Fucoidan composition and preparation method thereof

Abstract

The invention relates to a fucoidan composition and a preparation method thereof. The fucoidan composition comprises the following components in percentage by weight: 20-30% of nemacystus japonicus fucoidan, 20-30% of almacystus japonicus fucoidan, 20-30% of 25-35% of sporophyll carbohydrate gum of the undaria pinnatifida in the Tasmanni Australian sea area; 25-35% of fucus saccharide gum in the Australian Tasmanni subsea area; 5 to 10% of microalgae fucoidan; and 1-5% of microalgae fucoxanthin. According to the fucoidan composition disclosed by the invention, the effective inhibition rate of the prepared fucoidan composition on human colon cancer, human pancreatic cancer, human liver cancer, human prostatic cancer, human breast cancer or human lung cancer and the like reaches 89% or above through an optimized formula of multiple components.

Description

Technical Field

[0001] This invention relates to the field of health product technology, specifically to a fucoidan composition and its preparation method. Background Technology

[0002] Fucoidan is an anionic, water-soluble heteropolysaccharide containing sulfate groups, found in brown algae and some marine invertebrates. Its main components, besides L-fucose and sulfate, include galactose, mannose, xylose, glucose, glucuronic acid, and protein. Long-term scientific research has confirmed that fucoidan possesses multiple physiological benefits, including anti-tumor activity, immune enhancement, antioxidant effects, and regulation of blood pressure and lipids, while remaining safe and non-toxic. Fucoidan and its products can be used as functional foods or nutritional supplements. However, currently, fucoidan products on the domestic market have limited ingredient variety and are expensive, making them unaffordable for most ordinary consumers. Summary of the Invention

[0003] In view of this, the main objective of the present invention is to provide a fucoidan composition and its preparation method. The problem to be solved is to optimize the formulation of multiple components so that the prepared fucoidan composition can achieve an effective inhibition rate of more than 90% against human colon cancer, human pancreatic cancer, human liver cancer, human prostate cancer, human breast cancer, or human lung cancer.

[0004] To achieve the above objectives, the present invention provides a fucoidan composition containing, by weight percentage: 20-30% fucoidan from Okinawa, Japan; 25-35% fucoidan from Undaria pinnatifida, Australia; 25-35% fucoidan from Tasmania, Australia; 5-10% fucoidan from microalgae; and 1-5% fucoxanthin from microalgae.

[0005] Preferably, the fucoidan composition contains the following components by weight percentage: 30% fucoidan from Okinawa, Japan; 30% fucoidan from Undaria pinnatifida, Australia; 30% fucoidan from Tasmania, Australia; 8% fucoidan from microalgae; and 2% fucoxanthin from microalgae.

[0006] Preferably, the fucoidan composition contains the following components by weight percentage: 30% fucoidan from Okinawa, Japan; 30% fucoidan from Undaria pinnatifida, Australia; 30% fucoidan from Tasmania, Australia; 5% fucoidan from microalgae; and 5% fucoxanthin from microalgae.

[0007] Preferably, the fucoidan composition contains the following components by weight percentage: 30% fucoidan from Okinawa, Japan; 35% fucoidan from Tasmanian waters, Australia; 25% fucoidan from Tasmanian waters, Australia; 7% fucoidan from microalgae; and 3% fucoxanthin from microalgae.

[0008] Preferably, the fucoidan composition contains the following components by weight percentage: 30% fucoidan from Okinawa, Japan; 25% fucoidan from Undaria pinnatifida, Australia; 35% fucoidan from Tasmania, Australia; 9% fucoidan from microalgae; and 1% fucoxanthin from microalgae.

[0009] To achieve the above objectives, the present invention also provides a method for preparing a fucoidan composition, comprising the following steps:

[0010] The fucoidan composition is obtained by mixing the following ingredients evenly: Japanese Okinawa seaweed fucoidan, Australian Tasmanian seaweed spore leaf fucoidan, Australian Tasmanian seaweed fucoidan, microalgae fucoidan, and microalgae fucoxanthin.

[0011] Preferably, the microalgae fucoidan is prepared by the following steps:

[0012] Under indoor, industrialized, pollution-free conditions, *Phaeodactylum tricornutum* is cultivated. After 14-18 days of cultivation, when it matures, most of the brine is removed by centrifugation, and the microalgae slurry is collected. Then, 6-8 parts of pure water are added to 1 part of the microalgae slurry; the mixture is stirred and heated to 80-85℃, kept at this temperature for 30-40 minutes, and then cooled to 25-30℃, repeated 3-4 times. The cooled *Phaeodactylum tricornutum* solution is then frozen at -10-15℃ for 24-30 hours and freeze-dried. The internal temperature of the freeze dryer is adjusted to 25-30℃, and the vacuum pump is turned on to begin freeze-drying, thus obtaining the microalgae fucoidan.

[0013] Preferably, the freeze-drying temperature is 25-30°C and the time is 12-16 hours.

[0014] Preferably, the microalgal fucoxanthin is prepared by the following steps:

[0015] Microalgae are cultivated under indoor industrial pollution-free conditions. When they mature in 14-18 days, most of the brine is removed using a centrifuge and the microalgae sludge is collected. Then, 6-8 parts of pure water are added to 1 part of the microalgae sludge. The mixture is stirred and heated to 80-85℃ and kept at that temperature for 30-40 minutes. Then, the temperature is lowered to 25-30℃, and this process is repeated 3-4 times. The cooled microalgae solution is then frozen at -10-15℃ for 24-30 hours and freeze-dried to obtain the microalgae fucoxanthin.

