Polar component with anti-depression effect of cordyceps militaris fermented gastrodia elata product and preparation method of polar component

Abstract

The invention discloses a polar component with an anti-depression effect of a cordyceps militaris fermented gastrodia elata product and a preparation method of the polar component. The polar component comprises a petroleum ether polar component obtained by extracting the cordyceps militaris fermented gastrodia elata product with petroleum ether, ethyl acetate and n-butyl alcohol, and an extracted water-phase component, the invention provides two brand-new polar components obtained by extracting a cordyceps militaris fermented gastrodia elata product, and pharmacological studies show that the two components have an anti-depression effect, can be applied to preparation of anti-depression medicines, decoction pieces or supplements, and have wide application prospects. The invention provides a basis for further research and development of traditional Chinese medicine antidepressants with remarkable curative effects and ideal antidepressant effects, and has good application prospects.

Description

Technical Field

[0001] The invention relates to two polar components with antidepressant effects obtained by fermenting Gastrodia elata with Cordyceps militaris, as well as a preparation method and application thereof, belonging to the technical field of medicines. Background Art

[0002] Depression is a common mental illness characterized by low mood, loss of interest, unhappiness, guilt, low self-esteem, sleep or appetite disturbances, low energy, and difficulty concentrating. This condition can severely impact patients' daily lives, causing physical and emotional distress to themselves and their families. With the intensification of multiple stressors in modern society, depression has become a common illness, with its incidence rapidly increasing. Depression is poised to become the second most burdensome disease globally, second only to cardiovascular disease, for families and society.

[0003] Currently, treatment for depression typically relies primarily on medication, supplemented by other therapies such as psychological counseling. While medication has achieved considerable success in saving patients and alleviating their suffering, according to the World Health Organization, less than 25% of depressed patients worldwide receive effective treatment. Despite this, global sales of antidepressant drugs reach over $10 billion annually. This highlights the significant social and economic value of developing antidepressant drugs, but also demonstrates the long road ahead. In addition to traditional Western medications, existing literature and research have revealed the antidepressant potential and advantages of traditional Chinese medicines, botanicals, and medicinal fungi. This has made it possible to identify new and more effective components and ingredients within these medicines. However, the major challenge currently is the limited success of developing effective traditional Chinese medicines, botanicals, and fungi, resulting in suboptimal antidepressant efficacy from conventional medications. This is largely due to the complex pathogenesis of depression and its unclear etiology, as well as the limited depth and breadth of research on these drugs. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide two antidepressant polar components of a Cordyceps militaris fermentation product of Gastrodia elata with antidepressant effects, as well as a preparation method and application thereof, thereby providing a new option for antidepressant drug treatment.

[0005] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0006] The product of Cordyceps militaris fermentation with Gastrodia elata has two polar components with antidepressant effects. These two polar components are prepared by further extracting the product of Cordyceps militaris fermentation with Gastrodia elata with solvents of different polarities (oily ether, ethyl acetate and n-butanol), and the effective antidepressant active components are obtained through classic pharmacological experimental screening.

[0007] The fermentation strain in this study was Cordyceps militaris B1528; Cordyceps militaris belongs to the class Hypocreales, order Cordycepsaceae, and genus Cordyceps. The strain is B1528, and the strain accession number is CCTCC M 2019789. It was deposited in the China Center for Type Culture Collection on October 9, 2019.

[0008] The method for preparing the two polar components of the Cordyceps militaris fermentation product having antidepressant effects comprises the following steps:

[0009] (1) Preparation of fermentation medium: Take 200 g of potatoes, boil the juice and remove the residue, add 20 g of soluble starch, 1 g of peptone, and 40 g of Gastrodia elata, and make up to 1000 mL with water. Prepare the mixture according to the proportions and put it into 500 mL Erlenmeyer flasks, 200 ml per bottle. Sterilize at 121°C for 30 min and cool for later use.

