Method of extracting DNA from fish and using the extracted DNA in osteoporosis and osteoarthritis

Abstract

SOLUTION: To provide a method for extracting DNA from fish and a method for using a DNA extract.EFFECT: The DNA extract has an effect of improving or treating osteoarthritis or osteoporosis. In addition, the DNA extract exhibits free radical scavenging activity, antioxidant activity, antidepressant activity, and black hair promoting activity.SELECTED DRAWING: None

Description

[Technical Field]

[0001]

[0001] The present invention relates to a method for extracting DNA from fish, particularly from fish testes, and to the use of the DNA extract, particularly by oral administration, in osteoporosis and osteoarthritis. [Background technology]

[0002]

[0002] DNA mixtures purified from salmon sperm DNA are known to exhibit several pharmacological activities. Such DNA mixtures are commonly known as polydeoxyribonucleotides (PDRNs). PDRNs contain a mixture of deoxyribonucleotide polymers ranging from 50 to 2000 bp (Pizzino et al., "Adenosine Receptor Stimulation Improves Glucocorticoid-Induced Osteoporosis in a Rat Model," Frontiers in Pharmacology, September 5, 2017, Vol. 8, Paper 558). PDRNs are registered drugs that exhibit tissue repair, anti-ischemic, and anti-inflammatory activities. These activities suggest the use of PDRNs in regenerative medicine and for the treatment of diabetic leg ulcers (Squadrito et al., "Pharmacological Activity and Clinical Use of PDRNs," Frontiers in Pharmacology, April 26, 2017, Vol. 8, Paper 224). Another type of DNA mixture purified from salmon contains DNA with a high base pair length, for example, exceeding 20,000 bp. This type of DNA mixture, obtained from salmon testes, has been reported to have bone regeneration activity (Sato A, Kajiya H, Mori N, Sato H, Fukushima T, et al. (2017) Salmon DNA Accelerates Bone Regeneration by Inducing Osteoblast Migration. PLOS ONE 12(1):e0169522). The DNA mixture was prepared as a DNA disc and implanted into critically sized calvarial bone defects (CSDs). The discs biodegraded within three days of implantation. However, the DNA discs induced bone-forming cells, including both MSC-like cells and osteoblast-like cells, to migrate to the defect site, replacing the fibrous connective tissue in the bone defect. Micro-CT scans showed new bone formation in the defect area that received the DNA disc. H&E staining results were consistent with the new bone regeneration observed in the DNA disc-implanted specimens by micro-CT.

[0003]

[0003] Also known is a commercially available PDRN product, PRF001 (PRP salmon DNA, Pharma Research Product, Gangneung, Korea), which is a DNA polymer fragment with a molecular weight of 50 to 1,500 kDa extracted from adult salmon testes. PDRN products have been reported to have anti-inflammatory activity in vitro and to have protective effects against osteoarthritis in a rat model (Ra HJ et al., Effects of salmon DNA fraction in vitro and in a monosodium iodoacetate-induced osteoarthritis rat model. Korean J Physiol Pharmacol. March 2018;22(2):163-172). In vitro, the levels of inflammatory markers IL-1β, TNF-α, COX-2, iNOS, and PGE2 were reportedly significantly suppressed by PRF001 treatment. In vivo, PRF001 administration reduced inflammatory mediators and cytokines, such as IL-1β, p-Erk1 / 2, NF-kB, TNF-α, COX-2, PGE2, as well as MMP3 and MMP7, in rats with sodium iodoacetate (MIA)-induced osteoarthritis. The anti-inflammatory properties of PRF001 may contribute to its protective effects in osteoarthritis by regulating cytokines / signaling substances.

[0004]

[0004] PDRN has also been reported to produce similar effects to hyaluronic acid (HA) in relieving pain in knee osteoarthritis when administered intra-articularly, while avoiding the common side effects of HA injections (Kim MS, Cho RK, In Y. The efficacy and safety of polydeoxyribonucleotide for the treatment of knee osteoarthritis: Systematic review and meta-analysis of randomized controlled trials. Medicine (Baltimore). 2019 September;98(39):e17386). The analgesic effects of PDRN in clinical settings may serve as a complement to other injection treatments.

[0005]

[0005] Food-grade PDRN products are known in the art. For example, it has been reported that oral administration of a commercially available PDRN food product, PRF002 (PRP salmon DNA; Pharma Research Product, Gangneung, Korea), can improve alcohol-induced gastric mucosal damage (Kim J et al., Amelioration of alcohol-induced gastric mucosa damage by oral administration of food-polydeoxyribonucleotides. Mol Med Rep. 2021 November;24(5):790). Due to its beneficial effects, regular intake of food PDRN products as dietary supplements is recommended. Furthermore, oral administration of PDRN can inhibit the expression of IL-1β, matrix metalloproteinase (MMP)-3, and MMP-7, as well as the production of inflammatory mediators such as COX-2, prostaglandin E2 (PGE2), and TNF-α. Oral administration of PDRN also exerts mucosal protective effects against IND-induced gastric disease (Tae-Hee Kim et al., Applications of Marine Organism-Derived Polydeoxyribonucleotide: Its Potential in Biomedical Engineering Mar. Drugs 2021, 19(6), 296).

