Chiral Coumarin Enantiomers From Prangos pabularia Roots: Absolute Configuration, Tyrosinase Activation, and Anti‐Vitiligo Potential in B16F10 Cells

ABSTRACT

In order to isolate and characterize enantiomeric compounds from the roots of Prangos pabularia the dichloromethane extract was extensively fractionated by column chromatography, flash, semi‐preparative high performance liquid chromatography (HPLC), and chiral HPLC. Six pairs of enantiomers were isolated from dichloromethane extracts using preparative chiral HPLC. The absolute configurations of the isolated isomers were identified using circular dichroism (CD). The anti‐vitiligo activities of the corresponding (S)‐ and (R)‐enantiomers were assessed by determining their tyrosinase activation rates. (S)‐Pangelin 9 (PPE19‐1) showed higher activity with 125.42% than (R)‐pangelin 10 (PPE19‐2) with 120.6%, while the positive control (8‐methoxypsoralen) resulted in 122.3%. The percentage of activation rate of (S)‐oxypeucidanin hydrate 1 (PPE4‐1) was 119.74%, while it was 110.01% for (R) oxypeucidanin hydrate 2 (PPE4‐2). We conclude that the (S, R) enantiomers of pangelin and oxypeucidanin hydrate (S, R) may be useful as pigmentation‐promoting agents for the treatment of vitiligo.

Six pairs of enantiomers were isolated from Prangos pabularia roots by preparative chiral HPLC. Anti‐vitiligo activity was evaluated through tyrosinase activation. (S)‐Pangelin showed the highest activity (125.42%), exceeding (R)‐pangelin (120.6%), and 8‐methoxypsoralen (122.3%). Pangelin and oxypeucidanin hydrate enantiomers may serve as pigmentation‐promoting agents for vitiligo treatment.

1. Introduction

The Prangos genus (Apiaceae) consists of 72 species, which are perennial plants distributed in the Mediterranean region and Western and Central Asia [1]. This genus is fairly well distributed from Afghanistan [2] to India (Kashmir), Pakistan, Iran, Iraq, Russia, Turkey, Central Asia, and Caucasian at an altitude ranging from 780 to 3300 m [3]. Plants of the Prangos genus are commonly found around Dras in Ladakh, the Banihal pass in Kashmir and the Zanskar range (Western Himalaya). The plants grow in light (sandy) and medium (loamy), slightly acidic to neutral (pH–6.3 to 7.3) well drained soils [4].

The ethnomedicinal applications of the Prangos genus vary depending on the country where these plants grow. Moreover, Prangos species are used as stimulants, aphrodisiacs, and natural fertilizers [5, 6]. Prangos species are widely used in traditional medicines for their aphrodisiac, coagulant, carminative, and tonic effects [7, 8]. The whole plant of Prangos pabularia has medicinal properties and is used antioxidant [9], diuretic [10], antibacterial [11], antifungal [12], cytotoxic herb. The root of this plant is rich in coumarins and furanocoumarins [13]. Coumarins are important secondary plant metabolites in plants with various biological properties such as antitumor, anti‐HIV, antimicrobial, and anti‐inflammatory activities [14].

According to the pharmacological investigation coumarins are exciting central nervous effects and inhibitory effects on tyrosinase, that catalyzes the tyrosine oxidation to produce of melanin and other pigments in plants and animals. Tyrosinase is known to be a multifunctional copper‐containing enzyme from the oxidase superfamily, and it is the key protein involved in the biosynthesis of the large biological pigment, melanin [15].

A significant correlation has been observed between melanocytes, tyrosinase (TYR), and the manifestation of vitiligo. In the human skin, melanocytes are situated in the basal layer of the epidermis, with the melanosomes within melanocytes comprising the specific sites required for melanogenesis. The primary role of melanocytes is to produce melanin by oxidizing tyrosine, a process that is carried out by enzymes specific to melanocytes. Melanin is stored in melanosomes and transferred to neighboring keratinocytes to protect the skin from UV radiation. Keratinocytes are essential in maintaining melanocyte homeostasis and melanogenesis, and they form a functional and structural unit [16].

In regulating the expression of MITF the Wnt signaling pathway plays a crucial role. GSK‐3β is a key enzyme involved in this pathway, and it facilitates the ubiquitination and subsequent degradation of β‐catenin. Within the Wnt signaling pathway, phosphorylated (p) AKT triggers the phosphorylation of GSK3‐β, leading to its inhibition. Consequently, β‐catenin accumulates in the cytoplasm and is subsequently translocated to the nucleus, where it promotes the expression of MITF [17].

The substituents of coumarins (e.g., prenyl substitution) have chiral carbons, which produce enantiomers in the plant. Enantiomers with different biological activities have been encountered in bioactive natural products from medicinal plants and other sources [18, 19].

