Application of Cistanche tubulosa flavones and Cistanche deserticola tannins in the preparation of drugs for treating senile dementia

Abstract

The application discloses application of a trichosanthes kirilowii flavone and a cistanche deserticola tannin in preparation of a medicine for treating senile dementia and belongs to the technical field of medicines. The trichosanthes kirilowii flavone and the cistanche deserticola tannin are combined and used, and a synergistic effect is achieved in the treatment of senile dementia, and the pharmacodynamic activity is significantly improved. The application provides a new choice for clinical medicines for treating senile dementia.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of Cynomorium tannin and Cistanche flavonoids in the preparation of drugs for treating Alzheimer's disease. Background Technology

[0002] Alzheimer's disease (AD) is a neurodegenerative disease that begins with episodic memory decline and is accompanied by cognitive impairment. Its incidence is closely related to age and is the most common type of dementia, accounting for 50%-75% of cases. After age 65, the probability of developing Alzheimer's disease doubles every five years. Studies estimate that approximately 440 million people worldwide currently suffer from Alzheimer's disease, and this number is projected to triple by 2050. In the United States, the annual cost of treating dementia exceeds $600 billion. In the UK and Wales, AD is the leading cause of death among the elderly, accounting for 11.6% of all deaths. The prevalence of AD is higher in women than men and increases with age. In my country, the prevalence of AD is as high as 4.8%, showing regional differences; the number of patients in the south is significantly lower than in the north, and the age of onset in the east is significantly lower than in the west. In conclusion, Alzheimer's disease imposes a severe economic burden on society and families and has become a pressing healthcare problem that cannot be ignored in my country and worldwide.

[0003] Currently, neither drug nor non-drug treatments for Alzheimer's disease (AD) meet the needs of clinicians and patients' compliance. On the one hand, traditional AD medications such as acetylcholinesterase inhibitors (e.g., rivastigmine, galantamine, donepezil), N-methyl-D-aspartate receptor antagonists (e.g., memantine), and serotonin-based drugs for controlling AD mood symptoms only alleviate symptoms to a certain extent and have little impact on the disease course. While recent biologics, such as anti-amyloid monoclonal antibodies like adunatumab, have shown significant efficacy in early-stage patients, their significant side effects and high cost limit their therapeutic efficacy. Traditional Chinese medicine preparations have a slow onset of action and cannot meet the needs of some patients with accompanying serious illnesses for rapid physical recovery and prevention of secondary diseases. Therefore, how to alleviate cognitive impairment and improve the decline in daily living activities in AD patients is a key issue in ensuring the efficacy of clinical drugs for AD and improving the quality of life for the elderly. Research and development of anti-aging and cognitive-enhancing traditional Chinese medicines with clear material basis, well-defined efficacy, and minimal adverse reactions are urgently needed.

[0004] *Cynomorium songaricum* Rupr. is a perennial, holoparasitic herb belonging to the genus *Cynomorium* in the family Cynomoraceae, often parasitizing the roots of plants in the genus *Nitraria* in the family Cynomoraceae. Medicinal *Cynomorium* (Herba Cynomorii) refers to the dried, fleshy stem of *Cynomorium*, used as the whole herb in Mongolian medicine. *Cynomorium* was first recorded in *Ben Cao Yan Yi Bu Yi*, also known as *Suo Yang Dan Xi Xin Fa*, a name derived from its medicinal properties of "locking in yang energy, ensuring longevity," and is also called the "elixir of immortality" (*Pharmacology of Chinese Medicine*). *Cynomorium* is sweet and astringent in taste, slightly warm, and non-toxic, entering the liver, kidney, and large intestine meridians. It has the effects of tonifying kidney yang, nourishing essence and blood, and moistening the intestines to relieve constipation. It is commonly used to treat kidney yang deficiency, essence and blood deficiency, infertility, weakness of the lower back and knees, and constipation due to intestinal dryness. It has long been regarded as a precious medicinal material in Tibetan, Mongolian, and traditional Chinese medicine. Clinical data show that many well-known AD formulas, such as Yizhi Jiannao Granules, Dihuang Yinzi, Huanshao Dan, and Guilingji, contain Cynomorium songaricum, and their effects of tonifying essence and kidneys, generating marrow, and improving intelligence are clearly evident. Modern research indicates that the main pharmacologically active substances in Cynomorium songaricum are saponins, triterpenoids, steroids, polysaccharides, and tannins.

