Composition, crocins active site, and uses thereof

Abstract

A composition, and a crocins active site extracted from Gardenia jasminoides Ellis, mainly comprising the following ingredients: crocetin di--D-gentiobioside, crocetin--D-glucopyranosyl--D-gentiobioside, crocetin di--D-glucopyranoside, 13Z-crocetin di--D-gentiobioside, neocrocin B, crocetin mono--D-gentiobioside, 13Z-crocetin-8-O--D-gentiobioside, 13Z-crocetin-8-O--D-gentiobioside, and crocetin mono--D-glucopyranoside. Pharmacological experiment results show that the crocins active site can effectively improve learning and memory injuries in mice induced by scopolamine and amyloid protein.

Claims

1. A method of improving a subject's learning and memory ability, which comprises administering to the subject who is in need thereof a composition comprising therapeutically effective amounts of crocetin mono--D-gentiobioside, crocetin di--D-gentiobioside and neocrocin B, wherein said neocrocin B has the following structural formula ##STR00010##

2. A method of improving a subject's learning and memory ability, which comprises administering to the subject who is in need thereof a composition comprising a therapeutically effective amount of a crocins active site extracted from Gardenia jasminoides Ellis, wherein the crocins active site comprises crocetin mono--D-gentiobioside, crocetin di--D-gentiobioside and neocrocin B, wherein said neocrocin B has the following structural formula ##STR00011##

3. The method according to claim 2, wherein an ultra-performance liquid chromatography (UPLC) characteristic chromatogram of the crocins active site comprises 9 chromatographic peaks, wherein the nine chromatographic peaks comprise peaks for crocetin mono--D-gentiobioside, crocetin di--D-gentiobioside, crocetin--D-glucopyranosyl--D-gentiobioside, crocetin di--D-glucopyranoside, 13Z-crocetin di--D-gentiobioside, neocrocin B, 13Z-crocetin-8-O--D-gentiobioside, 13Z-crocetin-8-O--D-gentiobioside and crocetin mono--D-glucopyranoside, wherein a retention time of crocetin mono--D-gentiobioside is set as 1, wherein values of relative retention time are obtained for the various other chromatographic peaks respectively, and wherein the retention time of crocetin di--D-gentiobioside is 0.380.02, the retention time of crocetin--D-glucopyranosyl--D-gentiobioside is 0.480.02, the retention time of crocetin di--D-glucopyranoside is 0.600.02, the retention time of 13Z-crocetin di--D-gentiobioside is 0.780.02, the retention time of neocrocin B is 0.890.02, the retention time of crocetin mono--D-gentiobioside is 1.00, the retention time of 13Z-crocetin-8-O--D-gentiobioside is 1.130.02, the retention time of 13Z-crocetin-8-O--D-gentiobioside is 1.140.02, and the retention time of crocetin mono--D-glucopyranoside is 1.190.02.

4. The method according to claim 3, wherein the UPLC characteristic chromatogram of the crocins active site is established by reverse-phase ultra-performance liquid chromatography under a chromatographic condition in which octadecylsilane chemically bonded silica is a stationary phase, an acetonitrile-water solution containing 0.1% of formic acid is a mobile phase, and gradient elution is performed, wherein a flow velocity is 0.6 mL/min, a detection wavelength is 440 nm, and a temperature of a chromatographic column is 35 C.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 is characteristic chromatogram of Gardenia sourced crocins active site determined by liquid phase analysis with UPLC.

(2) FIG. 2 is a chromatographic peak assignment chromatogram of compound 1 isolated from Gardenia sourced crocins active site under the same UPLC condition.

(3) FIG. 3 is a chromatographic peak assignment chromatogram of compound 2 isolated from Gardenia sourced crocins active site under the same UPLC condition.

(4) FIG. 4 is a chromatographic peak assignment chromatogram of compound 3 isolated from Gardenia sourced crocins active site under the same UPLC condition.

(5) FIG. 5 is a chromatographic peak assignment chromatogram of compound 4 isolated from Gardenia sourced crocins active site under the same UPLC condition.

(6) FIG. 6 is a chromatographic peak assignment chromatogram of compound 5 isolated from Gardenia sourced crocins active site under the same UPLC condition.

(7) FIG. 7 is a chromatographic peak assignment chromatogram of compound 6 isolated from Gardenia sourced crocins active site under the same UPLC condition.

(8) FIG. 8 is a chromatographic peak assignment chromatogram of compound 7 isolated from Gardenia sourced crocins active site under the same UPLC condition.

(9) FIG. 9 is a chromatographic peak assignment chromatogram of compound 8 isolated from Gardenia sourced crocins active site under the same UPLC condition.