[0006] However, there remains a need for the development of novel DNA extracts from fish and medical applications of such DNA extracts. Summary of the Invention

[0007]

[0007] The present disclosure provides a novel method for extracting DNA from fish testes, DNA extracts obtained from this method, and methods of using such DNA extracts to treat or ameliorate osteoarthritis or osteoporosis, to remove free radicals, to reduce oxidative stress, to treat or ameliorate depression, and to promote black hair formation.

[0008]

[0008] In one embodiment, the fish used in this extraction method is a deep-sea fish, such as mackerel or salmon. In a preferred embodiment, the fish is mackerel.

[0009] In one embodiment, fish testes are homogenized to obtain a homogenate. In one embodiment, the fish testes are frozen and thawed prior to the homogenization step. In an embodiment in which the fish testes are frozen, the freezing temperature is −10 to −80° C. In a preferred embodiment, the freezing temperature is −10 to −40° C. In a preferred embodiment, the freezing temperature is −10 to −20° C.

[0010] In one embodiment, the fish testes are washed with a weak acid prior to the homogenization step. In a preferred embodiment, the weak acid is citric acid.

[0011] In one embodiment, the homogenate is incubated with an acid. In a preferred embodiment, the acid is HCl. In a more preferred embodiment, the HCl is 0.01 to 1 M HCl, preferably 0.05 to 0.5 M HCl. In a preferred embodiment, the incubation is for 30 to 240 minutes, preferably 30 to 180 minutes.

[0012] In one embodiment, the homogenate is centrifuged to collect the pellet. In a preferred embodiment, the centrifugation is at 20 g to 15,000 g for 30 seconds to 90 minutes, preferably at 30 g to 12,000 g for 15 to 60 minutes.

[0013] In one embodiment, the pellets are resuspended in water and the pH of the suspension is adjusted to pH 6.0 to 9.0, preferably pH 7.0 to 8.0. In a preferred embodiment, the ratio of pellets to water is 1:1 to 1:15, preferably 1:5 to 1:12, by weight.

[0014]

[0014] In one embodiment, the suspension is heated. In a preferred embodiment, the heating is carried out at 60 to 150°C, preferably 70 to 130°C, for 5 to 150 minutes, preferably 15 to 120 minutes.

[0015] In one embodiment, the suspension is cooled after heating, and a proteinase and / or nuclease is added to form a mixture. In a preferred embodiment, the proteinase and / or nuclease is one or more of Alcalase, Flavorzyme, proteinase, nuclease, pepsin, and trypsin. In a more preferred embodiment, the proteinase and / or nuclease is trypsin. In a preferred embodiment, the proteinase and / or nuclease is incubated with the suspension for 0.5 to 48 hours, preferably 0.5 to 24 hours.

[0016] In one embodiment, the proteinase and / or nuclease is inactivated by heating. In a preferred embodiment, the proteinase and / or nuclease is inactivated by heating at 50 to less than 120°C, preferably 70 to 100°C, for 10 to 70 minutes, preferably 15 to 60 minutes.

[0017] In one embodiment, the DNA extract is obtained by separating the mixture to obtain a supernatant containing the DNA. In a preferred embodiment, the separation is by filtration or centrifugation.

[0018] In one embodiment, the DNA extract undergoes a step to remove fishy flavor. In a preferred embodiment, the fishy flavor is removed with diatomaceous earth. In a preferred embodiment, the fishy flavor is removed with cyclodextrin.

[0019]

[0019] The present disclosure relates to a DNA extract produced by the above-described method. In one embodiment, the DNA extract comprises DNA molecules having DNA base pairs between 10 and 2000 bp, preferably between 10 and 500 bp, and more preferably between 50 and 250 bp.

[0020]

[0020] The present disclosure further relates to methods of using the DNA extract. In one embodiment, the present disclosure provides a method for treating or ameliorating osteoarthritis or osteoporosis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the DNA extract. In one embodiment, administration of the DNA extract reduces osteoarthritis pain. In a preferred embodiment, the DNA extract reduces one or more of inflammation, pannus formation, cartilage damage, and bone resorption. In one embodiment, administration of the DNA extract increases limb support. In one embodiment, administration of the DNA extract increases cancellous bone formation, surface area, and / or thickness.

[0021] In one embodiment, the present disclosure provides a method for scavenging free radicals in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a DNA extract. In a preferred embodiment, the free radical is an NO free radical.

[0022]

[0022] In one embodiment, the present disclosure provides a method for reducing oxidative stress in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a DNA extract.

[0023] In one embodiment, the present disclosure provides a method for treating or ameliorating depression in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a DNA extract, hi a preferred embodiment, the administration of the DNA extract inhibits monoamine oxidase (MAO).

[0024] In one embodiment, the present disclosure provides a method for promoting black hair formation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a DNA extract. In a preferred embodiment, administration of the DNA extract increases tyrosinase activity.

