Results From Acanthopanax Trifoliatus L. Merr.

Figure 4.30. NOESY spectrum of PK7

***Comments on the chemical composition of the Flatleaf Pine species

This is the first time in Vietnam and the second time in the world (the first since 1966) that the chemical composition of Pinus krempfii Lecomte has been published. The results of the thesis show that Pinus krempfii Lecomte contains many flavonoids, a class of substances commonly found in this genus. In addition to 6 flavonoids, a lignan compound, isolariciresinol, has been isolated and identified. Of the 7 isolated compounds, galangin and isolariciresinol are the two compounds isolated for the first time from this species.

According to the 1966 document, some substances were only identified by gas chromatography-mass spectrometry (GC-MS) and 1 H-NMR spectrum, so it is very possible that they were present in very small amounts that were not collected during the extraction and separation process - this explains why compared to the 1966 publication, some compounds were not identified.

According to many published documents, flavonoid compounds have many valuable biological activities and antioxidant activity is a typical activity of this class of substances.

Recent research results show that pinostrobin and tectochrysin both have the ability to inhibit breast cancer cells, while strobopinin and crytostrobin have activity against Staphylococcus aureus (ATCC 25923) and aeruginosa (ATCC 27853) bacteria [95], [96], [97]. With the two compounds isolated for the first time from this species, galangin, in addition to its good antibacterial ability, also has the ability to inhibit redox enzymes belonging to the polyphenol oxidase (OPD) group and isolariciresinol has a very strong antioxidant ability, even higher than vitamin E and butylated hydroxytoluene (BHT) [89], [98]. At a concentration of 2.5 μg/ml, while vitamin E only inhibited zymosan, the compound that activates polymorphonuclear leukocytes (PMNL) to produce free oxygen radicals, by 18.7%, the compound isolariciresinol could inhibit it up to 23.8%.

4.2. Results from Acanthopanaxtrifoliatus L. Merr.

4.2.1. Compounds isolated and identified from the species Acanthopanax

From the CH 2 Cl 2 extract of the stem and leaves of Acanthopanax cylindricalis, three lupane-framed triterpene compounds were isolated and identified, of which two compounds AT1 and AT2 were obtained with quite high content, used as raw materials for the later transformation process, and a new compound AT3 , discovered for the first time in nature.

4.2.1.1. 24-nor-11α-hydroxy-3-oxo-lup-20(29)-ene-28-oic acid (AT1)

Compound AT1 was isolated as a white solid. ESI-MS mass spectrum gave molecular ion peaks at m/z 479.2 [M+Na] + and 455.3 [ MH] - . Combined with 13 C - NMR spectral data , DEPT predicted the molecular formula of AT1 to be C29H44O4 .

The 1 H-NMR spectrum of AT1 gave signals of four quaternary methyl groups [δ H

0.998 (3H, s ); 1.02 (3H, s ); 1.20 (3H, s ); 1.70 (3H, s )], a secondary methyl group [δ H 0.997 (3H, d , J = 6.0 Hz), two olefinic protons [δ H 4.77 (1H, s ); 4.64 (1H, s )], and an oxymethine group [δ H 4.02 (1H, dt , J = 10.5 and 5.0 Hz)]. The 13 C-NMR and DEPT spectra gave signals for 29 carbons including a ketone group (δ C 214.3 s ), a carboxylic acid group (δ C 181.6), two olefinic carbons (δ C 110.3 and 149.7), an oxymethine carbon (δ C 70.6), five methyl groups, nine methylene carbons, six methine carbons and four

quaternary carbon. From the above spectral data, it is possible to predict that compound AT1 is a 24-nor-lupane triterpene compound. Comparing the 1 H, 13 C-NMR spectral data of this compound with the spectral data in references [19], [22] ( Table 4.9 ), the structure of compound AT1 was determined to be 24-nor-11 α -hydroxy-3-oxo-lup-20(29)-ene-28-oic acid.

Table 4.9. 1 H-, 13 C-NMR spectral data of AT1 and

24-nor-11α-hydroxy-3-oxo-lup-20(29)-ene-28-oic acid [19]

Location

AT1		24-nor-11 α -hydroxy-3-oxo-lup- 20(29)-ene-28-oic acid
δ H a,b	δ C a,c	δH #	δ C #
1		42.3		43.0
2		37.6		38.1
3		214.3		212.7
4		45.0		45.1
5		53.2		53.8
6		22.0		22.3
7		33.9		34.4
8		42.1		42.3
9		53.9		54.4
10		38.1		38.6
11	4.02 (1H, dt , J = 10.5; 5.0)	70.6	4.10 (1H, dt , J = 10.5; 5.5)	70.0
12		37.8		38.3
13		37.3		37.7
14		42.4		42.8
15		29.6		30.2
16		32.1		32.9
17		56.3		56.6
18		48.6		49.4
19		46.7		47.5
20		149.7		150.9
21		30.5		31.3
22		36.9		37.4
23	0.997 (3H, d , J = 6.0)	12.1	1.04 (3H, d , J = 6.5)	12.4
24	-	-	-	-

