Application of Dendrobium huoshanense protocorms in anti-fatigue and kidney-tonifying and aphrodisiac effects

Abstract

This invention discloses the application of Dendrobium huoshanense protocorms in anti-fatigue and kidney-tonifying and aphrodisiac effects. Combining Dendrobium huoshanense protocorms with Cistanche deserticola, American ginseng, Rubus idaeus, Cornus officinalis, and Lycium barbarum significantly enhances the anti-fatigue and kidney-tonifying and aphrodisiac effects of the formula. Compared with Cistanche deserticola alone, adding Dendrobium huoshanense protocorms significantly prolongs the exhaustive swimming time in mice, reduces serum urea nitrogen and lactic acid levels, increases liver glycogen content, and increases muscle glycogen content. Compared with Cistanche deserticola alone, adding Dendrobium huoshanense protocorms significantly increases serum superoxide dismutase activity, reduces serum malondialdehyde content, increases serum testosterone content, increases penile nitric oxide synthase activity, and increases penile nitric oxide and cyclic guanosine monophosphate content in penile tissue.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically involving the application of Dendrobium huoshanense protocorms in anti-fatigue and kidney-tonifying and aphrodisiac effects. Technical Background

[0002] With increasing life and work pressures, more and more people are in a sub-healthy state. Sub-health is considered the early stage of many chronic diseases and is also a reversible stage. Alleviating sub-health symptoms can prevent the occurrence and development of many chronic diseases. Fatigue and kidney yang deficiency are the main symptoms of sub-health.

[0003] In Traditional Chinese Medicine (TCM), fatigue falls under the category of "exhaustion syndrome," primarily caused by deficiency of Qi and blood, and imbalance of the internal organs. Fatigue, as a non-specific symptom, exists in many chronic diseases. If fatigue is not addressed promptly, it can accumulate and lead to overwork, resulting in chronic fatigue syndrome, which includes weakened immunity, endocrine disorders, organic organ lesions, and sexual dysfunction.

[0004] Erectile dysfunction (ED) is a significant symptom of kidney yang deficiency. Traditional Chinese medicine (TCM) believes that the kidneys govern the body's oxygen supply, and that the foundation of human health lies in the kidneys, while the driving force of sexual function is essence (jing). Kidney yang deficiency is the primary pathogenesis of ED, and kidney-tonifying and aphrodisiac TCM herbs can be used to treat ED. Modern research indicates that nitrogen (NO) is a crucial neurotransmitter in the smooth muscle of the corpus cavernosum. In penile tissue, nitric oxide synthase (NOS) catalyzes the synthesis of NO from arginine. NO enters the smooth muscle cells of the corpus cavernosum and activates guanylate cyclase, converting GTP to cGMP. cGMP acts as a second messenger, regulating intracellular ion concentration, causing relaxation of the corpus cavernosum smooth muscle, increased blood flow, and penile erection. When cGMP is hydrolyzed by phosphodiesterase type 5 (PDE5), this pathway is interrupted, and erection ceases. Disruption of the NOS / NO / cGMP signaling pathway, including decreased NOS activity and reduced NO and cGMP levels, is a significant pathogenesis of ED. Activating the NOS / NO / cGMP signaling pathway is an important approach to treating ED.

[0005] Cistanche deserticola, American ginseng, raspberry, cornus officinalis, and wolfberry are all traditional Chinese medicinal herbs that are used for warming the kidneys and strengthening yang. These herbs not only offer the therapeutic benefits of traditional Chinese medicine but also possess the safety characteristics of food, aligning with people's pursuit of health. However, the combined effects of Cistanche deserticola, American ginseng, raspberry, cornus officinalis, and wolfberry in combating fatigue and tonifying the kidneys and strengthening yang are relatively mild and require a longer period of use to take effect.

