Use of an active part of dendrobium nobile flower in preparation of an auxiliary protective agent for ionizing radiation hazards

Abstract

The present application relates to a new use of active parts of Dendrobium officinale flowers, and particularly relates to an application of the active parts of Dendrobium officinale flowers in preparation of an auxiliary protective agent for ionizing radiation hazards; the active ingredients of the Dendrobium officinale flowers are equivalent to the effects of andolium capsules, can be used for improving ionizing radiation hazards, and develop a new use of the Dendrobium officinale flowers, and provide a new selection for radiation protective agents.

Description

Technical Field

[0001] This invention relates to a novel use of the active components of Dendrobium officinale flowers, specifically to the application of the active components of Dendrobium officinale flowers in the preparation of an auxiliary protective agent against ionizing radiation hazards. Background Technology

[0002] Ionizing radiation refers to radiation carrying enough energy to ionize atoms or molecules, causing them to become free. Its wavelength is less than 100 nm, and it includes cosmic rays, X-rays, and radiation from radioactive materials. [1-2] Ionizing radiation has important applications in nuclear power generation, nuclear medicine diagnosis and treatment, food irradiation sterilization, polymer material modification, and environmental remediation. However, with the increasing application of ionizing radiation in research and technology, the number of metabolic diseases, acute hematopoietic syndromes, and immune system diseases caused by it is also rising. Recently, a research team led by David B. Richardson of the University of California, Irvine, studied decades of data from the International Nuclear Workers Study (INWORKS) cohort of over 300,000 people, confirming that the harm of chronic low-dose radiation is far greater than previously thought. [3] Therefore, the research and development of radiation protection agents (especially those targeting the immune system) has always been a focus of attention.

[0003] Existing anti-radiation drugs mainly include cytokines, sulfur-containing compounds, and hormones. However, their clinical use is limited due to their low efficacy or serious adverse reactions. Therefore, it is particularly important to develop an economical, efficient, and non-toxic radiation protection agent.

[0004] Dendrobium officinale is a plant belonging to the Orchidaceae family. Dendrobium officinale The dried stems of Kimum et Migo have the effects of nourishing the stomach and promoting the production of body fluids, as well as nourishing yin and clearing heat. [4] Throughout history, physicians and traditional Chinese medicine texts have highly praised the medicinal value of Dendrobium officinale. The Daoist medical classic, the *Daozang*, lists it as the "first of the nine great immortal herbs of China," and it is popularly known as the "life-saving immortal herb." Due to its unique environmental requirements, over-harvesting by humans, and extremely low reproductive capacity, wild Dendrobium officinale has become an endangered species in my country. (Dendrobium officinale flower...) Dendrobium officinale Flower Dendrobium officinale (DOF) is a by-product of the production process of Dendrobium officinale. It has a long history of consumption in my country. People often dry its flowers to make flower tea, or directly squeeze fresh juice from the flowers or use them as a flower dish.

[0005] According to literature review, Dendrobium officinale flowers contain various bioactive components such as polysaccharides, flavonoids, and anthocyanins. Modern pharmacological and clinical studies have demonstrated that Dendrobium officinale flowers have hepatoprotective, hypoglycemic, hypotensive, and antioxidant effects. [5]It has potential development and application value in the fields of medicine and health care. However, there are no reports on the auxiliary protective effect of Dendrobium officinale flowers against the hazards of ionizing radiation. Summary of the Invention

[0006] The purpose of this invention is to reduce acute radiation damage caused by gamma rays when the extract of Dendrobium officinale flower is administered orally. Therefore, it can be used in the preparation of oral radiation protection agents.

[0007] The application of an active component of Dendrobium officinale flower in the preparation of an auxiliary protective agent against ionizing radiation hazards.

