TWI692361B - Use of antrodia salmonea mycelium in alleviation of cancer cachexia - Google Patents

Abstract

The present invention relates to the use of an Antrodia salmonea mycelium composition in the alleviation of cancer cachexia symptoms, including Anorexia, easy feeling full, fatigue, body weight loss and physical weakness. The Antrodia salmonea mycelium composition of present invention can be used as a nutritional supplement to delay cancer cachexia, or as an auxiliary drug for cancer cachexia treatment.

Description

Application of Xiangshanzhi mycelium in delaying cancer cachexia

The present invention relates to a mycelium composition of Shanshanzhi used for delaying cancer cachexia. More particularly, the present invention relates to a mycelia composition of Shanxiangzhi used for improving or reducing the symptoms of cancer cachexia, including anorexia, susceptibility to satiety, fatigue, body weight loss and weakness.

Cancer has been ranked as the top ten cause of death for 34 years in a row. The main cause of 40% of cancer deaths is cancer cachexia. Clinically, more than 50 to 80% of cancer patients suffer from cachexia (Argilés, JMet al., Nature reviews Cancer , 14 (11): 754-762, 2014), which is mainly a proinflammatory cytokine secreted by malignant tumors Excessive amount of secretion leads to an increase in the body's inflammatory index and metabolic imbalance caused by physical condition wasting diseases, in which weight loss and body tissue loss can be regarded as important indicators of cancer cachexia prognosis. The concomitant occurrence of cancer cachexia not only reduces the patient's quality of life, but his physical condition is too weak to be able to bear the side effects caused by chemotherapy drugs. It often allows physicians to reduce the drug dose or even terminate the course of treatment after evaluation. There are adverse effects. How to improve or reduce the condition of cancer cachexia patients is an urgent problem to be solved in medical treatment.

It is known from the current literature that proinflammatory factors secreted by malignant tumor cells cause systemic inflammatory response and abnormal metabolism in the host. Among them, cytokines and interleukins -1 (interleukin, IL-1), IL-6 and tumor necrosis factor-α (tumor necrosis factor, TNF-α), will affect the central nervous system endocrine control of appetite and cause patients to appear anorexia symptoms, and enhance the body Thermogenesis in brown adipose tissue, which leads to increased energy consumption in the body, excessive loss of muscle tissue, reduced lipid synthesis in lipid tissue (lipogenesis), and promoted lipolysis (Tsoli, M .& Robertson, G., Trends in Endocrinology & Metabolism, 24(4), 174-183, 2013) and other patients with abnormal metabolism in the body. Therefore, the clinical symptoms of cancer cachexia include anorexia, susceptibility to satiety, fatigue, body weight loss and weakness.

Among the many therapeutic drugs, progesterone drugs are considered to be the most effective and safest drugs for treating cancer cachexia. Progesterone drugs, in addition to rapidly promoting appetite, can also suppress cancer cells to produce proinflammatory factors and proteolytic factors, which can slow down the continuous consumption of patients' body tissues. In addition, drugs under research include thalidomide, pentoxifylline, and melatonin, which mainly act on the release of cytokines; in addition, clenbuterol, which acts on muscles, and Antagonize protein consumption. Current clinical research indicates that the first-line treatment of cancer cachexia is synthetic progestin drugs, such as megestrol and medroxyprogesterone, and antioxidants and chemotherapy can also be used in combination.

Antrodia cinnamomea has been proven by research to have many therapeutic effects, such as anti-cancer, liver protection, antioxidant, anti-inflammatory, etc., especially the research literature on Antrodia cinnamomea extract and special active ingredients, such as triterpenoids, benzene compounds, polyphenols , Zhuoquinol, Antrodia acid, etc. have anti-inflammatory and anti-cancer effects. Therefore, research is expected to use Antrodia cinnamomea fruit bodies or their extracts to develop adjuvant therapeutic agents for cancer, and even to reduce cancer cachexia. However, Antrodia camphorata grows in the inner cavity of the trunk of Antrodia camphorata, and Antrodia camphorata is currently a conservation species in Taiwan. Antrodia camphorata is expensive, which is not conducive to commercial applications.