(10) FIG. 10 is a chromatographic peak assignment chromatogram of compound 9 isolated from Gardenia sourced crocins active site under the same UPLC condition.

(11) FIG. 11 is a diagram showing the protective effect of Gardenia sourced crocins active site GJ-4 on learning and memory impairment in mice caused by scopolamine.

(12) FIG. 12 is a diagram showing the protective effect of Gardenia sourced crocins active site GJ-4 on learning and memory impairment in mice caused by intracerebroventricular A.sub.25-35 injection (step-down test).

(13) FIG. 13 is a diagram showing the protective effect of Gardenia sourced crocins active site GJ-4 on learning and memory impairment in mice caused by intracerebroventricular A.sub.25-35 injection (Morris water maze).

DETAILED DESCRIPTION OF EMBODIMENTS

(14) The technical solutions of the present disclosure will be further explained with reference to examples below, but the present disclosure is not limited to these examples.

Example 1: Method of Preparing Gardenia Sourced Crocins Active Site

(15) 40.0 kg of dry Gardenia fruit was taken and pulverized appropriately, then 4 times of 60% ethanol was used for extraction for 3 times by heating under reflux, 2 hours each time. The extracting solution was combined. The solvent was evaporated at reduced pressure and 6.2 kg of total Gardenia extract was obtained. The extract was dissolved in an appropriate amount of water and centrifuged. Then it was subjected to macroporous resin open column chromatography (20.090 cm). Then it was eluted respectively with 4 times column volume of water, 30%, 50%, 70% and 95% ethanol. The eluent from respective part was collected. The solvent was recycled at reduced pressure. About 4.5 kg was obtained from the combination of water elution and 30% ethanol elution, 710.0 g from 50% ethanol elution, 150.0 g from 70% ethanol elution, 112.0 g from 95% ethanol elution. The one from 70% ethanol elution is the Gardenia sourced crocins active site GJ-4.

Example 2: Isolation and Identification of Main Ingredients in the Gardenia Sourced Crocins Active Site

(16) FIG. 1 shows the UPLC characteristic chromatogram of the Gardenia sourced crocins active site prepared in Example 1. With the help of the characteristic chromatogram, the structures of 9 compounds, i.e. crocetin di--D-gentiobioside, crocetin--D-glucopyranosyl--D-gentiobioside, crocetin di--D-glucopyranoside, 13Z-crocetin di--D-gentiobioside, neocrocin B, crocetin mono--D-gentiobioside, 13Z-crocetin-8-O--D-gentiobioside, 13Z-crocetin-8-O--D-gentiobioside and crocetin mono--D-glucopyranoside were identified by isolation means including ODS column chromatography and RP-HPLC preparative liquid chromatograph and by analysis and identification methods including UV, MS and NMR.

(17) The compounds as isolated were assigned under the same chromatographic conditions as the UPLC characteristic chromatogram of the Gardenia sourced crocins active site. See FIGS. 2-10 for the particular assignment process.

2.1 Isolation Process

(18) The obtained Gardenia sourced crocins active site was isolated by column chromatography on silica gel eluted with chloroform, methanol and water at a ratio of 8:2:0.2 to give compound 6 (about 7.0 g) and eluted with chloroform, methanol and water at a ratio of 9:1:0.1 to give compound 9 (136.5 mg). It was subjected to open column chromatography on ODS eluted with 50% methanol-water to give compound 1 (545.1 mg), eluted with 55% methanol-water to give compound 2 (143.7 mg) and eluted with 50% methanol-water to give compound 3 (315.7 mg). It was isolated by preparative high performance liquid column chromatography on ODS eluted with 60% methanol-water to give compound 4 (265.7 mg, t.sub.R=16.6 min), eluted with 68% methanol-acid water (0.1% CH.sub.3COOH) to give compound 5 (520.9 mg, t.sub.R=9.5 min), isolated with 42% acetonitrile-acid water (0.1% CH.sub.3COOH) to give compound 7 (8.0 mg, t.sub.R=17.9 min) and compound 8 (16.0 mg, t.sub.R=21.5 min).

2.2 Structural Analysis of Compounds

2.2.1 Compound 1

(19) Red amorphous powder. HR-ESI-MS gives m/z 999.3680 [M+Na].sup.+ (calculated value, 999.3685), formula determined as C.sub.44H.sub.64O.sub.24, calculated degree of unsaturation, 13.

