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Abstract

Four lignans, asarinin (1), horsfieldin (2), 5-(4-(3,4,5-trimethoxyphenyl)hexahydrofuro[3,4-c]furan-1-yl)benzo[d][1,3]dioxole (3), 5-(4-(3,5-dimethoxyphenyl)hexahydrofuro[3,4-c]furan-1-yl)benzo[d][1,3]dioxole (4), and four non-alkaloid compounds, piperonylic acid (5), hesperidin (6), syringin (7), and β-sitosterol (8) were isolated from the stem bark of Zanthoxylum rhetsa grown in Vietnam. Their chemical structures were elucidated by spectroscopic analysis and compared with the references. Except for compound 6, all the remaining compounds (1–5, 7, and 8) were isolated for the first time from Z. rhetsa. The isolated compounds were tested for their cytotoxic activity against three human cancer cell lines, LU-1, Hep-G2, and KB. Compound 5 showed moderate cytotoxic activity against all three cell lines with IC₅₀ values ranging from 48.13 to 49.06 µg mL⁻¹. Notably, compound 6 demonstrated selective cytotoxicity against LU-1, Hep-G2, and KB cancer cell lines (IC₅₀ 66.48–67.41 µg mL⁻¹) while non-toxic toward normal Vero cell. Compound 6 also exhibited its ability to inhibit main protease (M^pro) enzyme in vitro with an IC₅₀ value of 55.49 µg mL⁻¹ and in silico with the binding affinity toward targeted protein of −14.36 kcal mol⁻¹.

Keywords

cytotoxic lignan normal Vero cell Rutaceae

Introduction

Zanthoxylum rhetsa (Roxb.) DC. belonging to the Rutaceae family, is a flowering and aromatic plant with a small wood, thorny bark of trunk and branches, and spicy fruits. This plant distributed in India, Thai Lan, Myanmar, Lao, and Vietnam.^1,2 Z. rhetsa is an indispensable herb in Thai and Mong ethnic groups living in Northwest Vietnam, which use young leaves as a spice and fruits as the culinary soul of this region.^1–3 In folk medicine, fruit, stem, or root barks are used to treat diarrhea, malaria, rheumatism, and worms; essential oil of fruits is used as an antibacterial agent and prevention of cholera.¹ Recent studies found that Z. rhetsa contains alkaloids, lignans, coumarins, triterpenoids,^4–8 and monoterpenoids in the essential oil.^9–12 In our previously publication, we have reported the chemical constituents and biological activities of essential oils from the barks, leaves, and fruits of this plant.¹³ In this paper, we report the isolation and the structure elucidation, as well as the biological activities of secondary metabolites from the stem bark of Z. rhetsa.

Results and discussion

NMR analysis of secondary metabolites from stem bark of Z. rhetsa

Compound 1 was obtained as a white amorphous powder. The ¹H NMR spectrum of 1 presented the signals of two set ABX-type aromatic protons of two 1,3,4-trisubstituted phenyl groups at δ_H 6.90 (1H, d, J = 1.5 Hz, H-2), 6.81 (1H, d, J = 8.0 Hz, H-5), 6.87 (1H, m, H-6) and δ_H 6.92 (1H, d, J = 1.5 Hz, H-2′), 6.79 (1H, d, J = 8.0 Hz, H-5′), 6.88 (1H, m, H-6′); two methylenedioxy groups at δ_H 5.97 (2H, s, OCH₂O)/5.98 (2H, s, OCH₂O); two oxymethylene groups at δ_H 3.19 (1H, t, J = 8.5 Hz, H_a-9)/3.77 (1H, t, J = 8.5 Hz, H_b-9) and 4.11 (1H, d, J = 9.0 Hz, H_a-9′)/3.81 (1H, t, J = 8.5 Hz, H_b-9′); four methine groups at δ_H 4.38 (1H, d, J = 7.0 Hz, H-7), 4.82 (1H, d, J = 6.5 Hz, H-7′), 2.85 (1H, m, H-8), and 3.39 (1H, m, H-8′). The carbon-13 nuclear magnetic resonance spectroscopy (¹³C NMR), distortionless enhanced polarization transfer (DEPT), and two-dimensional 1H–13C heteronuclear single quantum coherence (HSQC) spectra of 1 showed 20 carbons, including 4 methylene, 10 methine, and 6 non-protonated carbons. The two-dimensional 1H–13C heteronuclear multiple bond correlation (HMBC) showed the correlations between H-7 (δ_H 4.38) and C-1 (δ_C 137.0)/C-2 (δ_C 107.2)/C-6 (δ_C 120.2)/C-8 (δ_C 55.7)/C-9′ (δ_C 71.5); between H-7′ (δ_H 4.36) and C-1′ (δ_C 133.9)/C-2′ (δ_C 107.1)/C-6′ (δ_C 119.6)/C-8′ (δ_C 50.8)/C-9 (δ_C 70.1). Combining the above data of 1 with those in the work of Li et al.¹⁴ (Tables 1 and 2) allowed for the determination of the chemical structure of compound 1 was asarinin (Figure 1). Asarinin was found in Zanthoxylum armatum,¹⁵ Zanthoxylum piperitum,¹⁶ Zanthoxylum myriacanthum var. pubescens,¹⁷ but this is the first time that asarinin was isolated from Z. rhetsa.

Table 1.

