A method for producing salidroside from rhizoma et radix rhodiolae crenulatae adventitious roots in suspension culture and a culture medium

Abstract

This invention provides a method and culture medium for producing rhodioloside from adventitious roots of Rhodiola rosea via suspension culture. The method employs a segmented liquid suspension culture technique, comprising: establishing a sterile seedling system for Rhodiola rosea, obtaining sterile seedlings through specific sterilization and culture conditions; liquid culture and acclimatization of adventitious roots, shortening the growth cycle to 35-40 days and improving growth vigor by utilizing optimized culture media and acclimatization strategies; and optimizing the culture for efficient accumulation of rhodioloside, transferring adventitious roots to a specific induction medium and culturing them on a shaker for 8-10 days, ultimately achieving a rhodioloside content ≥0.6%. This invention eliminates the need for natural root collection, avoiding damage to wild Rhodiola rosea resources. It features a short production cycle, high efficiency, stable quality, and no heavy metal or pesticide residues, enabling large-scale industrial production to meet the demands of the pharmaceutical and health product markets. Simultaneously, it reduces reliance on wild resources, contributing to the sustainable development and utilization of endangered traditional Chinese medicine.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method and culture medium for producing rhodioloside by suspension culture of adventitious roots of Rhodiola rosea. Background Technology

[0002] Rhodiola crenulata (Hook.f. & Thomson) H. Ohba is a perennial herbaceous plant belonging to the genus Rhodiola in the family Crassulaceae. It typically grows in grassy slopes, thickets, and rock crevices at altitudes of 2800–5600 meters, preferring high-altitude, cool mountain environments and stony soils. Rhodiola crenulata is listed as Endangered (EN) on the IUCN Red List of Threatened Species. Due to its long-term exposure to harsh environments characterized by oxygen deficiency, low temperatures, strong ultraviolet radiation, and large diurnal temperature variations, Rhodiola crenulata has developed exceptional resilience and adaptability.

[0003] The roots of Rhodiola rosea are rich in various active ingredients, including abundant rhodioloside, starch, protein, flavonoids, and a small amount of volatile oil. They possess properties that help treat physical weakness, delay aging, combat fatigue, and resist hypoxia, making them widely applicable in the medical and health fields. However, because Rhodiola rosea primarily grows in high-altitude, cold mountainous areas at elevations of 4000m–5100m, this unique growing environment presents significant challenges to its collection and cultivation. Furthermore, due to its immense value, over-harvesting has led to a sharp decline in the population of Rhodiola rosea. Rhodiola rosea is listed as an endangered (EN) species on the IUCN Red List of Threatened Species, severely limiting its exploitation and utilization.

[0004] Currently, the development of Rhodiola rosea mainly relies on the extraction of rhodioloside from its natural roots. However, due to its long growth cycle and limited distribution area, resources are insufficient to meet market demand. The harsh environment of high-altitude mountainous areas increases the difficulty of collection, while over-harvesting has led to its endangerment and affected sustainable utilization. At the same time, wild growth conditions make it difficult to control the purity and stability of active ingredients.

[0005] To address the aforementioned issues, the suspension culture technology of adventitious roots has gradually become a research hotspot in recent years. Adventitious roots grow much faster than natural plant roots, significantly increasing biomass accumulation. They also possess a high capacity for producing secondary metabolites, effectively accumulating target components such as rhodioloside. Through liquid suspension culture, adventitious roots are not constrained by environmental conditions, allowing for the artificial regulation of secondary metabolite accumulation. Furthermore, the culture process is free from environmental pollution, and the products are free of heavy metals, pesticide residues, and toxic side effects. This represents a crucial approach to the sustainable utilization of rare and endangered medicinal plants whose roots are used in traditional medicine, aligning with the development direction of modern green medicine.

[0006] Therefore, it is necessary to provide a liquid suspension culture method based on the adventitious roots of Rhodiola rosea to achieve efficient production of rhodioloside, thereby solving problems such as limited resources, difficult collection, endangerment due to over-harvesting, and uncontrollable component quality, and ensuring the sustainable development and utilization of Rhodiola rosea. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a method and culture medium for producing rhodioloside through suspension culture of Rhodiola rosea adventitious roots. This invention primarily utilizes MS as the basal medium, regulating the growth of adventitious roots and the accumulation of rhodioloside by altering the carbon source, hormones, and pH. By adjusting the composition and culture conditions of the Rhodiola rosea adventitious root culture medium, the most favorable culture method for adventitious root growth and rhodioloside accumulation is derived. This invention not only overcomes the problems of limited natural resources, difficult harvesting, and unstable component quality, but also ensures a green and safe production process, free of heavy metals and pesticide residues, meeting market demand and promoting the sustainable development and utilization of Rhodiola rosea.