[0010] (2) Fermentation culture conditions: The inoculum size of Cordyceps militaris was 5 mL, the culture temperature was set to be constant at 25°C, the shaker speed was constant at 200 RPM, and the culture was carried out in the dark for 12 days for fermentation.

[0011] (3) Preparation of fermentation products: After the fermentation is completed, the fermentation products are placed in a vacuum freeze dryer for freeze drying. The freeze-dried solids are crushed with a grinder, passed through a 120-mesh stainless steel sieve, and stored at low temperature for later use.

[0012] (4) Preparation of different polar components (petroleum ether, ethyl acetate, n-butanol, water) of the fermentation product of Cordyceps militaris and Gastrodia elata. Take the prepared lyophilized powder of the fermentation product and add an equal amount of warm water (40°C) and mix evenly. Then transfer it into a 2L conical flask. Add 1L of fermentation product to each conical flask, and then add 1L of petroleum ether. Mix the petroleum ether and fermentation product evenly. Place it on an ultrasonic table and perform ultrasonic-assisted extraction 3 times, each time for 40 minutes. After ultrasonication, let it stand and separate. After separation, the upper pale white turbid liquid is the petroleum ether extraction part, and the lower layer is the remaining part after petroleum ether extraction.

[0013] The upper layer is collected into a container. The remaining lower layer is then extracted with ethyl acetate and n-butanol in the same manner as above. The remaining portion after the n-butanol extraction is the aqueous phase.

[0014] The extraction solution containing substances of different polarities obtained by extraction with petroleum ether, ethyl acetate and n-butanol and the aqueous phase after extraction are concentrated under reduced pressure until they are odorless, thereby obtaining extracts of components of different polarities.

[0015] The present invention also discloses an application of a petroleum ether polar component of a product of Cordyceps militaris fermentation with Gastrodia elata and a water phase component after extraction in the preparation of a therapeutic antidepressant drug and an antidepressant adjuvant. The petroleum ether polar component of the product of Cordyceps militaris fermentation with Gastrodia elata and the water phase component after extraction can be used to prepare the therapeutic antidepressant drug and the antidepressant adjuvant.

[0016] Beneficial effects: Compared with the existing technology, the present invention provides two new polar components extracted from the products of Cordyceps militaris fermentation of Gastrodia elata. Pharmacological studies have shown that these two components have antidepressant effects and can be used to prepare drugs, herbal slices or supplements for treating depression. This provides a basis for further research and development of traditional Chinese medicine antidepressant drugs with significant efficacy and ideal antidepressant effects, and has good application prospects.

[0017] The present invention utilizes biotransformation technology, especially the technology of transforming Chinese medicine by fermentation of medicinal fungi, to find new antidepressant components, ingredients or new Chinese medicine slices.

[0018] Gastrodia elata (Gastrodia elata Bl.), the dried tuber of the orchid family, is a traditional Chinese medicine (TCM) designated by the National Health Commission as a health food and a medicinal food. It boasts a sweet, moist, and mild yang energy, and is listed as a top-grade medicinal herb in the Shennong's Herbal Classic. It has the effects of extinguishing wind and relieving spasms, calming liver yang, and dispelling wind and unblocking meridians. It is used to treat infantile convulsions, epilepsy, headaches, and dizziness, as well as limb numbness, hand and foot paralysis, and rheumatic pain. Modern pharmacological research has also confirmed its antidepressant, neuroprotective, and anti-aphrodisiac properties, including intellectual enhancement, brain protection, analgesic, sedative, hypnotic, anti-epileptic, and anti-vertigo properties.

[0019] Cordyceps militaris (L.) Fr., also known as Cordyceps militaris, is the model species of the Cordyceps genus and a new resource food approved by the Ministry of Health. It possesses high medicinal value. Years of chemical and pharmacological experiments have demonstrated that Cordyceps militaris contains active substances such as cordyceps polysaccharides, cordycepic acid, cordycepin, sterols, nucleosides, and trace elements and various amino acids essential to the human body. These substances exhibit diverse pharmacological activities, including immune regulation, anti-tumor, antiviral, anti-infective, antioxidant, anti-fatigue, anti-aging, blood sugar reduction, and liver and kidney protection. Notably, cordycepin from Cordyceps militaris has recently been found to exhibit rapid antidepressant effects.