Xiangshanzhi was identified as Antrodia in 2004 and is a unique fungus in Taiwan (Xie Yunrong, Xiangshanzhi mycelium extract alone and combined with anti-cancer drugs on human hepatocellular carcinoma cell apoptosis Impact, Taichung, Taiwan, 2011). Its fruiting body is salmon pink, parasitic in the hollow trunk of withered fragrant Chinese fir, similar in appearance to Antrodia camphorata (Chang, TT & Chou, WN, Botanical Bulletin of Academia Sinica. 45 : 347-352, 2004). It has been reported in the literature that both Shanshanzhi mycelium and Xiangshanzhi solid-state fermentation products have good antioxidant capacity (Enhe, Master Thesis, Department of Food and Applied Biotechnology, National ZTE University, Taichung, Taiwan, 2014). Another literature discusses the suppression of the survival rate of hematological cancer cells by Xiangshanzhi whole liquid and its possible mechanism. It was found that Xiangshanzhi whole liquid has the ability to induce apoptosis of blood cancer cells at a concentration that does not harm normal smooth muscle cells; In animal experiments, Xiangshanzhi whole liquid has the ability to inhibit growth and induce apoptosis of blood cancer tumors in nude mice (Chang, CTet al., Journal of ethnopharmacology , 196 : 9-19, 2017).

Many studies have pointed out that Xiangshanzhi has anti-inflammatory and anti-cancer effects. Therefore, the present invention hopes that the development of Xiangshan mycelium can replace Antrodia camphorata as a nutritional supplement to delay cancer cachexia, or as an adjunct therapeutic drug for cancer cachexia. . The present invention evaluates the relevant physiological parameters of Xiangshanzhi mycelium for delaying cancer cachexia, inhibiting tumor growth and activity fatigue by administering Xiangshanzhi mycelium to mice with cancer-like cachexia induced by injection of tumor cells Improvement and other effects.

Therefore, one aspect of the present invention relates to the use of the mycelia of Xiangshanzhi for the preparation of a pharmaceutical composition for alleviating the symptoms of cancer cachexia. The symptoms of cancer cachexia include (but are not limited to) anorexia, susceptibility to satiety, fatigue, body weight loss and weakness.

In some specific embodiments of the present invention, the mycelia of Xiangshanzhi is a liquid cultured mycelium of Xiangshanzhi. In some specific embodiments of the present invention, the cedar lucidum mycelium liquid-cultured in the cedar cedar mycelium system is dried, crushed, and sieved to obtain the cedar cedar mycelium powder.

Another aspect of the invention relates to the use of Xiangshanzhi mycelium for the preparation of cancer cachexia adjunct therapeutic drugs. The adjuvant therapy includes (but is not limited to) improving the loss of body tissue of cancer cachexia patients, improving the survival rate of cancer cachexia patients and inhibiting the tumor growth of cancer cachexia patients.

Another aspect of the present invention relates to the use of Xiangshanzhi mycelium for the preparation of inhibiting cancer cell metastasis in cancer cachexia patients.

Figure 1 shows the effect of Mycelia of Shanshanzhi on the average food intake of Lewis lung cancer cell-induced cancer cachexia mice. The experimental value is expressed as mean ± SD (n=6~9). #p <0.05 is the value compared to the control group without tumor induction (CON); *p <0.05 is the value compared to the group without tumor treatment (TC).

Figure 2 shows the effect of Mycelia of Shanshanzhi on the body weight of mice with cancer cachexia induced by Lewis lung cancer cells. The experimental value is expressed as mean ± SD (n=6~9). #p <0.05 is the value compared to the control group without tumor induction (CON); *p <0.05 is the value compared to the group without tumor treatment (TC).

Figure 3 shows the effect of the mycelia of Shanxiangzhi on the tumor volume of mice with cancer cachexia induced by Lewis lung cancer cells. The experimental value is expressed as mean ± SD (n=6~9). *p <0.05 is the value compared with the tumor-inducing (TC) group. Tumor volume (cm ³ ) = L × W × H ×π/6

Figure 4 shows the effect of Mycelia of Shanshanzhi on the tumor weight and tumor-free weight of mice with cancer cachexia induced by Lewis lung cancer cells. The experimental value is expressed as mean ± SD (n=6~9). In the overall weight internal standard, different capital letters indicate that there are significant differences between the groups ( p <0.05). Different lowercase letters in the internal standard of tumor weight indicate significant differences between groups ( p <0.05).