[0008] The active fraction of Dendrobium officinale flower was prepared by the following steps: 4 g of Dendrobium officinale flower was weighed, 200 mL of purified water was added, and the mixture was boiled in a water bath for 1 h. After extraction three times, the filtrates were combined and most of the water was removed by vortexing. The extract was then evaporated to dryness in a water bath. The extract was added to 100 mL of purified water to make a suspension, and then extracted with ethyl acetate three to four times until extraction was complete. The concentrated extract was the ethyl acetate fraction extract.

[0009] Ionizing radiation is gamma rays.

[0010] This invention uses dried Dendrobium officinale flowers as raw material to prepare the ethyl acetate fraction (DOF-EA) of the boiling water extract of Dendrobium officinale flowers, and studies the auxiliary protective effect of DOF-EA against ionizing radiation hazards. The protective effect of the active components of Dendrobium officinale flowers against acute radiation injury in mice was investigated by whole-body irradiation with gamma rays.

[0011] Key points of the invention

[0012] 1. Preparation of active components from Dendrobium officinale flowers;

[0013] 2. An active component of Dendrobium officinale flower has an auxiliary protective effect on the blood system of mice with acute ionizing radiation injury;

[0014] 3. An active component of Dendrobium officinale flower can improve DNA damage in the spleen, thymus, and bone marrow; and improve oxidative damage caused by ionizing radiation.

[0015] Beneficial effects: 1. Gamma rays can cause DNA molecule breaks. Cells have the ability to repair this damage, but if the damage is too severe and exceeds the cell's repair capacity, it can lead to cell death within weeks. The mechanism by which chromosomes can be protected and repaired—that is, protected against radiation damage—is not fully understood. Therefore, finding radiation shielding agents is a pressing technical challenge in this field.

[0016] 2. Andolin Capsules: Nourish Qi and blood, strengthen the body and detoxify. Primarily treats Qi and blood deficiency syndromes, suitable for symptoms such as decreased white blood cell count, weakened immune function, loss of appetite, fatigue, dizziness, and shortness of breath caused by radiotherapy and chemotherapy. It also has an improving effect on increased lymphocyte micronucleus rate caused by radiation damage during tumor radiotherapy and can be used for radiation injury.

[0017] 3. The active ingredient of Dendrobium officinale flower described in this invention has an effect comparable to that of Andolin capsules and can be used to improve the harm of ionizing radiation, opening up new uses for Dendrobium officinale flower and providing a new option for radiation protection agents. Specifically, DOF-EA was unexpectedly found to significantly increase the immune organ index of radiation-damaged mice, protect the mouse blood system, and reduce bone marrow DNA damage, while rutin alone had no effect, indicating that the active ingredient in this invention is not rutin. Attached Figure Description

[0018] Figure 1 HPLC chromatogram of the ethyl acetate fraction of the aqueous extract of Dendrobium officinale flowers; Note: Peak 1 – Caffeic acid glucoside; Peak 2 – Coumaroyl glucoside; Peak 3 – Caffeic acid; Peak 4 – p-Coumaric acid; Peak 5 – Rutin; Peak 6 – Isoquercetin; Peak 7 – Quercetin-3-O-malonyl glucoside; Peak 8 – Kaempferol rutin; Peak 9 – Astragaloside; Figure 2 DOF-EA Figure 2 Effects on immune organ indices in radiation-damaged mice: A represents the spleen index, and B represents the thymus index; Note: Compared with the normal group, ## p<0.05; compared with the model group, ** p<0.05; no significant difference in ns, n=6.

[0019] Figure 3 HE staining of mouse spleen tissue sections; Note: A is blank control group, B is model group, C is DOF-EA group, D is rutin group, E is endorphin group, black circle is white pulp, yellow circle is splenic trabeculae, red pulp (RP), white pulp (WP), central artery (CA), splenic corpuscle (Scor) (scale bar: 100 μm).

[0020] Figure 4 Effect of DOF-EA on changes in bone marrow DNA content in mice; Note: Compared with the normal group, ## p<0.05; compared with the model group, ** p<0.05; no significant difference in ns, n=6.