[0005] Cistanche deserticola Ma is the dried, fleshy stem with scale-like leaves of Cistanche deserticola YCMa or Cistanche tubulosa (Schenk) Wight, both belonging to the Orobanchaceae family. First recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), it is used to treat five types of fatigue and seven types of internal injuries, tonify the middle jiao (spleen and stomach), relieve cold and heat pain in the stem, nourish the five internal organs, regulate yin, benefit essence and qi, and treat abdominal masses in women. It is sweet, salty, and warm in nature, and enters the kidney and large intestine meridians. According to the *Compendium of Materia Medica* and *Rihua Zi Bencao*, Cistanche deserticola can be used to treat kidney yang deficiency, depletion of essence and blood, cold pain in the lower back and knees, tinnitus, blurred vision, leukorrhea, frequent urination, metrorrhagia, infertility, and constipation. Modern research shows that Cistanche deserticola is a commonly used drug in traditional Chinese medicine for treating brain neuropathies. It has the effects of tonifying the kidneys and replenishing essence, calming wind and unblocking the meridians. It can effectively promote brain angiogenesis and nerve function recovery, while nourishing brain cells and maintaining brain function. It has significant effects on improving brain aging and AD-related cognitive impairment. Its main effective components are Cistanche deserticola phenylethanol glycosides, iridoids, lignans, alkaloids and flavonoids.

[0006] There are currently no reports on the use of the active ingredients of Cynomorium songaricum in combination with those of Cistanche deserticola for the treatment of Alzheimer's disease. Summary of the Invention

[0007] The purpose of this invention is to provide novel pharmaceutical applications of Cynomorium tannins and Cistanche deserticola flavonoids, specifically their use in the preparation of remedies for treating and / or preventing Alzheimer's disease. Another object of this invention is to provide a pharmaceutical composition for treating and / or preventing Alzheimer's disease.

[0008] This invention provides the use of Cynomorium tannin and Cistanche deserticola flavonoids in the preparation of medicaments for treating and / or preventing Alzheimer's disease. The preferred mass ratio of Cynomorium tannin to Cistanche deserticola flavonoids is 1–45:1–150, more preferably 12–24:35–60, and even more preferably 28:40.

[0009] The present invention also provides a pharmaceutical composition for treating and / or preventing Alzheimer's disease, comprising: cynomorium tannin and cistanche flavonoids. The mass ratio of cynomorium tannin to cistanche flavonoids is preferably 1–45:1–150, more preferably 12–24:35–60, and even more preferably 28:40.

[0010] The present invention also provides the use of the above-described pharmaceutical composition in the preparation of medicaments for treating and / or preventing Alzheimer's disease.

[0011] The present invention also provides the use of the above-described pharmaceutical composition in the preparation of a drug having a nootropic effect.

[0012] This invention also provides a drug for treating and / or preventing Alzheimer's disease or a nootropic drug, comprising the above-mentioned pharmaceutical composition, and may also include excipients acceptable in pharmaceutical manufacturing. The drug can be an oral or injectable formulation. It can be prepared into an oral or injectable formulation by mixing Cynomorium songaricum tannin and Cistanche deserticola flavonoids with excipients and following conventional manufacturing processes. When mixed with commonly used excipients acceptable in oral formulations, such as disintegrants, lubricants, excipients, binders, and fillers, it can be prepared into oral solid pharmaceutical formulations such as tablets, capsules, soft capsules, granules, and pills according to conventional manufacturing processes. When mixed with commonly used solubilizers, emulsifiers, wetting agents, diluents, preservatives, stabilizers, and flavoring agents, it can be prepared into oral liquid formulations such as aqueous solutions and syrups according to conventional manufacturing processes.