Enantiomers are interesting and important natural compounds. Chirality corresponds to the relationship between the “left and right hands”, which mirror each other. Even if they have the same structure, they cannot overlap. Enantiomers have the same physical and chemical properties [20]. Substances such as amino acids, proteins, DNA, and polysaccharides in living organisms are abundant chiral compounds [21]. Chirality is widely involved in materials, drugs, biochemistry, agriculture, and many other fields closely related to human daily life [22]. Chiral molecules usually exist in one enantiomeric form in living organisms. Chirality represents the most essential properties of life processes from the molecular level. Molecules that are enantiomers of each other have different physiological activities in living organisms [23, 24]. Therefore, the purity of chiral compounds is one of the most important aspects to the living organism. Chiral drugs are widely used for the treatment of various diseases [25, 26]. Investigation of chiral compounds confirmed that a pair of enantiomers have different activities; one enantiomer is effective, and its mirror molecule is ineffective or even toxic [27]. Life systems tend to produce/utilize only one molecule of an enantiomeric pair [28]. It has been shown by scientists, for example, that humans only take in D‐glucose and L‐amino acids as nutrients. The fact that a pair of enantiomers can exert totally different bioactivities was recognized back in the 1960s when the “Phocomelia infants’ event” caused by the (S)‐enantiomer of the synthetic drug thalidomide taught the pharmaceutical industry an important lesson [29]. For many years, however, natural product workers failed to recognize the widespread occurrence of enantiomers in nature and failed to explore the differences in bioactivity between pairs of enantiomers. Fortunately, as data on natural enantiomers increase in scope, more and more biological properties of different classes of enantiomeric pairs have also been reported [30, 31, 32], and this has provided more examples with which to investigate the differences in bioactivity among enantiomers [14, 33]. Reducing of the side effects the purity of chiral drugs is very important and it's one of the big problems for researchers and a big issue in a word. In fact, most chiral compounds in nature usually occur as racemates [34]. A racemate is an equal mixture of pairs of enantiomers. A single pure enantiomer can be obtained by different methods of separations of natural and synthetic compounds [35]. However, there are still many problems to be solved in asymmetric synthesis, such as low yield, high cost, and by‐products. In contrast, enantiomer separation is an ideal method to obtain chiral drugs. At present, a large number of pure chiral drugs are obtained by enantiomer separation [35].

Previous our investigation resulted on the isolation of new coumarins Yuganin A, B [3, 36], and five unidentified compounds, including two prenylated coumarin glucosides, two prenylated furanocoumarin glucosides, and a benzofuran glucoside with the evaluation of their anti‐vitiligo effects of P. Pabularia [37].

In our previous publication we were reported about the high potential of anti‐vitiligo activity of coumarins isolated from the roots of P. pabularia [37]. To conduct a comprehensive study of this issue, we discovered that this plant contains isomers of the active compounds. The formation of enantiomeric coumarins is not as common as that of other compounds, such as lignans and flavonoids. Therefore, the biosynthesis of these enantiomeric secondary metabolites has not been deeply investigated [33]. According to the published material we also decided to study the enantiomers effect in these coumarins as it was not reported before us.

In the present investigation, six pairs of enantiomeric coumarins were isolated and separated from the roots of P. pabularia. Their absolute configurations were established, and the biological activities of the individual enantiomers toward B16F10 melanoma cells were evaluated.

2. Results and Discussion

We found enantiomer mixtures of furano‐coumarins in the extract of P. pabularia. Based on ${[\alpha ]}_{\mathrm{D}}^{20}$ values, (R) and (S) stereoisomers were confirmed in this extract, as the value was close to zero. To the best of our knowledge, no such analyses have been previously published, and the separation of enantiomers from P. pabularia and their bioactivity are reported here for the first time.

2.1. Structure Elucidation of Isolated Compounds

The structures of isolated compounds were identified based on spectroscopic data including HRESIMS, 1D, and 2D NMR data comparison with reported data.

2.1.1. Oxypeucedanin (PPE 4)

HRESIMS m/z 287.0905 [M + H]⁺ (calcd for C₁₆H₁₄O₅ ⁺ 287.0914).¹Н NMR (CDCl₃, 400 MHz): 6.32 (1H, d, J = 9.8 Hz, H‐3), 8.20(1H, dd, J = 9.8; 0.5 Hz, H‐4), 7.19 (1H, br.s., H‐8), 6.96 (1H, dd, J = 2.3; 0.9 Hz, H‐11), 7.62 (1H, d, J = 2.3 Hz, H‐12), 4.44 (2H, dd, J = 7.2; 6.5 Hz, H‐13), 3.24 (1H, dd, J = 6.5; 4.4 Hz, H‐14), 1,41 (3H, s., H‐16), 1,34 (3H, s., H‐17); ¹³СNMR (CDCl₃, 100 MHz): 161.6 (C‐2), 113.3 (C‐3), 139.1 (C‐4), 148.5 (C‐5), 114.4 (C‐6), 158.2 (C‐7), 95.0 (C‐8), 152.7 (C‐9), 107.6 (C‐10), 104.6 (C‐11), 145.4 (C‐12), 72.5 (C‐13), 61.2 (C‐14), 58.5 (C‐15), 19.2 (C‐16), 24.8 (C‐17) [38].