[0016] Preferably, the freeze-drying temperature is 25-30°C and the time is 12-16 hours.

[0017] By means of the above technical solution, the fucoidan composition and its preparation method provided by the present invention have at least the following advantages:

[0018] This invention utilizes a variety of fucoidan compositions to activate immunity, repair damaged cells, inhibit abnormal cell proliferation, promote the growth of healthy cells, induce mutated cells to restart their self-apoptosis mechanism, provide antioxidant effects, inject repair functions into body cells, and alleviate the side effects of treatment.

[0019] This invention optimizes the formula by adding fucoidan and fucoxanthin from microalgae *Phaeodactylum tricornutum* grown indoors in a pollution-free industrial environment. This results in a fucoidan composition with a maximum effective inhibition rate of over 89% against cancer, specifically 91% against human colon cancer, 89% against human pancreatic cancer, 90% against human liver cancer, 96% against human prostate cancer, 90% against human breast cancer, and 91% against human lung cancer. This is significantly higher than the highest inhibition rate (86%) of commercially available fucoidan products against cancer and is applicable to all major cancers, with inhibition rates exceeding those of commercially available fucoidan products.

[0020] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below. Attached Figure Description

[0021] Figure 1 The effect of the fucoidan composition of Example 1 of the present invention on the toxicity of human anterior colon cancer cells is shown;

[0022] Figure 2 The effect of the fucoidan composition of Example 1 of the present invention on the toxicity of human anterior pancreatic cancer cells is shown;

[0023] Figure 3 The effect of the fucoidan composition of Example 1 of the present invention on the toxicity of human prostate cancer cells is shown;

[0024] Figure 4 The effect of the fucoidan composition of Example 1 of the present invention on the toxicity of human breast cancer cells is shown;

[0025] Figure 5 The effect of the fucoidan composition of Example 1 of the present invention on the toxicity of human liver cancer cells is shown.

[0026] Figure 6 The effect of the fucoidan composition of Example 1 of the present invention on the cytotoxicity of human lung cancer cells is shown. Detailed Implementation

[0027] To further illustrate the technical means and effects adopted by the present invention to achieve its intended purpose, the following detailed description, in conjunction with preferred embodiments, provides a detailed explanation of the specific implementation, structure, features, and effects of a fucoidan composition and its preparation method according to the present invention. In the following description, different "embodiments" or "embodiments" do not necessarily refer to the same embodiment. Furthermore, specific features, structures, or characteristics in one or more embodiments can be combined in any suitable manner.

[0028] Unless otherwise specified, all materials or reagents listed below are commercially available.

[0029] Some embodiments of the present invention provide a fucoidan composition containing, by weight percentage: 20-30% fucoidan from Okinawa, Japan; 25-35% fucoidan from Undaria pinnatifida, Tasmania, Australia; 25-35% fucoidan from Tasmania, Australia; 5-10% fucoidan from microalgae; and 1-5% fucoxanthin from microalgae.

[0030] The functions of each component are as follows:

[0031] Fucoidan from Okinawa, Japan, exhibits a strong inhibitory effect on apoptosis in diseased cells (self-destruction of damaged cells). Fucoidan from the leaves of *Wagtail* spores in Tasmanian waters, Australia, can prevent cancer metastasis (inhibiting angiogenesis in diseased cells). Fucoidan from *Fucus vesiculosus* in Tasmanian waters, Australia, shows excellent efficacy against gynecological cancers and possesses outstanding antioxidant activity. However, commercially available fucoidan products often use only one type of fucoidan, which is somewhat insufficient. Microalgal fucoxanthin and microalgal fucoidan can penetrate deep into the cell nucleus, destroying cancer cells at their source.

[0032] Therefore, the fucoidan composition of the present invention combines five seaweed extracts—seaweed extract, wakame spore leaf extract, fucoidan, microalgae fucoxanthin, and fucoidan—to exert a synergistic effect, thereby enhancing the overall efficacy of the fucoidan composition. This makes the product of the present invention particularly outstanding, and is expected to cater to different types of cancer patients, providing more comprehensive and complete efficacy.

[0033] Considering that the product's ideal efficacy is to enhance the user's own ability to fight cancer cells, and to play a role in preventing disease before it occurs and preventing recurrence after recovery, it has an ideal inhibition rate for patients with different types of cancer.

[0034] Most products on the market contain only one or two types of fucoidan, at most three, such as fucoidan from Okinawa, Japan, fucoidan from wakame spore leaves in Tasmania, Australia, and fucoidan from Tasmania, Australia. Their individual fucoidan content is all above 35%, but the cancer inhibition rate of these products is all below about 85%, and the inhibition rate of individual cancers in different types is low, resulting in poor efficacy.

[0035] Based on previous experiments, this invention utilizes 20-30% fucoidan from Okinawa, Japan; 25-35% fucoidan from *Wakame spores* from Tasmanian waters; and 25-35% fucoidan from Tasmanian waters. These concentrations are sufficient. However, concentrations below 20% for Okinawa and below 25% for Tasmanian waters do not show higher inhibition rates against different types of cancer, and there are significant deviations in inhibition rates against individual cancers, with some showing lower inhibition rates. This invention, based on previous experiments, increases the fucoidan content by 5-10% and the fucoxanthin content by 1-5%, resulting in a maximum effective inhibition rate of 96% against atypical cells in cancer cell tests. Specifically, it achieves 91% inhibition against human colon cancer, 89% against human pancreatic cancer, 90% against human liver cancer, 96% against human prostate cancer, 90% against human breast cancer, and 91% against human lung cancer. The inhibition rates are all above 85% on average, and they have a good inhibitory effect on different types of cancer.

