Inhibitory effects of compounds from Phyllanthus amarus on nitric oxide production, lymphocyte proliferation, and cytokine release from phagocytes

Figures & data

Table 1 NMR spectroscopic data (500 MHz, CDCl₃) for compound 6 (δ in ppm)

Position	δC	δH, multiplicity (J in Hz)	HSQC	HMBC	NOESY
1	34.1	1.42, m	C-1, H-1	C-11	–
		1.45, m		C-11
2	18.1	1.39, m	C-2, H-2	C-5	–
		1.52, m		C-5
3	28.9	1.14, m	C-3, H-3	–	–
		1.35, m		–
4	27.8	1.60, m	C-4, H-4	–	–
		1.67, m		–
5	51.7	1.58, m	C-5, H-5	–	–
6	28.3	1.77, m	C-6, H-6	–	–
		1.83, m		–
7	76.4	3.45, m	C-7, H-7	–	H-24, 6
8	42.8	–		–	–
9	40.8	–		–	–
10	38.6	–		–	–
11	24.0	1.81, m	C-11, H-11	–	H-21
		1.91, m		–
12	122	5.58, d (5.5)	C-12, H-12	C-18, 17, 11, 9	H-26, 24, 6
13	141.9	–		–	–
14	39.3	–		–	–
15	19.9	0.83, m	C-15, H-15	–	–
		1.18, m		–
16	35.4	1.56, m	C-16, H-16	C-22, 19, 15, 8	–
17	44.3	1.46, m	C-17, H-17	–	H-25, 21
18	50.2	2.05, d (15)	C-18, H-18	–	–
19	60.0	0.97 (s)	C-19, H-19	–	–
20	30.8	1.32	C-20, H-20	–	–
21	29.1	1.06, m	C-21, H-21	–	H-30, 17
		1.18, m		–
22	34.8	–		–	–
23	14.9	0.89, s	C-23, H-23	C-10, 5	H-25
24	25.5	1.11, s	C-24, H-24	C-26, 13, 9, 7	–
25	16.1	0.75, s	C-25, H-25	C-16, 10, 8	–
26	29.4	1.01, s	C-26, H-26	C-24, 13, 9, 7	–
27	15.7	0.97, s	C-27, H-27	C-22, 21, 17	H-28, 16
28	22.9	0.79, d (6.5)	C-28, H-28	C-29, 20, 19	–
29	21.9	0.85, s	C-29, H-29	C-28, 20, 19	–
30	17.9	0.86, s	C-30, H-30	C-18, 17	C-21, 16

Note: – indicates data not applicable.

Abbreviations: HMBC, heteronuclear multiple bond correlation; HSQC, heteronuclear single-quantum correlation; NMR, nuclear magnetic resonance; NOESY, nuclear Overhauser effect spectroscopy; s, singlet; d, doublet; m, multiplet.

Table 2 NMR spectroscopic data (500 MHz, MeOD) for compound 10 (δ in ppm)

Position	δC	δH (J in Hz)	HMBC	NOESY
1	164.1	–	–	–
2	128.7	–	–	–
3	132.1	7.78, d (8.5)	C-1, 4, 11	H-4, 5
4	115.6	6.92, d (9.0)	C-2	H-3, 6
5	115.6	6.92, d (9.0)	C-7	–
6	132.1	7.78, d (8.5)	C-8	–
7	127.6	–	–	–
8	164.1	–	–	–
9	114.4	–	–	–
10	103.4	–	–	–
11	191.4	9.76, s	C-2, 3	H-3

Note: – indicates data not applicable.

Abbreviations: HMBC, heteronuclear multiple bond correlation; NMR, nuclear magnetic resonance; NOESY, nuclear Overhauser effect spectroscopy.

Figure 1 Structures of new compounds isolated from P. amarus.

Abbreviation: P. amarus, Phyllanthus amarus.

Figure 2 HMBC (A) and NOESY (B) correlation of 7β,19α dihydroxy-urs-12-ene (6).

Abbreviations: HMBC, heteronuclear multiple bond correlation; NOESY, nuclear Overhauser effect spectroscopy.

