reduction independent from multiple functional groups present within the starting compound [20]. Figure 3 showed the chemoselective reduction of the C-Aspergillus niger is considered as a source of oxidoreductases enzymes. NAD(P)Hdependent oxidoreductases enzymes, such as alcohol dehydrogenase (ADH), can selectively control the locus of reduction independent from multiple functional groups present within the starting compound [20]. Figure 3 showed the chemoselective reduction of the C-1 carbonyl group of vulgarin in the presence of a C2=C3 double bond. Moreover, the selective reduction of the Δ 2,3 double bond producing dihydrovulgarin has been previously reported [15].

COX-Inhibitory Activity of Vulgarin and Its Metabolites
Interestingly, a selective COX-1 inhibitor was equipotent to COX-2 selective inhibitor (celecoxib) for inhibiting PG formation in an inflammatory exudate. These findings and other data propose that the functions of COX-1 and COX-2 might be more complicated than they have been initially estimated and that COX-1 inhibition may contribute to the inhibition of PG production in inflammatory exudates. Hence, a combined inhibition of COX-1 and COX-2 may lead to the more efficient inhibition of chronic inflammation as compared to the selective inhibition of COX-1 or COX-2 [21].
The anti-inflammatory activity of vulgarin and its metabolites was evaluated by measuring their ability to inhibit COX-1 and COX-2 enzymes and comparing their inhibitory activity with that of a reference compounds, celecoxib and indomethacin (Table 3) [22,23]. Based upon the % of inhibitory activity of tested compounds on COX-1 and COX-2, IC50 (µ M/mL) were determined as shown in Table 4.

COX-Inhibitory Activity of Vulgarin and Its Metabolites
Interestingly, a selective COX-1 inhibitor was equipotent to COX-2 selective inhibitor (celecoxib) for inhibiting PG formation in an inflammatory exudate. These findings and other data propose that the functions of COX-1 and COX-2 might be more complicated than they have been initially estimated and that COX-1 inhibition may contribute to the inhibition of PG production in inflammatory exudates. Hence, a combined inhibition of COX-1 and COX-2 may lead to the more efficient inhibition of chronic inflammation as compared to the selective inhibition of COX-1 or COX-2 [21].

General
Using CDCl3 solvent and TMS as internal standard for chemical shifts, 1 H-NMR and 13 C-NMR spectra were recorded on Bruker DRX 500 and 700 NMR spectrometer operating at 500 and 700 MHz and 125 and 175 MHz, respectively. Chemical shifts (δ) are expressed in ppm with reference to TMS resonance. For 13 C NMR spectra, the number of attached protons was determined by DEPT 135° and 2D NMR data were obtained using the standard pulse sequence of the Bruker DRX-500 for COSY, HMQC, HMBC, and NOESY. The accurate mass determination was achieved with a JEOL JMS-700 High-Resolution Mass Spectrophotometer (JEOL USA Inc., Peabody, MA, USA) in positive and negative modes.
Normal phase chromatography was carried out using silica gel 60-120 mesh (Alpha Chemika, Mumbai, India) packed by the wet method in the specific solvents. Media ingredients and solvents used for extraction and chromatographic separation were purchased from El-Nasr Company for Pharmaceutical Chemicals, Egypt. Analytical thin layer chromatography was performed on precoated silica gel 60 GF254 on aluminum sheets (Merck, Germany). Plates were developed in different solvent mixtures, and developed chromatograms were visualized under UV light 254 and 366, and spots were made visible

General
Using CDCl 3 solvent and TMS as internal standard for chemical shifts, 1 H-NMR and 13 C-NMR spectra were recorded on Bruker DRX 500 and 700 NMR spectrometer operating at 500 and 700 MHz and 125 and 175 MHz, respectively. Chemical shifts (δ) are expressed in ppm with reference to TMS resonance. For 13 C NMR spectra, the number of attached protons was determined by DEPT 135 • and 2D NMR data were obtained using the standard pulse sequence of the Bruker DRX-500 for COSY, HMQC, HMBC, and NOESY. The accurate mass determination was achieved with a JEOL JMS-700 High-Resolution Mass Spectrophotometer (JEOL USA Inc., Peabody, MA, USA) in positive and negative modes.
Normal phase chromatography was carried out using silica gel 60-120 mesh (Alpha Chemika, Mumbai, India) packed by the wet method in the specific solvents. Media ingredients and solvents used for extraction and chromatographic separation were purchased from El-Nasr Company for Pharmaceutical Chemicals, Egypt. Analytical thin layer chromatography was performed on precoated silica gel 60 GF 254 on aluminum sheets (Merck, Germany). Plates were developed in different solvent mixtures, and developed chromatograms were visualized under UV light 254 and 366, and spots were made visible by spraying with vanillin sulphoric spray reagent after warming in an oven preheated to 105 • C for 1 min.