[0036] In some optional embodiments, the fucoidan composition contains, by weight percentage, the following components: 30% fucoidan from Okinawa, Japan; 30% fucoidan from Undaria pinnatifida, Australia; 30% fucoidan from Tasmania, Australia; 8% fucoidan from microalgae; and 2% fucoxanthin from microalgae.

[0037] In some optional embodiments, the fucoidan composition contains, by weight percentage, the following components: 30% fucoidan from Okinawa, Japan; 30% fucoidan from Undaria pinnatifida, Australia; 30% fucoidan from Tasmania, Australia; 5% fucoidan from microalgae; and 5% fucoxanthin from microalgae.

[0038] In some optional embodiments, the fucoidan composition contains, by weight percentage, the following components: 30% fucoidan from Okinawa, Japan; 35% fucoidan from Undaria pinnatifida, Australia; 25% fucoidan from Tasmania, Australia; 7% fucoidan from microalgae; and 3% fucoxanthin from microalgae.

[0039] In some optional embodiments, the fucoidan composition contains, by weight percentage, the following components: 30% fucoidan from Okinawa, Japan; 25% fucoidan from Undaria pinnatifida, Australia; 35% fucoidan from Tasmania, Australia; 9% fucoidan from microalgae; and 1% fucoxanthin from microalgae.

[0040] Some embodiments of the present invention also provide a method for preparing a fucoidan composition, comprising the following steps:

[0041] Under indoor, industrial, pollution-free conditions, *Phaeodactylum tricornutum* and microalgae were cultivated separately. After 14-18 days of cultivation, when they matured, 98 wt% brine was removed using a centrifuge (16000-16500 r / min, centrifugation capacity 1100-1300 L / h) and the microalgae sludge was collected. Then, 6-8 parts of pure water were added to 1 part of the microalgae sludge; the mixture was stirred and heated to 80-85℃, kept at that temperature for 30-40 minutes, and then cooled to 25-30℃, repeated 3-4 times. The cooled microalgae solution was then frozen at -10-15℃ for 24-30 hours and freeze-dried. The internal temperature of the freeze dryer (vacuum degree 14-16 Pa) was adjusted to 25-30℃, and the vacuum pump was turned on to start the freeze-drying process. The freeze-drying process was completed after 12-16 hours.

[0042] The fucoidan composition is obtained by mixing the following ingredients evenly: Japanese Okinawa seaweed fucoidan (purchased in Japan), Australian Tasmanian seaweed spore leaf fucoidan (purchased in Australia), Australian Tasmanian seaweed fucoidan (purchased in Australia), microalgae fucoidan, and microalgae fucoxanthin.

[0043] Some embodiments of the present invention provide the application of a fucoidan composition in human colon cancer, human pancreatic cancer, human liver cancer, human prostate cancer, human breast cancer, or human lung cancer. The relevant data only represent the inhibition rate of the fucoidan composition against cancer cells in vitro; the actual inhibition rate when taken orally and exerted in vivo is affected by multiple factors, including but not limited to absorption rate and dosage.

[0044] The specific embodiments of the present invention will be described in further detail below with reference to examples, but this should not be construed as a limitation on the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention still fall within the scope of protection of the present invention.

[0045] Unless otherwise specified, all materials and reagents mentioned below are commercially available products well known to those skilled in the art; unless otherwise specified, all methods described are methods known in the art. Unless otherwise defined, the technical or scientific terms used should have the ordinary meaning understood by those skilled in the art to which this invention pertains.

[0046] Example 1

[0047] This embodiment provides a fucoidan composition containing the following components by weight percentage:

[0048] 30% fucoidan from Okinawa, Japan; 30% fucoidan from Undaria pinnatifida in Tasmanian waters, Australia; 30% fucoidan from Tasmanian waters, Australia; 8% fucoidan from microalgae; 2% fucoxanthin from microalgae.

[0049] The preparation method of the above-mentioned fucoidan composition includes the following steps:

[0050] Under pollution-free, industrial-scale indoor conditions, *Phaeodactylum tricornutum* and microalgae were cultivated and matured in 14 days. 98 wt% brine was removed using a centrifuge (16300 r / min, 1200 L / h), and the microalgae sludge was collected. Then, 6-8 parts of pure water were added to 1 part of the microalgae sludge; the mixture was stirred and heated to 80°C, held for 30 minutes, and then cooled to 30°C, repeated three times. The cooled *Phaeodactylum tricornutum* and microalgae solutions were frozen at -15°C for 24 hours and then freeze-dried. The internal temperature of the freeze dryer was adjusted to 30°C, the vacuum pump was turned on, and freeze-drying began. The freeze-drying process was completed after 16 hours. Subsequently, fucoidan from Okinawa, Japan, wakame spore leaf fucoidan from Tasmania, Australia, fucoidan from Tasmania, Australia, microalgae fucoidan, and microalgae fucoxanthin were mixed evenly in proportion to obtain the fucoidan composition.

[0051] Example 2

[0052] This embodiment provides a fucoidan composition containing the following components by weight percentage:

[0053] 30% fucoidan from Okinawa, Japan; 30% fucoidan from Undaria pinnatifida, Australia; 30% fucoidan from Tasmanian waters, Australia; 5% fucoidan from microalgae; 5% fucoxanthin from microalgae.