[0025] In one embodiment, the DNA extract in the above-described method is orally administered to a subject. In a preferred embodiment, the oral administration is in the form of a solid, such as a tablet, capsule, powder, or granule, or in the form of a solution, such as a suspension, liquid, or emulsion. In one embodiment, the DNA extract is used as a dietary supplement.

[0026]

[0026] The present invention is described in detail in the following sections. Other features, objects, and advantages of the present invention will be readily apparent from the detailed description of the invention and the claims. [Brief explanation of the drawings]

[0027] [Figure 1] FIG. 1 shows the results of 1.5% agarose gel electrophoresis of mackerel DNA extract and salmon DNA extract hydrolyzed with trypsin for 1 to 6 hours. [Figure 2] FIG. 1 is a diagram showing the procedures of animal experiments. [Figure 3] FIG. 1 shows changes in animal weight during the experiment. [Figure 4] Figure 1 shows the erythema or edema scores in animals after administration of MIA. Data for each group are shown as mean ± SEM. The erythema or edema scores for all animals in the same group were summed and divided by the number of animals. [Figure 5] Images of the knee joint after local stimulation. Scale bar = 1.0 cm. The numbers in parentheses below each image indicate the animal number. [Figure 6] Figure 1 shows the pain tolerance threshold in the joint. The maximum stress values ​​tolerated in the right hind paw (affected paw) are shown 2 and 4 weeks after administration. Data are shown as mean ± SEM. *: p<0.05 compared to the G1 (OVX + MIA + water) group (statistical analysis by Student's t-test). [Figure 7] This figure shows changes in hindlimb support strength. The support strengths of the left and right hindlimb were subtracted from each other to determine the variation. Data are shown as mean ± SEM. *: p<0.05 compared to the G1 (OVX + MIA + water) group (statistical analysis by Student's t-test). [Figure 8] This figure shows microtomography images of the femur. The degree of osteoporosis can be determined by the degree of bone density. Scale bar = 2.0 mm. The numbers in parentheses below each image indicate the animal number. [Figure 9] Microtomography images of femurs were analyzed for bone mineral density (A), bone surface area fraction (B), bone volume fraction (C), bone surface area density (D), trabecular thickness (E), and trabecular number (F). Data are shown as mean ± SEM. *: p<0.05, significant change compared to the G1 (OVX + MIA + water) group (statistical analysis by Student's t-test). [Figure 10]

[0033] Figure 1 shows microtomography images of knee joint tissue. Images are at 50x magnification and show the results of H&E staining. The numbers in parentheses below each image indicate the animal number. [Figure 11] 1 shows the results of knee joint tissue analysis. Evaluation items include the total osteoarthritis score (A) and inflammation, pannus formation, cartilage damage, and bone resorption for histopathological analysis of osteoarthritis (B). Data are shown as mean ± SEM. *: significant change compared to the G1 (OVX + MIA + water) group (p<0.05, Student's t-test). L: Composition L, M: Composition M, H: Composition H, and X: Composition X. [Figure 12]

[0033] Figure 1 shows the results of femoral tissue analysis. Evaluation items included trabecular thickness and trabecular area. Data are shown as mean ± SEM. *: significant change compared to the G1 (OVX + MIA + water) group (p<0.05, Student's t-test). L: Composition L, M: Composition M, H: Composition H, and X: Composition X. [Figure 13] Figure 1 shows microtomography images of femoral tissue. Images are at 50x magnification and show the results of H&E staining. The numbers in parentheses below each image indicate the animal number. [Figure 14] FIG. 1 shows the effect of DNA extracts in scavenging NO free radicals. [Figure 15] FIG. 1 shows the effect of DNA extracts on chelating ferrous ions. [Figure 16]FIG. 1 shows the inhibitory effect of DNA extract on monoamine oxidase (MAO). [Figure 17] FIG. 1 shows the effect of DNA extract on increasing tyrosinase activity. DETAILED DESCRIPTION OF THE INVENTION

[0028] [Detailed Description of the Invention]

[0044] In order to make the features, objects and advantages of the technology of the present invention more clearly understood, a detailed description of the technical method of the present invention is provided below. The examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way. [Example]

[0029] Example 1: Mackerel DNA extraction

[0045] Freshly collected testes from mackerel were frozen at -10 to -80°C and thawed at room temperature before use. The thawed testes were washed with citric acid and homogenized to obtain a milky or pulpy homogenate. 0.05M to 0.5M HCl was added to the homogenate and incubated for 30 to 180 minutes. The mixture was then centrifuged at 30 to 12,000 g for 15 to 60 minutes. The supernatant was removed by filtration. The pellet was collected and resuspended in distilled water (solids:water ratio = 1:5 to 1:12 by weight). The pH of the suspension was adjusted to approximately 7.0 to 8.0. The suspension was then heated at 70 to 130°C for 30 to 120 minutes for sterilization. After cooling, trypsin was added to the suspension and then incubated at 10 to 40°C for 0.5 to 24 hours. The enzyme was inactivated by heating at 70 to 100°C for 15 to 60 minutes. After enzyme inactivation, the mixture was either filtered or centrifuged at 30–12,000 g for 15–60 minutes. The supernatant containing the DNA extract was collected. To remove the fishy odor, each unit of supernatant (100–1,000 mL) was treated with 0.02–60% diatomaceous earth and / or cyclodextrin. The results of removing the fishy odor were evaluated by olfactory analysis. Supernatants that passed the analysis were lyophilized and stored at room temperature.

