Journal List > Nat Prod Sci > v.23(4) > 1060678

Nugroho, Woo, Park, Kwon, Jung, Lee, Kim, and Park: Comparative Study on the Content and Cytotoxicity of Pseudolaric Acid B in the Five Plant Parts of Pseudolarix kaempferi

Abstract

Pseudolaric acids of Pseudolarix kaempferi (Pinaceae) have been known as diterpenoids with potent anti-fungal-, anti-microbial, and cytotoxic activities. In the present study, the five MeOH extracts were prepared from the five plant part (root bark, stem bark, leaf, the inner part of root, and cone) to find the relation between the concentration of pseudolaric acids and cytotoxicity. Pseudolaric acids B and C were isolated from the root bark of P. kaempferi to use them as standard compounds. The five extracts were tested on cytotoxicity against six cancer cell lines, A549 (lung), HCT116 (colon), MDA-MB-231 (breast), SNU638 (stomach), and SK-hep-1 (liver) by SRB assay, but against K562 (leukemia) by SRB- or MTT assay. HPLC quantification were performed on a Shisheido Capcell PAK C18 column (5 µm, 4.6 mm × 250 mm) using 254 nm wavelength. The cytotoxicity (IC50, 0.36 µg/ml on K562 cell lines) of the root bark extract was potent and the content (101.1 mg/g extract) of pseudolaric acid B was very high in the root bark. These results suggest that the MeOH extract obtained from the root bark could be developed as the anti-cancer agent with a high quantity of pseudolaric acid B.

References

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Fig. 1.
Structure of pseudolaric acids B (R = acetyl) and C (R = H) isolated from P. kaempferi.
nps-23-265f1.tif
Fig. 2.
HPLC chromatograms of the MeOH extracts obtained from the five plant parts of P. kaempferi.
nps-23-265f2.tif
Table 1.
1H- and13C- NMR data of compounds 1 (pseudolaric acid B) and 2 (pseudolaric acid C) measured in CDCl3
Position Compound 1
Compound 2
13C 1H 13C 1H
1 33.3 1.86 (2H, m) 33.3 1.86 (2H, m)
2 24.3 1.81 (1H, m) 24.5 1.76 (1H, m)
    2.92 (1H, 6.0, 13.8)   2.16 (1H, m)
3 49.3 3.34 (1H, d, 6.0) 54.1 2.23 (1H, d, 6.0)
4 90.2 80.6
5 30.7 1.76 (1H, m) 35.2 2.01 (1H, m)
    3.10 (1H, dd, 6.0, 13.8)   2.14 (1H, m)
6 20.2 2.17 (1H, m) 19.8 2.59 (1H, m)
    2.92 (1H, 6.0, 15.0)   2.93 (1H, 6.0, 15.6)
7 134.5 134.1
8 141.6 7.23 (1H, dd, J=4.2, 9.0 Hz) 142.7 7.26 (1H, dd-like)
9 27.8 2.63 (1H, dd, J=4.2, 15.0 Hz) 27.3 2.60 (1H, dd-like)
    2.78 (1H, dd, 9.0, 15.0)   2.71 (1H, dd, 8.4, 14.4)
10 55.3 55.1
11 83.7 80.6
12 28.5 1.62 (3H, s) 28.6 1.60 (3H, s)
13 144.5 5.94 (1H, d, 14.4) 145.0 5.96 (1H, d, 14.4)
14 121.8 6.58 (1H, dd, 11.4, 14.4) 121.5 6.59 (1H, dd, 11.4. 14.4)
15 138.7 7.29 (1H, d, 11.4) 138.8 7.28 (1H, d, 11.4)
16 127.8 127.4
17 12.6 1.99 (3H, s) 12.6 2.00 (3H, s)
18 169.4 168.2
19 173.2 173.9
20 168.0 168.2
19-OCH3 52.3 3.74 (3H, s) 52.0 3.75 (3H, s)
4-OCOCH3 172.8    
4-OCOCH3 21.7 2.15 (3H, s)    
Table 2.
Cytotoxic activity (IC50) of the MeOH extracts obtained from the five parts of P. kaempferi on six cancer cell growth in vitro
Extract A5491) HCT116 MDA-MB-231 SNU638 SK-hep-1 K562
Root bark 1.582) 1.25 34.6 30.7 2.12 0.36
Stem bark 17.98 10.68 >50 >50 >50 2.74
Leaf >50 >50 >50 >50 >50 >50
Inner part of root 9.49 7.26 >50 3.16 >50 2.76
Cone 16.59 17.58 31.86 27.2 >50 13.22
Pseudolaric acid B 0.63 0.14 13.17 0.13 0.93 0.11
Pseudolaric acid C >25 >25 >25 >25 >25 >25

1) Cancer cell line: A549 (lung), HCT116 (colon), MDA-MB-231 (breast), SNU638 (stomach), SK-hep-1 (liver), and K562 (leukemia).

2) Unit: µg/ml.

Table 3.
Linearity of standard curves and detection/quantification limits for the standard compounds
Compound tR (min) Calibration equation (linear model)a Linear range (µg/ml) R2b LODc (µg/ml) LOQd (µg/ml)
Pseudolaric acid C 13.33 y = 900.16x + 108.96 7.81–250.0 0.999 0.10 0.34
Pseudolaric acid B 16.78 y = 1045.42x + 109.95 7.81–250.0 0.999 0.09 0.29

a y, peak area at 254nm; x, concentration of the standard (µg/ml);

b R2, correlation coefficient for 6 data points in the calibration curves (n = 4);

c LOD, limit of detection (S/N = 3);

d LOQ, limit of quantification (S/N = 10).

Table 4.
Content of pseudolaric acid C and pseudolaric acid B in the extracts of various parts of P. kaempferi
Plant parts Extract (mg/g) Dried plant material (mg/g)
Pseudolaric acid C Pseudolaric acid B Pseudolaric acid C Pseudolaric acid B
Leaf 0.14 (0.01) 0.61 (0.03) 0.018 (0.002) 0.076 (0.003)
Cone 0.21 (0.02) 0.38 (0.02) 0.022 (0.002) 0.041 (0.002)
Stem bark 8.59 (0.26) 9.45 (0.21) 0.195 (0.006) 0.215 (0.005)
Root bark 27.54 (0.27) 101.05 (0.75) 0.925 (0.009) 3.39 (0.025)
Inner part of root 5.02 (0.07) 5.88 (0.16) 0.233 (0.003) 0.273 (0.007)

The data was present as average of three determinations. SD value was added in the parentheses.

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