[0054] The preparation method of the above-mentioned fucoidan composition includes the following steps:

[0055] Under pollution-free, industrialized indoor conditions, *Phaeodactylum tricornutum* and microalgae were cultivated and matured in 14 days. 98% of the brine was removed using a centrifuge (16300 rpm, 1200 L / h), and the microalgae sludge was collected. Then, 6-8 parts of pure water were added to 1 part microalgae sludge; the mixture was stirred and heated to 80°C, held for 30 minutes, and then cooled to 30°C, repeated three times. The cooled *Phaeodactylum tricornutum* and microalgae solutions were frozen at -15°C for 24 hours and then freeze-dried. The internal temperature of the freeze dryer was adjusted to 30°C, the vacuum pump was turned on, and freeze-drying began. The freeze-drying process was completed after 16 hours. Subsequently, fucoidan from Okinawa, Japan, wakame spore leaf fucoidan from Tasmania, Australia, fucoidan from Tasmania, Australia, microalgae fucoidan, and microalgae fucoxanthin were mixed evenly to obtain the fucoidan composition.

[0056] Example 3

[0057] This embodiment provides a fucoidan composition containing the following components by weight percentage:

[0058] 30% fucoidan from Okinawa, Japan; 35% fucoidan from Undaria pinnatifida, Australia; 25% fucoidan from Tasmanian waters, Australia; 7% fucoidan from microalgae; 3% fucoxanthin from microalgae.

[0059] The preparation method of the above-mentioned fucoidan composition includes the following steps:

[0060] Under pollution-free, industrial-scale indoor conditions, *Phaeodactylum tricornutum* and microalgae were cultivated and matured in 14 days. 98 wt% brine was removed using a centrifuge (16300 r / min, 1200 L / h), and the microalgae sludge was collected. Then, 6-8 parts of pure water were added to 1 part microalgae sludge; the mixture was stirred and heated to 80°C, held for 30 minutes, and then cooled to 30°C, repeated three times. The cooled *Phaeodactylum tricornutum* and microalgae solutions were frozen at -15°C for 24 hours and then freeze-dried. The internal temperature of the freeze dryer was adjusted to 30°C, the vacuum pump was turned on, and freeze-drying began. The freeze-drying process was completed after 16 hours. Subsequently, fucoidan from Okinawa, Japan, wakame spore leaf fucoidan from Tasmania, Australia, fucoidan from Tasmania, Australia, microalgae fucoidan, and microalgae fucoxanthin were mixed evenly to obtain the fucoidan composition.

[0061] Example 4

[0062] This embodiment provides a fucoidan composition containing the following components by weight percentage:

[0063] 30% fucoidan from Okinawa, Japan; 25% fucoidan from Undaria pinnatifida, Australia; 35% fucoidan from Tasmanian waters, Australia; 9% fucoidan from microalgae; 1% fucoxanthin from microalgae.

[0064] The preparation method of the above-mentioned fucoidan composition includes the following steps:

[0065] Under pollution-free, industrial-scale indoor conditions, *Phaeodactylum tricornutum* and microalgae were cultivated and matured in 14 days. 98 wt% brine was removed using a centrifuge (16300 r / min, 1200 L / h), and the microalgae sludge was collected. Then, 6-8 parts of pure water were added to 1 part microalgae sludge; the mixture was stirred and heated to 80°C, held for 30 minutes, and then cooled to 30°C, repeated three times. The cooled *Phaeodactylum tricornutum* and microalgae solutions were frozen at -15°C for 24 hours and then freeze-dried. The internal temperature of the freeze dryer was adjusted to 30°C, the vacuum pump was turned on, and freeze-drying began. The freeze-drying process was completed after 16 hours. Subsequently, fucoidan from Okinawa, Japan, wakame spore leaf fucoidan from Tasmania, Australia, fucoidan from Tasmania, Australia, microalgae fucoidan, and microalgae fucoxanthin were mixed evenly to obtain the fucoidan composition.

[0066] Application examples

[0067] The fucoidan composition of Example 1 was used to test the proliferation properties of six types of cancer cells. Cancer cells according to Table 1 were added to pure water and fucoidan composition solutions of different concentrations, and the number of cancer cells was measured at 24, 48, and 72 hours to calculate the inhibition rate.

[0068] Table 1. Tests on the proliferation properties of the fucoidan composition of Example 1 in six cancer cells.

[0069]

[0070] 1. Method

[0071] 1.1. Preparation of the fucoidan composition (Fucoidan 3+2 extract) of Example 1

[0072] Weigh the fucoidan composition (Fucoidan 3+2 extract) powder from Example 1, dissolve it in cell culture medium to a concentration of 16 mg / mL, and sonicate for 1 hour until completely dissolved. Then centrifuge at 4000 rpm for 10 minutes, filter through a 0.22 μm sterile filter membrane, and store at 4°C for later use.

[0073] 1.2. Reagents

[0074] All cell culture reagents were purchased from Thermo Fisher Scientific, including RPMI 1640 medium (ATCC modified, catalog number A1049101), DMEM (catalog number 11965084), MEM (catalog number 11095080), fetal bovine serum (FBS, catalog number 10500064), antibiotic-antifungal agent (100×, catalog number 15240062), trypsin-EDTA (0.25%), and phenol red (catalog number 25200072).