[0021] Figure 5 Effect of DOF-EA on SOD content in mouse liver tissue; Note: Compared with the normal group, ## p<0.05; compared with the model group, ** p<0.05; no significant difference in ns, n=6. Detailed Implementation

[0022] Example 1

[0023] 1. Experimental Materials

[0024] (1) Reagents

[0025] Andolin capsules (Fujian Zhong'an Pharmaceutical Co., Ltd., National Drug Approval Number Z10970016), SOD reagent kit (Beyotime Biotechnology Co., Ltd.), PBS solution, and rutin standard.

[0026] Preparation of the ethyl acetate fraction of water extract from Dendrobium officinale flowers

[0027] Weigh 4 g of Dendrobium officinale flowers, add 200 mL of purified water, boil in a water bath for 1 h, extract 3 times, filter and combine the filtrates, remove most of the water by vortexing and evaporate to dryness in a water bath; add 100 mL of purified water to the extract to make a suspension, extract with ethyl acetate, extract 3 to 4 times until extraction is complete; concentrate the extract to obtain the ethyl acetate fraction extract.

[0028] Experimental methods

[0029] (1) HPLC analysis of the ethyl acetate fraction of the water extract of Dendrobium officinale flowers

[0030] Preparation of the test solution: Weigh 6 mg of the ethyl acetate extract of Dendrobium officinale flowers and dissolve it in 1 mL of 70% methanol; filter it through a 0.22 μm organic filter membrane to obtain the sample solution.

[0031] Preparation of reference solutions: Accurately weigh appropriate amounts of several standard chemicals such as rutin and isoquercitrin, add 1 mL of 70% methanol, sonicate and shake well to obtain a single reference stock solution of a certain concentration, and filter it through a 0.22 μm microporous membrane before injection.

[0032] HPLC conditions: A Thermo Fisher Scientific UTiMate 3000 HPLC system with a diode array detector was used for analysis. The chromatographic conditions were as follows: Thermo Acclaim C18 (4.6 × 250 mm, 5 µm) column; mobile phase: 0.1% formic acid water (A) - acetonitrile (B); elution gradient: 0–12 min, 9% B; 12–13 min, 9%–11% B; 13–27 min, 11% B; 27–28 min, 11%–12% B; 28–46 min, 12%–13% B; 46–47 min, 13%–14% B; 47–66 min, 14%–14.5% B; 66–80 min, 14.5%–16.5% B; 80–90 min, 16.5%–19% B; 90–110 min, 19%–60% B. The injection volume was 10 µL; the column temperature was 30 ℃; and the detection wavelength was 340 nm.

[0033] (2) Animal grouping and modeling

[0034] Thirty-five 6-8 week old SPF-grade male Kunming mice, weighing 18-20g, were purchased from Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd., certificate number: SCXK(Su)2023-0019. After one week of acclimatization feeding, they were randomly divided into 5 groups: normal control group (Control), radiation model group (Moedel), DOF-EA group, rutin group, and positive control drug Andoulin group, with 6 mice in each group.

[0035] (3) Animal administration

[0036] After one week of acclimatization, except for the normal control group and the radiation model group, the other groups were administered the corresponding drugs by gavage for 3 consecutive days, with the gavage volume remaining consistent. The research group had previously investigated the dosage of DOF-EA in vitro, and since the rutin content in DOF-EA was approximately 3.4%, the rutin group served as a control group for pharmacodynamic experiments. Therefore, the dosages for the rutin group, endorphin group, and DOF-EA group were 3.4 mg / kg, 550 mg / kg, and 100 mg / kg, respectively. The normal control group and the radiation model group were administered the same volume of purified water by gavage (the test samples were prepared using purified water as a solvent, and all animals were given clean-grade complete rat pellet feed and sterilized water freely during the experiment).