Figure 5 shows the effect of Mycelia of Shanshanzhi on the survival rate of mice with cancer cachexia induced by Lewis lung cancer cells. (n=9)

Figure 6 shows the effect of Mycelia of Shanshanzhi on the weight-bearing swimming time of Lewis lung cancer cell-induced cancer cachexia mice. The first (1 ^st ) weight-loaded swimming test was conducted on the 10th day (day10) after tumor transplantation; the second (2 ^nd ) trial was conducted on the 16th day (day16); the third The third (3 ^rd ) trial was conducted on the 22nd day (day22); for the fourth (4 ^th ) trial, the TC group was conducted on the 26th day (day26), while the remaining groups were conducted on the 27th day (day27) get on. #p <0.05 is the value compared to the control group without tumor induction (CON); *p <0.05 is the value compared to the group with tumor induction without drug treatment (TC) (n=6~9).

Figure 7 shows the effect of the mycelia of Shanshanzhi on the tumor tissue of mice with cancer induced by Lewis lung cancer. The scale bar in the figure is 60 μm.

Other features and advantages of the present invention will be further exemplified and described in the following embodiment examples, and the embodiment examples are only used as an auxiliary description and are not intended to limit the scope of the present invention.

Example 1: Preparation of Mycelia of Xiangshanzhi by Liquid Culture

Antrodia salmonea strains are preserved in PDA (Potato dextrose agar) medium at 4ºC . During inoculation, an inoculation knife is used to cut out the mycelium with strong peripheral activity, and three pieces of mycelium medium with a size of about 5mm×5mm are cut out as strains. Inoculate into 100ml of liquid culture medium (each liter contains: 20g glucose; yeast extract 5g/L; (NH ₄ ) ₂ SO ₄ 2g; KH ₂ PO ₄ 0.875g; K ₂ HPO ₄ 0.125g; MgSO ₄ .7H ₂ O 0.5g) in a 250ml Slotted Erlenmeyer flask, put a cotton plug on it, and shake culture at 25ºC, 100rpm for 14 days. Afterwards, a Waring blender homogenizer was used for low-speed homogenization for 15 seconds, 5ml of homogenized liquid was taken, and then inoculated into another 250ml flat-bottomed triangular flask containing 95ml of liquid culture medium, and capped with a breathable cap. The culture flask was shaken for 10 days at 25ºC and 100rpm. After that, the activated strain culture liquid was applied to a low-speed homogenizer with a Waring blender homogenizer for 15 seconds, and the resulting homogenized liquid was the inoculum.

The basic medium used in this study contains 20g test carbon source, 5g test nitrogen source, 0.875g KH ₂ PO ₄ , 0.125g K ₂ HPO ₄ and 0.5g MgSO ₄ per liter. 7H ₂ O. Take 10 mL of the liquid inoculum prepared as described above and inoculate it into a triangular flask containing sterilized 190 mL of liquid medium. Incubate the culture flask at 25ºC and 100rpm until the 10th day to collect all the fermentation broth. After filtering with filter paper, the precipitate on the filter paper was washed with deionized water several times until the filtrate appeared clear. After drying the mycelium in an oven at 50ºC, it is crushed by a mill and sieved (40mesh) to obtain Xiangshanzhi mycelium powder, which is then placed in a drying oven for use.

Example 2: Mycelia of Shanshanzhi delays tissue loss in C57BL/6J mice that induce cancer cachexia

The following experiment used Lewis lung cancer cell line (Lewis lung cancer carcinoma, LLC) to inoculate C57BL/6J mice as an animal model for inducing cancer cachexia. Eight-week-old male C57BL/6J mice (63 purchased from National Laboratory Animal Center Taiwan R.O.C) were used. Feeding with LabDiet 5001 feed, litter GLP Aspen Bedding (TAPVEI®) feeding control temperature at 23 ℃ ± 2 ℃, relative humidity is 70 ± 10%, light / dark cycle is 12 hours light / 12 hours light and dark, light time is 8:00~20:00, During the experiment free to eat and drink water.