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0.998 (3H, s )	13.8	1.06 (3H, s )	13.8
26	1.02 (3H, s )	17.0	1.06 (3H, s )	17.5
27	1.20 (3H, s )	14.5	1.06 (3H, s )	14.7
28		181.6		178.8
29	4.77 (1H, s ); 4.64 (1H, s )	110.3	4.58 (1H, m ); 4.78 (1H, m )	110.1
30	1.70 (3H, s )	19.4		19.6

a Measured in CDCl 3 , b 500 MHz, c 125 MHz

# Spectral data of 24-nor-11 α -hydroxy-3-oxo-lup-20(29)-ene-28-oic acid measured in pyridine – d5 [19]

[M+Na] +

[M+H] +

Figure 4.31. ESI-MS mass spectrum (positive ion) of AT1

Figure 4.32. 1 H-NMR spectrum of AT1 ( 500 MHz, CDCl 3 )

Figure 4.33. 13 C-NMR and DEPT spectrum of AT1 (125MHz, CDCl 3 )

4.2.1.2. 24-nor-3α,11α dihydroxy-lup-20(29)-ene-28-oic acid (AT2)

Compound AT2 was isolated as a white powder. Combining the 13 C- NMR and DEPT spectral data and the molecular ion peaks at m/z 481.1 [M+Na] + and 457.4 [MH] - in the ESI-MS mass spectrum, the molecular formula of AT2 was predicted to be C 29 H 46 O 4 .

From the ESI-MS, 1 H-, 13 C-NMR mass spectral data, it was shown that AT2 has the same skeleton structure as AT1 and is a lupane triterpene acid. However, compared with the spectrum of AT1 , the 1D-NMR spectrum of AT2 does not have the signal of the carbonyl group at the C-3 position but instead there is the appearance of an oxymethin group [δ H 3.97 (1H, s ), δ C

71.8]. The hydroxyl group is identified at the 3α position thanks to the signal of H-3 in singlet form because if it is 3β , it must be in dt form . Compare the spectral data of AT2 with the literature.

[19] ( Table 4.10 ), allowing to identify the main AT2 substance as 24-nor-3 α ,11 α -dihydroxy- lup-20(29)-ene-28-oic acid.

Table 4.10. 1 H-, 13 C-NMR spectral data of AT2 and

24-nor-3 α ,11 α dihydroxy-lup-20(29)-ene-28-oic acid [19]

Location

AT2		24-nor-3 α ,11 α -dihydroxy-lup- 20(29)-ene-28-oic acid
δ H a,b	δ C a,c	δH #	δ C #
1		32.1		35.0
2		29.2		30.7
3	3.72 (1H, s )	71.8	3.88 (1H, m )	70.9
4		34.7		36.6
5		44.9		45.6
6		20.4		17.8
7		34.2		35.8
8		42.2		42.7
9		53.4		54.1
10		38.5		39.3
11	3.97 (1H, dt , J =10.5;4.5)	70.9	4.10 (1H, dt , J = 11.0;5.0)	70.3
12		36.9		38.6
13		37.4		37.8
14		42.3		42.9
15		28.8		30.1
16		30.5		33.0
17		56.3		56.6
18		48.6		49.5
19		47.6		47.6
20		149.8		150.9
21		29.4		31.3
22		35.7		37.5
23	0.93 (3H, d , J = 7.0)	13.1	1.08 (3H, d , J = 6.5)	12.1
24	-	-	-	-
25	0.95 (3H, s )	14.5	0.98 (3H, s )	13.6
26	0.98 (3H, s )	17.2	1.05 (3H, s )	17.8
27	1.01 (3H, s )	16.9	1.11 (3H, s )	14.7
28		181.8		178.8
29	4.63 (1H, s ), 4.77 (1H, s )	110.1	4.60 (1H, m ); 4.79 (1H, m )	110.1
30	1.70 (3H, s )	19.4	1.65 (3H, s )	19.6

a Measured in CDCl 3 , b 500 MHz, c 125 MHz

# Spectral data of 24-nor-3 α ,11 α dihydroxy-lup-20(29)-ene-28-oic acid measured in pyridine [19]

[M+Na] +

[M+H] +

Figure 4.34. ESI-MS spectrum of AT2

Figure 4.35. 1 H-NMR spectrum of AT2 (500 MHz, CDCl 3 )

Figure 4.36. 13 C-NMR and DEPT spectrum of AT2 (125MHz, CDCl 3 )

4.2.1.3. 11α,23-dihydroxy-3-oxo-lup-20(29)-ene-28-oic acid (AT3)

The isolated compound AT3 was in the form of a white powder. The molecular formula of AT3 was C 30 H 46 O 5 determined by the molecular ion peak at m/z 487.3424 [M+H] + (theoretical calculation for the formula C 30 H 47 O 5 is 487.3423) in the high-resolution mass spectrum HR- ESI-MS. . The infrared spectrum showed the characteristic absorption peak of the hydroxyl group at ν max 3447cm -1 , the carbonyl group at ν max 1695cm -1 and the C=C group at ν max 1642cm -1 and 891cm -1 .

The 1 H-NMR spectrum of AT3 showed the presence of two olefinic protons at δ H 4.65 (1H, br s, H-29A), δ H 4.79 (1H, br s, H-29B), two doublets of the oxymethylene group