[0075] 1.3. Cell Culture

[0076] Human colon cancer cell lines (CT26, HT29, SW480, HCT116) were cultured in RPMI 1640 medium containing 10% FBS and 1× antibiotic; human pancreatic cancer cell lines (Capan-2, Miapaca-2, ASPC-1, PANC-1) were cultured in DMEM containing 10% FBS; human liver cancer cell lines (HEPG2, MHCC-97L, MHCC-97H) and human prostate cancer cell lines (LNCaP, C4-2B, 22RV1, PC-3) were cultured in DMEM containing 10% FBS; and human breast cancer cell lines (MCF-7, MA-MB-231, BT-459, MA-MB-453, MCF-10A) were cultured in RPMI 1640 medium.

[0077] Human hepatic stellate cells (LX2), prostate stromal muscle fibroblasts (WPMY-1), and normal pancreatic ductal epithelial cells (HPDE) were cultured in MEM or DMEM containing 10 wt% FBS. All cells were cultured at 37°C, 5% (v / v) CO2, and 95% humidity.

[0078] Human lung epithelial carcinoma cell lines such as HCC4006, HCC827, and H1975 were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 1× antibiotic-antifungal agent. Human lung epithelial carcinoma cell lines A549 and PC9 were cultured in DMEM medium containing 10% FBS and 1× antibiotic-antifungal agent. Human embryonic kidney cells HEK293 were cultured in MEM medium containing 10% fetal bovine serum (FBS) and 1× antibiotic-antifungal agent. Cells were cultured at 37°C, 5% CO2, and 95% humidity.

[0079] 1.4 Cytotoxicity assay

[0080] Cancer cells and normal cells in the logarithmic growth phase were separated at 2 × 10⁻⁶ cells per well. 3 Cells were seeded at a density of 100 μL into 96-well plates and cultured until cell adhesion was achieved. The cells were then divided into eight groups: a normal control group (NC), a blank control group, and groups receiving different concentrations of fucoidan (3+2). Except for the normal control group, the drug-treated groups were added 100 μL of complete culture medium containing different drug concentrations via medium exchange, and incubated for 24, 48, and 72 hours, respectively. Subsequently, the culture medium in the wells was removed, the wells were washed with phosphate-buffered saline (PBS), and 100 μL of 10 wt% CCK-8 complete culture medium was added via medium exchange, followed by incubation for 2 hours. Cell viability was measured at 450 nm and calculated using the following formula: Inhibition rate (IR) = {1 - [(Drug-treated group - Blank control group) / (Negative control group - Blank control group)]} × 100%. A concentration-inhibition rate fitting curve was obtained using a scatter plot of drug concentration versus corresponding inhibition percentage to find the drug concentration required to inhibit 50% of cells, i.e., IC50. 50 IC 50 The average value was obtained through at least three independent experiments.

[0081] 2. Results

[0082] 2.1. The toxic effect of the fucoidan composition (Fucoidan 3+2 extract) of Example 1 on colon cancer cells.

[0083] Figure 1 This demonstrates the effect of the fucoidan composition (Fucoidan 3+2 extract) of Example 1 on the survival rate of colon cancer cells. IC50 The values ​​(concentrations required to inhibit 50% cell growth) are shown in Table 2.

[0084] 24-hour treatment: All cell lines were sensitive to Fucoidan 3+2, IC50 50 The range was 12.05–40.1 mg / mL, indicating a preliminary inhibitory effect.

[0085] 48-hour processing: IC 50 The concentration was significantly reduced to 2.18–8.73 mg / mL, indicating enhanced inhibitory effect.

[0086] 72-hour processing: IC 50 Further reduction, HT29 cells are most sensitive (IC50). 50 =0.58 mg / mL), IC50 of CT26, SW480 and HCT116 50 The values ​​were 2.43, 2.17, and 3.58 mg / mL, respectively.

[0087] Table 2. IC50 of the fucoidan composition (Fucoidan 3+2 extract) of Example 1 against colon cancer cells 50 Value (mg / mL)

[0088]

[0089] 2.2. The toxic effect of the fucoidan composition (Fucoidan 3+2 extract) of Example 1 on pancreatic cancer cells.

[0090] Figure 2 This shows changes in the survival rate of pancreatic cancer cells. IC 50 The values ​​are shown in Table 3.

[0091] 24-hour processing: IC 50 The concentration ranges from 2.57 to 6.56 mg / mL.

[0092] 48-hour processing: IC 50 It decreased to 1.78–2.92 mg / mL.

[0093] 72-hour treatment: Miapaca-2 is the most sensitive (IC) 50 =0.79 mg / mL), IC50 of Capan-2, ASPC-1 and PANC-1 50 The concentrations were 1.65, 1.62, and 1.71 mg / mL, respectively.

[0094] Normal pancreatic cells HPDE:IC 50 The concentration decreased significantly with prolonged treatment time (24h: 0.23 mg / mL; 72h: 0.10 mg / mL).

[0095] Table 3. IC50 of the fucoidan composition (Fucoidan 3+2 extract) of Example 1 against pancreatic cancer cells 50 Value (mg / mL)

[0096]

[0097] 2.3 Cytotoxic Effect of the Fucoidan 3+2 Extract Composition of Example 1 on Prostate Cancer Cells

[0098] The fucoidan composition (Fucoidan 3+2 extract) in Example 1 showed the following cell viability (%) against prostate cancer cells: Figure 3 As shown. The IC50 of fucoidan 3+2 against prostate cancer cells in Example 1, calculated based on cell viability (%). 50 The (mg / mL) values ​​are shown in Table 3.

[0099] After 24 hours of treatment, all cell lines were sensitive to fucoidan 3+2, IC50 50 The concentration ranged from 2.08 to 7.02 mg / mL, indicating that the 3+2 treatment with fucoidan had a potential inhibitory effect at this time.