[0008] The technical means employed in this invention are as follows:

[0009] A method for producing rhodiolosides by suspension culture of adventitious roots of Rhodiola rosea includes the following steps:

[0010] Establishment of sterile seedling system: The terminal stems of Rhodiola rosea were disinfected and inoculated into regeneration medium, and cultured under preset light and temperature conditions to obtain sterile seedlings;

[0011] Adventitious root liquid culture and acclimatization: Vigorous roots were cut from sterile seedlings and transferred to adventitious root liquid culture medium. Under preset temperature, rotation speed and aeration conditions, adventitious roots were induced and the growth cycle was shortened through multiple acclimatizations.

[0012] Optimization of culture for efficient accumulation of rhodiolosides: The cultured adventitious roots were transferred to Rhodiola induction medium and cultured under preset temperature and rotation speed conditions to increase the content of rhodiolosides;

[0013] Adventitious root harvesting and testing: After harvesting and culturing, the adventitious roots were freeze-dried and the content of rhodioloside was determined.

[0014] Furthermore, the method for establishing the aseptic seedling system includes: rinsing the top stem of Rhodiola rosea with running water for 30 minutes; rinsing once with sterile water under sterile conditions, absorbing the water with absorbent paper, soaking in 75% alcohol for 1 minute, rinsing 6 times with sterile water, soaking the roots, leaves, and stems in 0.1% HgCl2 solution for 5 minutes, rinsing 6 times with sterile water, absorbing the water, inoculating into regeneration medium, and culturing under light at 20°C for 40 days to obtain aseptic seedlings.

[0015] Furthermore, the regeneration medium is formulated as follows: MS (sucrose-free, agar-based) medium is used as the base medium; 30 g / L sucrose, 2.0 mg / L 6-BA (6-benzylaminopurine), 0.25 mg / L IAA (indole-3-acetic acid), and 6 g / L agar are added; the pH is adjusted to 5.5-6.0.

[0016] Furthermore, the method for liquid culture and acclimatization of adventitious roots includes: cutting vigorous roots from sterile seedlings into 1cm segments and transferring them to liquid culture medium for adventitious roots; culturing them in the dark for 55-60 days at 12-15℃ with a shaking speed of 90rpm or an aeration rate of 0.5V / V·min; cutting off vigorous new adventitious roots and transferring them to liquid culture medium again, and performing multiple acclimatization processes to shorten the growth cycle of adventitious roots to 35-40 days.

[0017] Furthermore, the formulation of the adventitious root liquid culture medium is as follows: MS (without sucrose or agar) is used as the basal medium; 40 g / L sucrose, 1.0 mg / L NAA (naphthaleneacetic acid), 2.5 mg / L IBA (3-indolebutyric acid), 0.25 mg / L 6-BA, 1.5 mg / L potassium humate, 4.0 mg / L Vc (ascorbic acid), and 0.1-1.0 mg / L β-nicotinamide are added; the pH is adjusted to 5.5-6.0.

[0018] Furthermore, the optimized method for accumulating rhodiolosides includes: transferring adventitious roots from liquid culture to Rhodiola rosea induction medium; and harvesting after culturing in the dark for 8-10 days at 25°C and 150 rpm on a shaker.

[0019] Furthermore, the formulation of the Rhodiola rosea induction medium is as follows: 1 / 2 MS (excluding sucrose and agar) is used as the basal medium; 20 g / L sucrose, 1.0 mg / L NAA, 2.5 mg / L IBA, 0.25 mg / L 6-BA, 4 mg / L 2,4-D, 1.5 mg / L potassium humate, 4.0 mg / L vitamin C, and 30 mg / L phenylalanine are added; the pH is adjusted to 4.5.

[0020] Furthermore, after the adventitious roots were harvested and cultured, and then freeze-dried, the content of rhodioloside was detected to be ≥0.6%.

[0021] The present invention also provides a culture medium for the above-mentioned method of producing rhodioloside by suspension culture of adventitious roots of Rhodiola rosea, including a regeneration culture medium, the formulation of which is: MS (sucrose-free, agar-free) as the base medium, with the addition of 30 g / L sucrose, 2.0 mg / L 6-BA, 0.25 mg / L IAA, and 6 g / L agar; the pH value is adjusted to 5.5-6.0.