2.1.2. 8‐Oxypucedanin Hydrate (PPE‐9)

HRESIMS m/z 327.0825 [M + Na]⁺ (calcd for C₁₆H₁₆O₆Na 327.0839). ¹Н NMR (DMSO‐d6, 600 MHz): 6.43 (1H, d, J = 9.6 Hz, H‐3), 8.14 (1H, d, J = 9.6 Hz, H‐4), 7.66 (1H, s, H‐5), 7.09 (1H, d, J = 2.1 Hz, H‐11), 8.12 (1H, d, J = 2.1 Hz, H‐12), 4.38 (1H, dd, J = 10.1, 8.3 Hz, H‐13), 4.64 (1H, dd, J = 10.1, 2.4 Hz, H‐13), 3.67 (1H, ddd J = 8.3, 5.7, 2.4 Hz, H‐14), 1.07 (3H, s, H‐16), 1.15 (3H, s, H‐17), 5.02 (1H, d, J = 5.7 Hz, 14‐H), 4.39 (1H, s, 15‐H); ¹³С NMR (DMSO‐d6, 150 MHz): 159.8 (C‐2), 114.2 (C‐3), 145.3 (C‐4), 113.4 (C‐5), 125.8 (C‐6), 147.1 (C‐7), 131.6 (C‐8), 142.6 (C‐9), 116.4 (C‐10), 107.0 (C‐11), 147.7 (C‐12), 75.4 (C‐13), 76.9 (C‐14), 70.8 (C‐15), 24.4 (C‐16), 27.3 (C‐17) [39].

2.1.3. 5‐Oxypucedanin Hydrate (PPE14)

HRESIMS m/z 327.0839 [M + Na]⁺ (calcd for C₁₆H₁₆O₆Na 327.0825). ¹Н NMR (DMSO‐d6, 400 MHz): 6.33 (1H, d, J = 9.8 Hz, H‐3), 8.37 (1H, d, J = 9.8 Hz, H‐4), 7.32 (1H, s, H‐8), 7.30 (1H, d, J = 2.2; 1 Hz, H‐11), 8.01 (1H, d, J = 2.5 Hz, H‐12), 4.74 (1H, dd, J = 9.9; 2.1 Hz, H‐13), 4.27 (1H, dd, J = 9.7; 6.8 Hz, H‐13), 3.64 (1H, dd, J = 8.4; 2.1 Hz, H‐14), 1.17 (3H, s, H‐16), 1.10 (3H, s, H‐17); ¹³С NMR (DMSO‐d6, 100 MHz): 160.2 (C‐2), 112.1 (C‐3), 140.1 (C‐4), 152.1 (C‐5), 113.4 (C‐6), 157.6 (C‐7), 93.3 (C‐8), 145.9 (C‐9), 106.5 (C‐10), 105.5 (C‐11), 149.3 (C‐12), 74.9 (C‐13), 76.3 (C‐14), 70.7 (C‐15), 27.7 (C‐16), 24.2 (C‐17) [39].

2.1.4. 5‐Oxypucedanin Methanolate (PPE18)

HRESIMS m/z 341.0995 [M + Na]⁺ (calcd for C₁₇H₁₈O₆Na 341.0985). ¹Н NMR (DMSO‐d₆ , 400 MHz): 6.34 (1H, d, J = 9.8 Hz, H‐3), 8.36(1H, dd, J = 9.6; 10 Hz, H‐4), 7.35 (1H, s, H‐8), 7.27 (1H, dd, J = 2.4; 0.9 Hz, H‐11), 8.01 (1H, d, J = 2.4 Hz, H‐12), 4.65 (1H, dd, J = 10.0; 2.2 Hz, H‐13), 4.27 (1H, dd, J = 9.9; 8.4 Hz, H‐13), 3.75 (1H, dd, J = 8.3; 2.1 Hz, H‐14), 3.15 (3H, s, 15‐OMe), 1.16 (3H, d, J = 10.0 Hz, H‐16), 1.11 (3H, s, H‐17); ¹³С NMR (DMSO‐d₆ , 100 MHz): 160.2 (C‐2), 112.2 (C‐3), 140.0 (C‐4), 149.2 (C‐5), 113.5 (C‐6), 157.5 (C‐7), 93.5 (C‐8), 152.1 (C‐9), 106.6 (C‐10), 105.4 (C‐11), 146.0 (C‐12), 74.8 (C‐13), 74.5 (C‐14), 76.2 (C‐15), 49.0 (C‐16), 22.2 (C‐17), 19.6 (C‐18) [40].