[0100] After 48 hours of treatment, all cell lines were sensitive to fucoidan 3+2, IC50 50 The concentration ranged from 0.95 to 3.45 mg / mL, indicating that the 3+2 treatment with fucoidan had an inhibitory effect at this time.

[0101] After 72 hours of treatment, the IC50 values ​​of all cell lines were... 50 The values ​​were all lower than those in the 24- or 48-hour treatment groups, indicating that the longer the treatment time, the lower the effectiveness of the compound. PC-3 remains the most sensitive cell line to treatment with the fucoidan composition (Fucoidan 3+2 extract), with an IC50 value lower than that of the 24- or 48-hour treatment groups. 50 The value was 0.48 mg / mL. 22RV1 was the second most sensitive cell line to treatment with the fucoidan composition (Fucoidan 3+2 extract) of Example 1, IC50 value. 50 The value was 0.84 mg / mL. The IC50 value of LNCaP and C4-2B cells treated with a fucoidan composition (Fucoidan 3+2 extract) was... 50 The values ​​were similar, at 1.33 mg / mL and 1.49 mg / mL, respectively.

[0102] The fucoidan composition (Fucoidan 3+2 extract) in Example 1 showed the following cell viability (%) against WPMY-1 cells (a human prostatic myofibroblast cell line derived from the peripheral region of the prostate): Figure 3As shown. The fucoidan composition (Fucoidan 3+2 extract) of Example 1 showed its IC50 activity against WPMY-1 at 24, 48, and 72 hours. 50 The mg / mL values ​​were 1.73±0.44 mg / mL, 1.32±0.12 mg / mL, and 0.86±0.19 mg / mL, respectively.

[0103] The fucoidan composition (Fucoidan 3+2 extract) in Example 1 showed the following cell viability (%) against prostate cancer cells: Figure 3 As shown. The IC50 of the fucoidan composition (Fucoidan 3+2 extract) of Example 1 against prostate cancer cells, calculated based on cell viability (%). 50 The (mg / mL) values ​​are shown in Table 4.

[0104] After 24 hours of treatment, all cell lines were sensitive to the fucoidan composition (Fucoidan 3+2 extract) of Example 1, IC50 50 The concentrations ranged from 2.08 to 7.02 mg / mL, indicating that treatment with the fucoidan composition (Fucoidan 3+2 extract) had a potential inhibitory effect at this time.

[0105] After 48 hours of treatment, all cell lines were sensitive to the fucoidan composition (Fucoidan 3+2 extract), IC50... 50 The concentration ranged from 0.95 to 3.45 mg / mL, indicating that the treatment with the fucoidan composition (Fucoidan 3+2 extract) had an inhibitory effect.

[0106] After 72 hours of treatment, the IC50 values ​​of all cell lines were... 50 The values ​​were all lower than those in the 24- or 48-hour treatment groups, indicating that the longer the treatment time, the lower the effectiveness of the compound. PC-3 remained the most sensitive cell line to treatment with the fucoidan composition (Fucoidan 3+2 extract) of Example 1, IC50 value. 50 The value was 0.48 mg / mL. 22RV1 was the second most sensitive cell line to treatment with the fucoidan composition (Fucoidan 3+2 extract) of Example 1, IC50 value. 50 The value was 0.84 mg / mL. The IC50 value of LNCaP and C4-2B cells treated with the fucoidan composition (Fucoidan 3+2 extract) of Example 1 was... 50 The values ​​were similar, at 1.33 mg / mL and 1.49 mg / mL, respectively.

[0107] The fucoidan composition (Fucoidan 3+2 extract) in Example 1 showed the following cell viability (%) against WPMY-1 cells (a human prostatic myofibroblast cell line derived from the peripheral region of the prostate): Figure 3As shown. The fucoidan composition (Fucoidan 3+2 extract) of Example 1 showed its IC50 activity against WPMY-1 at 24, 48, and 72 hours. 50 The mg / mL values ​​were 1.73±0.44 mg / mL, 1.32±0.12 mg / mL, and 0.86±0.19 mg / mL, respectively.

[0108] Table 4. IC50 of the fucoidan composition (Fucoidan 3+2 extract) of Example 1 against different prostate cancer cells 50 value

[0109]

[0110] 2.4 Cytotoxic Effects of the Fucoidan 3+2 Extract Composition of Example 1 on Breast Cancer Cells

[0111] The fucoidan composition (Fucoidan 3+2 extract) in Example 1 showed the following effect on the cell viability (%) of breast cancer cells: Figure 4 As shown. The IC50 of the fucoidan composition (Fucoidan 3+2 extract) against breast cancer cells, calculated based on cell viability (%). 50 The (mg / mL) values ​​are shown in Table 5.

[0112] After 24 hours of treatment, all cell lines were sensitive to the fucoidan composition (Fucoidan 3+2 extract) of Example 1, IC50 50 The concentrations ranged from 2.31 to 8.61 mg / mL, indicating that treatment with the fucoidan composition (Fucoidan 3+2 extract) had a potential inhibitory effect at this time.

[0113] After 48 hours of treatment, all cell lines were sensitive to the fucoidan composition (Fucoidan 3+2 extract) of Example 1, IC50 50 The concentration ranged from 1.68 to 5.52 mg / mL, indicating that the treatment with the fucoidan composition (Fucoidan 3+2 extract) had an inhibitory effect at this time.