The ¹H NMR data of compounds 1, 2, 3, 4, and horsfieldin.¹⁸

C	1	2	3	4	Horsfieldin
C	δ_H^a,b (J = Hz)	δ_H^a,b (J = Hz)	δ_H^a,b (J = Hz)	δ_H^a,b (J = Hz)	δ_H^d (J = Hz)
1	–	–	–	–	–
2	6.90 (d, 1.5)	6.90 (d, 1.5)	6.71 (s)	6.92 (s)	6.97 (s)
3	–	–	–	–	–
4	–	–	–	7.00 (s)	–
5	6.81 (d, 8.0)	6.79 (d, 8.0)	–	–	6.90 (s)
6	6.87 (m)	6.88 (dd, 1.5/8.0)	6.71 (s)	6.92 (s)	6.80 (d, 8.0)
7	4.38 (d, 7.0)	4.39 (d, 7.0)	4.85 (d, 6.0)	4.85 (d, 6.0)	4.44 (d, 7.0)
8	2.85 (m)	2.86 (m)	2.87 (m)	3.41 (m)	2.89 (m)
9	3.19 (t, 8.5), 3.77 (t, 8.5)	3.83 (m), 3.22 (m)	3.83 (m), 3.20 (m)	4.12 (dd, 1.0/9.25), 3.83 (d, 3.0)	3.86 (m), 3.33 (m)
1′	–	–	–	–	–
2′	6.92 (d, 1.5)	7.00 (d, 1.5)	6.71 (s)	6.87 (d, 1.5)	6.91 (d, 8.0)
3′	–	–	–	–	–
4′	–	–	–	–	–
5′	6.79 (d, 8.0)	6.80 (d, 8.0)	6.79 (d, 8.0)	6.79 (d, 7.5)	6.85 (d, 8.0)
6′	6.88 (m)	6.84 (dd, 1.5/8.0)	6.85 (dd, 1.0/8.0)	6.90 (dd, 1.5/7.5)	6.80 (d, 8.0)
7′	4.82 (d, 6.5)	4.83 (d, 6.0)	4.40 (d, 9.0)	4.39 (d, 7.0)	4.87 (d, 5.0)
8′	3.39 (m)	3.40 (m)	3.45 (m)	2.86 (m)	3.33 (m)
9′	4.11 (d, 9.0), 3.81 (t, 8.5)	4.10 (m), 3.80 (m)	3.85 (m), 3.79 (m)	3.77 (t, 9.0), 3.20 (d, 9.0)	4.14 (d, 9.5), 3.86 (m)
OCH₂O	5.98 (s)	5.98 (s)	5.98 (s)	5.98 (s)	5.98 (s)
OCH₂O	5.97 (s)	–	–	–	–
3-OCH₃	–	–	3.83 (s)	3.80 (s)	–
4-OCH₃	–	3.85 (s)	3.84 (s)	–	3.94 (s)
5-OCH₃	–	–	3.71 (s)	3.81 (s)	–

Recorded in ^aacetone-d6, ^b500 MHz; [18]: in CDCl₃, ^d500 MHz.

Table 2.

¹³C NMR data of compounds 1, 2, 4, and horsfieldin.¹⁸

C	1	2	4	Horsfieldin
C	δ_C^a,c	δ_C^a,c	δ_C^a,c	δ_C^e
1	137.0	137.1	132.6	135.2
2	107.2	107.2	112.6	114.2
3	147.3	148.7	149.3	146.4
4	147.9	148.1	110.8	147.2
5	108.6	108.6	149.3	108.3
6	120.2	120.2	118.6	119.6
7	88.3	88.3	82.6	87.7
8	55.7	55.7	50.9	54.6
9	70.1	70.2	71.5	69.8
1′	133.9	131.3	137.1	130.3
2′	107.1	110.3	120.2	106.6
3′	148.4	147.9	147.9	148.0
4′	148.7	146.4	148.7	144.6
5′	108.6	115.5	108.6	108.2
6′	119.6	119.1	107.2	118.4
7′	82.5	82.7	88.3	82.0
8′	50.8	50.9	55.7	50.2
9′	71.5	71.5	70.2	71.0
OCH₂O	101.8	101.2	101.9	101.1
OCH₂O	101.8	–	–	–
3-OCH₃	–	–	56.1	–
4-OCH₃	–	56.3		56.0
5-OCH₃	–	–	56.1

Recorded in ^aacetone-d6, ^c125 MHz; [18]: inCDCl₃, ^e125 MHz.

Figure 1.

Chemical structures of compounds 1−8.