[0037] Mice were administered the drug continuously for 7 days, followed by gamma-ray irradiation on day 4: except for the blank control group, mice were placed in transparent plexiglass cages and their entire bodies were exposed to 6.5 Gy of gamma rays at an average dose rate of 0.9 Gy / min. [6] During the experiment, the mice's living conditions were observed and their weight was recorded.

[0038] (4) Detection indicators

[0039] On day 8, mice were euthanized by cervical dislocation after blood was collected from their eyeballs. The spleen and thymus were immediately removed, surface fat was removed, and the organs were rinsed with pre-cooled physiological saline to remove any remaining blood. The organs were then blotted dry with filter paper, accurately weighed, and their organ indices calculated. A portion of the spleen was fixed in 4% paraformaldehyde for 48 hours, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE) for histological observation. The number of white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), and platelets (PLT) in whole blood of the mice was measured using an automated blood cell analyzer. Liver tissue was also removed to measure the superoxide dismutase (SOD) content in liver homogenate.

[0040] Detection of DNA content in mouse bone marrow cells: Femur bones were harvested, and 1 mL of PBS solution was drawn into a syringe to flush out all bone marrow cells. The cells were then passed through a syringe with a No. 4 needle to ensure thorough dispersion in the suspension. DNA content was measured at 260 nm using a UV spectrophotometer.

[0041] Statistical processing

[0042] Data were analyzed using GraphPad Prism 8.0 software. t-tests were used to analyze various indicators of mouse peripheral blood cells. Results are expressed as mean ± standard deviation (X ± S). One-way ANOVA was used to test for differences among multiple groups in immune organ coefficient, DNA quantification, and SOD detection results. P < 0.05 was considered statistically significant.

[0043] Experimental results

[0044] (1) Component identification of DOF-EA

[0045] Under these liquid chromatography conditions, the main chromatographic peaks of each fraction of Dendrobium officinale flower extract were well separated (see...). Figure 1 And identified the compounds corresponding to 9 chromatographic peaks (see...). Figure 1 Among them, peaks 1, 2, 3, and 4 belong to phenylpropanoid compounds, while peaks 5, 6, 7, 8, and 9 are flavonoid glycoside compounds.

[0046] (2) Results of routine blood tests

[0047] Four days after irradiation, compared with the normal control group, the peripheral blood WBC level of mice in the model group was significantly reduced, and the differences were statistically significant (P < 0.05). Compared with the model group, the peripheral blood WBC level of mice in the DOF-EA group and the Andolin group was significantly increased, and the differences were statistically significant (P < 0.05). Compared with the model group, RBC, HGB, and PLT levels were not significantly increased, and the differences were not statistically significant (results are shown in Table 1). This indicates that DOF-EA can improve peripheral leukopenia in mice caused by acute radiation injury.

[0048] Blank group - 5.87±1.21 10.26±0.41 14.92±0.70 669.00±105.60 Model group - <![CDATA[0.07±0.01 ** ]]> 10.46±1.15 15.23±1.02 <![CDATA[480.00±39.96 * ]]> DOF-EA Group 100 mg / kg <![CDATA[0.14±0.03 # ]]> 10.37±1.15 14.53±1.10 589.50±79.91 Rutin Group 3.4 mg / kg 0.06±0.01 10.76±1.28 12.55±7.06 602.50±179.60 Anduolin Group 550 mg / kg <![CDATA[0.09±0.00 # ]]> 10.78±0.65 15.45±0.54 669.50±220.90

[0049] Note: Compared with the normal group, * P < 0.1, ** P < 0.05; compared with the model group, # P < 0.05; n = 6

[0050] (3) Immune organ index

[0051] Compared with the normal group, the spleen and thymus of Kunming mice atrophied after gamma irradiation, and the immune organ indices decreased significantly. After administration of DOF-EA and Andolin capsules, both indices increased significantly (P < 0.05), but the rutin group showed no significant therapeutic effect (see results below). Figure 2 ).