[0114] After 72 hours of treatment, the IC50 values ​​of all cell lines were... 50 The values ​​were all lower than those in the 24- or 48-hour treatment groups, indicating that the longer the treatment time, the lower the effectiveness of the compound. BT-459 remains the most sensitive cell line to treatment with the fucoidan composition (Fucoidan 3+2 extract) of Example 1, IC50. 50 The value was 0.55 mg / mL. MA-MB-231 was used to treat the second most sensitive cell line with the fucoidan composition (Fucoidan 3+2 extract) of Example 1, with an IC50 value of 0.55 mg / mL. 50The value was 1.01 mg / mL. The IC50 value for MCF-10A cells was... 50 The value was 1.77 mg / mL, while the IC50 value of MCF-7 and MA-MB-453 cells treated with the fucoidan composition (Fucoidan 3+2 extract) of Example 1 was 1.77 mg / mL. 50 The values ​​were 3.37 mg / mL and 2.59 mg / mL, respectively.

[0115] Table 5. Effects of the fucoidan composition (Fucoidan 3+2 extract) of Example 1 on IC50 in different breast cancer cells. 50 value

[0116]

[0117] 2.5 Effect of the fucoidan composition (Fucoidan 3+2 extract) from Example 1 on the cytotoxicity of liver cancer cells

[0118] The fucoidan composition (Fucoidan 3+2 extract) in Example 1 showed the following cell viability (%) against liver cancer cells: Figure 5 As shown. IC 50 The value is the concentration of a drug or compound required to inhibit 50% cell growth or viability. The IC50 value of the fucoidan composition (Fucoidan 3+2 extract) against liver cancer cells, calculated based on cell viability (%). 50 The (mg / mL) values ​​are shown in Table 6.

[0119] After 24 hours of treatment, all cell lines were sensitive to the fucoidan composition (Fucoidan 3+2 extract) of Example 1, IC50 50 The concentrations ranged from 7.01 to 37.07 mg / mL, indicating that treatment with the fucoidan composition (Fucoidan 3+2 extract) had a potential inhibitory effect at this time.

[0120] After 48 hours of treatment, all cell lines were sensitive to the fucoidan composition (Fucoidan 3+2 extract) of Example 1, IC50 50 The concentration ranged from 2.75 to 7.49 mg / mL, indicating that the treatment with the fucoidan composition (Fucoidan 3+2 extract) had an inhibitory effect at this time.

[0121] After 72 hours of treatment, the IC50 values ​​of all cell lines were... 50 The values ​​were all lower than those in the 24- or 48-hour treatment groups, indicating that the longer the treatment time, the lower the effectiveness of the compound. HEPG2 was the cell line most sensitive to treatment with the fucoidan composition (Fucoidan 3+2 extract) of Example 1, IC50 value. 50The value was 0.79 mg / mL. The IC50 value of MHCC-97L and LX2 cells treated with the fucoidan composition (Fucoidan 3+2 extract) of Example 1 was... 50 The values ​​were similar, at 1.01 mg / mL and 1.75 mg / mL respectively, while the IC50 of MHCC-97H cells was... 50 The value was 4.06 mg / mL.

[0122] Table 6. IC50 of the fucoidan composition (Fucoidan 3+2 extract) of Example 1 against different hepatocellular carcinoma cells. 50 value

[0123]

[0124] 2.6 Cytotoxic Effects of the Fucoidan 3+2 Extract Composition of Example 1 on Lung Cancer Cells

[0125] The fucoidan composition (Fucoidan 3+2 extract) in Example 1 showed the following effect on the cell viability (%) of lung cancer cells: Figure 6 As shown. IC 50 The value is the concentration of a drug or compound required to inhibit 50% cell growth or viability. The IC50 value for the fucoidan composition (Fucoidan 3+2 extract) against lung cancer cells, calculated based on cell viability (%). 50 The (mg / mL) values ​​are shown in Table 7.

[0126] After 24 hours of processing, except for the ICs PC9 and A549 50 Apart from concentrations greater than 20 mg / mL, all cell lines showed similar sensitivity to the fucoidan composition (Fucoidan 3+2 extract) of Example 1, indicating that the treatment may be ineffective at this point.

[0127] After 48 hours of treatment, H1975 was the most sensitive cell line to the fucoidan composition (Fucoidan 3+2 extract) of Example 1, IC50. 50 The value was 2.62 mg / mL. The IC50 values ​​for PC9 and HCC4006 cells were... 50 The values ​​were similar, at 3.33 mg / mL and 3.47 mg / mL, respectively. The IC50 values ​​for HCC827 cells were... 50 The value was 4.21 mg / mL, while A549 cells had the lowest sensitivity, IC50 was 4.21 mg / mL. 50 The value was 7.32 mg / mL.

[0128] After 72 hours of treatment, the IC50 values ​​of all cell lines were... 50 The values ​​all increased, indicating that the longer the treatment time, the lower the effectiveness of the compound. H1975 remains the most sensitive cell line, with an IC50 value of [missing value]. 50The value was 4.75 mg / mL. The IC50 values ​​for PC9 and HCC4006 cells were... 50 The values ​​were similar, approximately 6 mg / mL, while the IC50 values ​​for HCC827 and A549 cells were similar. 50 The values ​​were 8.23 ​​mg / mL and 11.8 mg / mL, respectively.