Compound 2 was obtained as a white amorphous powder. The ¹H NMR and ¹³C NMR data of 2 were almost the same as those of 1, suggested that the structure of 2 might be a lignan. The ¹H NMR spectrum of 2 showed two set ABX-type aromatic protons of two 1,3,4-trisubstituted phenyl groups at δ_H 6.90 (1H, d, J = 1.5 Hz, H-2)/6.79 (1H, d, J = 8.0 Hz, H-5)/6.88 (1H, dd, J = 1.5/8.0 Hz, H-6) and δ_H 7.00 (1H, d, J = 1.5 Hz, H-2′)/6.80 (1H, d, J = 8.0 Hz, H-5′)/6.84 (1H, dd, J = 1.5/8.0 Hz, H-6′); two oxymethylene groups at δ_H 3.22 (1H, m, H_a-9)/3.83 (1H, m, H_b-9) and 4.10 (1H, m, H_a-9′)/3.80 (1H, m, H_b-9′); four methine groups at δ_H 4.39 (1H, d, J = 7.0 Hz, H-7)/4.83 (1H, d, J = 6.0 Hz, H-7′)/2.86 (1H, m, H-8)/3.40 (1H, m, H-8′). However, in the ¹H NMR spectrum of 2, there are only one methylenedioxy group at δ_H 5.98 (2H, s, OCH₂O) instead of two methylenedioxy groups of 1, in addition to the appearance of a methoxy group at δ_H 3.85 (3H, s, 4-OCH₃). The ¹³C NMR, DEPT, and HSQC spectra of 2 showed 20 carbon atoms, including one methyl, three methylene, ten methine, and six non-protonated carbons; in which the methoxy carbon displayed at δ_C 56.3 (4-OCH₃). In the HMBC spectrum, there were the correlations between H-7 (δ_H 4.39) and C-1 (δ_C 137.1)/C-2 (δ_C 107.2)/C-6 (δ_C 120.2)/C-8 (δ_C 55.7)/C-9′ (δ_C 71.5); between H-7′ (δ_H 4.83) and C-1′ (δ_C 131.3)/C-2′ (δ_C 110.3)/C-6′ (δ_C 119.1)/C-8′ (δ_C 50.9)/C-9 (δ_C 70.2). All the data of 2 were compared with those of horsfieldin¹⁸ and showed a similarity (Tables 1 and 2). Therefore, compound 2 was identified as horsfieldin (Figure 1). Horsfieldin was found in Z. armatum,¹⁵ but this is the first time this compound was isolated from Z. rhetsa.

Compound 3 was obtained as a white amorphous powder. The ¹H NMR spectra of 3 were the same as those of 1 and 2, suggesting that compound 3 might be also a lignan. However, in the ¹H NMR spectrum of 3, there were three singlet signals of three methoxy groups at δ_H 3.83 (3H, s, 3-OCH₃), 3.84 (3H, s, 4-OCH₃), and 3.71 (3H, s, 5-OCH₃) instead of one singlet signal of one methoxy group of 2. In the aromatic region, there were two singlet signals of two para-coupled protons at δ_H 6.71 (2H, s, H-2, and H-6). The above spectral data of 3 (Table 1) were compared with those reported in the work of Zhang et al.¹⁸ and showing a similar, so that, compound 3 was identified as 5-(4-(3,4,5-trimethoxyphenyl)hexahydrofuro[3,4-c]furan-1-yl)benzo[d][1,3]dioxole (Figure 1).

Compound 4 was obtained as a white amorphous powder. The ¹H NMR and ¹³C NMR spectra of 4 were the same as those of compounds 1, 2, and 3. However, the marked difference between these compounds were that, the ¹H NMR spectrum of 4 displayed the proton signals of one set ABX-type aromatic protons of one 1,3,4-trisubstituted phenyl group at δ_H 6.87 (1H, d, J = 1.5 Hz, H-2′)/6.79 (1H, d, J = 7.5 Hz, H-5′)/6.90 (dd, J = 1.5/7.5 Hz, H-6′); one 1,3,5-trisubstituted phenyl group at δ_H 6.92 (1H, s, H-2)/7.00 (1H, s, H-4)/6.92 (1H, s, H-6); two oxymethylene groups at δ_H 4.12 (1H, dd, J = 1.0/9.25 Hz, H_a-9)/3.83 (1H, d, J = 3.0 Hz, H_b-9) and 3.77 (1H, t, J = 9.0 Hz, H_a-9′)/3.20 (1H, d, J = 9.0 Hz, H_b-9′); four methine groups at δ_H 4.85 (1H, d, J = 6.0 Hz, H-7)/4.39 (1H, d, J = 6.0 Hz, H-7′)/3.41 (1H, m, H-8)/2.86 (1H, m, H-8′); one methylenedioxy group at δ_H 5.98 (2H, s, OCH₂O) and two methoxy groups at δ_H 3.80 (3H, s, 3-OCH₃)/3.81 (3H, s, 5-OCH₃). The ¹³C NMR and DEPT spectra of 4 displayed 21 carbons, including two methyl, three methylene, ten methine, and six non-protonated carbons, in which two methoxy carbons at δ_C 56.1 (3-OCH₃) and 56.1 (5-OCH₃). The HMBC spectrum of 4 showed correlations between H-7 (δ_H 4.85) and C-1 (δ_C 132.6)/C-2 (δ_C 112.6)/C-6 (δ_C 118.6)/C-8 (δ_C 50.9)/C-9 (δ_C 71.5)/C-9′ (δ_C 70.2); between H-7′ (δ_H 4.39) and C-1′ (δ_C 137.1)/C-2′ (δ_C 120.2)/C-6′ (δ_C 107.2)/C-9′ (δ_C 70.2); between protons of methoxy group at δ_H 3.80 (3-OCH₃)/3.81 (5-OCH₃) and C-3 (δ_C 149.3)/C-5 (δ_C 149.3). The above spectral data of 4 were similar to those reported in the work of Zhang et al.¹⁸ (Tables 1 and 2), so that, compound 4 was identified as 5-(4-(3,5-dimethoxyphenyl)hexahydrofuro[3,4-c]furan-1-yl)benzo[d][1,3]dioxole (Figure 1).