2.1.5. Pengilin (PPE19)

HRESIMS m/z 309.0733 [M + H]⁺ (calcd for C₁₆H₁₄O₅Na 309.0722). ¹Н NMR (DMSO‐d₆ , 400 MHz): 6,34 (1H, d, J = 9.8 Hz, H‐3), 8.29(1H, dd, J = 9.8; 0.5 Hz, H‐4), 7,35 (1H, s, H‐8), 7.30 (1H, dd, J = 2.4; 1.0 Hz, H‐11), 8,01 (1H, d, J = 2.4 Hz, H‐12), 4.41 (1H, m, H‐13), 4.48 (1H, dd, J = 8.9, 3.1 Hz, H‐14), 1.74 (3H, s, H‐16), 5.13 (2H, m, H‐17); ¹³С NMR (DMSO‐d₆ , 100 MHz): 160.1 (C‐2), 112.3 (C‐3), 139.8 (C‐4), 148.9 (C‐5), 113.5 (C‐6), 157.4 (C‐7), 93.5 (C‐8), 152.0 (C‐9), 106.6 (C‐10), 105.3 (C‐11), 146.0 (C‐12), 75.4 (C‐13), 72.7 (C‐14), 144.8 (C‐15), 18.5 (C‐16), 112.0 (C‐17) [41].

2.1.6. 8‐Oxypeucedanin Methanolate (PPE23)

HRESIMS m/z 341.0995 [M + Na]⁺ (calcd for C₁₇H₁₈O₆Na 341.0984). ¹Н NMR (DMSO‐d₆ , 600 MHz): 6,43 (1H, d, J = 9.6 Hz, H‐3), 8.14 (1H, d, J = 9.6 Hz, H‐4), 7.66(1H, s, H‐5), 7.09 (1H, d, J = 2.1 Hz, H‐11), 8.12 (1H, d, J = 2.4 Hz, H‐12), 4.75(1H, dd, J = 10.2, 2.1 Hz, H‐13), 4.38 (1H, dd, J = 10.2, 8.2 Hz, H‐13), 3.79 (1H, d, J = 7.5 Hz, H‐14), 3.14 (3H, s, H‐16), 1.15 (3H, s, H‐17), 1.08 (3H, s, H‐18); ¹³С NMR (DMSO‐d₆ , 150 MHz): 159.8 (C‐2), 114.2 (C‐3), 145.26 (C‐4), 113.7 (C‐5), 125.8 (C‐6), 147.1 (C‐7), 131.5 (C‐8), 142.6 (C‐9), 116.4 (C‐10), 107.0 (C‐11), 147.7 (C‐12), 75.2 (C‐13), 74.8 (C‐14), 76.2 (C‐15), 48.9 (C‐16), 22.1 (C‐17), 19.8 (C‐18) [42].

The ¹H and ¹³C NMR and HRESIMS spectrum of isolated enantiomers are displayed in the Figures S8–S25.

2.2. Determination of the Absolute Configuration

The absolute configuration of chiral compounds is usually determined through single‐crystal x‐ray diffraction, asymmetric synthesis, nuclear magnetic resonance (NMR), and the chiral optical method. For determining the absolute configuration of compounds, the ECD calculation is considered a powerful tool, as its sample consumption is low and recyclable. By comparing the calculated ECD spectra with the experimental ECD spectra, the stereoisomers of compounds were identified. We determined the absolute configuration of the separated stereoisomers by comparing their experimental circular dichroism (CD) spectra with theoretical electronic circular dichroism (ECD) spectra.

As shown by the results the experimental CD spectrum of compound 1 (PPE4‐1) was consistent with the theoretical CD spectrum of (S) configuration which confirmed the (S) configuration of this compound. The Figure 1 demonstrated the experimental and calculated CD spectra of compound 1 (PPE4‐1) and 2 (PPE4‐2) and their absolute configurations.

FIGURE 1.

The experimental and calculated CD spectra and the absolute configuration of compound 1 (PPE4‐1) and 2 (PPE4‐2).

The experimental CD spectrum of compound 3 (PPE9‐1) was matched with the calculated CD of (R) configuration which indicated that this compound has (R) configuration.

The experimental and calculated CD spectrum of compound 3 (PPE9‐1) and 4 (PPE9‐2) and their absolute configurations are illustrated at the Figure 2.

FIGURE 2.

The experimental and calculated CD spectra and the absolute configuration of compound 3 (PPE9‐1) and 4 (PPE9‐2).