[0020] The applicants have discovered that fungal transformation of traditional Chinese medicines can enhance their efficacy, produce new ingredients, increase the content of active ingredients, and degrade macromolecules and less readily absorbed components. This invention utilizes the fermentation product of Gastrodia elata fermented with Cordyceps militaris, extracting it with solvents of varying polarity to produce new polar components. These components were then evaluated for their antidepressant efficacy. The results showed that the petroleum ether polar component of the fermentation product and the aqueous phase fraction after extraction exhibited significant antidepressant effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the timetable for the drug treatment and mouse behavioral experiments of the present invention;

[0022] Figure 2 This is a diagram showing the effects of different extraction parts of the Cordyceps militaris fermented Gastrodia elata product on mice tested by the forced swimming test of the present invention;

[0023] Figure 3 It is a diagram of the tail suspension experiment results of the present invention.

[0024] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. DETAILED DESCRIPTION

[0025] 1 Experimental Materials

[0026] 1.1 Experimental animals and drugs

[0027] Healthy SPF-grade male ICR mice aged 5 weeks and weighing 18-20 g were selected and provided by Sibeifu (Beijing) Biotechnology Co., Ltd. The experimental animal license number is SCXK (Beijing) 2019-0010. The animals were kept in a room with a temperature of 20°C-25°C and a humidity of 50%. Solid pellet maintenance feed and sterile water (sterilized in an autoclave) were fed regularly every day, and the breeding environment was kept quiet and noiseless.

[0028] 1.2 Experimental Reagents

[0029] Table 1 Main reagents

[0030]

[0031] 1.3 Experimental instruments

[0032] Table 2 Main experimental instruments

[0033]

[0034] 1.4 Preparation of different extraction parts of Cordyceps militaris fermentation products of Gastrodia elata

[0035] (1) Preparation of fermentation medium: 200 g of potatoes were boiled and the residue removed. 20 g of soluble starch, 1 g of peptone, and 40 g of Gastrodia elata were added. The volume was made up to 1000 mL with water. The mixture was prepared according to the proportions and placed in 500 mL Erlenmeyer flasks, 200 mL per bottle. The mixture was sterilized at 121°C for 30 min and then cooled for later use.

[0036] (2) Fermentation conditions: The inoculum size of Cordyceps militaris was 5 mL, the culture temperature was set to 25°C, the shaker speed was set to 200 RPM, and the culture was carried out in the dark for 12 days.

[0037] (3) Preparation of fermentation products: After the fermentation is completed, the obtained fermentation products are placed in a vacuum freeze dryer for freeze drying. The freeze-dried solids are then crushed with a grinder, passed through a 120-mesh stainless steel sieve, and stored at low temperature for later use;

[0038] (4) Preparation of different polar components (petroleum ether, ethyl acetate, n-butanol, water) of Gastrodia elata fermentation product: Take the lyophilized powder of the fermentation product prepared in step (3) above, add an equal amount of 40°C warm water, mix evenly, and transfer to a 2L conical flask. Add 1L of fermentation product to each conical flask, and then add 1L of petroleum ether. Mix the petroleum ether and fermentation product evenly, place on an ultrasonic table, and perform ultrasonic-assisted extraction 3 times, each time for 40 minutes. After ultrasonication, let it stand and separate.

[0039] After the separation is completed, the upper pale white turbid liquid is the petroleum ether extraction part, and the lower layer is the remaining part after the petroleum ether extraction. The upper layer is collected into a container; then the remaining lower layer is extracted with ethyl acetate and n-butanol in the same way as above, and the remaining part after the n-butanol extraction is the aqueous phase after extraction;

[0040] The extracts containing substances of varying polarity obtained by extraction with petroleum ether, ethyl acetate, and n-butanol, as well as the aqueous phase after extraction, were concentrated under reduced pressure until odorless. This yielded extracts containing components of varying polarity, and the extraction yield of each polarity of the fermentation product was calculated. Extraction yield (%) = total weight of the extract obtained after extraction / weight of the lyophilized fermentation product powder × 100%.

