(31) the การประดิษฐ์ relates to a method of producing the composition according to the
above (26), ซึ่งประกอบรวมด้วย the method of the above (30);
(32) the การประดิษฐ์ relates to a food of producing the composition according to the above
(27), ซึ่งประกอบรวมด้วย the method of the above (30);
(33) the การประดิษฐ์ relates to a method of producing the cosmetic according to the above 15 (28), ซึ่งประกอบรวมด้วย the method of the above (30);
(34) the การประดิษฐ์ relates to a method of producing the medicine according to the above
(23), ซึ่งประกอบรวมด้วย the method of the above (30);
Effect of the การประดิษฐ์
20 [0011] This การประดิษฐ์ discovered a remarkable anti-การสร้างหลอดเลือดใหม่ action in an ethanol
extract from ผลปาล์มใบเลื่อย, and also a remarkable anti-การสร้างหลอดเลือดใหม่ action in a รงควัตถุสีแดง of ผลปาล์มใบเลื่อย purified from the ethanol extract.
BRIEF DESCRIPTION OF THE DRAWINGS
25 [0012] รูป 1: A picture of tube formation under the presence of test substance to
the HUVEC cell on the Matrigel.
(a): Effect of 100ug/mL รงควัตถุสีแดง treatment for the tube formation. There
is almost no tube formation. (b), (c), (d), (e): lOug/mL, 114/mL, 0.1ug/mL,
0.01gg/mL of รงควัตถุสีแดง treatment. (f): Effect of vehicle control treatment for the
30 tube formation. The whole length of the tube was measured from the picture. (g):
Effect of 3011M suramin treatment for the tube formation. There is almost no tube
formation. (h), (i), (j), (k): 1511M, 10µM, 31AM, liAM of suramin treatment. (1):
Vehicle control treatment.
- The whole length of the tube was measured from the picture.
35 รูป 2: Inhibition of รงควัตถุสีแดง for the tube formation. (a):the full length
of formed tube treated with each concentration of รงควัตถุสีแดง ((a) to (e) of Fig 1) is
indicated as % of control, when the full length of formed tube in vehicle control ((f) of Fig 1) is as 100%. Full length of the tube treated with 100ug/mL of รงควัตถุสีแดง
shows 20% of vehicle control and 101.1g/mL shows 80% of control. (b): the full length of formed tube treated with each concentration of suramin ((g) to (k) of Fig 1) is
5 indicated as % of control, when the full length of formed tube in vehicle control ((1) of
Fig 1) is as 100%. Full length of the tube treated with 30RM of รงควัตถุสีแดง shows
about 0% of vehicle control. Tube formation is completely inhibited by 30uM of
suramin treatment. *: remarkable inhibition of tube formation
รูป 3: Anti-tumor effect of รงควัตถุสีแดง from ปาล์มใบเลื่อย (A): Tumor
10 growth curve of human hepatocellular carcinoma on the heterograft nude mouse model.
"NYG-1 0.1 mg/kg" and "NYG-1 1mg/kg" mean that ปาล์มใบเลื่อย รงควัตถุ in saline is administrated intraperitoneally at amount of 0.1 mg or 1 mg per 1 kg of body weight, respectively. "Control" is saline only. "Day post injection" means the period of the day after the last injection (day 0). "Tumor growth (%)" was calculated by using the
15 tumor size of "day 7" as 100% of each group (Control, NYG-1 0.1mg/kg and NYG-1
1mg/kg). (B): Suppressed VEGF expression of human hepatocellular carcinoma on
the heterograft nude mouse model. "VEGF expression (%)" means an amount of vascular endothelial growth factor (VEGF) in a supernatant after centrifugation of tumor homogenate.
20 รูป 4: TLC analysis of purified ปาล์มใบเลื่อย รงควัตถุสีแดง. Leanl : Purified
ปาล์มใบเลื่อย รงควัตถุสีแดง (Before hydrolysis), Lean2: Purified ปาล์มใบเลื่อย red
รงควัตถุ (After hydrolysis), Lean3: cyanidin(standard), Lean4: deiphinidin (standard),
Lean5: peonidin (standard), Lean6: petunidin (standard), Lean7: malvidin (standard).
Cellulose TLC glass plate (Merck) is attached an airtight container, at room temperature, 25 with acetate: hydrochloride: water =30:3:10 as the developing solvent. The plates
were analyzed visually.
รูป 5: Structural formula of โปรแอนโธไซยานิดินfrom grape (condensed
tannin) as an embodiment of โปรแอนโธไซยานิดิน(condensed tannin)
รูป 6: Cytotoxicity of NYG-1 on a normal hepatocyte. The longitudinal 30 axis is absorbance at 595mn/Ref 650nm, and it is indicated as % of control.
รูป 7: Effect of NYG-1 on the growth of HCC cells (HepG2 and MHC97L).
The longitudinal axis is absorbance at 595nm/Ref 650nm, and it is indicated as % of control.
รูป 8: Cytotoxicity of NYG-1 and suramin on a vascular endothelial cell. 35 The longitudinal axis is absorbance at 595nrn/Ref 650nm, and it is indicated as % of
control.
รูป 9: NYG-1 suppresses การสร้างหลอดเลือดใหม่ at low dose. รูป 10: NYG-1 suppresses migration of HuVECs.