[0030] Example 2: Characterization of DNA extracts

[0046] The DNA obtained in Example 1 was analyzed for DNA size. The DNA extract was treated with trypsin for 1 to 6 hours. After trypsin treatment, the extract was subjected to 1.5% agarose gel electrophoresis, and the OD 260 The DNA concentration was assessed by detecting the absorbance of the DNA. For comparison, DNA obtained from salmon according to the extraction procedure in Example 1 was used. The results are shown in Figure 1, which shows that the DNA base pair distribution is between 10 bp and 500 bp. This DNA base pair range overlaps with the known range of 50 to 2,000 bp for DNA.

[0031]

[0047] The DNA extracts were further evaluated for DNA purity. The OD of the supernatant containing the mackerel DNA extract subjected to different periods of trypsin digestion as described above was 260 and O.D. 280の The results are shown in Table 1 below.

[0032] [Table 1]

[0033]

[0048] The OD shown in Table 1 260 / OD 280 From the results, OD 260 / OD 280 It can be seen that the value is between 1.60 and 1.70.

[0034] Example 3: Removal of fishy odor from DNA extracts Example 3.1: Removal of Fishy Odor by Use of Diatomaceous Earth (DE) and / or Cyclodextrin (CD)

[0049] Different amounts of DE and / or CD were added to the trypsin-digested DNA extract, and the mixture was incubated at room temperature or 30-60°C for 18-36 hours, followed by centrifugation. The supernatant was collected and the effectiveness of the fishy odor removal was evaluated by olfactory analysis. The experimental design is shown in Table 2 below.

[0035] [Table 2]

[0036]

[0050] In the olfactory analysis, at least two panelists evaluate the fishy odor. The results are shown in Table 3 below, which show that the addition of 5-20% DE and / or CD can eliminate the fishy odor.

[0037] [Table 3]

[0038] Example 4: Evaluation of the effects of DNA extracts on osteoporosis and osteoarthritis in animal models

[0051] 4.1 Grouping and treatment of experimental animals

[0052] Female SD rats were ovariectomized (performed by BioLASCO, Taiwan) and transferred to an animal room for four weeks for recovery and adaptation. The rats were weighed and divided into five groups (groups 1-5) based on their weight. Each group contained five female rats. After grouping, there was no significant difference in animal weight between groups (P<0.05). Five female rats of the same age as the grouped animals, but which underwent the same surgical procedure without ovariectomy, were further divided into a sham control group (group 0).

[0039]

[0053] [Table 4]

[0040]

[0054] [Table 5]

[0041]

[0055] 4.2 Experimental procedure

[0056] After ovariectomy and 4 weeks of acclimation, rats were injected with MIA (sodium iodoacetate) into the knee joint of their right hind leg on day 1. Treatment was carried out from day 1 to day 29. After that, the rats were sacrificed and samples were collected. A flowchart of the experimental procedure is shown in Figure 2.

[0042]

[0057] 4.2.1 Induction of osteoarthritis (OA)

[0058] On the first day of the experiment, the ovariectomy was performed on female rats in groups 1 to 5. Each animal was anesthetized using isoflurane and then injected with 50 μL of MIA saline (4 mg / 50 μL) into the right knee joint using a 26G needle. After completion, the rats were returned to their cages for recovery. Animals in group 0 underwent the same surgery but were injected with normal saline to serve as normal controls.

[0043]

[0059] 4.2.2 Administration of Test Composition

[0060] Starting on the day of MIA induction (Day 1), rats were orally fed (gavaged) once daily with water or food containing the test composition for 28 days. The first day of administration is defined as "Day 1 of the experiment" or D1 (Day 1).

[0044]

[0061] 4.3 Experimental results

[0062] 4.3.1 Animal Observation (SOP-TM201)

[0063] During the experiment, the animals were observed once daily. During the experiment, only swelling of the knee joint was observed for 3 days (~day 3) after the injection of MIA to induce OA. However, this is a known toxic reaction induced by MIA. No other abnormalities were observed.

[0045]

[0064] 4.3.2 Animal weight (Precisa, XB6200D)

[0065] During the experiment, the animals' weights were measured once a week. After ovariectomy of female rats, the weights of animals in groups 1 to 5 were significantly higher than those of animals in group 0 due to hormonal changes. During the experiment, the weights of animals in group 1 did not differ compared to groups 2 to 5. The growth curves of each group were similar (Figure 3).

[0046]

[0066] 4.3.3 Evaluation of local OA reactions

[0067] During the experiment (after the introduction of MIA), the right knee was observed three times a week to evaluate erythema and edema.

[0047] [Table 6]

[0048]

[0068] The local irritation reaction at the injection site of MIA can be seen in Figure 4. A photograph of the local site is shown in Figure 5.