[0052] (4) Pathological examination results of spleen tissue

[0053] In the normal control group, the spleen structure was intact, with clear white pulp (WP) and red pulp (RP) structures. The splenic corpora (Scor) lymphoid nodules were neatly arranged and clearly structured, and the central artery (CA) and periarterial lymphoid sheath (PALS) were clearly visible. In the model group, the boundary between the red and white pulp was unclear, with severe red pulp congestion, white pulp atrophy, significant lymphocyte depletion, increased megakaryocytes, and interstitial fibrosis. In the DOF-EA and Andolin groups, the boundary between the red and white pulp in the spleen was relatively good, showing mild white pulp degeneration with a small amount of lymphocyte loss, and no obvious red pulp congestion. Compared with the model group, the rutin group had virtually no therapeutic effect (see results). Figure 3 ).

[0054] DNA content detection results

[0055] The absorbance results showed that compared with the normal control group, the DNA absorbance of the model group was significantly reduced. The DOF-EA and Andolin groups showed highly significant differences compared with the model group, while the rutin group showed no difference. This indicates that DOF-EA can increase the DNA content of bone marrow cells in irradiated mice and improve radiation-induced DNA damage (see results below). Figure 4 ).

[0056] Liver SOD content test results

[0057] The results showed that on day 4 after irradiation, the SOD activity in the liver of mice in the model group was significantly lower than that in the blank control group (P < 0.05). Compared with the model group, the SOD activity in the liver of mice in the DOF-EA and Andolin intervention groups was significantly increased (P < 0.05), while there was no significant difference in the rutin group (see results below). Figure 5 ).

[0058] References:

[0059] [1] International Atomic Energy Agency (IAEA). Radiation, Humans and the Environment. Atomic Energy Press. 2006.

[0060] [2] GB / T17857 Medical Radiology Terminology

[0061] [3]Richardson DB, Leuraud K, Laurier D, Gillies M, Haylock R, Kelly-Reif K, Bertke S, Daniels RD, Thierry-Chef I, Moissonnier M, Kesminiene A,Schubauer-Berigan MK. Cancer mortality after low dose exposure to ionisingradiation in workers in France, the United Kingdom, and the United States(INWORKS): cohort study. BMJ. 2023 Aug 16;382:e074520.

[0062] [4] National Pharmacopoeia Commission. Pharmacopoeia of the People's Republic of China: Part I [S]. Beijing: China Medical Science and Technology Press, 2015: 124-126.

[0063] [5]Zhou H, Zhou L, Li B, Yue R. Anti-cyclooxygenase, anti-glycation, and anti-skin aging effect of Dendrobium officinale flowers' aqueous extract and its phytochemical validation in aging. Front Immunol. 2023 Mar 17;14:1095848.

[0064] [6]Liu D, Zhuang B, Wei M, Yuan T, Li J, Deng P, Du L, Yuan B, Jin Y. Oral konjac glucomannan for prevention of ionizing radiation-induced injury by regulating gut microbiota and increasing short chain fatty acids. Int JBiol Macromol. 2023 Jun 15;240:124402.

Claims

1. The application of an ethyl acetate extract of Dendrobium officinale flowers in the preparation of an auxiliary protective agent against ionizing radiation hazards, characterized in that, The boiling water ethyl acetate extract was prepared by the following steps: 4 g of Dendrobium officinale flowers were weighed, 200 mL of purified water was added, and the mixture was boiled in a water bath for 1 h. After extraction three times, the filtrates were combined and most of the water was removed by vortexing. The extract was then evaporated to dryness in a water bath. The extract was added to 100 mL of purified water to make a suspension, and then extracted with ethyl acetate three to four times until extraction was complete. The concentrated extract was the boiling water ethyl acetate extract.

2. The application according to claim 1, characterized in that... Ionizing radiation is gamma rays.

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