[0041] 2 Experimental methods

[0042] The method reported in the literature was followed. After the mice were fed adaptively, each group of mice was given the corresponding extracts of different extracts of Cordyceps militaris fermented Gastrodia elata products and fluoxetine hydrochloride by gavage at 9:00 am every day during the entire experiment for 14 consecutive days. Starting from the 14th day, each group of rats underwent behavioral experiments to preliminarily screen the effective extracts of Cordyceps militaris fermented Gastrodia elata products for antidepressant effects, such as Figure 1 shown.

[0043] 2.1 Animal grouping and drug administration

[0044] After 5 days of adaptive feeding, SPF male ICR mice were randomly divided into a blank group, a positive drug group, a petroleum ether fraction group, an ethyl acetate fraction group, an n-butanol fraction group, and a post-extraction aqueous phase group, with 8 mice in each group. Except for the blank group, mice in the remaining treatment groups received the corresponding drug by gavage. The drug was dissolved in sterile water, and each mouse received a dose of 0.4 ml per gavage. The optimal rat gavage dose previously determined by the research team was converted to a mouse dose. The extraction site was administered once daily for 14 consecutive days. The dosing concentration schedule is shown in the table.

[0045] Table 3 Dosage regimen for rats in each experimental group

[0046]

[0047] 2.2 Forced swimming test

[0048] Mice were placed in an open cylindrical container (11 cm diameter, 30 cm deep, filled with 25°C water, 20 cm high) and forced to swim for 6 minutes. The time the mouse remained afloat in the water without struggling or escaping was defined as immobility time. After recording, the mice were kept still for 5 minutes. After the experiment, the mice were wiped dry with a towel, dried, and moved to a dry cage.

[0049] 2.3 Tail suspension test

[0050] One hour after dosing on day 14, the mice were suspended upside down with tape from their tails on a hook used for tail suspension tests, with their heads approximately 20 cm above the surface. Side panels were placed on either side of the suspension to block the animals' line of sight. The mice were suspended for 6 minutes, and the duration of immobility during the last 5 minutes was recorded. Depression models exhibiting depressive-like behaviors will demonstrate increased immobility. In most cases, the TST is used to assess antidepressant responses.

[0051] 2.4 Statistical analysis

[0052] The experimental data were statistically analyzed using SPSS26 software. The data were expressed as mean ± standard deviation (mean ± SD). When the data conformed to the normal distribution, one-way ANOVA was used; when the data did not conform to the normal distribution, non-parametric test methods were used. The results were considered to be significantly different when P < 0.05, and extremely significantly different when P < 0.01.

[0053] 3 Experimental results

[0054] 3.1 Preparation results of different extraction parts of Cordyceps militaris fermentation Gastrodia elata products

[0055] 600 g of freeze-dried powder of the fermentation product was extracted with the help of ultrasonic wave with petroleum ether, ethyl acetate and n-butanol. The remaining part was the aqueous phase after extraction. After vacuum concentration, the extracts of different extraction parts were 4.5 g, 9.6 g, 11.7 g and 606.1 g, respectively. The extraction rates were 0.75%, 1.6%, 1.95% and 101.02%, respectively.