Compound 5 was obtained as white amorphous powder. The ¹H NMR spectrum of 5 presented proton signals of one ABX system aromatic ring at δ_H 7.41 (1H, d, J = 2.0 Hz, H-2)/6.93 (1H, d, J = 8.5 Hz, H-5)/7.65 (1H, dd, J = 2.0/8.5 Hz, H-6); one methylenedioxy group at δ_H 6.11 (2H, s, OCH₂O). The ¹³C NMR spectrum of 5 showed six carbons of one ABX aromatic ring at δ_C 125.4 (C-1)/109.9 (C-2)/148.8 (C-3)/152.6 (C-4)/108.7 (C-5)/126.1 (C-6); one methylenedioxy at δ_C 103.0 (OCH₂O) and one carboxyl carbon at δ_C 166.9 (C-7). The HMBC spectrum of 5 exhibited correlations between proton of methylenedioxy group (δ_H 6.11) and carbons C-3 (δ_C 148.8), C-4 (δ_C 152.6); between protons H-2 (δ_H 7.41)/H-6 (δ_H 7.65) and carbon C-7 (δ_C 166.9). Based on the above spectral data, 5 was identified as piperonylic acid (Figure 1). This is the first time that piperonylic acid was isolated from Z. rhetsa.

Compound 6 was obtained as white amorphous powder. The ¹H NMR spectrum of 6 showed two doublet signals of meta-coupled aromatic protons at δ_H 6.13 (1H, d, J = 2.5 Hz, H-6)/6.12 (1H, d, J = 2.5 Hz, H-8) and three other aromatic protons of an ABX system at δ_H 6.94 (1H, d, J = 3.0 Hz, H-2')/6.93 (1H, dd, J = 3.0/8.0 Hz, H-5')/6.90 (1H, J = 8.0 Hz, H-6'); one signal of oxymethine proton at δ_H 5.48 (1H, dd, J = 3.0/12.5 Hz, H-2); two characteristic signals of two anomeric protons at δ_H 4.97 (1H, d, J = 7.5 Hz, H-1'')/4.52 (1H, s, H-1'''), proved two sugar units in 6. In addition, the ¹H NMR of 6 showed one singlet signal of one methoxy group at δ_H 3.78 (3H, s, OMe), one doublet signal of one methyl group at δ_H 1.08 (3H, d, J = 6.5 Hz, H-6'''). The ¹³C NMR, DEPT, and HSQC spectra of 6 displayed signals of 16 methine, two methylene, two methyl, and eight non-protonated carbons, in which one carbonyl carbon at δ_C 197.1 (C-4), two anomeric carbons at δ_C 99.5 (C-1'')/100.6 (C-1'''), one oxymethylene carbon at δ_C 65.0 (C-6''), and one methyl carbon at δ_C 17.8 (C-6'''). The coupling constant values of two anomeric protons were typical for β-glucopyranose and α-rhamnopyranose. The chemical shift of the methylene group of β-glucopyranose was higher than normal, predicted α-rhamnopyranose was linked to β-glucopyranose through an ether linkage at this methylene group. The HMBC spectrum displayed the correlations between protons of the methoxy group (δ_H 3.78) and carbon C-4' (δ_C 147.9), proving the position of the methoxy group at C-4' of flavanone skeleton; between protons H-2' (δ_H 6.94)/H-6' (δ_H 6.90) and carbon C-2 (δ_C 78.4). Moreover, the HMBC correlations between protons of methylene group H-6_b'' (δ_H 3.81) and anomeric carbon C-1''' (δ_C 100.6), between anomeric proton H-1''' (δ_H 4.52) and carbon C-6'' (δ_C 65.0) confirmed the presence of rutinose unit. The rutinose moiety was located at C-7 of flavanone by HMBC correlation between anomeric proton H-1'' (δ_H 4.97) with carbon C-7 (δ_C 165.1). In comparing the NMR data of 6 with those of hesperidin,¹⁹ compound 6 was identified as hesperidin (Figure 1).

Compound 7 was obtained as white amorphous powder. The ¹H NMR spectrum of 7 showed a singlet signal of two meta-coupled protons at δ_H 6.77 (2H, s, H-2,6), two olefinic protons of one double bond with configuration trans at δ_H 6.57 (1H, d, J = 15.5 Hz, H-7) and 6.57 (1H, dd, J = 15.5/5.5 Hz, H-8), one methylene group at δ_H 4.24 (2H, dd, J = 5.5/1.2 Hz, H-9), two methoxy groups at δ_H 3.88 (3H, s, 3-OMe) and 3.88 (3H, s, 5-OMe). Besides, the ¹H NMR spectrum of 7 also displayed one anomeric proton at δ_H 4.88 (1H, d, J = 7.5 Hz, H-1') and other oxymethine groups with δ_H values in the range of 3.23–3.50 ppm, together one oxymethylene group at δ_H 3.81 (1H, m)/3.69 (1H, m), confirmed the presence of one β-D-glucopyranoside unit. The ¹³C NMR, DEPT, and HSQC spectra of 7 displayed signals of two methyl, two methylene, nine methine, and four non-protonated carbons, in which two olefinic carbons at δ_C 131.2 (C-7) and 130.1 (C-8), two methine carbons at δ_C 105.5 (C-2,6), three non-protonated carbons that are bound directly to oxygen at δ_C 154.4 (C-3,5) and 135.9 (C-4), one non-protonated carbon at δ_C 135.3 (C-1), and two methoxy groups at δ_C 57.0 (3-OCH₃, 5-OCH₃). The presence of β-D-glucopyranose was confirmed by signals of one anomeric carbon at δ_C 105.4 (C-1') and one oxymethylene carbon at δ_C 62.6 (C-6'). Comparison of the above data with those reported in the work of Hossin et al.,²⁰ compound 7 was identified as syringin (Figure 1). Syringin was found in Zanthoxylum ailanthoides,²¹ but this is the first time this compound was isolated from Z. rhetsa.

