A method for extracting active components from saussurea involucrata

Abstract

The present application relates to a kind of methods for extracting active ingredients of Saussurea, including anisodine and umbelliferone, characterized in that, the extraction method includes the following steps: (1) obtain Saussurea paste; (2) 3-10% mass of ammonium bicarbonate is added in Saussurea paste, and the extract is obtained under the condition of ultrasonic power 200-400W, ultrasonic frequency 30-50kHz, room temperature for 30-50 minutes; (3) the extract is concentrated to relative density 1.1-1.3g / ml under reduced pressure to obtain extract infusion; (4) 0.8-1.5 times volume of cyclohexane is added in extract infusion for extraction, and the supernatant is concentrated under reduced pressure to obtain the final Saussurea extract. By using the method of the present application, the content of anisodine and umbelliferone in Saussurea extract can be effectively improved.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical processing technology, and more specifically to a method for extracting active ingredients from snow lotus, wherein the active ingredients include scopolamine lactone and umbelliferone. Background Technology

[0002] Snow lotus (Saussurea DC.) belongs to the genus Saussurea in the tribe Tribe (Cynareae Less.) of the family Compositae. It is a precious traditional Chinese medicine commonly used in high-altitude and cold regions. Records of snow lotus can be found as early as the 8th century in the ancient Tibetan medical text *Yue Wang Yao Zhen*. Later, it was also documented in *Western Medical Canon*, *Northwest Regions Records*, *Lan Liuli*, *Jingzhu Materia Medica*, and the Qing Dynasty's *Compendium of Materia Medica Supplement*. It is a commonly used ethnic medicine in the high-altitude regions of Tibet, Xinjiang, Qinghai, Gansu, Yunnan, and Sichuan. *Xinjiang Traditional Chinese Medicine* states that snow lotus is warm in nature, slightly bitter, and functions to dispel wind and dampness, possessing special efficacy in treating rheumatic diseases. Clinical pharmaceutical research has proven that the whole snow lotus plant, when used medicinally, possesses functions such as promoting blood circulation, warming the uterus and dispersing blood stasis, dispelling cold and dampness, strengthening muscles and boosting yang, stopping bleeding, and reducing swelling. Clinically, it is mainly used to treat pain caused by acute and chronic rheumatoid arthritis, cough due to lung cold, abdominal pain due to uterine cold, amenorrhea, retained placenta, impotence, and measles that fail to erupt. In recent years, snow lotus, as a traditional Chinese medicine, has received considerable attention for its anti-inflammatory, analgesic, anti-early pregnancy, and anti-aging effects. With the continuous deepening of basic research, it has been discovered that snow lotus has effects such as scavenging free radicals, anti-fatigue, enhancing immunity, antibacterial, antispasmodic, sedative, antihypertensive, anti-gastric ulcer, and anti-tumor properties.

[0003] Snow lotus typically grows below the snow line in high-altitude areas. The climate there is highly variable, with unpredictable temperatures and alternating periods of rain and snow. The highest average monthly temperature is 3-5℃, and the lowest average monthly temperature is -19 to 21℃. Annual precipitation is approximately 800 mm, and the frost-free period is only about 50 days. The soil is primarily alpine meadow soil, with an organic matter content of 8.5-11% and a nitrogen content of 4.5-10%. Under these harsh high-altitude conditions, snow lotus grows slowly, taking at least 4-5 years to flower and bear fruit.

[0004] The cells of the snow lotus accumulate large amounts of soluble sugars, proteins, and lipids, which lowers the freezing point of the protoplasm. When the temperature drops below the freezing point of the protoplasm, water from the protoplasm seeps into the intercellular spaces and the spaces where the plasmolysis occurs, freezing thereafter. Meanwhile, the protoplast gradually shrinks without being damaged. When the weather warms up, the ice melts, the water is reabsorbed by the protoplast, and the cells return to normal.

[0005] Nearly a hundred compounds have been isolated from snow lotus, among which the main active ingredients include costunolide, dehydrocostunolide, lignans, arctiin, snow lotus flavonoids, apigenin, p-hydroxyacetophenone, umbelliferone, various secondary metabolites, jaceosidin, hispidulin, and snow lotus polysaccharides, all of which are effective components with various pharmaceutical activities. Currently, snow lotus products are widely used not only in medicine but also in the cosmetics and health product industries. Scopolamine and umbelliferone are the most important active components in snow lotus. Umbelliferone has significant antibacterial, hypotensive, sedative, and antispasmodic effects; scopolamine has antirheumatic, anti-inflammatory, analgesic, expectorant, and antitumor effects, and its clinical efficacy in treating acute and chronic bronchitis is 96.6%.