(20) .sup.1H-NMR (600 MHz, in DMSO-d.sub.6) suggests characteristic alkenyl hydrogen signal [ 7.35 (2H, d, J=10.8 Hz), 6.87 (2H, dd, J=7.8, 2.4 Hz), 6.82 (2H, d, J=15.0 Hz), 6.67 (2H, dd, J=15.0, 12.6 Hz), 6.53 (2H, br.d, J=9.6 Hz),]; 4 pairwise overlapping saccharide end group signals [ 5.42 (2H, d, J=8.4 Hz), 4.17 (2H, d, J=7.8 Hz)] and 4 pairwise overlapping methyl hydrogen signals [ 2.00 (6H, s), 1.97 (6H, s)] of crocetin.

(21) By comparison with the documentation.sup.[11], it is determined that compound 1 is crocetin di--D-gentiobioside. See Table 1 for .sup.13C-NMR of compound 1.

2.2.2 Compound 2

(22) Red amorphous powder. HR-ESI-MS gives m/z 837.3166 [M+Na].sup.+ (calculated value, 837.3157), formula determined as C.sub.38H.sub.54O.sub.19, calculated degree of unsaturation, 12.

(23) .sup.1H-NMR (600 MHz, in DMSO-d.sub.6) suggests characteristic alkenyl hydrogen signal [ 7.35 (2H, d, J=11.4 Hz), 6.86 (2H, dd, J=8.4, 3.0 Hz), 6.82 (1H, d, J=14.4 Hz), 6.81 (1H, d, J=15.0 Hz), 6.66 (2H, dd, J=15.0, 12.0 Hz), 6.54 (2H, br.d, J=8.4 Hz),], 4 methyl hydrogen signals [ 1.99 (6H, s), 1.97 (6H, s)] and 3 saccharide end group proton signals [ 5.42 (2H, d, J=7.8 Hz), 4.17 (1H, d, J=7.8 Hz)] of crocetin.

(24) By comparison with the documentation.sup.[11], it is determined that compound 2 is crocetin--D-glucopyranosyl--D-gentiobioside. See Table 1 for .sup.13C-NMR of compound 2.

2.2.3 Compound 3

(25) Red amorphous powder. ESI-MS (positive) gives m/z 675 [M+Na].sup.+, 1327 [2M+Na].sup.+. It is speculated that its molecular weight is 652.

(26) .sup.1H-NMR (600 MHz, in DMSO-d.sub.6) suggests characteristic alkenyl hydrogen signal [ 7.35 (2H, d, J=11.4 Hz), 6.86 (2H, dd, J=8.4, 3.0 Hz), 6.81 (2H, d, J=15.0 Hz), 6.67 (2H, dd, J=15.0, 11.4 Hz), 6.54 (2H, br.d, J=9.6 Hz),]; 2 overlapping saccharide end group signals [ 5.42 (2H, d, J=7.8 Hz)] and 4 pairwise overlapping methyl hydrogen signals [ 2.00 (6H, s), 1.97 (6H, s)] of crocetin.

(27) By comparison with the documentation.sup.[12], it is determined that compound 3 is crocetin di--D-glucopyranoside. See Table 1 for .sup.13C-NMR of compound 3.

2.2.4 Compound 4

(28) Red amorphous powder. HR-ESI-MS gives m/z 999.3665 [M+Na].sup.+ (calculated value, 999.3685), formula determined as C.sub.44H.sub.64O.sub.24, calculated degree of unsaturation, 13.

(29) Compound 4 is an isomer of compound 1. By comparing their .sup.1H-NMR (600 MHz, in DMSO-d.sub.6), there is a big change in the alkenyl hydrogen area of compound 4, and the rest of signals are substantially consistent with compound 1. In .sup.13C-NMR (150 MHz, in DMSO-d.sub.6) of compound 4, the configuration change in the double bond on the 13th site breaks the highly symmetric structure of the compound. Each of many overlapping alkenyl carbons signals becomes 2 signals, the methyl carbon signal on the 20th site shifts to 20.0 toward the low field, and the end group hydrogen signal of the saccharide connected with the carbon on the 8th site changes from 5.42 to 5.44.

(30) By comparison with the documentation.sup.[11], it is determined that compound 4 is 13Z-crocetin di--D-gentiobioside. See Table 1 for .sup.13C-NMR of compound 4.

2.2.5 Compound 5

(31) Red amorphous powder. ESI-MS (positive) gives m/z 1011 [M+Na].sup.+ and indicates that the molecular weight of the compound is 988.HR-ESI-MS gives 989.3642[M+H].sup.+ (calculated value, 989.3654), formula determined as C.sub.48H.sub.60O.sub.22, calculated degree of unsaturation, 19.