รูป 11: Monitoring of body weight of a mouse treated by NYG-1. The longitudinal axis is body weight (g) and the horizontal axis is days.
5 รูป 12: NYG-1 at low dose can suppress a tumor growth in a heterologous
transplantation model.
รูป 13: NYG-1 reduces the tumor size in a orthotropic liver tumor transplantation mouse. A is non-treated, and B is NYG-1 treated.
รูป 14: NYG-1 induces a phosphorylation of Erkl/2 in HepG2.
10 รูป 15: NYG-1 induces a phosphorylation of Erk1/2 in HepG2. Left-hand
column is normal oxygen conditions and right-hand column is low oxygen conditions.
BEST MODE FOR CARRYING OUT THE การประดิษฐ์
[0013] ผลปาล์มใบเลื่อย means the fruit belonging to the familyArecaceae genus
15 Trachycarpus. ที่ควรใช้คือ, it is a fruit of Serenoa serrulate or Serenoa repens. The saw
palmetto fruit was obtained from a market.
[0014] ที่ควรใช้คือ, a ผลปาล์มใบเลื่อย is a dried ผลปาล์มใบเลื่อย. The dried saw
palmetto fruit contains, but not limited to, not more than 10% w/w of water„ and
ที่ควรใช้คือ not more than 5% of water. The dried ผลปาล์มใบเลื่อย can obtain from a 20 fresh ผลปาล์มใบเลื่อย by dehydrating and drying by using any methods (e.g., solar
drying, heat drying, vacuum drying, vacuum-freeze drying). ที่ควรใช้คือ, the dried saw
palmetto fruit is in a form of milling or powdering.
[0015] Ethanol includes, but not limited to a solvent ที่รวมถึง ethanol, ที่ควรใช้คือ, it is
aqueous ethanol ที่รวมถึง not less than 30%v/v ethanol. ที่ควรใช้กว่าคือ, it includes 25 80 to 100%v/v ethanol, and even ที่ควรใช้กว่าคือ, it includes 90 to 95%v/v/ ethanol.
[0016] An extraction of ผลปาล์มใบเลื่อย with ethanol is conducted by adding
ethanol to a ผลปาล์มใบเลื่อย and stirring for a certain period of time at room
temperature (15°C to 28°C) or under heating.
[0017] The extraction heating temperature may be below the boiling point of ethanol, 30 or below 120°C in an airtight container. A preferred extraction temperature is 90 to
100. ที่ควรใช้กว่าคือ, it is 95 to 105°C. A preferable heating method is, but not
limited to, heating in an oil bath.
[0018] The extraction time is, but not limited to, from 5 min. to 24hrs, ที่ควรใช้คือ from
15 min. to 3hrs, ที่ควรใช้กว่าคือ from 30 min. to 2hrs.
35 [0019] The extraction may be conducted, but not limited to, one or two times or more.
The extraction may be conducted, but not limited to, with stirring. The preferred
stirring method was by use of a magnetic stirrer and stirring bar, or a motor driven
stirrer. The agitation rate was generally, from 100 to 600 rpm but ที่ควรใช้คือ from 200 to 500rpm.
[0020] The used amount of ethanol to ผลปาล์มใบเลื่อย in extraction is, but not
5 limited to, 300 parts or more of ethanol, ที่ควรใช้คือ 500 to 2000 ethanol and even
ที่ควรใช้คือ 750 to 1500 ethanol to 100 parts by weight of ปาล์มใบเลื่อย.
[0021] Alternatively, the water content of the ของผสม of ผลปาล์มใบเลื่อย and
ethanol in extraction is not more than 70%w/w, ที่ควรใช้คือ less than 20%w/w and even
ที่ควรใช้คือ 10%w/w. In the case of the extraction for 100g of dried ผลปาล์มใบเลื่อย 10 with 10% water and 1000mL of 90%v/v ethanol (density is 0.8222 at 15°C, 90v/v%
(according to International Organization of Legal Metrology (OIML))), the water
content at extraction (%) was calculated as [weight of water in ปาล์มใบเลื่อย l Og +
weight of water in ethanol 100g] / [weight of ปาล์มใบเลื่อย 100g + weight of ethanol
822g] * 100 =12%.
15 [0022] After the ethanol extraction, it is separated to an ethanol extract and a solid
residue to obtain an ethanol extract. A separation method is ที่ควรใช้คือ, but not limited to, filtration or centrifugation.
[0023] The filtration method for the ethanol extract is ที่ควรใช้คือ, but not limited to, by
using a membrane (nylon mesh, paper etc.), filter (Nutsche's filter, Buchner funnel etc.). 20 In addition, combined use of both coarse filtration and fine filtration is preferable.
[0024] The pore size of the coarse filter is, but not limited to, 500 to 5011m, ที่ควรใช้คือ
300 to 100gm.
[0025] The pore size of the fine filter is, but not limited to, 10 to lum, ที่ควรใช้คือ7.5 to
2.5pm and even ที่ควรใช้คือ 6 to 4pm. In addition, it was preferable to use celite or 25 other diatomaceous earth overlaid on a filter paper as a filter aid.
[0026] The filtration method includes, but not limited to, natural dripping, pressure
filtration or vacuum filtration.