Compound 8 was obtained as white needles. The ¹H NMR spectrum of 8 exhibited signals of five methyl groups at δ_H 1.01 (3H, s)/0.92 (3H, d, J = 6.4 Hz)/0.85 (3H, t, J = 7.6 Hz)/0.84 (3H, d, J = 7.0 Hz)/0.82 (3H, d, J = 7.0 Hz)/0.68 (3H, s), one methine group at δ_H 5.35 (1H, d, J = 5.0 Hz) and one methylene groups at δ_H 1.98–2.27 ppm. The ¹H NMR spectral data of 8 and β-sitosterol were the same.²² Therefore, compound 8 was identified as β-sitosterol (Figure 1).

Cytotoxic activity of isolated compounds

Compounds 1–6 were selected to evaluate for their cytotoxic activity against three human cancer cell lines (KB, HepG-2, and LU-1). Compounds 7 and 8 have not investigated for biological activity because their small amounts were only enough to measure the spectrum to determine the chemical structures.

The results showed that compounds 5 and 6 exhibited moderate cytotoxic activity in all three cancer cell lines, with IC₅₀ values ranging from 48.13 to 67.41 µg mL⁻¹ (Table 3).

Table 3.

The cytotoxic activity of compounds 1–6.

Compounds	IC₅₀ (µg mL⁻¹)
Compounds	KB	LU-1	HepG2	Vero
1	>100	>100	>100	nt
2	>100	>100	>100	nt
3	>100	>100	>100	nt
4	>100	>100	>100	nt
5	48.13	49.06	48.29	nt
6	67.41	66.48	67.09	>100
Ellipticine*	0.42	0.35	0.34	1.91

Positive control; nt: not determined.

Because hesperidin (6) is present with high content in the trunk bark of Z. rhetsa (about 0.017% dried weight of raw material), this compound was selected to assess for its cytotoxic on normal Vero cells. The results in Table 3 showed that hesperidin (6) was non-toxic to Vero cells (IC₅₀ > 100 µg µL⁻¹). These results demonstrated that hesperidin (6) was selective cytotoxicity against cancer cell lines without cytotoxicity to normal Vero cells.

Enzyme inhibitory activities of hesperidin (6)

Compound hesperidin (6) was also assessed for its potential inhibitory effect on angiotensin-converting enzyme-2 (ACE-2) and main protease (M^pro) enzyme. The obtained results (Table 4) showed that hesperidin (6) was active against M^pro enzyme—a critical enzyme in the virus replication, with IC₅₀ value of 55.49 µg mL⁻¹. Transcription is the main antiviral activity of hesperidin as a potential medicinal source for preventing the virus.

Table 4.

Enzyme inhibition of hesperidin (6).

Compound	IC₅₀ (µg mL⁻¹)
Compound	ACE-2	M^pro
Hesperidin (6)	>100	55.49
Positive control*	0.034	80**

Positive control for the test of ACE-2 was MLN-4760; positive control for the test of M^pro was GC376; **in nM.

Docking using Autodock

Autodock4 is an open-source package that can predict rapidly the binding affinity of ligands toward a specific protein/enzyme target. In computation, binding free energy (ΔG) is calculated to estimate binding affinity of the ligand–target complex. It was reported by Gohlke et al. that ligand calculated partial charge with the PM6 method has been shown to greatly increase docking accuracy and cluster population of the most accurate docking.²³ According to the criteria of AutoDock4, lower binding free energy values indicate higher stability for the complex and favoring interaction of ligand within the binding site when it predicts the docking pose.^24,25 Table 5 presents the docking results of studied compounds.

Table 5.

Docking results of hesperidin and reference ligand with lists of interacting residues.

Compounds	Dock score (kcal mol⁻¹)	Interacting residues
Hesperidin	−14.36	His41, Leu141, Ser144, Cys145, Met165, Glu166, Pro168, Ala191, Gln192
PF-07321332	−12.41	His41, Met49, Pro52, Tyr54, Cys145, His163, Met165, Glu166, Arg188, Gln189, Thr190

As previous studies have reported, the active site of SARS-CoV-2 M^pro is constituted by key residues as follows: His41, Met49, Asn142, Gly143, Cys145, His164, Met165, Glu166, Lru167, Pro168, His172, Gln189, Thr190, and Ala191.²⁶ Binding conformation analysis of PF-07321332 indicate that five H-bonding were created by this compound with Cys145, Glu166, Arg188, Gln189, and Thr190, four among them are essential residues in the active binding region of SARS-CoV-2 M^pro. Besides, the interaction was further strengthened by hydrophobic bonds with His41, Met49, Pro52, Tyr54, His163, and Met165 (Figure 2). Regarding dock pose of hesperidin, the binding affinity of this ligand toward targeted protein was observed to be higher than the reference ligand (−14.36 and −12.41 kcal mol⁻¹, respectively). The hydrophobic pockets formed with hesperidin involved residues His41, Met165, Pro168, and Ala191 while Leu141, Ser144, Cys145, Glu166, and Gln192 were the four essential residues creating hydrogen bonds with the ligand. These finding are in agreement with other studies published previously,^27,28 thus, hesperidin could be assumed as a potential inhibitor of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) M^pro.