(32) In the .sup.1H-NMR (600 MHz, in DMSO-d.sub.6) chromatogram of compound 5, the low field area shows a group of trans-alkenyl hydrogen signals [ 7.44 (1H, d, J=15.6 Hz, H-3), 6.16 (1H, d, J=16.2 Hz, H-2)]; a group of intercoupling aromatic proton signals [ 7.03 (1H, d, J=1.8 Hz, H-5), 6.98 (1H, dd, J=8.4, 1.8 Hz, H-9) 6.74 (1H, d, J=7.8 Hz, H-8)]. In combination with .sup.13C-NMR (150 MHz, in DMSO-d.sub.6) signals, 148.5 (C-7), 145.6 (C-6), 125.2 (C-4), 121.6 (C-9), 115.7 (C-8) and 114.9 (C-5), it indicates that there is 1,3,4-trisubstituted benzene ring in the structure. The HMBCs of the alkenyl hydrogen proton signals H-3/C-4, C-5, C-9, C-1; H-2/C-1, C-4 are long-range correlated. It indicates that it contains a caffeoyl segment of C.sub.6-C.sub.3.

(33) The saccharide end group proton signals [ 5.42 (1H, d, J=7.8 Hz, H-1) and 4.17 (1H, d, J=7.8 Hz, H-1)] indicate that the two glucose residues both have a configuration. In the HMBC chromatography, the correlated peaks H-6/C-1 and H-1/C-6 indicate that the two glucosyl groups are in 1.fwdarw.6 bond and thus form a gentiobiosyl. The hydrolytic derivatization experiment of saccharide indicate that the glucose has a D configuration as its absolute configuration.

(34) By removing 2 glucose residues and 1 caffeoyl segment of C.sub.6-C.sub.3 and comparing with known documentation, the characteristic crocetin signals in the structure may be assigned.

(35) By .sup.1H-.sup.1H COSY, HSQC and HMBC chromatograms, it is identified that there is structural segment of 3-caffeoylquinic acid in the structure, and it is speculated by HMBC chromatogram that the 4th site of the caffeic acid is bonded with crocetin.sup.[13].

(36) By searching, compound 5 is a new compound that has never been reported and is named neocrocin B. See Table 1 for .sup.13C-NMR of compound 5.

2.2.6 Compound 6

(37) Red amorphous powder. HR-ESI-MS gives [M+Na].sup.+ of 675.2625 (calculated value, 675.2629), formula determined as C.sub.32H.sub.44O.sub.14, calculated degree of unsaturation, 11.

(38) .sup.1H-NMR (600 MHz, in DMSO-d.sub.6) suggests characteristic alkenyl hydrogen signal, 2 saccharide end group signals and 4 methyl hydrogen signals of crocetin.

(39) By comparison with the documentation.sup.[14], it is determined that compound 6 is crocetin mono--D-gentiobioside. See Table 1 for .sup.13C-NMR of compound 6.

2.2.7 Compound 7

(40) Red amorphous powder. ESI-MS (positive) gives m/z 675 [M+Na].sup.+, m/z 1327 [2M+Na].sup.+ and indicates that the molecular weight is 652. HR-ESI-MS gives [M+Na].sup.+ of 675.2617 (calculated value, 675.2629), formula determined as C.sub.32H.sub.44O.sub.14, calculated degree of unsaturation, 11.

(41) Compound 7 is a cis geometrical isomer of compound 6. But the difference lies in that compound 6 has two types of cis geometrical isomers as the compound 6 itself has an asymmetric structure. By .sup.1H, .sup.13C-NMR and two-dimensional nuclear magnetic data analysis, it is determined that compound 7 is 13Z-crocetin-8-O--D-gentiobioside. See Table 1 for .sup.13C-NMR of compound 7.

2.2.8 Compound 8

(42) Red amorphous powder. ESI-MS (positive) gives m/z 675 [M+Na].sup.+, m/z 1327 [2M+Na].sup.+ and indicates that the molecular weight is 652. HR-ESI-MS gives [M+Na.sup.+] of 675.2617 (calculated value, 675.2629), formula determined as C.sub.32H.sub.44O.sub.14, calculated degree of unsaturation, 11.

(43) Compound 8 is another geometrical isomer of compound 6. By one-dimensional and two-dimensional nuclear magnetic data analysis, compound 8 is identified as 13Z-crocetin-8-O--D-gentiobioside. By searching, compound 8 is a new compound that has never been reported. See Table 1 for its .sup.13C-NMR.