[0013] This invention combines tannins from Cynomorium songaricum with flavonoids from Cistanche deserticola, resulting in a synergistic effect in the treatment of Alzheimer's disease. The efficacy of the drug is significantly enhanced, providing a new option for clinical medication in the treatment of Alzheimer's disease. Attached Figure Description

[0014] Figure 1 The escape latency period of Alzheimer's mice in each group. Compared with the normal group, $$ P<0.01; compared with the model group, & P<0.05, && P<0.01; compared with the tannin group (10 groups), # P<0.05; compared with the Cistanche deserticola flavonoid group (9 groups), * P<0.05.

[0015] Figure 2Number of times Alzheimer's mice crossed the platform in each group. Compared with the normal group, $$ P<0.01; compared with the model group, & P<0.05, && P<0.01; compared with the tannin group (10 groups), # P<0.05; compared with the Cistanche deserticola flavonoid group (9 groups), * P<0.05.

[0016] Figure 3 AChE activity in brain tissue of Alzheimer's mice in each group. Compared with the normal group, $$ P<0.01; compared with the model group, & P<0.05, && P<0.01; compared with the tannin group (10 groups), # P<0.05; compared with the Cistanche deserticola flavonoid group (9 groups), * P<0.05.

[0017] Figure 4 ChAT activity in brain tissue of Alzheimer's mice in each group. Compared with the normal group, $$ P<0.01; compared with the model group, & P<0.05, && P<0.01; compared with the tannin group (10 groups), # P<0.05; compared with the Cistanche deserticola flavonoid group (9 groups), * P<0.05.

[0018] Figure 5 The Ach content in the brain tissue of Alzheimer's mice in each group. Compared with the normal group, $$ P<0.01; compared with the model group, & P<0.05, && P<0.01; compared with the tannin group (10 groups), # P<0.05; compared with the Cistanche deserticola flavonoid group (9 groups), * P<0.05. Detailed Implementation

[0019] In the early stages, this invention compared the anti-Alzheimer's disease effects by combining the main active components of Cynomorium songaricum and Cistanche deserticola one by one (such as Cynomorium saponins + Cistanche deserticola phenylethanol glycosides, Cynomorium saponins + Cistanche deserticola iridoids, ... Cynomorium triterpenes + Cistanche deserticola phenylethanol glycosides, Cynomorium triterpenes + Cistanche deserticola iridoids, and many other combinations). The results showed that, under the same ratio conditions, Cynomorium tannins combined with Cistanche deserticola flavonoids had the best therapeutic effect.

[0020] The present invention will be further described in detail below with reference to embodiments and test examples, but the implementation of the present invention is not limited thereto.

[0021] Example 1: Tablets

[0022] The prescription is as follows:

[0023]

[0024] Preparation method: Take the prescribed amount of raw materials and excipients, mix them evenly, pass them through an 80-mesh sieve, then add 95% ethanol to make a soft mass, granulate it through a 16-mesh screen; dry it at 60℃ and then granulate it, then add 50g of magnesium stearate, mix it evenly, compress it into 1000 tablets, and coat it to obtain the final product.

[0025] Example 2 Capsules

[0026] The prescription is as follows:

[0027]

[0028] Preparation method: Take the prescribed amount of raw materials and excipients, mix them evenly, pass them through an 80-mesh sieve, then add 95% ethanol to make a soft mass, granulate it through a 16-mesh screen; dry it at 60℃, then granulate it and fill it into capsules, making a total of 1000 capsules.

[0029] Example 3 Soft Capsules

[0030] The prescription is as follows:

[0031]

[0032] Preparation: Take the prescribed amount of Cistanche deserticola flavonoids and Cynomorium songaricum tannins, dry them at 60℃, mix them thoroughly, pulverize them into fine powder, add soybean oil and mix well to make 1000 soft capsules.

[0033] Example 4 Oral Liquid

[0034] The prescription is as follows:

[0035]

[0036] Preparation method: Take the prescribed amount of raw materials and excipients, mix them evenly, add 1000mL of purified water, stir to dissolve, mix well, filter, fill into 1000 bottles of oral liquid, and sterilize to obtain the final product.