Establishment of an animal model of tumor-induced cachexia: A mouse lung cancer cell line (Lewis lung carcinoma cell, LLC) is suspended in 100 μL of PBS with a cell volume of 5 x 10 ⁵ and implanted into the right hind leg of the mouse by subcutaneous injection Tumors are induced at the site, and the treated animals are hereinafter referred to as C57BL/6J cancer-induced cachexia mice. The date of injecting LLC cells was set as the starting day of the experiment (Day 0, Day 0), after which the average food intake and water intake were calculated every two days, and the body weight and tumor size of the mice were recorded. During the experiment, the size of the tumor is measured once every two days using a vernier caliper. The calculation formula of the tumor volume (cm ³ ) is: tumor length (L)×width (W)×height (H)×π÷6.

Before inducing cancer cachexia, a non-induced control group (CON group) was divided based on body weight, a total of 9 animals. After induction, the other mice were divided into 6 groups according to the first weight-bearing swimming time and tumor volume, and each group of 9 mice. The following are the doses and administration reagents for each group: (1) tumor control group ( tumor control, TC), only 0.1 mL of sterilized RO water is given; (2) positive control group (positive control, PC), administered with 10 mg/kg body weight of paclitaxel (taxol) and 6 mg/kg body weight of cisplatin; (3) Antrodia cinnamomea mycelium group (AC), administered 369mg/kg body weight of Antrodia cinnamomea mycelium; (4) Low-dose Xiangshanzhi mycelium group (low dosage of Antrodia salmonea, LAS), administered 170mg/ kg body weight Shanxiangzhi mycelium; (5) medium dosage of Antrodia salmonea (MAS), administered 340mg/kg bodyweight Shanshanzhi mycelium; and (6) high dose In the high dosage of Antrodia salmonea (HAS) group, 1700 mg/kg body weight of Mycelia mycelia was administered. Among them, only the PC positive control group was administered by intraperitoneal injection, and the frequency of administration was once a week; the other groups were administered by tube feeding, the frequency of administration was once a day, and continuous feeding for 17 days.

The results in Figure 1 show that on the eighth day after induction, the food intake of the cancer cachexia group began to decrease significantly, among which the food intake of the PC group and the AC group was less than that of the other groups, while the food intake of the LAS, MAS and HAS groups Food intake did not change significantly during the subsequent experiment. On the 15th day after induction, the PC group was significantly lighter than the CON and TC groups, which may be related to the side effects of chemotherapy drugs (paclitaxel and cisplatin). Since the 19th day after induction, the body weight of the TC group was significantly higher than that of the CON group. It is speculated that this is related to the significant increase in tumor weight caused by rapid tumor growth, but it must still be combined with the measurement of tumor volume trends, tumor weight, and deduction of tumor weight. Body weight is used as evidence (see Example 3 below). For the groups administered with AC, LAS, MAS, and HAS, the weight of mice with cancer cachexia induced during the experiment was not significantly different from that of the CON group, indicating that administration of low, medium, and high doses of Xiangshan mycelium can Delay anorexia and weight loss of cancer cachexia (see Figure 2).

Example 3: Mycelia of Shanshanzhi inhibits tumor growth in C57BL/6J mice that induce cancer cachexia

On day 7 after injection of LLC lung cancer cells, a macroscopically observable mass appeared in C57BL/6J mice that induced cancer cachexia. Since the eleventh day of inducing cancer cachexia, the tumor volume of mice in PC, AC, LAS, MAS and HAS groups was significantly smaller than that in TC group. The tumor volume measurement results on the 27th day showed that the tumor suppression effect was as follows: the PC group was the best, the LAS, MAS, and HAS groups were the second, the AC group was the worse, and the dosage of Mycelia mycelia was administered. There was no significant difference in the inhibitory effect on tumor growth (see Figure 3). These results show that the administration of low, medium, and high doses of Mycelia of Shanxiangzhi has the effect of inhibiting tumor growth, and its tumor suppressing effect is more significant than Antrodia cinnamomea.

Further comparing the results of removing the tumor tissue after sacrifice, as shown in Figure 4, the tumor in the TC group was significantly heavier than the other groups (p<0.05); the tumor in the PC group was significantly lighter than the other groups (p<0.05), There was no significant difference in tumor weight between AC, LAS, MAS and HAS groups. After deducting the weight of the tumor, the body weight of the LAS, MAS, and HAS groups was significantly higher than that of the TC, PC, and AC groups, but significantly lower than that of the CON group (p<0.05). Comparing this result with the measurement results of the weight change of each group of mice in Example 2 (Figure 3), it is shown that the weight of the mice in the TC group increased significantly compared with the other groups during the experiment, which is actually due to the tumor in the body The rapid growth of tissues does not result from an increase in body tissues. Based on the above results, the administration of Mycelia of Xiangshanzhi can effectively inhibit the growth of cachexia tumors in mice induced by Lewis lung cancer, and delay the symptoms of body tissue loss caused by cachexia.