[0049]

[0069] Macroscopic observation revealed some degree of local irritation at the joint injection site. On Day 1, each MIA-administered group showed a score of 1.0 to 2.4, indicating moderate to low irritation of the MIA in the joint cavity. By Day 3, the irritation decreased across all groups compared to Day 1. The mean irritation scores for G1 (OVX + MIA + water), G2 (OVX + MIA + L), G3 (OVX + MIA + M), G4 (OVX + MIA + H), and G5 (OVX + MIA + X) were 1.0, 0.8, 0.8, 0.6, and 0.4, respectively, which were not statistically significant. In the latter half of the experiment, the mean irritation score was less than 0.6, which is negligible under a rating system with a total score of 8. Therefore, the test composition can dose-dependently ameliorate the irritation caused by MIA. The sham group (G0: water) showed no local irritation throughout the experimental period.

[0050]

[0070] 4.3.4 Assessment of osteoarthritis (OA) pain

[0071] Measurements of the pain index, i.e., gf (gram-force) values, of the right hind paw were performed using a Pressure Application Measurement (PAM) device (Ugo Basile, Italy, catalogue number 38500) before administration of the test composition (week -1) and after 2 and 4 weeks of administration.

[0051]

[0072] The results of the evaluation of the test composition on MIA-induced osteoarthritis pain are shown in FIG.

[0052]

[0073] The PAM device compresses the muscles around the knee joint to measure the pain threshold of arthritis in rats. A lower pain threshold indicates a higher pain index (stronger pain), while a higher pain threshold indicates a lower pain index (weaker pain). Before the experiment, the pain threshold of each group of animals was between 920 and 1152 gf. After injecting water or MIA solution into the knee joint, the pain threshold decreased, indicating that injection into the joint cavity induces pain. After MIA was injected into the joint cavity, two weeks after treatment with the control or test compositions, the mean pain thresholds of each group [G2 (OVX+MIA+L): 776.40±65.79, G3 (OVX+MIA+M): 851.66±112.86, G4 (OVX+MIA+H): 879.28±90.33, and G5 (OVX+MIA+X): 806.16±94.63] were higher than the mean pain threshold of the disease control group [G1 (OVX+MIA+water): 612.12±68.40], and the difference between G4 and G1 reached statistical significance. After MIA was injected into the joint cavity, four weeks after treatment with the control or test compositions, the mean pain thresholds of each group (G2: 824.20±18.57, G3: 695.00±134.54, G4: 831.44±188.05, G5: 806.16±94.63) were higher than the mean pain threshold of the disease control group (G1: 976.80±228.94), but this was not statistically significant. Based on the above results, it can be seen that the test composition (OVX+MIA+H Company) can improve the pain of MIA-induced arthritis.

[0053]

[0074] 4.3.5 Evaluation of hindlimb support

[0075] Two and four weeks after administration of the test composition, the supporting strength (unit: g) of the hind limbs was measured as an index of arthritis pain using a Librae Incapacitance Tester for rats (WP, Taiwan).

[0054]

[0076] The evaluation results of hindlimb support are shown in FIG.

[0055]

[0077] The change in the support strength of both hind limbs caused by unloading the right hind limb to relieve pain after arthritis was induced was evaluated by measuring the difference in the weight that both hind limbs could support. If the arthritic limb can support a weight similar to that supported by the normal limb, the test composition is considered to have a therapeutic effect on the arthritic limb. The disease control group (G1: OVX + MIA + water) showed a clear increase in the support strength of both limbs compared to the normal control group (G0: sham + PBS + water), and the difference reached statistical significance. Two and four weeks after arthritis induction, the change in support strength of both hind limbs increased 15-fold and 20-fold, respectively, indicating successful induction of the arthritis model.

[0056]

[0078] Two weeks after the induction of arthritis and the administration of the test composition, each test group showed a significant decrease in the supporting strength of both limbs compared with the disease control group (G1), and the decrease reached statistical significance. Test groups G2 (OVX+MIA+L), G3 (OVX+MIA+M), G4 (OVX+MIA+H), and G5 (OVX+MIA+X) showed pain relief of 28%, 34%, 38%, and 42%, respectively, compared with G1.

[0057]

[0079] Four weeks after the induction of arthritis and administration of the test composition, each test group showed a significant decrease in the supporting strength of both limbs compared to the disease control group (G1: OVX + MIA + water). Except for G2, G3 to G5 reached statistical significance. Test groups G2, G3, G4, and G5 showed pain relief of 45%, 52%, 72%, and 74%, respectively, compared to G1.

[0058]

[0080] 4.3.6 Femoral microcomputed tomography evaluation

[0081] The femur of the left hind limb was fixed in a fixative (10% formalin) and subjected to microtomography to evaluate bone parameters such as bone mineral density and cancellous bone.

[0059]

[0082] The femoral microtomography images are shown in Figure 8. The results of the analysis of the femoral microtomography images are shown in Figure 9.

[0060]

[0083] From the femoral microtomography images in Figure 8, it can be seen that the bone mass of the femur in the normal control group (G0) is densely packed with only small holes. In the osteoporosis control group (G1: OVX + MIA + water), the bone mass is low, with large holes and some scattered spongy bone. In the test groups G2 to G5, the bone mass is generally denser than in G1, the holes are small, and the spongy bone dispersed within the holes is abundant and distinct.