[0006] The protocorm of *Dendrobium huoshanense* is an embryogenic tissue obtained through plant tissue culture using the stem tip meristem as explants. The cells of the protocorm possess both the ability to replicate and proliferate, and the ability to differentiate into complete plants, exhibiting characteristics of stem cells and thus constituting a type of pluripotent stem cell from *Dendrobium huoshanense*. In 2024, the National Health Commission issued an announcement including the protocorm of *Dendrobium huoshanense* in the list of new food ingredients. This invention applies the protocorm of *Dendrobium huoshanense* to a formula containing *Cistanche deserticola*, American ginseng, raspberry, cornus officinalis, and wolfberry, significantly enhancing the formula's anti-fatigue and kidney-tonifying effects. Summary of the Invention

[0007] The purpose of this invention is to provide a novel application of Dendrobium huoshanense protocorms to enhance the anti-fatigue and kidney-tonifying effects of a formula containing Cistanche deserticola, American ginseng, raspberry, Cornus officinalis, and Lycium barbarum. The Dendrobium huoshanense protocorms used in this invention are undifferentiated protocorms; differentiated Dendrobium huoshanense buds or plants have not been found to enhance the anti-fatigue and kidney-tonifying effects of the formula.

[0008] The application of Dendrobium huoshanense protocorms in anti-fatigue and kidney-tonifying and aphrodisiac effects includes the following steps:

[0009] 1. Add 3 parts by weight of dried Dendrobium nobile protocorms to the formula consisting of 5 parts by weight of Cistanche deserticola, 5 parts by weight of American ginseng, 3 parts by weight of raspberry, 2 parts by weight of Cornus officinalis, and 1 part by weight of wolfberry. Grind the powder into powder for later use.

[0010] 2. The ultrafine powder was added to a gastric juice simulation solution with pH 2.0 and pepsin activity of 2000 U / mL at a material-to-liquid ratio of 1:50. After stirring at 37℃ for 2 hours, the pH was adjusted to 6.8 with 1 mol / L NaOH solution to inactivate the pepsin. Then, trypsin and pancreatic amylase were added until the activity of each was 100 U / mL. After stirring at 37℃ for another 2 hours, the mixture was heated to 95℃ and held for 5 minutes to inactivate the enzymes. The supernatant was filtered and collected. The supernatant was concentrated and dried to obtain the biomimetic extract.

[0011] 3. Bionic extracts can be used in traditional Chinese medicine preparations for anti-fatigue and kidney tonification and aphrodisiac effects.

[0012] The beneficial technical effects of this invention are as follows: compared with the formula using Cistanche deserticola alone, adding Dendrobium huoshanense protocorms can significantly prolong the exhaustive swimming time of mice, reduce the content of urea nitrogen and lactic acid in mouse serum, increase the content of liver glycogen in liver tissue, and increase the content of muscle glycogen in muscle tissue; compared with the formula using Cistanche deserticola alone, adding Dendrobium huoshanense protocorms can significantly increase the activity of superoxide dismutase in the serum of rats with kidney yang deficiency, reduce the content of malondialdehyde in the serum, increase the content of testosterone in the serum, increase the activity of nitric oxide synthase in penile tissue, and increase the content of nitric oxide and cyclic guanosine monophosphate in penile tissue. Attached Figure Description

[0013] Figure 1 Dendrobium huoshanense protocorm. Detailed Implementation

[0014] The present invention will be further described below with reference to specific embodiments. The features and advantages of the present invention will become clearer as the description unfolds, but the exemplary embodiments are merely illustrative and do not constitute any limitation on the scope of the invention.

[0015] Example 1

[0016] Preparation of Cistanche deserticola biomimetic extract: 5 parts of Cistanche deserticola, 5 parts of American ginseng, 3 parts of raspberry, 2 parts of Cornus officinalis, and 1 part of Lycium barbarum were weighed according to weight and ultra-finely pulverized for later use. The ultra-fine powder was added to a gastric juice simulation solution with pH 2.0 and pepsin activity of 2000 U / mL at a material-to-liquid ratio of 1:50. After stirring at 37℃ for 2 hours, the pH was adjusted to 6.8 with 1 mol / L NaOH solution to inactivate pepsin. Then, trypsin and pancreatic amylase were added until the activity of each was 100 U / mL. After stirring at 37℃ for another 2 hours, the mixture was heated to 95℃ and held for 5 minutes to inactivate the enzymes. The supernatant was filtered and collected. The supernatant was concentrated and dried to obtain the Cistanche deserticola biomimetic extract.