[0056] 3.2 Forced swimming test to test the effects of different extracts of Cordyceps militaris fermented Gastrodia elata on mice The results are as follows Figure 2 (Compared with the blank group: * P<0.05, ** P < 0.01), by Figure 2As shown, after 14 days of oral gavage, the immobility time of mice in the positive drug group was significantly reduced in the forced swim test compared with the blank group (P<0.01). Among the extracts of the Cordyceps militaris fermented Gastrodia elata product, the petroleum ether and aqueous extracts significantly reduced the immobility time of mice in the behavioral despair model, but the ethyl acetate and n-butanol extracts did not. There was no statistically significant difference between the extracts of the Cordyceps militaris fermented Gastrodia elata product and the fluoxetine extract (P>0.05). This suggests that among the extracts of the Cordyceps militaris fermented Gastrodia elata product, the petroleum ether extract and the aqueous extract after extraction were effective in the tail suspension test, with no difference in effect from the positive drug, while the ethyl acetate and n-butanol extracts were ineffective.

[0057] 3.3 Tail suspension test The results are as follows Figure 3 (Note: Compared with the blank group: * P<0.05, ** P < 0.01), by Figure 3 It can be seen that after 14 days of gavage, in the tail suspension test, the immobility time of the positive drug group was significantly reduced compared with the blank group; the various extraction parts of the Cordyceps militaris fermented Gastrodia elata product can also reduce the immobility time of the model mice, and there is no significant statistical significance between the various extraction parts of the Cordyceps militaris fermented Gastrodia elata product and the fluoxetine group (P>0.05), indicating that the various extraction parts of the Cordyceps militaris fermented Gastrodia elata product are effective in the tail suspension test, and the effect is no different from that of the positive drug.

[0058] 4 Discussions

[0059] The present invention used three solvents of varying polarity—petroleum ether, ethyl acetate, and n-butanol—to extract the product of Cordyceps militaris fermented with Gastrodia elata using ultrasound. The results showed that the aqueous phase of the product had the highest extraction yield, followed by the n-butanol-extracted portion, and the petroleum ether-extracted portion had the lowest. This is likely because n-butanol and the aqueous phase have relatively strong polarity, resulting in a wide solubility range and the ability to enrich most components.

[0060] This study utilized a behavioral despair model in animals to evaluate the effects of three different polarity fractions and their aqueous extracts from a Cordyceps militaris-fermented Gastrodia elata product on the behavioral despair model. The results showed that, 14 days after oral gavage, the petroleum ether extract and aqueous extract of the Cordyceps militaris-fermented Gastrodia elata product significantly reduced immobility time in the forced swim test in all treatment groups compared to the control group. However, the ethyl acetate and n-butanol extracts failed to reduce immobility time in the model mice, suggesting that the petroleum ether extract and aqueous extract have the potential to improve depression.

[0061] In the tail suspension test, the immobility time of each extract fraction of the Cordyceps militaris-fermented Gastrodia elata product was significantly reduced compared to the blank control. According to the TST and FST results, the petroleum ether fraction and the aqueous fraction exhibited antidepressant effects. This suggests that the antidepressant active substances of the Cordyceps militaris-fermented Gastrodia elata product are primarily concentrated in the petroleum ether extract fraction and the aqueous fraction after extraction. This provides a scientific basis for screening the active ingredients and mechanism of action of the Cordyceps militaris-fermented Gastrodia elata product.

[0062] 4.1 Behavioral despair model and depression drug screening

[0063] The forced swim test (FST) is one of the most commonly used methods for assessing depressive-like and antidepressant-like behaviors. The FST involves placing rodents in an inescapable water-filled cylinder. After an initial escape attempt, rodents become immobile upon realizing they cannot escape. This test results in increased immobility time in the animals, which is considered a passive stress response strategy or a depressive-like behavior, termed behavioral despair. The FST is easy to perform and has proven reliable in the laboratory. It has been shown to be useful for detecting substances with antidepressant properties because these drugs shift from passive coping to active coping, i.e., reducing immobility. Therefore, we used this method to test the antidepressant effects of different extract fractions. The results showed that the immobility time of mice in the fluoxetine, petroleum ether, and post-extraction aqueous phase groups was significantly reduced. However, the immobility time in the ethyl acetate and n-butanol groups was not significantly different from that in the control group, suggesting that the petroleum ether extract fraction and post-extraction aqueous phase have potential antidepressant effects.