Figure 2.

Docking conformation of hesperidin and PF-07321332 within the active site of SARS-CoV-2 main protease suggested by molecular docking simulation.

Conclusion

This study reports on the isolation, elucidation of chemical structures and cytotoxic activity of four lignans (asarinin (1), horsfieldin (2), 5-(4-(3,4,5-trimethoxyphenyl)hexahydrofuro[3,4-c]furan-1-yl)benzo[d][1,3]dioxole (3), and 5-(4-(3,5-dimethoxyphenyl)hexahydrofuro[3,4-c]furan-1-yl)benzo[d][1,3]dioxole (4)), and four non-alkaloid compounds (piperonylic acid (5), hesperidin (6), syringin (7), and β-sitosterol (8)) from the trunk bark of Z. rhetsa grown in Vietnam. Hesperidin (6) is not only presented in high amounts in this plant but also exhibited selective cytotoxicity against LU-1, Hep-G2, and KB cancer cell lines (IC₅₀ 66.48–67.41 µg mL⁻¹), non-toxic toward normal Vero cells and inhibits the M^pro enzyme with an IC₅₀ value of 55.49 µg mL⁻¹. Besides, hesperidin (6) showed converting enzyme inhibitory activity in vitro and in silico. Further studies are needed to evaluate other activities of hesperidin to effectively exploit this active ingredient for public health care application on the basis of natural products.

Experimental

General

The NMR spectra, including ¹H NMR (500 MHz) and ¹³C NMR (125 MHz), DEPT, COZY (two-dimensional 1H–1H correlated spectroscopy), HSQC, and HMBC were recorded using a Bruker DRX 500 spectrometer. Column chromatography (CC) was performed on silica gel (Kieselgel 60, 70–230 mesh and 230–400 mesh, Merck) or RP-18 resins (30–50 μm, Fuji Silysia Chemical Ltd.). For thin-layer chromatography (TLC), pre-coated silica gel 60 F254 (0.25 mm, Merck) and RP-18 F254S (0.25 mm, Merck) plates were used.

Plant materials

The trunk bark of Z. rhetsa was collected at Thuan Chau, Son La, Vietnam, and identified by Dr Nguyen Quoc Binh, Vietnam Museum of Nature (VMN), Vietnam Academy of Science and Technology (VAST). The voucher specimen was deposited at the Institute of Natural Products Chemistry (INPC), VAST.

Extraction and isolation

The dried powdered stem bark of Z. rhetsa (5.0 kg) was extracted with hot methanol three times (each 10 L, 2 days) at 50 °C using an ultrasound-assisted technique, then removed solvent in vacuo to yield the methanol extract (270.0 g). In the methanol extract, there is a white solid. This solid was decanted and then purified on a reversed-phase RP-18 silica gel CC to yield compound 6 (300 mg). The rest of methanol extract was suspended in water (2.0 L) and successively partitioned with n-hexane, ethyl acetate (EtOAc) to yield n-hexane (ZR/H, 92.0 g), and EtOAc (ZR/E, 85.0 g) fractions. The fraction ZR/E was chromatographed on a normal phase silica gel CC, eluting with CH₂Cl₂:CH₃OH gradient system (100:0–0:100, v:v) to give four fractions, ZR/E1–ZR/E4. ZR/E1 and ZR/E2 have streaks of substance on TLC that were the same as ZR/H, grouped them and denoted ZR/H. ZR/H was chromatographed on a normal phase silica gel CC, eluting with n-hexane:EtOAc gradient system (100% n-hexane, 50:1, 40:1, 30:1, 20:1, 10:1, 1:1, 100% EtOAc, v:v), to give five fractions, ZR/H1–ZR/H5. ZR/H2 was further purified on a silica gel CC using n-hexane:acetone (3:1, v/v) to yield compound 8 (6.0 mg). ZR/H3 was chromatographed on a normal phase silica gel CC, eluting with n-hexane:EtOAc (5:1, v:v) to give three smaller fractions, ZR/H3/1–ZR/H3/3. ZR/H/3/1 was recrystallized from dichloromethane:methanol (3:1, v:v) and then washed with methanol to yield compound 1 (15.0 mg). ZR/H4 and ZR/H5 have the same spots on TLC, so these two fractions were combined and chromatographed on a normal phase silica gel CC, eluting with n-hexane:acetone (6:1, v:v) to give three smaller fractions, ZR/H4+5/1–ZR/H4+5/3. ZR/H4+5/1 was purified by reversed-phase RP-18 silica gel CC, eluting with methanol:water (1:1, v:v), to yield compound 5 (10.0 mg). ZR/H4+5/3 was chromatographed on a reversed-phase RP-18 silica gel CC, eluting with acetone:water (1:1, v:v) to yield compounds 2 (15.0 mg), 3 (12.0 mg), and 4 (9 mg). ZR/E4 was loaded on a normal phase silica gel CC using dichloromethane:methanol (8:1, v:v) to give three smaller fractions, ZR/E4/1–ZR/E4/3. Compound 7 (5.0 mg) was obtained from ZR/E4/1 fraction using reversed-phase RP-18 silica gel CC with methanol:water (1:1, v:v).

Asarinin (1): white amorphous powder, ¹H NMR (500 MHz, acetone-d6) and ¹³C NMR (125 MHz, acetone-d6); see Tables 1 and 2.