[0017] Preparation of biomimetic extract of Dendrobium huoshanense protocorm + Cistanche deserticola: 5 parts by weight of Cistanche deserticola, 5 parts of American ginseng, 3 parts of raspberry, 2 parts of Cornus officinalis, 1 part of Lycium barbarum, and 3 parts of dried Dendrobium huoshanense protocorm were weighed and ultra-finely pulverized for later use. The ultra-fine powder was added to a gastric juice simulation solution with pH 2.0 and pepsin activity of 2000 U / mL at a material-to-liquid ratio of 1:50. After stirring at 37℃ for 2 hours, the pH was adjusted to 6.8 with 1 mol / L NaOH solution to inactivate pepsin. Then, trypsin and pancreatic amylase were added until the activity of each was 100 U / mL. After stirring at 37℃ for another 2 hours, the mixture was heated to 95℃ and held for 5 minutes to inactivate the enzymes. The supernatant was filtered and collected. The supernatant was concentrated and dried to obtain the biomimetic extract of Dendrobium huoshanense protocorm + Cistanche deserticola.

[0018] Example 2

[0019] Verification experiment on the enhancement of the anti-fatigue efficacy of the formula of Cistanche deserticola, American ginseng, Rubus idaeus, Cornus officinalis, and Lycium barbarum by Dendrobium huoshanense protocorms: Mice were randomly divided into a blank control group (group 0), a Cistanche deserticola formula group (group 1), and a Dendrobium huoshanense protocorm + Cistanche deserticola formula group (group 2). The blank control group was administered distilled water by gavage, the Cistanche deserticola formula group was administered a biomimetic extract of the Cistanche deserticola formula by gavage, and the Dendrobium huoshanense protocorm + Cistanche deserticola formula group was administered a biomimetic extract of the Dendrobium huoshanense protocorm + Cistanche deserticola formula by gavage. The dosage of each extract was 900 mg / kg, and the gavage volume was 0.2 ml. Each experimental group was administered the drug once a day for 30 consecutive days. On day 30 after the first administration, the exhaustive swimming time of mice was measured; the serum urea nitrogen and lactic acid content were measured; the liver glycogen content in liver tissue was measured; and the muscle glycogen content in muscle tissue was measured.

[0020] The results of the verification experiment on the enhancement of the anti-fatigue efficacy of the formula of Cistanche deserticola, American ginseng, raspberry, cornus officinalis, and wolfberry by the protocorm of Dendrobium huoshanense are shown in Tables 1, 2 and 3.

[0021] Table 1 Results of the mouse exhaustive swimming time test

[0022] Group Swimming time (s) Group 0 196.79±21.31 Group 1 <![CDATA[227.33±23.71 a ]]> Group 2 <![CDATA[259.73±22.63 ab ]]>

[0023] Note: a indicates a significant difference from the blank control group (P≤0.05), b indicates a significant difference from the Cistanche deserticola group (P≤0.05).

[0024] Table 2 Results of urea nitrogen and lactate content tests in mouse serum

[0025] Group Blood urea nitrogen content (mmol / L) Lactic acid content (mmol / L) Group 0 8.71±0.31 24.87±1.91 Group 1 <![CDATA[7.27±0.63 a ]]> <![CDATA[23.39±1.72 a ]]> Group 2 <![CDATA[5.98±0.54 ab ]]> <![CDATA[22.02±1.85 ab ]]>

[0026] Note: a indicates a significant difference from the blank control group (P≤0.05), b indicates a significant difference from the Cistanche deserticola group (P≤0.05).