[0006] Long-term research has been conducted to extract active ingredients from snow lotus. For example, Chinese authorized patent CN103550275B discloses a method for extracting snow lotus essence, including the following steps: (1) pretreatment of snow lotus and purification of water; (2) extraction of pretreated snow lotus and purified water; (3) coarse filtration of cooled snow lotus soup followed by fine separation; (4) further filtration of the finely filtered liquid using a microporous membrane with a pore size of 0.25-0.45um; (5) concentration of the purified liquid using a reverse osmosis membrane at room temperature; (6) further concentration of the snow lotus soup concentrated by the reverse osmosis membrane under reduced pressure; (7) finally obtaining the essence extract. For example, Chinese authorized patent CN104147071B discloses a method for extracting effective components from snow lotus, characterized by the following steps: (1) Take completely dried snow lotus, remove impurities manually, chop into pieces about 2mm long and wide, add soft water, and grind into a paste in a mixer to obtain snow lotus paste; (2) Heat the snow lotus paste to 50-60℃, add cellulase under heat preservation conditions for hydrolysis to obtain snow lotus enzymatic hydrolysate; heat preservation time is 1-3 hours; (3) Heat the snow lotus enzymatic hydrolysate to 80-90℃ and keep it warm for 4-6 minutes to inactivate the added cellulase; (4) Filter the inactivated snow lotus enzymatic hydrolysate through a 400-mesh sieve and take the filtrate for later use; (5) Heat the filtrate to above 60℃, then add food-grade maltodextrin and concentrated whey protein, stir to dissolve and homogenize to form a uniform and stable snow lotus solution. (6) A homogeneous and stable snow lotus solution is spray-dried to form snow lotus powder in microcapsule form. However, the above method is not specifically for the extraction of scopolamine and umbelliferone, which results in a limited content of scopolamine and umbelliferone in the extract, thus limiting its application. Summary of the Invention

[0007] Based on the above background technology, the technical problem to be solved by the present invention is to provide a method for extracting active ingredients from snow lotus, so as to increase the content of hyoscyamine butylide and umbelliferone in the extract. To achieve the objective of the present invention, the following technical solution is proposed: This invention relates to a method for extracting active ingredients from snow lotus, the active ingredients including scopolamine lactone and umbelliferone, characterized in that the extraction method includes the following steps: (1) Take a dried and whole snow lotus, remove impurities, chop it slightly, add 1-12 times the weight of the snow lotus in deionized water, and blend it in a mixer until it becomes a paste to obtain snow lotus paste.

[0008] (2) Add 3-10% ammonium bicarbonate to the snow lotus paste and extract for 30-50 minutes under ultrasonic power of 200-400W, ultrasonic frequency of 30-50kHz and room temperature to obtain the extract. (3) Concentrate the extract under reduced pressure until the relative density is 1.1-1.3 g / ml to obtain the extract paste; (4) Add 0.8-1.5 times the volume of cyclohexane to the extract for extraction, and concentrate the supernatant under reduced pressure to obtain the final snow lotus extract.

[0009] In a preferred embodiment of the present invention, in step (1), the snow lotus is shredded into fragments with a length and width of 1-3 mm using a shredder.

[0010] In a preferred embodiment of the present invention, in step (2), the amount of ammonium bicarbonate is 4-6% by mass. This further facilitates the extraction of the active ingredients scopolamine and umbelliferone.

[0011] In a preferred embodiment of the present invention, in step (2), the power of the ultrasonic wave is 250-350W.

[0012] In a preferred embodiment of the present invention, in step (4), the volume ratio of cyclohexane to extract is 1:1.

[0013] In a preferred embodiment of the present invention, the content of hyoscyamine lactone in the final snow lotus extract is 0.7-0.9 mg / g.

[0014] In a preferred embodiment of the present invention, the content of umbelliferone in the final snow lotus extract is 1.4-1.7 mg / g.

[0015] In another aspect of the invention, the invention also relates to the final snow lotus extract obtained by the above-described extraction method.

[0016] In another aspect of the invention, the effects of the final snow lotus extract of the invention on antibacterial, anti-inflammatory, and analgesic properties are also involved.

[0017] In another aspect of the invention, the application of the final snow lotus extract of the invention in cosmetics, health products, pharmaceuticals, and food is also described. Beneficial effects

[0018] Compared with existing technologies, the method of this invention can effectively increase the content of scopolamine and umbelliferone in snow lotus extract. While the mechanism is not entirely clear, it is speculated that the decomposition of ammonium bicarbonate under ultrasonic waves produces ammonia, and the introduction of ammonia facilitates the dissolution of scopolamine and umbelliferone. Implementation

[0019] To further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0020] Unless otherwise specified, all reagents involved in the embodiments of this invention are commercially available products and can be purchased through commercial channels. Example

[0021] (1) Take a dried and completely dried snow lotus, remove impurities by hand, and chop it into pieces about 2mm in length and width using a pulverizer. Then add 10 times the weight of the snow lotus to deionized water (i.e., the weight of the snow lotus: the weight of the deionized water = 1:10), and grind it into a paste in a mixer to obtain snow lotus paste.

[0022] (2) Add 5% ammonium bicarbonate to the above snow lotus paste and extract for 40 minutes under ultrasonic power of 300W, ultrasonic frequency of 40kHz and room temperature to obtain the extract. (3) The extract was concentrated under reduced pressure until the relative density was 1.2 g / ml to obtain the extract paste; (4) Add an equal volume of cyclohexane to the extract for extraction, and concentrate the supernatant under reduced pressure to obtain the final snow lotus extract. Example

[0023] Same as Example 1, except that the amount of ammonium bicarbonate is 8%. Example

[0024] Same as Example 1, except that the amount of ammonium bicarbonate is 3%. Example

[0025] Same as Example 1, except that the ultrasonic power is 400W.