2.2.9 Compound 9

(44) Red amorphous powder. HR-ESI-MS gives 513.2095 [M+Na].sup.+ (calculated value, 513.2101), 1003.4303 [2M+Na].sup.+, formula determined as C.sub.26H.sub.34O.sub.9, calculated degree of unsaturation, 10.

(45) .sup.1H-NMR (600 MHz, in DMSO-d.sub.6) suggests characteristic alkenyl hydrogen signal, 1 saccharide end group signal and 4 methyl hydrogen signals of crocetin.

(46) By comparison with the documentation.sup.[11], it is determined that compound 9 is crocetin mono--D-glucopyranoside. See Table 1 for .sup.13C-NMR of compound 9.

2.3 UPLC-Q/TOF-MS Analysis of Gardenia Sourced Crocins Active Site

2.3.1 Chromatographic Condition

(47) BEH C18 (3.0 mm150 mm, 1.7 m); mobile phase: solvent A (water, 0.1% formic acid) and solvent B (acetonitrile, 0.1% formic acid), gradient elution (0 min-20% B, 0.5 min-20% B, 19 min-50% B, 20 min-100% B, 23 min-100% B, 24 min-20% B), flow rate: 0.6 mL/min, column temperature: 35 C., detection wavelength: 440 nm.

2.3.2 Mass Spectrum Condition

(48) Positive ion mode of electrospray, capillary voltage: 2.0 kV; solvent-removing stream: N.sub.2, flow rate 600 L/h, solvent-removing temperature 300 C.; taper hole stream: N.sub.2, flow rate 50 L/h; ion source temperature: 100 C.; Extractor: 4.00 V; colliding gas:argon. See Table 2 for the mass spectrometry of 9 main chromatographic peaks.

(49) TABLE-US-00001 TABLE 1 NO. Pos. 1 2 3 4 5 6 7 8 9 8, 8 166.2 166.3 166.2 166.2 166.2 166.2 166.1 169.3 166.2 167.0 169.2 169.2 166.2 169.1 9, 9 125.3 125.3.sup.a 125.4 125.9 125.4 125.2 125.8 128.1 125.3 125.4.sup.a 125.5 126.1 127.1 127.2 125.0 127.0 10, 10 139.9 140.0.sup.a 139.8 140.1 140.0 139.9 140.1 137.9 139.8 139.9.sup.a 140.0 139.0 138.0 137.9 140.0 138.0 11, 11 123.9 124.0 123.9 125.1 123.9.sup.a 123.8 124.9 125.5 123.8 123.7 124.0.sup.a 124.3 124.0 123.5 124.3 12, 12 144.6 144.7.sup.a 144.5 136.8 144.7 144.7 136.8 135.3 144.6 144.6.sup.a 144.7 144.0 143.3 143.2 144.7 143.3 13, 13 136.9 137.0 136.9 136.5 136.9.sup.a 137.0 134.8 135.2 137.0 135.2 136.8.sup.a 136.7 136.4 136.2 136.7 14, 14 136.0 136.1 136.0 134.6 136.1 136.0 134.5 133.7 136.0 135.9 135.7 135.3 135.1 135.9 135.3 15, 15 132.0 132.1 132.0 130.8 132.1 132.1 130.6 131.1 132.1 131.1 131.9 131.6 130.8 130.3 131.6 19, 19 12.7 12.7 12.7 12.7 12.7 12.7 12.7 12.8 12.7 12.8 12.8 12.8 12.7 12.8 20, 20 12.6 12.6 12.6 20.0 12.6 12.6 20.0 20.1 12.6 12.5 12.6 12.5 12.5 12.5 12.5 1.sup. 94.5 94.6 94.6 94.5 94.5 94.5 94.6 94.5 94.6 2.sup. 72.5 72.6.sup.a 72.5 72.5 72.5 72.5 72.5 72.4 72.5 3.sup. 76.3 76.3 76.4 76.3 76.3 76.3 76.2 76.2 76.5 4.sup. 69.2 69.3 69.5 69.2 69.2 69.2 69.2 69.2 69.5 5.sup. 76.3 76.3 77.8 76.3 76.3 76.3 76.3 76.3 77.9 6.sup. 67.9 68.0 60.5 67.9 67.9 67.9 67.9 67.9 60.6 1 103.1 103.1 103.1 103.1 103.1 103.1 103.1 2 73.5 73.5 73.5 73.5 73.5 73.4 73.5 3 76.8 76.8 76.8 76.8 76.8 76.8 76.8 4 70.0 70.0 70.0 70.0 70.0 70.0 70.0 5 76.9 76.9 76.9 76.9 76.9 76.9 76.9 6 61.0 61.0 61.0 61.0 61.0 61.0 61.0 1 94.6 73.6 2 72.5.sup.a 37.4 3 76.5 67.5 4 69.6 74.2 5 77.9 66.3 6 60.6 37.6 7 174.8 .sup.1 165.6 .sup.2 113.6 .sup.3 145.6 .sup.4 125.2 .sup.5 114.9 .sup.6 145.6 .sup.7 148.5 .sup.8 115.7 .sup.9 121.6 .sup.ameans signals could be interchangeable with the corresponding position in one compound.