As the experimental CD spectrum of compound 5 (PPE14‐1) has the same shape as the calculated CD spectrum of (R) configuration it's suggested that this compound belong (R) configuration.

The experimental and calculated CD spectrum of compound 5 (PPE14‐1) and 6 (PPE14‐2) and their absolute configurations are illustrated at the Figure 3.

FIGURE 3.

The experimental and calculated CD spectra and the absolute configuration of compound 5 (PPE14‐1) and 6 (PPE14‐2).

The absolute configuration of compound 7 (PPE18‐1) was assigned as (R) based on the good agreement between its experimental CD spectrum and the calculated CD spectrum of the (R)‐enantiomer.

The Figure bellow demonstrated the experimental and calculated CD spectra of compound 7 (PPE18‐1) and 8 (PPE18‐2) and their absolute configurations are illustrated at the Figure 4.

FIGURE 4.

The experimental and calculated CD spectra and the absolute configuration of compound 7 (PPE18‐1) and 8 (PPE18‐2).

The experimental CD spectrum of compound 9 (PPE19‐1) matched well with the theoretical CD spectrum of (S) enantiomer, indicating that the compound possesses the (S)‐configuration.

The Figure 5 demonstrated the experimental and calculated CD spectra of compound 9 (PPE19‐1) and 10 (PPE19‐2) and their absolute configurations.

FIGURE 5.

The experimental and calculated CD spectra and the absolute configuration of compound 9 (PPE19‐1) and 10 (PPE19‐2).

Compound 11 (PPE23‐1) found (S) configuration as it was matched with the theoretical CD spectrum of (S) configuration.

The experimental and calculated CD spectra of compound 11 (PPE23‐1) and 12 (PPE23‐2) and their absolute configurations are demonstrated at Figure 6.

FIGURE 6.

The experimental and calculated CD spectra and the absolute configuration of compound 11 (PPE23‐1) and 12 (PPE23‐2).

2.3. The Bioactivity Tests of Individual Stereoisomers

The bioactivity tests of individual stereoisomers (enantiomers) were carried out after their separation from the racemic mixture. Twelve individual compounds were obtained which were then individually tested for their effects towards B16F10 melanoma cells. This analysis resulted in the same effect for both the (R) and (S) enantiomers. The activation rate of tyrosinase of the compound 1 (S)‐oxypeucidanin hydrate (PPE4‐1) was 119.74%, while it was 110.1% for compound 2 (R)‐oxypeucidanin hydrate (PPE4‐2). For compound 9 (S)‐pangelin (PPE19‐1), this activation was 125.42% and 120.63% for compound 10 (R)‐pangelin (PPE19‐2). Thus, the differences were minimal. The activation rates of the tyrosinase by the other enantiomers are illustrated in Figure 7.

FIGURE 7.

The effect of separated enantiomers on tyrosinase activation.

2.4. The Effect of Compound 9 ((S)‐Pangelin) on the Cell Viability of B16F10melanoma Cells

There was a weak potential of compound 9 (PPE19‐1) at high concentrations enhancing melanin synthesis and tyrosinase activity. Western blot analyses detected the effects of melanogenesis‐related proteins and signaling pathway‐related proteins.

Negative control was choosing DMSO (0.1%), 8‐MOP was using as a positive control and the activity of compound 9 was detected at concentrations (1) 1 µM, (10) 10 µM, (50) 50 µM. The viability of B16F10 cells was 92.26% ± 0.041% and 79.89% ± 0.009% at concentrations of 1–50 µM without significant effect on cell morphology. This concentration range (1–50 µM) was considered as optimal concentration range (Figure 8).

FIGURE 8.

Effect of different concentrations of (S)‐pangelin on B16F10 cell viability and cell morphology The effect of different concentrations (0–200 µM) of (S)‐pangelin on B16 F10 melanoma and HaCaT cells for 48 h. Cell viability was measured by the CCK‐8 assay. The morphological changes in the (S)‐pangelin‐treated (0–50 µM) B16F10 cells as determined by microscopy (100 × magnification). (B‐NC) 0.1% DMSO, (B‐1) 1 µM, (B‐10)10 µM, (B‐50) 50 µM of (S)‐pangelin. CCK‐8, cell counting kit‐8; DMSO, dimethylsulfoxide; NC, negative control.

Tyrosinase activity and melanin contents were measured by treating B16F10 cells with 1, 10, and 50 µM compound 9 for 24 and 48 h. This result indicated that compound 9 promoted melanin synthesis and tyrosinase activity in a dose‐dependent manner. When the concentration of compound 9 was 50 µM, the melanin content level was significantly upregulated compared to the blank control group (180% ± 9.76%; p < 0.0001), and the tyrosinase activity was (223.41% ± 7.33%; p < 0.0001), and the differences were statistically significant which were 1.28 and 1.49 times higher than those noted in the positive control group. Both groups exhibited significantly higher values compared with those of the blank control group, and statistically significant promotion were observed (Figure 9).