Example 4. Mycelia of Shanshanzhi improves the survival rate of Lewis lung cancer cell-induced cancer cachexia C57BL/6J mice

On the 26th day after the induction of cancer cachexia, the tumor-bearing tumor of the mice in the TC group had reached the humane end point standard. The humane end point standard was the average tumor diameter of 2 cm. The results in Figure 5 show that, since the 21st day after induction, tumor-bearing mice appear to be unable to bear tumors and die. The first group of dead mice was the TC and MAS groups. To the day of sacrifice, the survival rate of each group was as follows: CON and LAS group (100%)>PC group (88.9%)>AC, MAS and HAS group (77.8%)>TC group (66.7%). It is indicated that the administration of Antrodia cinnamomea mycelium and the low, medium and high doses of Xiangshanzhi mycelium can increase the survival rate of cancer cachexia mice compared with the TC group, in which the low dose (170mg/kg body weight) of Xiangshanzhi is given Mycelium, no mice died during the experiment, the best effect.

Example 5: Mycelia of Shanshanzhi improves the fatigue and physical weakness of C57BL/6J mice that induce cancer cachexia

In this example, the weight-loaded swimming test was used to evaluate the effect of Xiangshanzhi mycelium on improving cancer cachexia due to decreased physical strength due to decreased physical endurance. Before the formal weight-bearing swimming test, mice were first acclimated to swimming. The first weight-bearing swimming test was performed before the sample was fed (Day 10), and the second weight-bearing swimming test was performed one week after the sample was fed (Day 16), and the sample was fed two weeks later. (Day 22) Conduct the third weight-bearing swimming test and the last weight-bearing swimming test the day before sacrifice (Day 26). Weigh the weight of the mouse before the experiment, tie the tail of the mouse with a nickel wire with a weight of 5% of the body weight, and then put it into a water bottle with a diameter of 11 cm and a height of 25 cm. The height of the water is 20 cm and the water temperature is controlled at At 27±1℃, the mice were forced to swim and struggle until their physical exertion was exhausted and sinked. The time from when the mouse entered the water until the head entered all the water for 3 seconds and could not rise to the surface was the swimming time.

The results in Figure 6 show that in the first weight-bearing swimming test, the swimming time of the tumor-bearing mice group was shorter than that of the CON group, and there was a significant difference between the TC group and the CON group. In the second test, due to the increased tumor burden of the tumor-bearing mice group, there was a tendency to fatigue, and swimming performance began to show differences between the groups. The swimming time of TC, LAS and HAS groups was significantly shorter than that of CON group, and the swimming time of PC group was significantly longer than that of TC group. In the third trial, the swimming time of the AC and TC groups was significantly shorter than that of the CON group, and the swimming time of the PC, MAS, and HAS groups was significantly longer than that of the TC group. In the weight-bearing swimming test sacrificed the day before, the tumor-bearing mice had a larger tumor burden, and the swimming time was significantly shorter than that of the CON group with no tumor. The average swimming time of the PC, AC, LAS, MAS, and HAS groups was longer than that of the TC group, but there was no difference between the groups. The PC group can effectively delay the fatigue caused by cachexia due to the inhibition of tumor growth by chemotherapy drugs; give Antrodia cinnamomea mycelium and low, medium and high doses Compared with the TC group, the mycelia of Xiangshanzhi also showed the effect of effectively delaying the occurrence of fatigue symptoms of cancer cachexia.