[0022] Furthermore, it also includes an adventitious root liquid culture medium, the formulation of which is: MS (without sucrose or agar) as the basal medium, with the addition of 40 g / L sucrose, 1.0 mg / L NAA, 2.5 mg / L IBA, 0.25 mg / L 6-BA, 1.5 mg / L potassium humate, 4.0 mg / L vitamin C, and 0.1-1.0 mg / L β-nicotinamide, and the pH is adjusted to 5.5-6.0;

[0023] Furthermore, it also includes a Rhodiola rosea induction medium, the formulation of which is: based on 1 / 2 MS (without sucrose and agar), 20 g / L sucrose, 1.0 mg / L NAA, 2.5 mg / L IBA, 0.25 mg / L 6-BA, 4 mg / L 2,4-D, 1.5 mg / L potassium humate, 4.0 mg / L vitamin C, and 30 mg / L phenylalanine, and the pH is adjusted to 4.5.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] 1. This invention employs segmented liquid suspension culture to achieve efficient proliferation of adventitious roots and enrichment of secondary metabolites. Through repeated domestication and optimization of the growth environment of adventitious roots, the growth cycle of adventitious roots is shortened from 55-60 days in the traditional method to 35-40 days, thereby increasing the yield of rhodioloside.

[0026] 2. This invention precisely optimizes the culture medium composition, using MS or 1 / 2 MS medium, and adds specific hormones and inducing factors such as NAA, IBA, 6-BA, 2,4-D, potassium humate, vitamin C, β-nicotinamide, and phenylalanine to promote the rapid growth of adventitious roots and the efficient accumulation of rhodioloside. This enables adventitious roots to synthesize rhodioloside efficiently in a short time, ultimately obtaining adventitious roots with a rhodioloside content ≥0.6%.

[0027] 3. This invention uses liquid suspension culture technology, which enables large-scale and stable production of adventitious roots rich in rhodioloside under conditions such as temperature, rotation speed and aeration rate controlled by a shaker. It is characterized by high efficiency, standardization and strong controllability, which greatly improves the feasibility of industrial production compared with traditional wild harvesting methods.

[0028] In summary, this invention has significant advantages in increasing the yield of rhodioloside, shortening the production cycle, improving production stability, and achieving sustainable development. The adventitious roots obtained are rich in rhodioloside and free of heavy metals and pesticide residues, making them suitable for large-scale industrial production. They can be used not only as active pharmaceutical ingredients for pharmaceutical research on anti-fatigue, antioxidant, and immune-enhancing effects, but also as raw materials for functional foods or health products, meeting market demand for rhodioloside and providing a new path for the sustainable development and utilization of Rhodiola rosea. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 This is a photograph of the sterile seedlings cultured using the sterile seedling system of the present invention in Example 1.

[0031] Figure 2 This is a photograph of the adventitious roots of Rhodiola rosea cultivated using the method of the present invention in Example 1.

[0032] Figure 3 The chromatogram shows the detection of rhodioloside in the adventitious roots of Rhodiola rosea cultivated using the method of the present invention in Example 1.

[0033] Figure 4 This is a photograph of the adventitious roots of Rhodiola rosea grown for 60 days without domestication, as shown in Comparative Example 1.

[0034] Figure 5 The chromatogram shows the detection of rhodioloside in the adventitious roots of uncultured plants in Comparative Example 1.

[0035] Figure 6 This is a photograph of the adventitious roots of Rhodiola rosea after 40 days of domestication, as shown in Comparative Example 2.

[0036] Figure 7 The chromatogram shows the detection of rhodioloside in adventitious roots after domestication in Comparative Example 2. Detailed Implementation

[0037] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] Example 1

[0039] This invention provides a method for producing rhodioloside through suspension culture of adventitious roots of Rhodiola rosea, comprising the following specific steps:

[0040] (1) Establishment of a sterile seedling system for Rhodiola rosea:

[0041] Rhodiola rosea regeneration medium was prepared according to the following formula: 4.74 g / L MS (sucrose-free, agar-free), 30 g / L sucrose, 2.0 mg / L 6-BA, 0.25 mg / L IAA, and 6 g / L agar. The medium was sterilized and ready for use. The top stems of Rhodiola rosea were rinsed with running water for 30 min, then transferred to a sterile environment and rinsed once with sterile water. The water was then blotted dry with absorbent paper, soaked in 75% alcohol for 1 min, rinsed 6 times with sterile water, and then soaked in 0.1% HgCl2 for 5 min. The roots, leaves, and stems were rinsed 6 times with sterile water, blotted dry, and inoculated onto the regeneration medium. The medium was then cultured at 20°C under light for 40 days to obtain regenerated sterile seedlings (e.g., ...). Figure 1 (As shown).