(50) TABLE-US-00002 TABLE 2 Measured Calculated Mass error Elemental NO. t.sub.R Selected Ion mass mass (ppm) composition Fragmentation Identification 976.3770, 999.3676; 652.2716, 1* 5.79 [M + Na].sup.+ 999.3676 999.3685 0.9 C.sub.44H.sub.64O.sub.24 675.2599; 329.1764, 311.1641, crocetin di--D-gentiobioside 293.1528 2* 7.35 [M + Na].sup.+ 837.3118 837.3157 4.7 C.sub.38H.sub.54O.sub.19 814.3245, 837.3118; 675.2667; crocetin--D-glucopyranosyl- 329.1737, 311.1633, 293.1537 -D-gentiobioside 3* 9.15 [M + Na].sup.+ 675.2614 675.2629 2.2 C.sub.32H.sub.44O.sub.14 652.2706, 675.2614; 513.2049; crocetin 329.1725, 311.1632, 293.1535 di--D-glucopyranoside 4* 11.71 [M + Na].sup.+ 999.3680 999.3685 0.5 C.sub.44H.sub.64O.sub.24 976.3772, 999.3680, 1953.7651; 13Z-crocetin 652.2731; 329.1740, 311.1644, di--D-gentiobioside 293.1530 5* 13.56 [M + H].sup.+ 989.3597 989.3654 5.8 C.sub.48H.sub.60O.sub.22 988.3539, 989.3597, 1976.7084; neocrocin B 665.2532; 827.3113 6* 15.09 [M + Na].sup.+ 675.2620 675.2629 1.3 C.sub.32H.sub.44O.sub.14 652.2720, 675.2620; crocetin 329.1744, 311.1635, 293.1530 mono--D-gentiobioside 7* 17.08 [M + Na].sup.+ 675.2632 675.2629 0.4 C.sub.32H.sub.44O.sub.14 652.2731, 675.2632; 13Z-crocetin-8-O--D- 329.1759, 311.1651, 293.1541 gentiobioside 8* 17.51 [M + Na].sup.+ 675.2625 675.2629 0.6 C.sub.32H.sub.44O.sub.14 652.2675, 675.2625; 13Z-crocetin-8-O--D- 329.1741, 311.1641, 293.1539 gentiobioside 9* 18.03 [M + Na].sup.+ 513.2098 513.2101 0.6 C.sub.26H.sub.34O.sub.9 490.2192, 513.2098, 1003.4275; crocetin mono--D- 329.1747, 311.1643, 293.1534 glucopyranoside *indicates the comparison with control products (which are monomeric compounds obtained by isolation)

Example 3: Gardenia Sourced Crocins Active Site GJ-4 Improves Learning and Memory Impairment in Mice Induced by Scopolamine (Step-Down Test)

3.1 Principle of the Step-Down Test

(51) The device used in the step-down test was a rectangular reflection box with a size of 10 cm10 cm60 cm. The reflection box was partitioned into 5 compartments by black plastic plates and provided with copper grid on its floor at an interval of 0.5 cm. The grid could be energized. The voltage strength was controlled by a transformer. A wooden platform with a height and a diameter both being 4.5 cm was provided at the right corner in each compartment. During the time of test, 36 V alternating current was supplied. When shocked, the mice normally responded by jumping onto the safe platform to avoid the harmful stimulus. No power was supplied on the first day. Mice were put in the reflection box and were free for 5 min to get familiar with the environment. 24 h later, the copper grid was energized (with 36 V alternating current). The timings when the respective groups of mice first jumped onto the safe platform after being shocked (response time) and error times that they jumped from the safe platform within 5 min (basic error times) were recorded as the learning test scores. The next day, the above process was repeated. The timings when the respective groups of mice first jumped from the safe platform (latent period) and times of being shocked within 5 min (error times) were recorded as the memory test scores. In test, if the mice stayed on the safe platform for more than 5 min, the latent period was taken as 5 min.

