Biosynthesis of Novel Shikonin Glucosides by Enzymatic Glycosylation

Abstract

Shikonin, a natural naphthoquinone, has attracted much attention due to its various biological activities. Two shikonin glucosides, shikonin-1′,8-di-O-β-D-glucopyranoside (1) and shikonin-1′-O-β-D-glucopyranoside (2), were biosynthesized through in vitro enzymatic glycosylation and their structures were elucidated using spectroscopic techniques. The water-solubility and stability of compounds 1 and 2 were significantly higher than those of the parent compound. Furthermore, compound 2 showed moderate cytotoxicity against six cancer cell lines, with IC₅₀ values ranging from 36.10 to 67.47 µM. This research indicated that in vitro enzymatic glycosylation of shikonin is an effective strategy to improve it water solubility and chemical stability.

Introduction

Natural products are an important source for the discovery and development of drugs.^1,2) Shikonin is a naphthoquinone isolated from Lithospermum erythrorhizon Siebold & Zuccarini that reportedly has a range of biological activities, including anticancer, antimicrobial, and anti-inflammatory.^3–6) However, its poor water solubility and toxicity restrict its use as a drug. Many efforts have been made to chemically modify shikonin in order to synthesize derivatives that would yield more effective therapeutic agents.^7–11) The glycosyltransferase (GT) YjiC is a member of the GT1 family found in Bacillus licheniformis DSM-13. YjiC transfers a sugar moiety from nucleoside diphosphate (NDP)-sugar to an acceptor substrate and has recently been used to biosynthesize many novel glycosides from natural products, such as apigenin, phloretin, bavachinin, anthraquinone, and resveratrol both in vitro and in vivo.^12–17) In this study, we report the successful enzymatic biosynthesis of the novel shikonin glucosides shikonin-1′,8-di-O-β-D-glucopyranoside (1) and shikonin-1′-O-β-D-glucopyranoside (2), and their water solubility, chemical stability, and cytotoxicity across six cancer cell lines (Fig. 1).

Fig. 1. Chemical Structures of Shikonin, Compounds 1 and 2

Results and Discussion

Effects of Reaction Conditions on the Glucosylation of Shikonin

Shikonin was subjected to in vitro glucosylation and the reaction mixture was analyzed by HPLC, which showed two product peaks (compounds 1 and 2). Optimization of the glucosylation reaction conditions showed that the most effective reaction occurred at pH 6.0 and 6 h incubation time with 3 mM of uridine-5′-diphosphate (UDP)-Glc, which yielded 0.32 mg/L and 1.09 mg/L of compounds 1 and 2, respectively (Fig. 2). These results indicate that GT YjiC has high substrate specificity for shikonin.

Fig. 2. Production of Novel Shikonin Glucosides under Different Reaction Conditions

(A) Effect of buffer pH on in vitro glucosylation of shikonin. (B) Effect of incubation time on the in vitro glucosylation of shikonin. (C) Effect of UDP-Glc concentration on in vitro glucosylation of shikonin.

Identification of Novel Shikonin Glucosides

Compound 1 was obtained as a red powder and its molecular formula C₂₈H₃₆O₁₅ was established by high resolution-electrospray ionization (HR-ESI)-MS (m/z 611.1962 [M−H]⁻) and NMR analysis. The NMR spectra of 1 were very similar to those of shikonin, except for the additional signals from two glucosyl moieties (Table 1). The two glucosyl moieties of 1 were both identified as being in the β-conformation, based on the coupling constant of their anomeric protons (J = 6.0 and 6.6 Hz, respectively). Additionally, the heteronuclear multiple bond correlation (HMBC) correlations from δ_H 4.55 (d, 1H, J = 6.0 Hz, H-1″) to δ_C 75.8 (C-1′), and δ_H 4.29 (d, 1H, J = 6.6 Hz, H-1‴) to δ_C 169.8 (C-8) suggested that the glucosyl moieties were attached to C-1′ and C-8, respectively (Fig. S2). Further, comprehensive HMBC analysis of 1 permitted the complete assignments of its carbons and protons. Therefore, the structure of 1 was identified as shikonin- 1′,8-di-O-β-D-glucopyranoside.