[0061]

[0084] The images were further analyzed by microtomography. The results showed that, compared with the G0 normal control group, the bone mineral density (BMD), bone volume fraction (BV / TV), bone surface area density (BS / TV), trabecular thickness (TB / TH), and trabecular number (TB / N) of the osteoporosis control group G1 were 19.9%, 28.3%, 36.5%, 89.0%, and 31.7%, respectively, of the G0 group. Furthermore, the bone surface area ratio (BS / BV) of G1 was 130% of that of G0. All of the above results reached statistical significance. The analytical results were consistent with the images, indicating the successful establishment of an osteoporosis disease model.

[0062]

[0085] Compared with the G1 disease model group, the mean values ​​of BMD, BV / TV, BS / TV, TB.TH, and TB.N in G2, G3, and G4 were higher than those in G1, while the value of BS / BV was lower than that in G1. However, the above differences did not reach statistical significance. The above results indicate that the test compositions of G2 (OVX+MIA+L), G3 (OVX+MIA+M), and G4 (OVX+MIA+H) have the potential to improve osteoporosis.

[0063]

[0086] Compared with the G0 normal control group, the BMD, BV / TV, BS / TV, TB.TH, and TB.N values ​​of G5 were 167%, 141%, 130%, 106%, and 133% of those of G1, respectively, while the bone surface area ratio (BS / BV) value was 90.7% of that of G1. All of the above differences reached statistical significance. The results demonstrated that the test composition of G5 (OVX+MIA+X Company) was effective in treating osteoporosis.

[0064]

[0087] 4.3.7 Histopathological analysis of the knee joint cavity

[0088] After the experiment, live test animals were randomly euthanized by carbon dioxide (CO2) (SOP-TM210). Blood was collected, and the carcasses were dissected. The appearance of all tissues and organs in the thoracic and abdominal cavities, as well as changes at the injection site, were macroscopically examined and recorded. In addition, the left and right hind limbs were collected and fixed for subsequent processing and evaluation.

[0065]

[0089] Histological images of the knee joint cavity are shown in Figure 10. The results of histopathological analysis of the knee joint cavity are shown in Figure 11.

[0066]

[0090] The knees (including the lateral bones) of all animals in the control and test groups were fixed, decalcified, trimmed, dehydrated, wax-immersed, embedded, and sliced ​​(4-6 μm), then stained with H&E to prepare tissue section biopsies. Histopathological analysis was performed by a specialized veterinarian using a microscope (Leica DM2700M, USA). Four indices of arthritis were evaluated in the histopathological analysis: inflammation, pannus formation, cartilage damage, and bone resorption. Each indices was classified into five levels according to severity: 0 (no response), 1 (slight response), 2 (low response), 3 (moderate response), and 4 (highly severe response).

[0067]

[0091] The histopathological analysis of the knee joint cavity showed that only a slight inflammatory reaction was observed in the G0 normal control group (Sham + PBS + water) (average score 0.4). The scores of the other three indices were 0. The total score for the overall arthritis was also 0.4, which was determined to be negligible. Therefore, the animals were healthy and did not have arthritis.

[0068]

[0092] The G1 disease model control group (OVX + MIA + water) had scores of 2.6 (each score between slight and severe), 3.2 (each score between moderate and severe), 2.8 (each score between slight and severe), and 2.2 (each score between slight and moderate). The total arthritis score was 10.8, which is between slight and moderate arthritis. This score was approximately 27 times more severe than the normal control group, indicating that the arthritis induction model was successful.

[0069]

[0093] The scores of inflammation, pannus formation, cartilage damage, and bone resorption, as well as the total score of arthritis, for G2 (OVX+MIA+L) and G3 (OVX+MIA+M) were lower than those for G1. However, this difference did not reach statistical significance. The test compositions for G2 (OVX+MIA+L) and G3 (OVX+MIA+M) reduced the arthritis response by 11% and 19%, respectively.

[0070]

[0094] The scores of inflammation, pannus formation, cartilage damage, and bone resorption for G4 (OVX+MIA+H) were 1.8, 1.8, 1.6, and 1.6, respectively. The total score of arthritis was 6.8. These scores were lower than those of G1. Furthermore, the scores of pannus formation and cartilage damage and the total score of arthritis were statistically significantly different from those of G1. The test composition of G4 (OVX+MIA+H) could significantly reduce the arthritis reaction by 37%.

[0071]

[0095] The scores of inflammation, pannus formation, cartilage damage, and bone resorption for G5 (OVX+MIA+X) are 1.8, 1.6, 1.2, and 1.2, respectively. The total score of arthritis is 5.8. These scores are lower than those of G1. Furthermore, the scores of pannus formation, cartilage damage, and bone resorption, as well as the total score of arthritis, are statistically significantly different from those of G1. The test composition of G5 (OVX+MIA+X) can significantly reduce arthritis reaction by 46%.

[0072]

[0096] 4.3.8 Histopathological analysis of femurs

[0097] The results of the histopathological analysis of the femur are shown in Figure 12. Histological images of the femur are shown in Figure 13.