FIGURE 9.

Effect of different concentrations of (S)‐pangelin on melanin content and TYR enzyme activity in B16F10 cells. Representation of the image indicating the cell precipitation and dissolved solution in the presence of 5% sodium hydroxide. Presentation of the melanin content following 48 h of treatment with (0–50 µM) of (S)‐pangelin. TYR activity following 24 h (S)‐pangelin treatment. Photoshop (Adobe Systems, Inc.) was used to determine the protein band density (*p < 0.05, ***p < 0.001, ****p < 0.0001). The data are shown as the mean ± SD and were analyzed by one‐way ANOVA followed by Tukey's test. All experiments were performed three times.

In order to deeper understanding of the mechanism of melanogenesis mediated by compound 9, Western blot analysis was used to detect the expression of proteins related to the melanogenesis signaling pathway.

Western blot analyses were used to detect the expression levels of melanogenesis‐related proteins. Compound 9 significantly promoted the expression levels of MITF, TYR, TRP1, and TRP2. As the concentration of the compound increased, the protein expression level also increased. A concentration of 50 µM significantly promoted the expression levels of all four proteins related to melanin synthesis (Figure 10).

FIGURE 10.

Effect of different concentrations of (S)‐pangelin on melanogenesis‐related genes in B16F10 cells. The protein levels of MITF, TYR, TRP‐1, and TRP‐2 were detected following 48 h of (S)‐pangelin treatment. Photoshop (Adobe Systems, Inc.) was used to determine the protein band density (***p < 0.001, ****p < 0.0001). The data are shown as the mean ± SD and were analyzed by one‐way ANOVA followed by Tukey's test. All experiments were performed three times. TYR, tyrosinase; MITF, microphthalmia‐associated transcription factor; and TRP, tyrosinase‐related protein.

GSK3β is a key protein in the AKT/GSK3β/β‐catenin signaling pathway and promotes ubiquitination and degradation of β‐catenin. Addition of a phosphate group to a serine/threonine residue at the N‐terminal end of β‐catenin, the phosphorylated β‐catenin is covalently modified by ubiquitination and degraded by the proteasome, maintaining low levels of β‐catenin in the cytoplasm. When GSK3β is phosphorylated by p‐Akt, APC/Axin/GSK3β function is inhibited, β‐catenin cannot be degraded in time and accumulates in the cytoplasm. When β‐catenin protein accumulates to a certain level in the cytoplasm, it can enter the nucleus to promote the expression of MITF (Figure 11).

FIGURE 11.

The compound (S)‐pangelin activated the Wnt signaling pathway via the phosphorylation of GSK‐3β, β‐catenin, and AKT. The expression levels of melanogenesis‐related signaling pathway proteins in the B16F10 cells treated with (0–50 µM) (S)‐pangelin for 48 h were analyzed compared with those of β‐actin. Photoshop (Adobe Systems, Inc.) was used to determine the protein band density (****p < 0.0001). The data are shown as the mean ± SD and were analyzed by one‐way ANOVA followed by Tukey's test. All experiments were performed three times.

MITF plays a crucial role as a primary target. It eventually leads to the upregulation of tyrosinase expression, promoting melanin synthesis. Activated cAMP induces phosphorylation of the cAMP response element binding protein CREB, and the p‐CREB can bind to the promoter region of MITF and upregulate MITF transcription to promote melanin production.

3. Materials and Methods

3.1. General Experimental Procedures

NMR spectra were recorded on a Varian MR‐400 (400 MHz for ¹H and 100 MHz for ¹³C) spectrometer in DMSO‐d₆ and CD₃OD. TMS (δ 0.00) signal was used as an internal standard for ¹H NMR shifts, and DMSO‐d₆ (39.52 ppm vs. TMS) signal was used as a reference for ¹³C NMR shifts. Column chromatographic separation was performed on Sephadex LH‐20 gel (Amersham Pharmacia Biotech, Sweden) and silica gel (100–200 mesh, Qingdao Haiyang Chemical Factory, China). HPLC analysis was performed using a DIONEX Ultimate 3000 HPLC system (Thermo–Fisher, USA) equipped with autosampler and coupled to variable UV wavelength detector. The chromatographic separation was performed using X‐Bridge TM C18 column (10 × 150 mm, particle size 5 µm, Waters), operated at 35°C. The separation of enantiomers was performed on preparative HPLC using a chiral column (CHIRALPAK). The HR‐ESI‐MS data were collected with a QStar Elite mass spectrometer (AB SCIEX, Framingham, MA, USA). The CD experiment was performed on a Chirascan machine. The absolute configuration was confirmed based on the TDDFT‐ECD method.