[0037] Example 5: Droplets

[0038] The prescription is as follows:

[0039]

[0040] Preparation method: Take the prescribed amount of Cistanche deserticola flavonoids and Cynomorium songaricum tannins, mix them evenly, add molten PEG-4000 and PEG-6000, stir evenly, drip in dimethyl silicone oil and solidify, and make 1000 pills in total.

[0041] Example 6 Granules

[0042] The prescription is as follows:

[0043]

[0044] Preparation method: Take the prescribed amount of raw materials and excipients, mix them evenly, pass them through an 80-mesh sieve, then add 95% ethanol to make a soft mass, granulate it through a 16-mesh screen; dry it at 60℃, then granulate it, and package it into 1000 bags.

[0045] Example 1: Pharmacodynamic test of the drug of the present invention in treating Alzheimer's disease.

[0046] 1. Experimental Materials

[0047] 1.1 Medicinal materials: Tannins from Cynomorium songaricum were purchased from Shaanxi Changyue Phytochemical Technology Co., Ltd., and flavonoids from Cistanche deserticola were purchased from Nanjing Puyi Biotechnology Co., Ltd.

[0048] 1.2 Positive control drug: Donepezil hydrochloride.

[0049] 1.3 Experimental animals: SPF grade C57 mice.

[0050] 1.4 Chemical Reagent: Aβ 1-42 Human-derived polypeptides and physiological saline; acetylcholinesterase (AChE), acetylcholinetransferase (ChAT), and acetylcholine (ACh) assay kits were purchased from Nanjing Jiancheng Biotechnology Institute.

[0051] 1.5 Experimental instruments: Morris water maze (XR-XM101), multi-functional microplate reader (Model 680), tissue homogenizer (H09028).

[0052] 2. Test Methods

[0053] 2.1 Drug group pair

[0054] To comprehensively and effectively screen the optimal drug compatibility and dosage ratio of the initially selected prescriptions, and to minimize the number of experiments, this experiment adopted the baseline method for prescription design. The baseline ratio was based on the effective doses of Cynomorium songaricum and Cistanche deserticola flavonoids in improving the latency period of the water maze platform in Alzheimer's disease mice (Cistanche deserticola: 4–45 mg, Cistanche deserticola flavonoids: 30–150 mg). The dosage of Cistanche deserticola flavonoids decreased by 8–28% and the dosage of Cynomorium songaricum increased by 8–28%, or the dosage of Cynomorium songaricum decreased by 8–38% and the dosage of Cistanche deserticola flavonoids increased by 8–38%, expanding to both sides until reaching the extremes. The two extremes were Cistanche deserticola flavonoids alone and Cynomorium songaricum alone, respectively. Several combinations of Cistanche deserticola flavonoids and Cynomorium songaricum tannins were grouped together. Based on the research objectives, the primary and secondary effects of the two drugs were used as evaluation indicators. Through comprehensive information analysis, the optimal selection of each combination ratio was carried out. The ratios of the two drugs are shown in Table 1.

[0055] Table 1. Baseline Design of Equal Proportion of Cistanche Flavonoids and Cynomorium Tannins

[0056]

[0057]

[0058] 2.2 Screening of optimal dosage through pharmacological testing

[0059] 2.2.1 Preparation of the test drug

[0060] Weigh out 1 to 10 doses of Cistanche deserticola flavonoids and Cynomorium songaricum tannins according to the dosage of the compositions in Table 1, add 10 mL of purified water to dissolve and mix well to obtain 1 to 10 drug composition solutions.

[0061] 2.2.2 Animal Model Preparation

[0062] After anesthetizing mice with ketamine (100 mg / kg, intraperitoneal injection), 5 μL of condensed Aβ was injected once into the lateral ventricle (0.3 mm posterior to Bregma, 1 mm lateral to him, and 2.5 mm subdurally) using a microsyringe. 1-42 (containing Aβ) 1-42 (410 pmol). The needle was left in place for 2 minutes, and an equal volume of physiological saline was administered to the lateral ventricle of the normal group mice. Aβ 1-42 Pretreatment: After dissolving in sterile physiological saline, pretreatment at 37℃ for 120h causes it to become aggregated.