Table 3 IC₅₀ values (μg/mL) of lymphocyte proliferation, cytokine release, and NO inhibitory activities of P. amarus extract and its chemical constituents on phagocytes (mean ± SEM, n=3)

Samples	Lymphocyte proliferation	Nitric oxide	TNF-α	IL-1β
P. amarus extract	0.52±1.05	4.84±3.14	12.09±3.06	7.58±3.21
Ethyl 8-hydroxy-8-methyl-tridecanoate (1)	3.78±1.43 (13.93±3.77)	0.25±0.01 (0.91±0.03)	–	–
Isolintetralina (2)	–	–	–	–
1-triacontanol (3)	2.36±0.20 (5.39±0.46)	1.01±0.28 (2.30±0.68)	–	–
Stigmasterola (4)	–	–	–	–
β-sitosterola (5)	–	–	–	–
7β,19α dihydroxy-urs-12-enea (6)	–	–	–	–
Hypophyllanthin (7)	0.93±0.38 (2.18±0.89)	1.36±0.01 (3.16±0.02)	12.75±3.45 (29.65±8.03)	–
Phyltetralin (8)	0.44±0.03 (1.07±0.07)	1.23±0.38 (2.95±0.93)	–	–
Phyllanthin (9)	0.76±0.13 (1.82±0.33)	2.49±0.81 (5.95±1.94)	17.00±5.47 (40.67±0.66)	–
1,7,8-trihydroxy-2-naphtaldehyde (10)	5.84±0.89 (28.67±4.37)	0.22±0.10 (1.07±0.31)	–	–
GA	0.96±0.69 (5.66±4.05)	4.12±0.80 (24.17±2.50)	13.92±4.65 (76.32±7.12)	35.28±3.62 (194.99±6.35)
EA	13.23±1.24 (43.77±1.54)	5.54±0.22 (18.36±1.09)	32.47±3.66 (105.95±6.65)	39.20±3.61 (134.10±5.02)
Ger	12.31±1.68 (22.60±1.32)	4.25±0.47 (7.81±0.31)	7.51±5.31 (8.66±2.51)	14.80±2.56 (16.41±0.74)
Cor	11.08±1.17 (17.41±2.12)	5.07±1.75 (7.98±2.05)	4.60±2.99 (7.39±2.53)	27.40±1.17 (43.19±2.53)
Predni	0.038±0.002 (0.09±0.06)	–	–	–
Dexa	–	0.14±0.76 (0.35±0.23)	0.77±0.27 (1.96±0.44)	0.26±1.02 (0.67±1.32)

Notes: IC₅₀ values (μM) are in parentheses.

a Not determined. – indicates data not applicable.

Abbreviations: Cor, corilagin; Dexa, dexamethasone; EA, ellagic acid; GA, gallic acid; Ger, geraniin; IC₅₀, half maximal inhibitory concentration; IL-1β, interleukin-1β; NO, nitric oxide; P. amarus, Phyllanthus amarus; Predni, prednisolone; SEM, standard error of the mean; TNF-α, tumor necrosis factor-α.

Figure 3 Percentage inhibition of NO release by P. amarus and its major compounds on LPS-stimulated RAW 264.7 cells.

Notes: Data are mean ± SEM (n=3). Significance of differences with respective control: *P<0.05. For Dexa, only percentage inhibition at 4 μg/mL is shown.
Abbreviations: Cor, corilagin; Dexa, dexamethasone; EA, ellagic acid; GA, gallic acid; Ger, geraniin; LPS, lipopolysaccharide; NO, nitric oxide; P. amarus, Phyllanthus amarus; SEM, standard error of the mean.

Figure 4 Percentage inhibition of T-cell proliferation by P. amarus and its major compounds on PHA-stimulated human PBMCs.

Notes: Data are mean ± SEM (n=3). Significance of differences with respective control: *P<0.05. For Predni, only percentage inhibition at 5 μg/mL is shown.
Abbreviations: Cor, corilagin; Dexa, dexamethasone; EA, ellagic acid; GA, gallic acid; Ger, geraniin; P. amarus, Phyllanthus amarus; PBMCs, peripheral blood mononuclear cells; PHA, phytohemagglutinin; Predni, prednisolone; SEM, standard error of the mean.

Figure 5 Percentage inhibition of TNF-α by P. amarus and its major compounds on LPS-stimulated human PBMCs.

Notes: Data are mean ± SEM (n=3). Significance of differences with respective control: *P<0.05. For Dexa, only percentage inhibition at 5 μg/mL is shown.
Abbreviations: Cor, corilagin; Dexa, dexamethasone; EA, ellagic acid; GA, gallic acid; Ger, geraniin; LPS, lipopolysaccharide; P. amarus, Phyllanthus amarus; PBMCs, peripheral blood mononuclear cells; SEM, standard error of the mean; TNF-α, tumor necrosis factor-α.

Figure 6 Percentage inhibition of IL-1β by P. amarus and its major compounds on LPS-stimulated human PBMCs.

Notes: Data are mean ± SEM (n=3). Significance of differences with respective control: *P<0.05. For Dexa, only percentage inhibition at 5 μg/mL is shown.
Abbreviations: Cor, corilagin; Dexa, dexamethasone; EA, ellagic acid; GA, gallic acid; Ger, geraniin; IL-1β, interleukin-1β; LPS, lipopolysaccharide; P. amarus, Phyllanthus amarus; PBMCs, peripheral blood mononuclear cells; SEM, standard error of the mean.