3.2. Plant Material

The roots of P. pabularia were authenticated by Dr. Dovudsho Navruzshoev after collecting from the Takob valley, Republic of Tajikistan and voucher sample (No. TAS 23659) has been deposited in the herbarium of Chinese‐Tajik Innovation Center for Natural Products.

3.3. Extraction and Isolation Procedures

The 3.4 kg of dried roots of P. pabularia were extracted with 95% ethanol for 24 h at 45°C and the extraction was repeated three times yielding 898 g of extract (23.5%). The crude extract was suspended in water and successively partitioned in four fractions: petroleum ether (35.40 g), dichloromethane (295 g), n‐butanol (171.5 g), and water‐soluble part (393 g). The separation protocols are available in the Supporting Information.

3.4. Separation of Isolated Compounds

Enantiomeric compounds were separated from the dichloromethane fraction using a solvent system consisting of hexane and ethanol at various ratios. A preparative HPLC equipped with chiral columns was employed for the chromatographic separations. Compounds 1 and 2 were separated from PPE4 using a hexane:ethanol (85:15) solvent mixture with retention times of 28.5 and 30.3 min, respectively. Compounds 3 and 4 were separated from a PPE9 sub‐fraction using an isocratic hexane:ethanol (75:25) mixture with retention times of 15.6 and 17.0 min, respectively. Compounds 5 and 6 were separated from PPE14 after 25.0 and 34.4 min using hexane:ethanol (95:5) as solvent. Compounds 7 and 8 were separated from PPE18 using hexane:ethanol (75:25) solvent, and their retention times were 20.8, and 22.2 min, respectively. Compounds 9 and 10 were obtained from PPE19, and compounds 11 and 12 were separated from PPE23 at 38.6 and 40.4 min with hexane:ethanol (93:7) solvent. Figure S1 shows the separation scheme and structures are demonstrated in Figures S2–S7.

3.5. Experimental CD Spectroscopy

The experimental CD spectra were fitted based on measured values using a Chirascan machine. The compounds of (+) and (−) stereoisomers were diluted with methanol. Then, the CD scanning spectra of the isolated isomers were obtained at 50 nm/min with a CD spectrometer, with a wavelength coverage of 200–400 nm. Two replicates were obtained for each of the stereoisomer samples. A simulated calculation of the ECD spectra was based on the Tmolex 3.3‐ZMM software. Initially, accurate 3D chemical structures of the coumarin's stereoisomers were built by preparing a structure tool module for the compounds. All conformers were optimized at PM6. The lowest‐energy conformers were chosen and further optimized based on density functional theory (DFT) with the B3LYP method in methanol. The electronically excited state was calculated using B3LYP‐D3(BJ)/6‐31G(d) of the time‐dependent density functional theory (TD‐DFT). After obtaining the low‐energy conformers, based on both the Boltzmann distribution and the geometrical optimization methods, the calculated ECD spectra of the isolated enantiomers were simulated and fitted. The absolute configuration was confirmed based on the TDDFT‐ECD method.

3.6. Cell Cultures

B16F10 melanoma cells were purchased from the Beijing Chinese Academy of Sciences (RRID: CVCL‐F936). The cells were maintained in Dulbecco's modified Eagle's medium (DMEM, Gibco Life Technologies, Paris, France), supplemented with 10% (v/v) FBS, penicillin G (100 U/mL), and streptomycin (100 mg/mL) (Gibco‐BRL, Grand Island, NY, USA) in 5% CO2 at 37°C.

3.7. Melanin Content Determination

The melanoma cells were cultured in HG‐DMEM, and then supplemented with 10% FBS, 100 mg/mL streptomycin and 100 U/mL penicillin. The cells were maintained in a humidified incubator with 5% CO₂ at 37°C, and they were sub‐cultured every 2 days to maintain logarithmic growth [43, 44]. Cells were seeded in a 96‐well plate at a density of 5 × 10³ cells/well. After 24 h of incubation, different concentrations of the test compounds were added to each well of the plate. After the plate was incubated for an additional 48 h, the attached cells were incubated with MTT (0.5 mg/mL, 1 h) and subsequently solubilized in DMSO. The absorbance at 550 nm was then measured using a micro‐plate reader (SpectraMax M2 Multi‐Mode Microplate Reader, Molecular Devices, Sunnyvale, CA, USA) to calculate the percentage cell viability. The analysis was replicated three times for each compound. DMSO was used as negative control and for positive control was using 8‐MOP [45] as well.