Horsfieldin (2): white amorphous powder, ¹H NMR (500 MHz, acetone-d6) and ¹³C NMR (125 MHz, acetone-d6); see Tables 1 and 2.

(5-(4-(3,4,5-trimethoxyphenyl)hexahydrofuro[3,4-c]furan-1-yl)benzo[d][1,3]dioxole) (3): white amorphous powder, ¹H NMR (500 MHz, acetone-d6) and ¹³C NMR (125 MHz, acetone-d6); see Table 1.

5-(4-(3,5-dimethoxyphenyl)hexahydrofuro[3,4-c]furan-1-yl)benzo[d][1,3]dioxole (4): white amorphous powder, ¹H NMR (500 MHz, acetone-d6) and ¹³C NMR (125 MHz, acetone-d6); see Tables 1 and 2.

Piperonylic acid (5): white amorphous powder. ¹H NMR (500 MHz, acetone-d6) δ (ppm): 7.41 (1H, d, J = 2.0 Hz, H-2), 6.93 (1H, d, J = 8.5 Hz, H-5), 7.65 (1H, dd, J = 2.0/8.25 Hz, H-6), 6.11 (2H, s, OCH₂O). ¹³C NMR (125 MHz, acetone-d6) δ (ppm): 125.4 (C-1), 109.9 (C-2), 148.8 (C-3), 152.6 (C-4), 108.7 (C-5), 126.1 (C-6), 166.9 (C-7), and 103.0 (OCH₂O).

Hesperidin (6): white amorphous powder, ¹H NMR (500 MHz, CD₃OD), δ_H (ppm): 5.48 (1H, dd, J = 3.0/12.5 Hz, H-2), 2.78 (1H, dd, J = 3.0/17.0 Hz, H_a-3), 3.25 (1H, m, H_b-3), 6.13 (1H, d, J = 2.5 Hz, H-6), 6.12 (1H, d, J = 2.5 Hz, H-8), 6.94 (1H, d, J = 3.0 Hz, H-2'), 6.93 (1H, dd, J = 3.0/8.0 Hz, H-5'), 6.90 (1H, J = 8.0 Hz, H-6'), 3.78 (3H, s, OMe), 4.97 (1H, d, J = 7.5 Hz, H-1''), 3.23 (1H, m, H-2''), 3.26 (1H, m, H-3''), 3.14 (1H, m, H-4''), 3.53 (1H, t, J = 9.0 Hz, H-5''), 3.43 (1H, m, H_a-6''), 3.81 (1H, m, H_b-6''), 4.52 (1H, s, H-1'''), 3.63 (1H, m, H-2'''), 3.42 (1H, m, H-3'''), 3.14 (1H, m, H-4'''), 3.40 (1H, m, H-5'''), 1.08 (3H, d, J = 6.5 Hz, H-6'''). ¹³C NMR (125 MHz, CD₃OD), δ (ppm): 78.4 (C-2), 42.0 (C-3), 197.1 (C-4), 163.0 (C-5), 96.4 (C-6), 165.1 (C-7), 95.6 (C-8), 162.5 (C-9), 103.3 (C-10), 130.9 (C-1'), 114.1 (C-2'), 146.5 (C-3'), 147.9 (C-4'), 112.1 (C-5), 117.9 (C-6), 55.7 (MeO-4'), 99.5 (C-1''), 73.0 (C-2''), 76.3 (C-3''), 69.6 (C-4''), 75.5 (C-5''), 65.0 (C-6''), 100.6 (C-1'''), 70.3 (C-2'''), 70.7 (C-3'''), 72.9 (C-4'''), 68.3 (C-5'''), and 17.8 (C-6''').

Syringin (7): white amorphous powder. ¹H NMR (500 MHz, CD₃OD), δ_H (ppm): 6.77 (2H, s, H-2, H-6), 6.57 (1H, d, J = 15.5 Hz, H-7), 6.57 (1H, dd, J = 15.5/5.5 Hz, H-8), 4.24 (2H, dd, J = 5.5/1.2 Hz, H-9), 4.88 (1H, d, J = 7.5 Hz, H-1′), 3.50 (1H, m, H-2′), 3.43 (1H, m, H-3′), 3.43 (1H, m, H-4′), 3.23 (1H, m, H-5′), 3.81 (1H, m, H_a-6′), 3.69 (1H, m, H_b-6′), 3.88 (3H, s, 3-OCH₃), 3.88 (3H, s, 5-OCH₃). ¹³C NMR (125 MHz, CD₃OD), δ (ppm): 135.3 (C-1), 105.5 (C-2), 154.4 (C-3), 135.9 (C-4), 154.4 (C-5), 105.5 (C-6), 131.2 (C-7), 130.1 (C-8), 63.6 (C-9), 105.4 (C-1'), 75.7 (C-2'), 75.8 (C-3'), 71.4 (C-4'), 78.4 (C-5'), 62.6 (C-6'), 57.0 (3-OCH₃), and 57.0 (5-OCH₃).

β-sitosterol (8): white needles, ¹H NMR (500 MHz, acetone) δ (ppm): 0.68 (3H, s, CH₃-18), 1.01 (3H, s, CH₃-19), 0.81 (3H, d, J = 7.0 Hz, CH₃-26), 0.86 (3H, d, J = 7.0 Hz, CH₃-27), 0.83 (3H, t, J = 7.3 Hz, CH₃-29), 0.92 (3H, d, J = 10.0 Hz, CH₃-21), 3.52 (1H, m, H_-3), 5.35 (1H, d, J = 5.0 Hz, H-6).