3.2 Scheme of Step-Down Test

(52) ICR mice, male, 160 mice, divided into 8 groups, 20 in each group, respectively control group, model group, donepezil (5 mg/kg) group, memantine (5 mg/kg) group, GJ-4 (12.5 mg/kg) group, GJ-4 (25 mg/kg) group, GJ-4 (50 mg/kg) group and GJ-4 (100 mg/kg) group. The mice were administrated for 7 consecutive days in advance. Then the mice were trained in step-down test on day 5 and day 6. On day 7, after 1 h of administration, the model group and the administrated groups were administrated with scopolamine (2 mg/kg) by intraperitoneal injection respectively. 30 min later, they were put in behavior test by step-down method. The timing when the mice first jumped down (latent period) and times of jumping down within 5 min (error times) were recorded. See FIG. 11 for the data.

(53) The test result shows that Gardenia sourced crocins active site GJ-4 shows a good improvement effect in animal dementia induced by scopolamine. GJ-4 can obviously elongate the step-down latent period for mice and reduce the step-down error times. 25 mg/kg, 50 mg/kg and 100 mg/kg dosage groups exhibit a certain dose-effect relationship. Medium and high dosage groups have an equivalent efficacy with the positive control drug, donepezil. And the test result is reproducible. No toxic reaction associated with administration was observed in any dosage groups in the test.

Example 4: Gardenia Sourced Crocins Active Site GJ-4 Improves Learning and Memory Impairment in Mice Induced by Intracerebroventricular A.SUB.25-35 .Injection (Step-Down Test and Morris Water Maze Test)

4.1 Intracerebroventricular Injection Operation for Mice, Grouping and Administration

(54) 5 g/L A.sub.25-35 was prepared with sterile triple distilled water and placed in an incubator at 37 C. and left to stand for 7 days to aggregate. Then it was cryopreserved in a refrigerator at 20 C. After being adaptively fed for two days, ICR mice were anesthetized with 4% chloral hydrate (10 mg/kg) by intraperitoneal injection. Then they were fixed on a stereotaxic apparatus. The head skin of the mice was cut open along the center line with surgical scissors to expose the bregma and the lambdoidal suture. The skull was drilled with an electric drill at the left paracele at a spot 2 mm behind the bregma, 2 mm to the left of the center line, and 1.7 mm under the cerebral dura mater to an extent that the meninx would not be damaged, and then 2 L A.sub.25-35 was injected to the left paracele of the mice, 10 g/mouse. The administration was done within 1 min. The injection needle was left there for 3 min and then pulled out slowly. Then the incision was sutured with surgical suture. The mice were administrated with ampicillin (5 mg/kg) by intramuscular injection and then put into a cage. For the sham group, the mice were injected with 2 L sterile triple distilled water in the left paracele at a spot 2 mm behind the bregma, 2 mm to the left of the center line, and 1.7 mm under the cerebral dura mater. After operation, the mice injected with A.sub.25-35 in the paracele were randomly divided into a model group, GJ-4 (25 mg/kg) group, GJ-4 (50 mg/kg) group, GJ-4 (100 mg/kg) and donepezil (5 mg/kg) group, 15 mice in each group. After operation, the mice were allowed to rest for 3 days. Each group was intragastrically administrated with drugs of corresponding dosage. The sham group and the model group were administrated with the same dosage of saline, once a day, for 12 consecutive days.

4.2 Behavior Test

4.2.1 Step-Down Test

(55) On day 7 after administration, the mice were tested for learning and memory abilities by step-down test. The device used in the step-down test is a rectangular reflection box with a size of 10 cm10 cm60 cm. The reflection box is partitioned into 5 compartments with black plastic plates and provided with copper grid on its floor at an interval of 0.5 cm. The grid can be energized. The voltage strength is controlled by a transformer. A wooden platform with a height and a diameter both being 4.5 cm is provided at the right corner in each compartment. During the time of test, 36 V alternating current is supplied. When shocked, the mice normally respond by jumping onto the safe platform to avoid the harmful stimulus. No power was supplied on the day 5 after administration. Mice were put into the reflection box and were free for 5 min to get familiar with the environment. 24h later, the copper grid was energized (with 36 V alternating current). The timings when the respective groups of mice first jumped onto the safe platform after being shocked (response time) and error times that they jumped from the safe platform within 5 min (basic error times) were recorded as the learning test scores. On day 7 after administration, the above process was repeated. The timings when the respective groups of mice first jumped from the safe platform (latent period) and times of being shocked within 5 min (error times) were recorded as the memory test scores. In test, if the mice stayed on the safe platform for more than 5 min, the latent period was taken as 5 min. See FIG. 12 for the result.