[0027] Table 3. Results of experiments on liver glycogen and muscle glycogen content in mouse tissues.

[0028] Group Liver glycogen content (mg / g) Muscle glycogen content (mg / g) Group 0 3.39±0.59 0.41±0.09 Group 1 <![CDATA[4.56±0.67 a ]]> <![CDATA[0.52±0.11 a ]]> Group 2 <![CDATA[5.45±0.63 ab ]]> <![CDATA[0.63±0.13 ab ]]>

[0029] Note: a indicates a significant difference from the blank control group (P≤0.05), b indicates a significant difference from the Cistanche deserticola group (P≤0.05).

[0030] The experimental results in Tables 1, 2, and 3 show that, compared with the formula containing Cistanche deserticola alone (Group 1), the formula containing Dendrobium huoshanense protocorms and Cistanche deserticola (Group 2) can significantly prolong the exhaustive swimming time of mice (P≤0.05), significantly reduce the blood urea nitrogen content in mouse serum (P≤0.05), significantly reduce the lactic acid content in mouse serum (P≤0.05), significantly increase the liver glycogen content in liver tissue (P≤0.05), and significantly increase the muscle glycogen content in muscle tissue (P≤0.05).

[0031] Example 3

[0032] A verification experiment on the effect of Dendrobium huoshanense protocorms on enhancing the kidney-tonifying and aphrodisiac efficacy of a formula containing Cistanche deserticola, American ginseng, Rubus idaeus, Cornus officinalis, and Lycium barbarum was conducted using rats as experimental animals. Rats were randomly divided into a blank control group (group 0), a kidney-yang deficiency model group (group M), a Cistanche deserticola formula group (group 1), and a Dendrobium huoshanense protocorms + Cistanche deserticola formula group (group 2). The blank control group and the kidney-yang deficiency model group were administered distilled water by gavage. The Cistanche deserticola formula group was administered a biomimetic extract of the Cistanche deserticola formula by gavage. The Dendrobium huoshanense protocorms + Cistanche deserticola formula group was administered a biomimetic extract of the Dendrobium huoshanense protocorms + Cistanche deserticola formula by gavage. The dosage of all extracts was 900 mg / kg, and the gavage volume was 1.0 ml. Administration was twice daily for 30 consecutive days.

[0033] Starting from day 1 of the experiment, rats in the kidney-yang deficiency model group, the Cistanche deserticola formula group, and the Dendrobium huoshanense protocorm + Cistanche deserticola formula group were subcutaneously injected with hydrocortisone injection at a dose of 10 mg / kg, once daily for 15 consecutive days, to establish a kidney-yang deficiency rat model. On day 30 after the first administration, serum superoxide dismutase activity and malondialdehyde content were measured; serum testosterone content was measured; penile nitric oxide synthase activity was measured; and penile nitric oxide and cyclic guanosine monophosphate (cGMP) content were measured.

[0034] The results of the verification experiment on the effect of Dendrobium huoshanense protocorm on enhancing the kidney-tonifying and aphrodisiac efficacy of the formula of Cistanche deserticola, American ginseng, raspberry, Cornus officinalis, and wolfberry are shown in Tables 4, 5, 6, and 7.

[0035] Table 4. Results of superoxide dismutase activity and malondialdehyde content in rat serum.

[0036] Group Superoxide dismutase activity (U / mL) Malondialdehyde content (mmol / mL) Group 0 717.12±20.69 7.58±0.69 Group M 652.34±19.76 9.32±1.01 Group 1 <![CDATA[688.59±19.93 c ]]> <![CDATA[8.67±0.73 c ]]> Group 2 <![CDATA[705.72±20.27 cb ]]> <![CDATA[7.81±0.81 cb ]]>

[0037] Note: c indicates a significant difference from the model group (P≤0.05), b indicates a significant difference from the Cistanche deserticola group (P≤0.05).