Cytotoxic assays

The cell lines, including the KB (human oral carcinoma), HepG-2 (human hepatocellular carcinoma), and LU-1 (human lung carcinoma), were obtained from Milan University, Italy, and Hawaii University, USA. The human cancer cytotoxic activity of the compounds (1–6) was evaluated by measuring cell viability using the sulforhodamine B (SRB) assay. The human cancer cell lines were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum and penicillin/streptomycin at 37 °C in a humidified 5% CO₂ atmosphere. Exponentially growing cells performed the experiments and the SRB assays: (1) treated human cancer cells (1 × 10⁵ cells mL⁻¹) for 2 days with 1, 10, 20, 50, and 100 µM of each compound and ellipticine, (2) performed all the experiments three times, an (3) determined the inhibitory concentration of 50% (IC₅₀) for each compound.

Enzyme inhibition assays

Compound (6) tested ability enzyme inhibition by measuring as the manufacturer’s introductions for the M^pro /3CL Protease, Untagged (SARS-CoV-2) Assay Kit (BPS Bioscience, CA) and ACE-2 inhibitor screening kit (MAK378, Merck KGaA, Darmstadt, Germany). It utilizes the ability of an active ACE-2 and M^pro to cleave a synthetic 4-methoxycoumarin-7-acetic acid (MCA) based peptide substrate to release a free fluorophore. Measuring the fluorescence intensity to assess the ability of the enzyme to lose peptidase activity when the presence of an inhibitor, MLN-4760, is a positive control. The enzyme inhibition assay is as follows: coincubate 10 μL of potent inhibitors (Sample, S) with 50 μL of diluted enzyme solutions (ACE-2 and M^pro) for 15 min, and then add 40 μL of the substrate. The ACE-2, M^pro, and substrate mixtures without sample or inhibitor exhibited the maximum enzyme activity (enzyme control, EC). Use a fluorescence microplate reader (Tecan Spark, SW) with λ_Ex/Em = 320/420 nm to measure the fluorescence at room temperature for 1-h intervals. All experiments were performed in triplicate. Calculate the enzyme inhibition of (7) using the formula

% Relative activity = Δ RFU (S) / Δ RFU (EC) \times 100

In which ΔRFU (S) and ΔRFU (EC) are the sample’s and control’s relative fluorescence units, respectively.

The inhibitory value is expressed as an IC₅₀ value (the concentration of test agents that cause 50% inhibition), which was obtained by nonlinear regression using the TableCurve 2D program (SPSS Statistical Software, Chicago, IL).

Ligand preparation

The studied compounds were prepared three-dimensional structure using MarvinSketch version 19.27.0 and PyMOL version 1.3r1.²⁹ Energy minimization were then conducted using MM2 force field and quantum chemical calculation were performed using PM6 semi-empirical method implemented in Gaussian 09.³⁰ In this study, PF-07321332, a known inhibitor of SARS-CoV-2 M^pro, was selected as reference ligand.³¹

Protein preparation

The X-ray crystal structure of SARS-CoV-2 M^pro was downloaded from the Protein Data Bank archive (PDB ID: 6LU7).³² The protein structure was prepared using the Graphical User Interface program named Autodock Tools (ADT).

Docking using Autodock

The molecular docking study utilizes AutoDock 4.2.6 with Lamarckian genetic algorithm (LGA) for searching the optimum dock pose together with scoring function to calculate the binding affinity. ADT was employed to set up and performed docking calculation. In this study, we performed the docking study assuming that having a rigid protein and consider the conformational space of the ligands to analyze the inductive effect of the hybrid compounds. ADT was utilized to prepare protein for docking simulations. The heteroatoms including water molecules were deleted, and polar hydrogen atoms and Kollman charges were added to the receptor molecule. All other bonds were allowed to be rotatable. In the docking analysis, the binding site was enclosed in a box with the number of grid points in x × y × z directions and a grid spacing of 0.375 Å. Initially, AutoGrid was run to generate the grid map of various atoms of the ligands and receptor. After the completion of the grid map, AutoDock was run using autodock parameters as follows: GA population size, 300; maximum number of energy evaluations, 25,000,000; and the number of generations, 27,000. A maximum of 50 conformers were considered for each molecule, and the root mean square (RMS) cluster tolerance was set to 2.0 Å in each run. The outputs from AutoDock modeling studies were analyzed using PyMOL,²⁹ Discovery Studio Visualizer³³, and LigPlus.³⁴ PyMOL was used to calculate the distances of hydrogen bonds as measured between the hydrogen and its assumed binding partner.

Supplemental Material

sj-docx-1-chl-10.1177_17475198231185704 – Supplemental material for Lignans and some other non-alkaloid compounds from the stem bark of Zanthoxylum rhetsa and their biological activities

Supplemental material, sj-docx-1-chl-10.1177_17475198231185704 for Lignans and some other non-alkaloid compounds from the stem bark of Zanthoxylum rhetsa and their biological activities by Tran Thi Tuyen, Pham Cao Bach, Do Huu Nghi, Pham Minh Quan, Pham Thi Hong Minh and Nguyen Thi Hong Van in Journal of Chemical Research

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research is funded by Vietnam Academy of Science and Technology (VAST) under grant numbers NCVCC07.10/22-23 and ĐL0000.07/22-23.

ORCID iD

Nguyen Thi Hong Van

Supplemental material

Supplemental material for this article is available online.

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