Table 1. ¹H- and ¹³C-NMR Data of Compounds 1 and 2 in CD₃ OD (δ in ppm)

No.	Compound 1		Compound 2
	δC	δH (J = Hz)	δC	δH (J = Hz)
1	181.2	—	183.3	—
2	151.1	—	152.0	—
3	136.0	7.41 (s)	137.8	7.17 (s)
4	180.9	—	183.2	—
5	170.5	—	174.5	—
6	133.0	7.62 (s)	132.7	7.25 (s)
7	133.2	7.73 (s)	134.8	7.19 (s)
8	169.8	—	174.0	—
9	112.5	—	113.3	—
10	113.6	—	114.3	—
1′	75.8	5.28 (m)	75.7	5.10 (br s)
2′	36.2	2.48–2.53 (m); 2.59–2.62 (m)	34.3	2.48–2.53 (m); 2.63–2.65 (m)
3′	120.4	5.25 (t, 15.6)	120.6	5.31 (t, 14.8)
4′	136.4	—	135.7	—
5′	25.6	1.65 (s)	26.0	1.66 (s)
6′	20.5	1.50 (s)	18.2	1.52 (s)
1″	105.8	4.55 (d, 6.0)	104.2	4.47 (d, 7.7)
2″	75.4	3.27 (m)	75.4	3.27 (m)
3″	78.1	3.38 (m)	78.1	3.28 (m)
4″	78.0	3.20 (m)	78.0	3.20 (m)
5″	71.7	3.26 (m)	71.7	3.26 (m)
6″	62.7	3.70 (m); 3.55 (m)	62.7	3.70 (m); 3.55 (m)
1‴	102.5	4.29 (d, 6.6)	—	—
2‴	75.2	3.47 (m)	—	—
3‴	78.2	3.29 (m)	—	—
4‴	78.6	3.41 (m)	—	—
5‴	72.0	3.38 (m)	—	—
6‴	63.4	3.87 (m); 3.60 (m)	—	—

Compound 2 was obtained as a red powder and its molecular formula C₂₂H₂₆O₁₀ was established by HR-ESI-MS (m/z 449.1447 [M−H]⁻). The ¹H- and ¹³C-NMR data of compound 2 were consistent with those reported for shikonin-1′-O-β-D-glucopyranoside.¹¹⁾ The relative positions of the glucose and alkyl chain were determined from HMBC correlations (Fig. S2). Therefore, the structure of 2 was identified as shikonin-1′-O-β-D-glucopyranoside.

Determination of Water-Solubility

Commonly, modifying natural products by introducing glucose moieties improves their water solubility and chemical stability.^18,19) The water solubilities of compounds 1 and 2 were found to be 15.20 mM and 10.05 mM, approximately 1520 and 1005 times higher than that of the parent compound, respectively (Table 2). These results showed that the enzymatic glucosylation of shikonin significantly enhances its water solubility.

Table 2. Solubility of Shikonin and Its Glucosides 1 and 2 in Water

Compounds	Water-solubility (mg/mL)	MW	Water-solubility (mM)	Relative solubility
1	9.303	612.2	15.20	>1520
2	4.523	450.2	10.05	>1005
Shikonin	<0.01	288.3	<0.01	1

Determination of pH and Temperature Stability

To clarify whether the glucosylation of shikonin enhanced its stability, the pH and temperature stabilities of compounds 1 and 2, and shikonin were determined using HPLC analysis. As expected, compounds 1 and 2 were more stable than shikonin at pH 6–10 and 50–100°C (Fig. 3). These results suggested that the enzymatic glucosylation of shikonin significantly enhances its stability under a range of different pH values and temperatures, which may lead to improvement in its bioavailability and biological activity.

Fig. 3. Stability of Shikonin and Its Glucosides 1 and 2

(A) Effect of pH of Tris–HCl buffer on the stability of shikonin and its glucosides (1 and 2). (B) Effect of temperature on the stability of shikonin and its glucosides (1 and 2).