[0063] 2.2.3 Animal grouping and administration

[0064] After modeling, experimental animals were randomly divided into a normal group, a model group, a donepezil positive control group, and drug groups 1-10, with 12 mice in each group. Mice were acclimatized for 3 days before drug administration. The normal and model groups were given physiological saline according to body weight, while the donepezil groups were given donepezil (1.3 mg / kg / day). Each drug group received the corresponding dose of drug via gavage at a dose of 0.25 mL / kg starting from the day of the experiment, for 28 consecutive days. Mice were housed in a constant temperature and humidity environment [(24±1)℃, (50±5)%], with 12-hour circulating light (08:00-20:00), and free access to food and water. During the drug administration period, the animals were weighed daily, and their condition was observed and recorded.

[0065] 2.2.4 Specimen Collection

[0066] After the last administration, mice were euthanized by cervical dislocation, and the apex of the heart was perfused with 0.1M phosphate buffer (pH 7.4). Brain tissue was harvested, immediately washed with pre-cooled physiological saline, and the surface moisture was blotted dry with filter paper, or flash-frozen in liquid nitrogen and stored at -80°C for later testing.

[0067] 2.2.5 Detection Indicators and Methods

[0068] (1) Behavioral testing

[0069] The water maze test is a sensitivity test used to identify spatial learning and memory impairments. The water maze pool is 0.5m high and 1.2m in diameter, with a platform diameter of 10cm. The water temperature is controlled at 21-22℃. The maze is divided into four quadrants of roughly equal size. Before the formal test, each mouse was trained twice a day for 2 minutes each time, for a total of 4 days. During each training session, the mice were placed in the water maze from different locations. The time it took for the mouse to find and stand on the hidden underwater platform after entering the water was recorded, i.e., the escape latency, expressed in seconds (s). If the mouse failed to find the hidden platform within 60 seconds of entering the water, it was gently guided to the platform with a long stick and allowed to stand on it for 30 seconds; the escape latency was recorded as 60 seconds. On the 5th day, the formal test was conducted. The mice were placed facing the pool wall and gently lowered into the water from the same location in the opposite quadrant. The time it took to reach the platform and the number of times it crossed the platform were observed.

[0070] (2) Detection of the cholinergic system

[0071] Remove the brain tissue stored at -80℃ and thaw it slowly at low temperature. Weigh an appropriate amount of tissue and add 9 times the tissue weight of pre-cooled physiological saline. Homogenize the tissue three times at low temperature for 2 minutes each time in a tissue homogenizer. Centrifuge at 3000 r / min and 4℃ for 10 minutes in a refrigerated centrifuge. Collect the supernatant and detect the content of Ach and the activities of AchE and ChAT in mouse brain tissue according to the instructions of Nanjing Jiancheng reagent kit.

[0072] 2.3 Statistical Methods

[0073] SPSS 17.0 software was used for statistical analysis. Data are expressed as mean ± standard deviation (x ± s). One-way ANOVA was used for comparison between groups. LSD test was used for groups with homogeneous variances and Tamhane's T2 test was used for groups with unequal variances.

[0074] 2.4 Experimental Results

[0075] (1) Results on the effect on escape latency in Alzheimer's mice are shown in […]. Figure 1 .

[0076] Depend on Figure 1 It was found that, compared with the normal group, the platform latency of mice in the model group was significantly increased in the water maze test (P<0.01); compared with the model group, the platform latency of mice in groups 1-10 of the composition was significantly decreased (P<0.05), of which group 7 of the composition was significantly decreased (P<0.01); compared with the tannin group of Cynomorium songaricum (10 groups) and the flavonoid group of Cistanche deserticola (9 groups), the platform latency of mice in groups 1-8 of the composition showed a decreasing trend, and the platform latency of mice in group 7 of the composition was significantly decreased (P<0.05).

[0077] (2) Results on the effect on the number of times Alzheimer's mice crossed the platform are shown in [the table]. Figure 2 .