[0129] Table 7. Effects of the fucoidan composition (Fucoidan 3+2 extract) of Example 1 on IC50 in different lung cancer cells. 50 value

[0130]

[0131] Epidermal growth factor receptor (EGFR) is a protein that plays an important role in regulating cell growth and division. It is a transmembrane receptor located on the cell surface and is activated by binding to ligands such as epidermal growth factor (EGF). EGFR mutation is the most common oncogenic driver in non-small cell lung cancer (NSCLC) and is associated with cellular sensitivity to EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib and erlotinib. H1975, PC9, HCC4006, and HCC827 are EGFR-mutant NSCLC cell lines derived from patients, while A549 is an EGFR wild-type NSCLC cell line. The results indicate that the fucoidan composition (Fucoidan 3+2 extract) of Example 1 inhibits the proliferation of NSCLC cell lines, suggesting that the fucoidan composition (Fucoidan 3+2 extract) of Example 1 has a stronger anti-proliferative effect on EGFR-mutant NSCLC cell lines than on the EGFR wild-type NSCLC cell line A549.

[0132] In summary, the experimental results show that the fucoidan composition (Fucoidan 3+2 extract) of Example 1, after treatment for 24, 48 and 72 hours, exhibited varying degrees of inhibitory effects on the proliferation of colon cancer, pancreatic cancer, prostate cancer, breast cancer, liver cancer cells and lung cancer cells.

[0133] In the above embodiments, the descriptions of each embodiment have different focuses. For parts not described in detail in a certain embodiment, please refer to the relevant descriptions in other embodiments.

[0134] The numerical range described in this invention includes all values ​​within this range, and also includes any range value composed of any two values ​​within this range. Different values ​​of the same indicator appearing in all embodiments of this invention can be arbitrarily combined to form a range value.

[0135] The technical features in the claims and / or specification of this invention can be combined, and the combination is not limited to the combinations obtained through reference in the claims. Technical solutions obtained by combining the technical features in the claims and / or specification are also within the scope of protection of this invention.

[0136] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A fucoidan composition, characterized in that, It contains the following components by weight percentage: 20-30% fucoidan from Okinawa, Japan; 25-35% fucoidan from Tasmanian waters, Australia; 25-35% fucoidan from Tasmanian waters, Australia; 5-10% fucoidan from microalgae; and 1-5% fucoxanthin from microalgae.

2. The fucoidan composition according to claim 1, characterized in that, The fucoidan composition contains the following components by weight percentage: 30% fucoidan from Okinawa, Japan; 30% fucoidan from Undaria pinnatifida, Australia; 30% fucoidan from Tasmania, Australia; 8% fucoidan from microalgae; and 2% fucoxanthin from microalgae.

3. The fucoidan composition according to claim 1, characterized in that, The fucoidan composition contains the following components by weight percentage: 30% fucoidan from Okinawa, Japan; 30% fucoidan from Undaria pinnatifida, Australia; 30% fucoidan from Tasmania, Australia; 5% fucoidan from microalgae; and 5% fucoxanthin from microalgae.

4. The fucoidan composition according to claim 1, characterized in that, The fucoidan composition contains the following components by weight percentage: 30% fucoidan from Okinawa, Japan; 35% fucoidan from the Tasmanian Seaweed, Australia; 25% fucoidan from the Tasmanian Sea, Australia; 7% fucoidan from microalgae; and 3% fucoxanthin from microalgae.

5. The fucoidan composition according to claim 1, characterized in that, The fucoidan composition contains the following components by weight percentage: 30% fucoidan from Okinawa, Japan; 25% fucoidan from Undaria pinnatifida, Australia; 35% fucoidan from Tasmania, Australia; 9% fucoidan from microalgae; and 1% fucoxanthin from microalgae.

6. A method for preparing the fucoidan composition according to any one of claims 1-5, characterized in that, Includes the following steps: The fucoidan composition is obtained by mixing the following ingredients evenly: Japanese Okinawa seaweed fucoidan, Australian Tasmanian seaweed spore leaf fucoidan, Australian Tasmanian seaweed fucoidan, microalgae fucoidan, and microalgae fucoxanthin.

7. The method for preparing the fucoidan composition according to claim 6, characterized in that, The microalgae fucoidan is prepared by the following steps: Under indoor, industrialized, pollution-free conditions, *Phaeodactylum tricornutum* is cultivated. After 14-18 days of cultivation, when it matures, most of the brine is removed by centrifugation, and the microalgae slurry is collected. Then, 6-8 parts of pure water are added to 1 part of the microalgae slurry; the mixture is stirred and heated to 80-85℃, kept at this temperature for 30-40 minutes, and then cooled to 25-30℃, repeated 3-4 times. The cooled *Phaeodactylum tricornutum* solution is then frozen at -10-15℃ for 24-30 hours and freeze-dried. The internal temperature of the freeze dryer is adjusted to 25-30℃, and the vacuum pump is turned on to begin freeze-drying, thus obtaining the microalgae fucoidan.

8. The method for preparing the fucoidan composition according to claim 6, characterized in that, The microalgal fucoxanthin is prepared by the following steps: Microalgae are cultivated under indoor industrial pollution-free conditions. When they mature in 14-18 days, most of the brine is removed using a centrifuge and the microalgae sludge is collected. Then, 6-8 parts of pure water are added to 1 part of the microalgae sludge. The mixture is stirred and heated to 80-85℃ and kept at that temperature for 30-40 minutes. Then, the temperature is lowered to 25-30℃, and this process is repeated 3-4 times. The cooled microalgae solution is then frozen at -10-15℃ for 24-30 hours and freeze-dried to obtain the microalgae fucoxanthin.

9. The method for preparing the fucoidan composition according to claim 7 or 8, characterized in that, The freeze-drying temperature is 25-30℃, and the time is 12-16 hours.

Images