Cytotoxicity

To understand the anti-tumor activity of the novel shikonin glucosides, we analyzed their anti-proliferative activities against six cancer cell lines using the 3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. As shown in Table 3, compound 2 exhibited moderate antiproliferative activity with IC₅₀ values ranging from 36.10 to 67.47 µM. Unfortunately, both the shikonin glucosides 1 and 2 exhibited weak cytotoxicity compared to the parent compound. It is reported that some cytotoxic glycoside exhibit less anticancer potential in vitro than the corresponding aglycone or after being inactivated by glycosylation.^20,21) Therefore, further studies on the in vivo anticancer activity and pharmacokinetic properties of shikonin glucosides 1 and 2 are required to fully understand their potential as therapeutic agents.

Table 3. Cytotoxicity (IC₅₀) of Shikonin and Compounds 1 and 2 (Mean ± S.D., n = 3) in Cancer Cell Lines

Compounds	MCF-7	MDA-MB-231	H1975	HNE1	SG7901	I-10
1	>100	>100	>100	>100	>100	>100
2	48.72 ± 1.93	57.39 ± 0.96	42.83 ± 1.13	67.47 ± 3.10	36.10 ± 0.87	36.86 ± 2.53
Shikonin	1.21 ± 0.17	1.15 ± 0.01	0.75 ± 0.02	2.99 ± 0.68	1.38 ± 0.04	1.07 ± 0.08

Experimental

General Experimental Procedures

Shikonin (purity >98% by HPLC) was purchased from the Chinese National Institutes for Food and Drug Control. UDP-Glc and other reagents were obtained from Sigma-Aldrich (U.S.A.). HPLC was carried out on Waters 2535Q equipped with a SunFire™ C₁₈ column (150 × 4.6 mm, 5 µm or 250 × 10 mm, 5 µm). NMR spectroscopic data were collected on a Bruker Avance 600 NMR spectrometer (Bruker, U.S.A.). HR-ESI-MS spectra were recorded on an Agilent 6538 Accurate Q-TOF mass spectrometer (Agilent Technologies, U.S.A.). The human breast cancer (MCF-7 and MDA-MB-231), non-small cell lung cancer (H1975), nasopharyngeal carcinoma (HNE1), and human angastric cancer (SG7901), and testicular cancer (I-10) cell lines were purchased from the Cell Bank of the Chinese Academy of Sciences in Shanghai (China).

Enzymatic Glycosylation and Purification of Shikonin Glucosides

Incubation time, buffer pH, and concentration of UDP-Glc were investigated to optimize the different parameters in reactions as previously described.²²⁾ The optimal glucosylation reaction was performed in a 50 mL reaction vessel containing 100 mM of Tris–HCl (pH 6), 1 mM of MgCl₂, 1.5 mM of shikonin, 3 mM of UDP-Glc, and 50 µg/mL of YjiC enzyme, which was incubated at 28°C for 6 h. The reaction mixture was extracted with EtOAc and the extract was purified by semi-prep HPLC with 25% acetonitrile in water (3.0 mL/min) to yield compounds 1 (4.3 mg) and 2 (14.1 mg), respectively.

Determination of Water Solubility

An excess of each compound (shikonin and compounds 1 and 2) was mixed with 0.8 mL of water in an Eppendorf tube, and its solubility was maximized using an ultrasonic cleaner for 1 h at room temperature. After sonication, the samples were centrifuged at 16099 × g for 10 min to remove insoluble material, and the water solubility was determined using HPLC analysis.

Determination of pH and Temperature Stability

Each compound was dissolved in 500 µL of Tris–HCl buffer at pH 6, 7, 8, 9, and 10 for 30 min at room temperature and analyzed by HPLC. To determine temperature stability, the compounds were incubated at 50, 60, 70, 80, 90, and 100°C in 500 µL of Tris–HCl buffer at pH 9 for 30 min. The pH and temperature stabilities were calculated as a percentage of the total peak area using HPLC analysis.