Plant Material
Artemisia judaica L. plant was collected from the Huraymila region, to the west of Riyadh city, in February 2019. The plant was identified by Dr. Mohammad Atiqur Rahman, a taxonomist of MAP-PRC at the Faculty of Pharmacy, King Saud University, Riyadh, Saudi Arabia. A voucher specimen (#16723) was kept in the herbarium in this center.

Extraction and Isolation of Vulgarin
The dried ground aerial parts (3 kg) of A. judaica were extracted by 95% ethanol (30 L) until exhaustion. The extract was evaporated under vacuum using rotatory evaporator, leaving a dark green residue which was subjected to liquid-liquid fractionated using solvents; The preliminarily screening showed that Aspergillus niger (ATCC 10549) was the most promising microorganism as it reproducibly produced several metabolites. Therefore, a preparative scale-up fermentation study was designed to isolate, identify, and study the possible mechanism of action.

Large Scale Fermentation
Aspergillus niger (ATCC 10549) was grown in five 250 mL Erlenmeyer flasks, each containing 50 mL of liquid media. A total of 200 mg of vulgarin in 1000 µL DMSO were evenly distributed among the 24 h old stage II culture. The incubation mixture was monitored, and fermentation was terminated after 12 days by extraction with CHCl 3 and concentration by evaporation under reduced pressure using a rotatory evaporator at 45 • C.

Isolation of Metabolites
The dried extract was applied on silica gel column using ethyl acetate/chloroform solvent system in gradient mode for elution analysis.
The first metabolite was eluted by 15% EtOAc/DCM, collected, pooled, and evaporated to give a pure metabolite (2) (20 mg). Silica gel GF 254 TLC plate showed a single pink spot, and then a violet spot at R f = 0.57 in 50% EtOAc /DCM solvent system after heating with vanillin sulphoric spray reagent.
The epimeric mixture (3a, b) was eluted by 20% EtOAc /DCM. Silica gel GF 254 TLC chromatogram showed brownish violet spot at R f = 0.46 in solvent system 50% EtOAc/DCM after heating with spray reagent. Evaporation of the collected fractions to dryness under reduced pressure yielded metabolite (3a, b) (8 mg).
Metabolite (3a) was eluted by 25% EtOAc /DCM. Silica gel GF 254 TLC chromatogram showed a brownish violet spot at R f = 0.46 in solvent system 50% EtOAc /DCM after heating with spray reagent. Evaporation of the collected fractions to dryness under reduced pressure yielded one metabolite (3a) (10 mg).

In Vitro COX-1 and COX-2 Enzyme Inhibitory Assay
The abilities of vulgarin and its metabolites to inhibit the conversion of arachidonic acid into PGH2 were evaluated using COX-1 Cayman human enzyme inhibitory assay kit (No. 701070), COX-2 Cayman human enzyme inhibitory assay kit (No. 701080, USA), and ROBONIK P2000 EIA reader. Evaluation of the data was performed by using Four Parameter Logistic Curve online data analysis tool of MyAssays Ltd. Procedures were carried out according to manufacturer's instructions [22,26] Celecoxib ® and indomethacin ® (Sigma-Aldrich, Burlington, MA, USA) were used as reference drugs. The selectivity indices (SI) of the tested/reference compounds (SI = IC 50 COX-1/IC 50 COX-2) were calculated [27].

Conclusions
Vulgarin, an eudesmanolides-type sesquiterpene lactone, was subjected to microbial transformation using several microbial strains. The most significant one was Aspergillus niger, as it reproducibly produced three metabolites in higher yields. The three metabolites were 1-epi-tetrahydrovulgarin (1α,4α-dihydroxy-5αH,6,11βH-eudesman-6,12-olide), 1α,4αdihydroxyeudesm-2-en-5αH,6,11βH-6,12-olide, and a C-1 epimeric mixture of the second metabolite. The second metabolite and its C-1 epimeric mixture were reported here for the first time to be obtained by the biotransformation of vulgarin, as selective reduction occurred at C-1. By evaluation of the anti-inflammatory activity using the human COX inhibitory assay, vulgarin showed potent activity. The structure-activity relationship of vulgarin indicated that the C 1 -α, β unsaturated carbonyl group is essential for the activity. Additionally, the reduction of the carbonyl group at C-1 to 2ry alcoholic group either αor β-increased the inhibitory activity toward COX-1 and decreased the inhibitory activity toward COX-2.