3.8. Cell Viability Preparation

Cell viability was detected using the Cell Counting Kit‐8 (CCK8) assay. Cell density 1 × 10⁴ cell/well, with a volume of 100 µL, were inoculated onto a 96‐well plate and incubated in a constant temperature incubator containing 5% CO₂ at 37°C for 24 h, then 100 µL of culture medium containing different concentrations of compounds to be tested were added and incubated for another 24 h, untreated cells served as the blank control. After 24 h, 10 µL CCK8 detection solution was added to each well and incubated for 2 h. The absorbance was measured at 450 nm. (Cell viability = treatment group OD₄₅₀/blank control group OD₄₅₀) × 100%.

3.9. Tyrosinase Activity Assay

To assess tyrosinase function in vitro, B16 cells were first plated (2 × 10⁵ cells/well) in 6‐well dishes and allowed to settle for 24 h [46]. Cells were then exposed to PMPP or left untreated, followed by another 24‐h incubation. Afterward, they were washed with cold PBS and lysed in a buffer containing Triton X‐100 and sodium deoxycholate. The lysates underwent thermal treatment at ‒80 °C (30 min), then clarified by centrifugation (12 000 × g, 15 min). For the enzymatic assay, 90 µL of the clarified extract was combined with 10 µL of 10 mM L‐DOPA in a 96‐well plate. The mixture was incubated for one hour at 37 °C in the dark. Optical density was read at 490 nm. Enzymatic activity was reported as a percentage compared to control wells: Tyrosinase activity (%) = (Sample OD₄₉₀/Control OD₄₉₀) × 100.

3.10. Western Blot Assay

Cells treated with PMPP were lysed on ice using RIPA buffer (pH 7.4) with protease inhibitors, including 1 M 4‐nitrophenyl phosphate, 1 M sodium fluoride, 10 mM PMSF, 100 mM benzamidine, 100 mM _D,L‐dithiothreitol, and 200 mM sodium orthovanadate. Lysis lasted 30 min for complete protein extraction. Lysates were collected and quantified via BCA assay. Equal protein (60 µg each) was run on 12% SDS–PAGE gels, transferred to PVDF membranes (Merck Millipore Ltd., Billerica, MA, USA), blocked with 5% milk in TBST for 1 h, then overnight at 4°C with primary antibodies. β‐actin served as a loading control. Membranes were washed thrice with TBST, then incubated for 1 h at room temperature with HRP‐conjugated secondary antibodies (1:2000). Protein bands were visualized using ECL Western blotting detection reagents (GE Healthcare, Parramatta, NSW, Australia) and captured with ChemiDoc MP Imaging system (Bio‐Rad Laboratories, Inc., Hercules, CA, USA). Data are from at least three independent replicates.

3.11. Statistical Analysis

All bioassays were performed in triplicate (n = 3 independent experiments). Data are presented as mean ± standard deviation (SD). All statistical analyses were carried out using GraphPad Prism version 9.0 (GraphPad Software, Inc., San Diego, CA, USA) (RRID: SCR‐002798). One‐way ANOVA and Tukey's multiple comparison tests were used to compare the statistical differences between the treatment groups. A p ≥ 0.05 was considered not significantly different, a* p < 0.05 was considered statistically different, a** p ≤ 0.01 was considered statistically significantly different, and a*** p ≤ 0.001 and a**** p ≤ 0.0001 were considered extremely significantly different.

4. Conclusions

The absolute configurations of six pairs of enantiomers were confirmed using CD and ECD spectroscopic analyses. The anti‐vitiligo assay demonstrated that all the tested enantiomers exhibited the potential to promote melanogenesis by increasing melanin production in B16F10 melanoma cells. The difference in biological activities between the (S)‐ and (R)‐enantiomers was negligible. Western blot analysis was used to detect the expression levels of melanogenesis‐related proteins. Compound 9 identified as (S)‐pangelin significantly promoted the expression levels of MITF, TYR, TRP1, and TRP2. Furthermore, this compound significantly enhanced melanin production in B16F10 cells. Preliminary mechanism investigation suggested that its melanogenic effect was associated with the activation of tyrosinase and melanogenesis‐related proteins, together with the regulation of the AKT/GSK3β/β‐catenin signaling pathway.

Author Contributions

Sunbula Atolikshoeva: methodology, investigation, data curation, and writing – original draft preparation. Sodik Numonov: resources, validation, writing – review and editing, and project administration. Jun Li: conceptualization and methodology. Jiangyu Zhao: formal analysis. Mayire Nueraihemaiti: data curation. Xin Xuelei: methodology and supervision. Maitinuer Maiwulanjiang: data curation and project administration. Isomiddin Gulmurodov: resources and data curation. Khurshed Bozorov: methodology. Ibrohimjon Askarov: conceptualization. Guangmin Yao: methodology. Haji Akber Aisa: project administration, supervision, and funding acquisition. All authors have read and agreed to the published version of the article.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File 1: cbdv71537‐sup‐0001‐SuppMat.docx

Data Availability Statement

The data supporting the structure confirmation of this study are available in the Supporting Information of this article.