[0073]

[0098] The right hind femurs of all animals in the control and test groups were fixed, decalcified, trimmed, dehydrated, wax-immersed, embedded, and sliced ​​(4-6 μm), then stained with H&E to prepare tissue section biopsies. Histopathological analysis was performed by a specialized veterinarian using a microscope (Leica DM2700M, USA). The histopathological analysis evaluated the morphology and microstructure of the femurs. The thickness (μm) and surface area (%) of the trabecular bone were analyzed using ImageJ software.

[0074]

[0099] Histopathological analysis of the femurs showed that in the G0 normal control group (Sham + PBS + water), a dense bone structure was observed in the microscopic images of the femurs. Trabecular bone accounted for 68.4% of the surface area. The average thickness of the trabecular bone was 223 μm, approximately 2.2 times that of the G1 disease model control group (OVX + MIA + water). These results indicate that the femurs were dense and consistent with the femoral structure expected in healthy animals.

[0075]

[0100] Microscopic images of the femur in the G1 disease model group (OVX + MIA + water) showed that the bone structure was rough. Numerous bubble-like structures were present between the trabecular bones. Trabecular bone accounted for approximately 50.6% of the surface area. This surface area was reduced by approximately 18%. The average thickness of the trabecular bone was 103 μm, only 0.5 times that of the normal control group G0. The results indicated osteoporosis in the femur. The osteoporosis induction model was successful. Furthermore, this degree of osteoporosis is suitable for use in evaluating the effects of health foods.

[0076]

[0101] Microscopic images of the femur in G2 (OVX+MIA+L) show that the bone structure is rough. Numerous foam-like structures are present between the cancellous bone. The cancellous bone accounts for approximately 56.8% of the surface area. This surface area is approximately 9.6% greater than that of G1. However, the average thickness of the cancellous bone is only 100 μm, similar to that of G1. Therefore, the test composition for G2 (OVX+MIA+L) does not exhibit effective amelioration or treatment effects for osteoporosis.

[0077]

[0102] Microscopic images of the femur in G3 (OVX+MIA+M) showed a better bone structure than G1 and G2, but some foamy structures were still present between the cancellous bone. The cancellous bone accounted for approximately 61.4% of the surface area. This surface area increased by approximately 10.8% compared to G1, but the difference was not statistically significant. The average thickness of the cancellous bone was 130 μm, approximately 1.3 times that of G1, which was statistically significant. Therefore, the test composition for G3 (OVX+MIA+M) exhibited an ameliorative or therapeutic effect on osteoporosis.

[0078]

[0103] Microscopic images of the femur in G4 (OVX+MIA+H) showed a better bone structure than G1 and G2, but some foamy structures were still present between the cancellous bone. The cancellous bone accounted for approximately 62.4% of the surface area. This surface area increased by approximately 11.8% compared to G1, but the difference was not statistically significant. The average thickness of the cancellous bone was 126 μm, approximately 1.2 times that of G1, which was statistically significant. Therefore, the test composition for G4 (OVX+MIA+H) exhibited an ameliorative or therapeutic effect on osteoporosis.

[0079]

[0104] Microscopic images of the femur in G5 (OVX+MIA+X) showed a better bone structure than G1 and G2, but there were still foam-like structures present between the cancellous bone. However, the number of foam-like structures was less than in G1. The cancellous bone accounted for approximately 60.3% of the surface area. This surface area increased by approximately 9.6% compared to G1, but the difference was not statistically significant. The average thickness of the cancellous bone was 126 μm, approximately 1.2 times that of G1, which was statistically significant. Therefore, the test composition for G5 (OVX+MIA+X) exhibits an improving or therapeutic effect on osteoporosis.

[0080]

[0105] 4.4 Conclusion

[0106] For comprehensive analysis, experiments were selected in which the data from the disease model control group G1 was significantly different from the data from the normal control group G0. The results are shown in Table 7 below.

[0081]

[0107] [Table 7]

[0082]

[0108] In the experimental conditions, the test compositions were orally administered for 28 days. The results show that Composition X has the effects of alleviating arthritis pain, repairing tissue damage caused by arthritis, increasing bone density, and increasing the thickness and number of cancellous bone. Composition H has the effects of alleviating arthritis pain, repairing tissue damage caused by arthritis, and increasing the thickness of cancellous bone. Composition M has the effects of alleviating arthritis pain and increasing the thickness of cancellous bone. Composition L has the effect of slightly alleviating arthritis pain. The therapeutic effects are in the following order: Composition X > Composition H > Composition M > Composition L.

[0083] Example 5: Evaluation of the antioxidant effect of DNA extracts

[0109] 5.1 Evaluation of the effect of scavenging NO free radicals

[0110] A standard sample for NO free radical was prepared by adding 8 μl of 10 mM NaNO to 492 μl of HO to form a 160 μM NaNO solution. 250 μL of the 160 μM NaNO solution was added to an Eppendorf containing 250 μL of HO to give a concentration of 80 μM, which was serially diluted to 40 μM, 20 μM, 10 μM, and 5 μM to measure a standard curve of NO concentrations, with pure water used as 0 μM.