MTT Assay

The six cancer (MCF-7, MDA-MB-231, H1975, HNE-1, SG7901, I-10) cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM) or RPMI 1640 medium containing 10% FBS (Hyclone, U.S.A.) and 1% penicillin/streptomycin (Gibco, U.S.A.). All cells were seeded in 96-well plates at a density of 3000 cells/well for overnight incubation. After adherence, the cells were treated with various concentrations of compounds for 72 h. The anti-proliferative activity was evaluated by standard MTT assay procedures.¹⁴⁾

Acknowledgments

This research was financially supported by the Education Department of Anhui Natural Science Research Project China under Grant (KJ2018A0232, KJ2018A1009); the Science and Technology Development Fund Project of Bengbu Medical College (BYKF1718).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

References

• 1) Ngo L. T., Okogun J. I., Folk W. R., Nat. Prod. Rep., 30, 584–592 (2013).
• 2) Newman D. J., Cragg G. M., J. Nat. Prod., 79, 629–661 (2016).
• 3) Yoon Y., Kim Y. O., Lim N. Y., Jeon W. K., Sung H. J., Planta Med., 65, 532–535 (1999).
• 4) Lu B., Gong X., Wang Z. Q., Ding Y., Wang C., Luo T. F., Piao M. H., Meng F. K., Chi G. F., Luo Y. N., Ge P. F., Yao Hsueh Hsueh Pao, 38, 1543–1553 (2017).
• 5) Bhakuni D. S., Dhar M. L., Dhar M. M., Dhawan B. N., Mehrotra B. N., Indian J. Exp. Biol., 7, 250–262 (1969).
• 6) Tanaka S., Tajima M., Tsukada M., Tabata M., J. Nat. Prod., 49, 466–469 (1986).
• 7) Ahn B. Z., Baik K. U., Kweon G. R., Lim K., Hwang B. D., J. Med. Chem., 38, 1044–1047 (1995).
• 8) Yang F., Chen Y., Duan W., Zhang C., Zhu H., Ding J., Int. J. Cancer, 119, 1184–1193 (2006).
• 9) Su Y., Xie J., Wang Y., Hu X., Lin X., J. Med. Chem., 45, 2713–2718 (2010).
• 10) He H., Bai L. P., Jiang Z. H., Bioorg. Med. Chem. Lett., 22, 1582–1586 (2012).
• 11) Hu X., Chen J., Su Y. H., Jiang Z., Wang Y. G., Patent CN102552296A (2012).
• 12) Gurung R. B., Kim E. H., Oh T. J., Sohng J. K., Mol. Cells, 36, 355–361 (2013).
• 13) Pandey R. P., Li T. F., Kim E. H., Yamaguchi T., Park Y. I., Kim J. S., Sohng J. K., Appl. Environ. Microbiol., 79, 3516–3521 (2013).
• 14) Dai Y., Ma T., Ge M., Li J., Huo Q., Li H. M., Zhang X., Liu H., Wu C. Z., J. Chin. Chem. Soc., 63, 376–378 (2016).
• 15) Ghimire G. P., Koirala N., Pandey R. P., Jung H. J., Sohng J. K., World J. Microbiol. Biotechnol., 31, 611–619 (2015).
• 16) Pandey R. P., Parajuli P., Shin J. Y., Lee J., Lee S., Hong Y. S., Park Y. I., Kim J. S., Sohng J. K., Appl. Environ. Microbiol., 80, 7235–7243 (2014).
• 17) Choi O., Lee J. K., Kang S. Y., Pandey R. P., Sohng J. K., Ahn J. S., Hong Y. S., J. Microbiol. Biot., 24, 614–618 (2014).
• 18) Gantt R. W., Peltier-Pain P., Thorson J. S., Nat. Prod. Rep., 28, 1811–1853 (2011).
• 19) Mandai T., Okumoto H., Oshitari T., Nakanishi K., Mikuni K., Hara K. J., Hara K. Z., Iwantani W., Amano T., Nakamura K., Tsuchiya Y., Heterocycles, 54, 561–566 (2001).
• 20) Cheng H., Cao X. H., Xian M., Fang L. Y., Cai T. B., Ji J. J., Tunac J. B., Sun D., Wang P. G., J. Med. Chem., 48, 645–652 (2005).
• 21) Yu C., Zhang Z., Zhang H., Zhen Z., Calway T., Wang Y., Yuan C. S., Wang C. Z., Oncol. Rep., 30, 2411–2418 (2013).
• 22) Dai Y., Zhang S., Liu D. C., Li H. M., Ma T., Huo Q., Wu C. Z., Phetochemistry. Lett, 23, 9–14 (2018).