[0026] Same as Example 1, except that 5% sodium bicarbonate is added instead of ammonium bicarbonate.

[0027] Similar to Example 1, except that step (2) is performed under rapid stirring at 600 rpm.

[0028] Same as Example 1, except that dichloromethane is used instead of cyclohexane for extraction.

[0029] Same as Example 1, except that diethyl ether is used instead of cyclohexane for extraction.

[0030] Experimental Example 5: Determination of Active Ingredients The determination of active ingredients shall be performed in accordance with the method described in CN104569259B, specifically: (1) Preparation of the test solution Take 1g of extract into a 10ml volumetric flask, add methanol to the mark, and shake well to obtain the test solution. (2) Determination of the content of scopolamine and umbelliferone The experimental conditions for high performance liquid chromatography are as follows: C 18 Chromatographic column: 250 mm × 4.6 mm, 5 μm; Mobile phase: Acetonitrile as mobile phase A, water as mobile phase B; Elution gradient: 0-17 min: 10%-60% A; 17-18 min: 60%-80% A; 18-24 min: 80% A; 24-30 min: 80%-95% A; 30-32 min: 95%-10% A; 32-42 min: 10% A; Elution time: 42 min; UV detector wavelength: 346 nm; Flow rate: 1.0 ml / min; Column temperature: 40 ℃; Sample injection volume: 10 μl; Preparation of scopolamine and umbelliferone standards: Scopolamine reference standard was prepared into reference solutions containing 0.05 mg, 0.04 mg, 0.03 mg, 0.02 mg, and 0.01 mg per ml, respectively; umbelliferone reference standard was prepared into reference solutions containing 0.05 mg, 0.04 mg, 0.03 mg, 0.02 mg, and 0.01 mg per ml, respectively.

[0031] High-performance liquid chromatography (HPLC) determination: Reference solutions of different concentrations were injected at 10 μL, and the peak areas of each standard solution were measured. Standard curves were plotted with the peak areas of each standard reference solution as the ordinate and the corresponding standard reference concentrations as the abscissa, and linear regression equations were calculated. Then, the test solution was subjected to HPLC determination to obtain the peak areas of scopolamine and umbelliferone. The contents of scopolamine and umbelliferone in the test sample were determined according to the standard curves, and the contents of scopolamine and umbelliferone in the test sample were calculated based on the mass of the test sample. The experimental results are shown in Table 1.

[0032] Table 1: Results of the determination of the content of scopolamine and umbelliferone (extraction was repeated 3 times and the results were determined separately). Example 1 0.86±0.11 1.63±0.19 Example 2 0.81±0.09 1.52±0.12 Example 3 0.80±0.08 1.47±0.18 Example 4 0.79±0.10 1.51±0.17 Comparative Example 1 0.48±0.13 0.84±0.14 Comparative Example 2 0.52±0.07 0.76±0.15 Comparative Example 3 0.61±0.09 0.92±0.10 Comparative Example 4 0.57±0.10 1.23±0.13 The above experimental results show that the method of the present invention can effectively increase the content of hyoscyamine and umbelliferone in snow lotus extract.

[0033] The preferred embodiments of the present invention have been described above, but are not intended to limit the invention. Those skilled in the art can make modifications and variations to the embodiments disclosed herein without departing from the scope and spirit of the invention.

Claims

1. A method for extracting active ingredients from snow lotus, said active ingredients comprising scopolamine lactone and umbelliferone, characterized in that, The extraction method includes the following steps: (1) Take a dried and whole snow lotus, remove impurities by hand, chop it with a pulverizer, add 1-12 times the weight of the snow lotus in deionized water, and grind it into a paste in a mixer to obtain snow lotus paste; (2) Add 3-10% ammonium bicarbonate to the snow lotus paste and extract for 30-50 minutes under ultrasonic power of 200-400W, ultrasonic frequency of 30-50kHz and room temperature to obtain the extract. (3) Concentrate the extract under reduced pressure until the relative density is 1.1-1.3 g / ml to obtain the extract paste; (4) Add 0.8-1.5 times the volume of cyclohexane to the extract for extraction, and concentrate the supernatant under reduced pressure to obtain the final snow lotus extract; The final snow lotus extract contained 0.7-0.9 mg / g of hyoscyamine lactone and 1.4-1.7 mg / g of umbelliferone.

2. The method according to claim 1, characterized in that, In step (1), the snow lotus is shredded into pieces with a length and width of 1-3 mm using a shredder.

3. The method according to claim 1, characterized in that, In step (2), the amount of ammonium bicarbonate is 4-6% by mass.

4. The method according to claim 1, characterized in that, In step (2), the ultrasonic power is 250-350W.

5. The method according to claim 1, characterized in that, In step (4), the volume ratio of cyclohexane to extract is 1:

1.

6. The final snow lotus extract obtained by the method described in any one of claims 1-5.