[0064] Another important test, based on similar assumptions and interpretations to the FST, is the tail suspension test (TST). In the TST, mice are suspended by their tails from a fixed location for a defined period of time, and their immobility time is measured. This model is effective for testing the antidepressant effects of drugs, as antidepressants significantly reduce the amount of immobility spent by experimental animals. The basic principles for measuring the absence of active coping behaviors are the same in the TST and FST; however, differences in their responses to some antidepressants suggest that different antidepressant classes may exhibit behavioral differences. Furthermore, it is important to note that the results of the TST are not affected by water temperature, as is the case with the FST. After the mice have been acclimated to tail suspension for 1 minute, the immobility time over a 5-minute period is measured to assess the antidepressant effect of the drug. Notably, our tail suspension test results showed that the immobility time of rats in the petroleum ether fraction group was shorter than that in the model group, thus concluding that the petroleum ether fraction and the aqueous phase after extraction have antidepressant effects.

[0065] 5. Summary

[0066] The above experimental results show that the active substances with antidepressant effects in the products of Cordyceps militaris fermented with Gastrodia elata are mainly concentrated in the petroleum ether part and the aqueous phase part after extraction.

[0067] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any person skilled in the art can, without departing from the scope of the technical solution of the present invention, make some changes or modifications to equivalent embodiments using the technical contents disclosed above. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. The product of Cordyceps militaris fermentation with Gastrodia elata has a polar component with antidepressant effect, characterized by: The invention comprises a petroleum ether polar component obtained by extracting a product of Cordyceps militaris and Gastrodia elata through petroleum ether, ethyl acetate and n-butanol, and a water phase component after the extraction.

2. The method for preparing the polar component with antidepressant effect from the Cordyceps militaris fermentation product of Gastrodia elata as claimed in claim 1, characterized in that: The following steps are involved: (1) Preparation of fermentation medium: 200 g of potatoes were boiled and the residue removed. 20 g of soluble starch, 1 g of peptone, and 40 g of Gastrodia elata were added. The volume was made up to 1000 mL with water. The mixture was prepared according to the proportions and placed in 500 mL Erlenmeyer flasks, 200 mL per bottle. The mixture was sterilized at 121°C for 30 min and then cooled for later use. (2) Fermentation conditions: The inoculum size of Cordyceps militaris was 5 mL, the culture temperature was set to 25°C, the shaker speed was set to 200 RPM, and the culture was carried out in the dark for 12 days. (3) Preparation of fermentation products: After the fermentation is completed, the obtained fermentation products are placed in a vacuum freeze dryer for freeze drying. The freeze-dried solids are then crushed with a grinder, passed through a 120-mesh stainless steel sieve, and stored at low temperature for later use; (4) Preparation of different polar components of Cordyceps militaris and Gastrodia elata fermentation products: Take the lyophilized powder of the fermentation product prepared in step (3), add an equal amount of 40°C warm water, mix evenly, and transfer to a 2L conical flask. Add 1L of fermentation product to each conical flask, and then add 1L of petroleum ether. Mix the petroleum ether and fermentation product evenly, place on an ultrasonic table, and perform ultrasonic-assisted extraction 3 times, each time for 40 minutes. After ultrasonication, let it stand and separate. After separation, the upper layer of pale white turbid liquid is the petroleum ether extraction part, and the lower layer is the remaining part after petroleum ether extraction; Collect the upper layer into a container; then continue to extract the remaining lower layer by adding ethyl acetate and n-butanol in the same way as above, and finally the remaining part after n-butanol extraction is the aqueous phase after extraction; The extraction solution containing substances of different polarities obtained by extraction with petroleum ether, ethyl acetate and n-butanol and the aqueous phase after extraction are concentrated under reduced pressure until they are odorless, thereby obtaining extracts of components of different polarities.

3. Application of the petroleum ether polar component and the extracted aqueous phase component of the Cordyceps militaris fermentation product of Gastrodia elata in the preparation of therapeutic antidepressant drugs and antidepressant adjuvants.

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