[0042] (2) Liquid culture and acclimatization of adventitious roots:

[0043] Prepare a liquid culture medium for adventitious roots of Rhodiola rosea according to the following formula: 4.74 g / L MS (excluding sucrose and agar), 40 g / L sucrose, 1.0 mg / L NAA, 2.5 mg / L IBA, 0.25 mg / L 6-BA, 1.5 mg / L potassium humate, 4.0 mg / L vitamin C, and 0.5 mg / L β-nicotinamide. Adjust the pH to 5.8 and sterilize before use.

[0044] Roots of *Rhodiola rosea* with vigorous growth, no browning, and strong meristem activity, grown on regeneration medium, were cut from sterile seedlings and inoculated into the aforementioned adventitious root liquid medium. The roots were cultured in the dark at 15°C and 90 rpm for 60 days until new adventitious roots emerged. Vigorous new adventitious roots were then transferred again to the liquid medium for further culture. After repeated acclimatization and three cycles of culture, the growth cycle of the adventitious roots was shortened to 35-40 days.

[0045] (3) Optimization of culture for efficient accumulation of rhodioloside in adventitious roots of Rhodiola rosea:

[0046] Using 1 / 2 MS (without sucrose or agar) as the basal medium, 20 g / L sucrose, 1.0 mg / L NAA, 2.5 mg / L IBA, 0.25 mg / L 6-BA, 4 mg / L 2,4-D, 1.5 mg / L potassium humate, 4.0 mg / L vitamin C, and 30 mg / L phenylalanine were added. The pH was adjusted to 4.5 to prepare Rhodiola rosea induction medium, which was then sterilized and set aside for use. The adventitious roots of Rhodiola rosea cultured in liquid in step (2) were transferred to the above Rhodiola rosea induction medium and cultured in the dark at 25°C and 150 rpm for 10 days to obtain adventitious roots (such as...). Figure 2 (As shown).

[0047] (4) After freeze-drying the adventitious roots of Rhodiola rosea obtained in (3), the content of rhodioloside was determined to be 0.63% using Waters UPLC H-Class ultra-high performance liquid chromatography, referring to the method in the 2020 edition of the Pharmacopoeia of the People's Republic of China. The chromatogram is shown below. Figure 3 As shown.

[0048] Comparative Example 1

[0049] Aseptic seedlings were established according to the method in Implementation Case 1. Roots of *Rhodiola rosea* species, exhibiting vigorous growth, no browning, and strong meristematic ability, grown on regeneration medium, were cut from the aseptic seedlings and inoculated into the aforementioned adventitious root liquid medium. The plants were cultured in the dark at 15°C and 90 rpm on a shaker for 60 days. After new adventitious roots had grown, they were recovered, freeze-dried, and the rhodioloside content was determined to be 0.12%. The adventitious roots were as follows... Figure 4 As shown, the chromatogram is as follows Figure 5 As shown.

[0050] Comparative Example 2

[0051] Aseptic seedlings were established according to the method in Implementation Case 1. Roots of *Rhodiola rosea* species, exhibiting vigorous growth, no browning, and strong meristematic ability, grown on regeneration medium, were cut from the aseptic seedlings and inoculated into the adventitious root liquid medium. The seedlings were cultured in the dark at 15°C and 90 rpm under shaking conditions for 60 days. After acclimatization, the roots were cultured for 40 days and then recovered and freeze-dried. The rhodioloside content was found to be 0.28%. The adventitious roots were as follows... Figure 6 As shown, the chromatogram is as follows Figure 7 As shown.

[0052] It can be seen that Comparative Example 1, where Rhodiola rosea was directly cut from sterile seedlings and cultured in adventitious root liquid culture medium for 60 days, yielded short adventitious roots with the lowest rhodioloside content. Comparative Example 2 involved acclimating the adventitious roots from Comparative Example 1 to better adapt them to the liquid culture system, resulting in longer adventitious roots with an increased rhodioloside content compared to Comparative Example 1. Example 1 further induced the acclimated adventitious roots to accumulate more rhodioloside, and liquid chromatography results also confirmed a higher Rhodiola rosea content. The results indicate that the culture method provided by this invention is most conducive to adventitious root growth and rhodioloside accumulation.

[0053] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for producing rhodioloside through suspension culture of adventitious roots of Rhodiola rosea, characterized in that, Includes the following steps: Establishment of a sterile seedling system: The terminal stems of Rhodiola rosea were disinfected and inoculated into a regeneration medium. The seedlings were cultured under pre-defined light and temperature conditions to obtain sterile seedlings. The regeneration medium consisted of MS basal medium, 30 g / L sucrose, 2.0 mg / L 6-BA, 0.25 mg / L IAA, and 6 g / L agar; the pH was adjusted to 5.5-6.