Example 6: Mycelia of Shanshanzhi inhibits tumor cell metastasis of C57BL/6J mice that induce cancer cachexia

In this example, the histopathological study was used to evaluate the effect of the mycelia of Shanshanzhi on the pathology of C57BL/6J mice that induce cancer cachexia. On the 27th day after induction by injection of LLC cell lines (Day27), the mice were suffocated with carbon dioxide and sacrificed. After sacrifice, remove the heart, liver, kidney, spleen, testicles, accessory testicles, quadriceps, gastrocnemius muscle, soleus muscle, abdominal fat tissue, peri-renal fat tissue, accessory testicular fat tissue and tumor tissue. After taking part of the tumor tissue, large lobe of liver and a kidney in 10% formalin solution for fixation, a rough tissue repair was carried out and sent to the National ZTE University Animal Disease Diagnostic Center for paraffin embedding and hematoxylin (hematoxylin)- Eosin (eosin) staining for tissue sectioning. The comprehensive evaluation results are listed in Table 1 below.

-：無明顯壞死。N：無可目測之腫瘤組織。¹：壞死程度依其嚴重程度分為五級：0=無影響；1=最低(<1%)；2=輕微(1-25%)；3=中度(26-50%)；4=中度/嚴重(51-75%)；5=嚴重/高度(76-100%)。²發生率：發生轉移小鼠/總試驗小鼠。

-: No obvious necrosis. N: There is no visible tumor tissue. ¹ : The degree of necrosis is divided into five levels according to its severity: 0=no effect; 1=lowest (<1%); 2=slight (1-25%); 3=moderate (26-50%); 4= Moderate/Severe (51-75%); 5=Severe/High (76-100%). ² Occurrence rate: metastatic mice/total test mice.

The pathological section results (Figure 7) show that the type of live cancer cells is round, epithelial, and has mitosis, and local acute cancer cell necrosis occurs in the central area of the tumor. In this experiment, cancer cells in group TC The percentage of survival was significantly lower than that of the PC, LAS, MAS, and HAS groups (p<0.05), and the proportion of cancer cell necrosis in the TC group was significantly higher than that of the PC, LAS, MAS, and HAS groups (p<0.05). Differentiation and proliferation cause insufficient supply of oxygen and nutrients for cancer cells in the central area of the tumor, leading to more cell necrosis. Although the proportion of cancer cells surviving in the treatment group given to the sample was more than that in the TC group, the tumor size of the treatment group was significantly smaller than that of the TC group according to the tumor volume. Therefore, it is speculated that the mechanism of the tumor suppression effect of the sample may be to inhibit the differentiation of cancer cells instead Directly cause cancer cell death.

According to the pathological section results of liver and kidney, no tumor metastasis was found in the kidney, and the number of mice with liver cancer cell metastasis was: 3 mice in the TC group and 1 mouse in the PC, AC, LAS, MAS, and HAS groups. It means that the incidence of liver cancer cell metastasis in the PC, AC, LAS, MAS, and HAS groups is significantly lower than that in the TC group (see Table 1), and the proportion of liver metastasis in the TC group is higher than that of other sample treatment groups. It is speculated that the sample can be delayed Tumor metastasis. The above results show that from the above results, the mechanism of Xiangshanzhi mycelia inhibiting tumors may be to inhibit the differentiation and proliferation of cancer cells, not by inducing apoptosis; and Xiangshanzhi mycelia has the ability to inhibit cancer cell metastasis. effect.

In summary, the administration of the mycelia of Shanshanzhi of the present invention can inhibit tumor growth and delay the loss of body tissue caused by cancer cachexia. The survival rate of mice shows that the mycelium of Xiangshanzhi It can delay the death of mice due to excessive tumor load, and the results of the weight-bearing swimming test prove that the administration of Antrodia cinnamomea mycelium and the low, medium and high doses of Xiangshanzhi mycelium can effectively delay the fatigue symptoms caused by cancer cachexia. Therefore, the mycelia composition of Xiangshanzhi of the present invention can be used to improve the physical condition of cancer cachexia patients and improve the quality of life of cancer patients, and can also be used as a cancer treatment auxiliary agent that inhibits tumor growth and metastasis.

Claims (4)

The utility model relates to an mycelium of Antrodia salmonea used for preparing a medicinal composition for alleviating the symptoms of cancer cachexia. The use according to claim 1, wherein the cancer cachexia symptoms are selected from at least one of anorexia, susceptibility to satiety, fatigue, body weight loss and frailty. The use according to any one of claims 1, wherein the mycelia of Shanshanzhi is a liquid cultured mycelium of Shanshanzhi. The use as claimed in claim 3, wherein the liquid cultured Shanxiangzhi mycelium system is dried, crushed and sieved to obtain a Shanshanzhi mycelium powder.

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