[0038] Table 5 Results of testosterone content test in rat serum

[0039] Group Testosterone content (nmol / L) Group 0 34.99±2.39 Group M 28.36±2.13 Group 1 <![CDATA[30.57±1.97 c ]]> Group 2 <![CDATA[33.02±2.21 cb ]]>

[0040] Note: c indicates a significant difference from the model group (P≤0.05), b indicates a significant difference from the Cistanche deserticola group (P≤0.05).

[0041] Table 6 Results of nitric oxide synthase activity assay in rat penile tissue

[0042] Group Nitric oxide synthase activity (U / mgprot) Group 0 1.69±0.27 Group M 1.31±0.14 Group 1 <![CDATA[1.45±0.19 c ]]> Group 2 <![CDATA[1.72±0.21 cb ]]>

[0043] Note: c indicates a significant difference from the model group (P≤0.05), b indicates a significant difference from the Cistanche deserticola group (P≤0.05).

[0044] Table 7 Results of nitric oxide and cyclic guanosine monophosphate content tests in rat penile tissue

[0045] Group Nitric oxide content (μmol / mgprot) Cyclic guanosine monophosphate (nmol / mg prot) Group 0 17.92±2.06 7.85±1.96 Group M 10.45±2.67 4.36±1.61 Group 1 <![CDATA[16.05±3.13 c ]]> <![CDATA[7.76±1.73 c ]]> Group 2 <![CDATA[18.37±2.72 cb ]]> <![CDATA[8.89±1.98 cb ]]>

[0046] Note: c indicates a significant difference from the model group (P≤0.05), b indicates a significant difference from the Cistanche deserticola group (P≤0.05).

[0047] The experimental results in Tables 4, 5, 6, and 7 show that, compared with the formula containing Cistanche deserticola alone (Group 1), the formula containing Dendrobium officinale protocorms and Cistanche deserticola (Group 2) significantly increased the activity of superoxide dismutase in rat serum (P≤0.05), significantly decreased the content of malondialdehyde in serum (P≤0.05), significantly increased the content of testosterone in serum (P≤0.05), significantly increased the activity of nitric oxide synthase in penile tissue (P≤0.05), significantly increased the content of nitric oxide in penile tissue (P≤0.05), and significantly increased the content of cyclic guanosine monophosphate in penile tissue (P≤0.05).

[0048] The above description is only a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made to the content of the present invention without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of Dendrobium huoshanense protocorms in the preparation of traditional Chinese medicine preparations with anti-fatigue and kidney-tonifying and aphrodisiac effects, characterized in that, The traditional Chinese medicine preparation includes Dendrobium huoshanense protocorms, Cistanche deserticola, American ginseng, Rubus idaeus, Cornus officinalis and Lycium barbarum. The Dendrobium huoshanense protocorms are undifferentiated Dendrobium huoshanense protocorms. The traditional Chinese medicine preparation is prepared by the following steps: (1) Add 3 parts by weight of undifferentiated Dendrobium huoshanense protocorms to a formula consisting of 5 parts by weight of Cistanche deserticola, 5 parts by weight of American ginseng, 3 parts by weight of Rubus idaeus, 2 parts by weight of Cornus officinalis and 1 part by weight of Lycium barbarum, and then pulverize it into ultrafine powder. (2) The ultrafine powder was added to a gastric juice simulation solution with pH 2.0 and pepsin activity of 2000 U / mL at a material-to-liquid ratio of 1:

50. After stirring at 37°C for 2 hours, the pH was adjusted to 6.8 with 1 mol / L NaOH solution to inactivate the pepsin. Then, trypsin and pancreatic amylase were added until the activity of each was 100 U / mL. After stirring at 37°C for 2 hours, the mixture was heated to 95°C and held for 5 minutes to inactivate the enzymes. The supernatant was filtered and collected. The supernatant was concentrated and dried to obtain the traditional Chinese medicine preparation.

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