0. Adventitious root liquid culture and acclimatization: Vigorous roots were cut from sterile seedlings and transferred to adventitious root liquid culture medium. Under preset temperature, rotation speed, and aeration conditions, adventitious roots were induced, and the growth cycle was shortened through repeated acclimatization. The adventitious root liquid culture medium consisted of MS basal medium, 40 g / L sucrose, 1.0 mg / L NAA, 2.5 mg / L IBA, 0.25 mg / L 6-BA, 1.5 mg / L potassium humate, 4.0 mg / L VLC, and 0.1-1.0 mg / L β-nicotinamide; the pH was adjusted to 5.5-6.

0. Optimization of culture for efficient accumulation of rhodiolosides: Adventitious roots were transferred to Rhodiola rosea induction medium and cultured under preset temperature and rotation speed conditions to increase the content of rhodiolosides. The Rhodiola rosea induction medium consisted of 1 / 2 MS basal medium, 20 g / L sucrose, 1.0 mg / L NAA, 2.5 mg / L IBA, 0.25 mg / L 6-BA, 4 mg / L 2,4-D, 1.5 mg / L potassium humate, 4.0 mg / L vitamin C, and 30 mg / L phenylalanine; the pH was adjusted to 4.

5. Adventitious root harvesting and testing: After harvesting and culturing, the adventitious roots were freeze-dried and the content of rhodioloside was determined.

2. The method for producing rhodioloside by suspension culture of adventitious roots of Rhodiola rosea according to claim 1, characterized in that, The method for establishing the aseptic seedling system includes: rinsing the top stem of Rhodiola rosea with running water for 30 minutes; rinsing once with sterile water under sterile conditions, absorbing the water with absorbent paper, soaking in 75% alcohol for 1 minute, rinsing 6 times with sterile water, soaking the roots, leaves and stems in 0.1% HgCl2 solution for 5 minutes, rinsing 6 times with sterile water, absorbing the water, inoculating into regeneration medium, and culturing under light at 20℃ for 40 days to obtain aseptic seedlings.

3. The method for producing rhodioloside by suspension culture of adventitious roots of Rhodiola rosea according to claim 1, characterized in that, The method for liquid culture and acclimatization of adventitious roots includes: cutting vigorous roots from sterile seedlings into 1cm segments and transferring them to liquid culture medium for adventitious roots; culturing them in the dark for 55-60 days at 12-15℃ with a shaking speed of 90rpm or an aeration rate of 0.5V / V·min; cutting off vigorous new adventitious roots and transferring them to liquid culture medium again, and performing multiple acclimatization processes to shorten the growth cycle of adventitious roots to 35-40 days.

4. The method for producing rhodioloside by suspension culture of adventitious roots of Rhodiola rosea according to claim 1, characterized in that, The method for optimizing the cultivation of highly efficient rhodioloside accumulation includes: transferring adventitious roots from liquid culture to Rhodiola induction medium; and harvesting after culturing in the dark for 8-10 days at 25°C and 150 rpm on a shaker.

5. The method for producing rhodioloside by suspension culture of adventitious roots of Rhodiola rosea according to claim 4, characterized in that, After harvesting and culturing, the adventitious roots were freeze-dried, and the content of rhodioloside was detected to be ≥0.6%.

6. A culture medium for producing rhodioloside using the suspension culture method of Rhodiola rosea adventitious roots according to any one of claims 1-5, characterized in that, The medium includes a regeneration medium, an adventitious root liquid medium, and a Rhodiola rosea induction medium. The regeneration medium consists of MS basal medium, 30 g / L sucrose, 2.0 mg / L 6-BA, 0.25 mg / L IAA, and 6 g / L agar, with the pH adjusted to 5.5-6.

0. The terminal stems of Rhodiola rosea are inoculated into the regeneration medium for culture. The adventitious root liquid culture medium consists of MS basal medium, 40 g / L sucrose, 1.0 mg / L NAA, 2.5 mg / L IBA, 0.25 mg / L 6-BA, 1.5 mg / L potassium humate, 4.0 mg / L vitamin C, and 0.1-1.0 mg / L β-nicotinamide, with the pH adjusted to 5.5-6.

0. The Rhodiola rosea induction medium consisted of 1 / 2 MS basal medium, 20 g / L sucrose, 1.0 mg / L NAA, 2.5 mg / L IBA, 0.25 mg / L 6-BA, 4 mg / L 2,4-D, 1.5 mg / L potassium humate, 4.0 mg / L vitamin C, and 30 mg / L phenylalanine, with the pH adjusted to 4.5.

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