4.2.2 Morris Water Maze Test

(56) On the second day after the step-down test (i.e. day 8 after administration), the mice were further tested for learning and memory abilities by Morris water maze test (the mice in the respective groups were tested for learning and memory abilities in spatial sense of position and sense of direction). The device used in Morris water maze test was a circular pool with a diameter of 120 cm and a depth of 40 cm, provided with a layer of black adhesive tape on its internal surface. The water temperature was kept at 23-25 C. The indoor temperature was kept at 26-28 C. The water tank was randomly divided into four quadrants. The platform stayed where it was during the time of test, i.e. in the center of the second quadrant, 1-2 cm lower than the water surface. Obvious marks were made on the walls in the room for the mice to identify directions. All the objects in the room were stationary during the test, so as not to interfere with the mice. The test lasted for 5 days, twice a day. For the first 4 days, place navigation test was conducted. The mice were gently put into the water from two quadrants in sequence, facing the pool walls, in a way to avoid stress and putting the head of the mice into water. In the meantime, the latent period within which the mice found the safe platform within 1 min was recorded. The mice were allowed to stay on the safe platform for 30 s and then taken out and put back into the cage. If the mice did not find the safe platform within 1 min, they were put on the safe platform and allowed to stay for 30 s. And the latent period was taken as 60 s. The average value of latent period within which the mice found the safe platform twice a day would be the swimming result of the mice on that day. Statistical analysis was conducted. See FIG. 13 for the results. On day 5, spatial probe test was conducted. The safe platform was removed. Mice were put into the water from a selected quadrant, with their heads facing the pool walls. The times that the mice crossed the position where the platform was within 1 min and the swimming time in the quadrant where the platform was were recorded. Statistical analysis was conducted. See FIG. 13.

(57) In the step-down test, Gardenia sourced crocins active site GJ-4 could obviously elongate the step-down latent period for mice and reduce the step-down error times. In the water maze test, GJ-4 obviously shortened the latent period within which the mice find the platform, increased times of crossing the platform and elongated the swimming time in the quadrant where the platform was in the meanwhile. The test result shows that GJ-4 shows a good improvement effect in learning and memory disorder in mice. Various dosage groups exhibit a certain dose-effect relationship. The high dosage groups have an equivalent or even better efficacy than the positive control drug, donepezil. No toxic reaction associated with administration was observed in any dosage groups in the test.

Example 5: Neuroprotection Effect of Crocin Monomer in Gardenia in SH-SY5Y Cell Damage Model Induced by L-Glutamic Acid

5.1 Culture Method of SH-SY5Y Nerve Cells

(58) SH-SY5Y nerve cells were cultured in DMEM medium (containing 5% fetal calf serum by volume fraction) which were placed in an incubator containing 5% CO.sub.2 under 37 C. They were subcultured once every 3-4 days. Cells in logarithmic phase were used for test

5.2 Screening Method of L-Glutamic Acid Damage Model

(59) SH-SY5Y cells were inoculated in a 96-well plate at a concentration of 510.sup.3 and continued to culture for 24 h. 100 L chemical liquid medium containing L-glutamic acid was added to the 96-well plate so that the L-glutamic acid had a final concentration of 160 mM, the drug had a final concentrations of 10 M, 1 M and 0.1 M. 3 parallel wells were arranged for each concentration. They were continued to culture for 24 h. 24 h later, the supernatant was pipetted out and discarded. 100 L MTT (0.5 mg/mL) was added into each well. They were further incubated for 4 h. Then the supernatant was pipetted out and discarded. 150 L DMSO was added into each well. They were vibrated for 10 min. A wavelength of 570 nm was selected. The absorbance value was measured on a microplate reader.sup.[13]. (effective rate %=(OD.sub.drugOD.sub.model)/(OD.sub.controlOD.sub.model)*100). See Table 3 for the results.

(60) TABLE-US-00003 TABLE 3 Drug concentration Viability of cells Compound (moL/L) with drug neocrocin B (5) 10.sup.5 40.03 3.91** 10.sup.6 27.63 5.36* 10.sup.7 9.89 2.93 crocetin mono--D- 10.sup.5 0.00 0.00 glucopyranoside (9) 10.sup.6 0.00 0.00 10.sup.7 0.00 0.00 crocetin di--D-glucopyranoside (3) 10.sup.5 0.00 0.00 10.sup.6 0.00 0.00 10.sup.7 0.00 0.00 crocetin--D-glucopyranosyl-- 10.sup.5 0.00 0.00 D-gentiobioside (2) 10.sup.6 0.00 0.00 10.sup.7 0.00 0.00 *P < 0.1, **P < 0.05, ***P < 0.01.