[0078] Depend on Figure 2 It was found that, compared with the normal group, the number of times mice in the model group crossed the platform was significantly reduced (P<0.01); compared with the model group, the number of times mice in groups 1-10 crossed the platform was significantly increased (P<0.05), among which group 7 of the composition showed a highly significant increase (P<0.01); compared with the tannin and flavonoid groups of Cynomorium songaricum, the number of times mice in groups 1-8 crossed the platform showed an increasing trend, and the number of times mice in group 7 of the composition crossed the platform was significantly increased (P<0.05).

[0079] (3) Results of the effect on AChE activity in brain tissue of Alzheimer's mice are shown in […]. Figure 3 .

[0080] Depend on Figure 3 It was found that, compared with the normal group, the AChE activity in the brain tissue of the model group mice was significantly increased (P<0.01); compared with the model group, the AChE activity in the brain tissue of mice in groups 1-10 of the composition group was significantly decreased (P<0.05), among which group 7 of the composition group showed a highly significant decrease (P<0.01); compared with the Cynomorium songaricum and Cistanche deserticola flavonoid groups, the AChE activity in the brain tissue of groups 1-8 of the composition group showed a decreasing trend, and the AChE activity in the brain tissue of mice in group 7 of the composition group was significantly decreased (P<0.05).

[0081] (4) Results of the effect on ChAT activity in brain tissue of Alzheimer's mice are shown in […]. Figure 4 .

[0082] Depend on Figure 4 It was found that, compared with the normal group, the ChAT activity in the brain tissue of the model group mice was significantly reduced (P<0.01); compared with the model group, the ChAT activity in the brain tissue of mice in groups 1 to 10 of the composition group was significantly increased (P<0.05 or P<0.01), with the largest increase in group 7 of the composition group (P<0.01); compared with the Cynomorium tannin and Cistanche deserticola flavonoid groups, the ChAT activity in the brain tissue of groups 1 to 8 of the composition group showed an increasing trend, and the ChAT activity in the brain tissue of mice in group 7 of the composition group was significantly increased (P<0.05).

[0083] (5) Results of the effect on Ach content in brain tissue of Alzheimer's mice are shown in […]. Figure 5 .

[0084] Depend on Figure 5 It can be seen that, compared with the normal group, the Ach content in the brain tissue of the model group mice was significantly reduced (P<0.01);

[0085] Compared with the model group, the Ach content in the brain tissue of mice in groups 1-10 of the composition group was significantly increased (P<0.05 or P<0.01), with the largest increase in group 7 of the composition group (P<0.01). Compared with the tannin and flavonoid groups of Cistanche deserticola, the Ach content in the brain tissue of groups 1-8 of the composition group showed an increasing trend, and the Ach content in the brain tissue of mice in group 7 of the composition group was significantly increased (P<0.05).

[0086] 3. Experimental Conclusions

[0087] (1) The above-mentioned 10 ratios of Cistanche deserticola flavonoids and Cynomorium songaricum tannins have an effect on Aβ. 1-42 The combination of Cistanche deserticola flavonoids and Cynomorium songaricum has a good therapeutic effect on cognitive impairment in mice with Alzheimer's disease, and the therapeutic effect of the combination of Cistanche deserticola flavonoids and Cynomorium songaricum tannins is better than that of Cistanche deserticola flavonoids and Cynomorium songaricum tannins alone.

[0088] (2) The optimal ratio of Cistanche deserticola flavonoids to Cynomorium tannins for the treatment of Alzheimer's disease is 40mg:28mg.

[0089] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A pharmaceutical composition for treating and / or preventing Alzheimer's disease, characterized in that: It is made from cynomorium tannin and cistanche flavonoids, with a mass ratio of cynomorium tannin to cistanche flavonoids of 28:

40.

2. The use of the pharmaceutical composition of claim 1 in the preparation of a medicament for treating and / or preventing Alzheimer's disease.

3. A drug for treating and / or preventing Alzheimer's disease, characterized in that: It is made from the pharmaceutical composition of claim 1 and excipients that are acceptable in pharmaceutical manufacturing.

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