[0084]

[0111] For test sample analysis, 50 μL of 10 mM sodium nitroprusside (SNP) or HO (blank) was added to 50 μL of 1 mg / mL sample or gallic acid (GA) (positive control) and preincubated at room temperature for 25–60 minutes. After preincubation, 50 μL of 2% sulfanilamide (SA) and 50 μL of 0.2% N-(1-naphthyl)ethylenediamine dihydrochloride (NED) were added to the test sample and incubated at room temperature for 10 minutes, protected from light. Samples were monitored at OD 540 nm. The results are shown in Figure 14, demonstrating that DNA extracts from salmon and mackerel testes exhibited NO scavenging effects, with the mackerel DNA extract demonstrating superior efficacy.

[0085]

[0112] 5.2 Evaluation of the chelating effect of ferrous ions

[0113] 90 μL of DNA extract sample or positive control (1 mg / mL EDTA-2Na) was treated with 20 μL of 0.3 mM FeCl₂·4H₂O, followed by the addition of 40 μL of 0.75 mM Ferrozine or H₂O (blank) and incubation at room temperature for 10 minutes. Samples were detected at OD 562 nm. The results are shown in Figure 15, demonstrating that DNA extracts from salmon and mackerel testes exhibited ferrous ion chelating effects, with mackerel DNA extract showing superior chelating effects.

[0086] Example 6: Evaluation of the antidepressant effect of DNA extracts

[0114] The DNA extracts were evaluated for their inhibitory effect on monoamine oxidase (MAO), an enzyme known to be associated with depression. 50 μL of sample or positive control (200 μM (+)-catechin) was treated with 50 μL of 0.1 mg / mL MAO-A, followed by the addition of 100 μL of 500 mM kynuramine to form a reaction mixture. The mixture was incubated at 37°C for 40 minutes, protected from light. After incubation, 10 μL of Amplex Red (0.5 mM) was added to the mixture, followed by 20 μL of horseradish peroxidase (HRP) (1 mg / mL) or HO (blank). The mixture was then incubated for an additional 5 minutes at room temperature, protected from light. Next, 0.3 mL of HO was added, followed by detection at OD 571 nm. The results are shown in Figure 16, demonstrating that DNA extracts from salmon and mackerel testes exhibited MAO inhibitory effects, with the mackerel DNA extract demonstrating superior efficacy.

[0087] Example 7: Evaluation of the dark hair promoting effect of DNA extract

[0115] Tyrosinase is a key enzyme known to be involved in the formation of melanin, a pigment important in the formation of dark hair. The effect of DNA extracts on increasing tyrosinase activity was evaluated. 450 μL of 0.1 M pH 6.8 phosphate buffer and 500 μL of 0.03% tyrosine were added to 450 μL of sample or positive control (ascorbic acid (vitamin C), 500 μg / ml) and preincubated at 37°C for 10 minutes. Next, 50 μL of 350 units / mL tyrosinase or HO (blank) was added to the reaction solution and incubated at 25°C for an additional 25 minutes. Samples were detected at OD 475 nm. The results are shown in Figure 17 and demonstrate that DNA extracts from salmon or mackerel testis exhibit a tyrosinase-activating effect.

Claims

1. A pharmaceutical composition for treating or ameliorating osteoarthritis or osteoporosis in a subject in need thereof, comprising a therapeutically effective amount of a DNA extract obtained from fish testes and having DNA base pairs between 10 and 2000 bp.

2. The composition of claim 1 , wherein the DNA extract is administered orally.

3. 10. The composition of claim 1, wherein administration of the DNA extract reduces osteoarthritis pain.

4. 4. The composition of claim 3, wherein the DNA extract reduces one or more of inflammation, pannus formation, cartilage damage, and bone resorption.

5. The composition of claim 1 , wherein administration of the DNA extract increases limb support.

6. The composition of claim 1 , wherein administration of the DNA extract increases cancellous bone formation, surface area, and / or thickness.

7. A pharmaceutical composition for scavenging free radicals in a subject in need thereof, comprising a therapeutically effective amount of a DNA extract obtained from fish testes and having DNA base pairs between 10 and 2000 bp.

8. The composition of claim 7 , wherein the DNA extract is administered orally.

9. 8. The composition of claim 7, wherein the free radical is an NO free radical.

10. A pharmaceutical composition for treating or ameliorating depression in a subject in need thereof, comprising a therapeutically effective amount of a DNA extract obtained from fish testes and having DNA base pairs between 10 and 2000 bp.

11. The composition of claim 10, wherein the DNA extract is administered orally.

12. 11. The composition of claim 10, wherein administration of the DNA extract inhibits monoamine oxidase (MAO).

13. A pharmaceutical composition for promoting black hair formation in a subject in need thereof, comprising a therapeutically effective amount of a DNA extract obtained from fish testes and having DNA base pairs between 10 and 2000 bp.

14. The composition of claim 13, wherein the DNA extract is administered orally.

15. 14. The composition of claim 13, wherein administration of the DNA extract increases tyrosinase activity.

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