Method for separating and purifying effective components of gaultheria yunnanensis

Abstract

The invention belongs to the technical field of gaultheria yunnanensis extraction, and provides a method for separating and purifying effective components of gaultheria yunnanensis. The method for separating and purifying the effective components of the gaultheria yunnanensis comprises the following steps: mixing gaultheria yunnanensis powder and a solvent, and carrying out ultrasonic extraction to obtain an extracting solution; extracting the extracting solution by adopting a mixed solvent to obtain an extraction phase and a raffinate phase; and removing the solvent from the raffinate phase to obtain the lignan effective component. And removing the solvent from the extract phase to obtain the salicylate effective component. According to the method disclosed by the invention, the gaultheria yunnanensis powder is extracted by using the mixed solvent of the ethyl acetate and the methanol, so that lignan active ingredients and salicylate active ingredients in the gaultheria yunnanensis can be extracted at the same time, and efficient co-extraction of multiple ingredients is realized; a mixed solvent of n-butyl alcohol and dichloromethane is adopted to extract an extracting solution, and lignan effective components and salicylate effective components can be efficiently separated at the same time only through one-time extraction.

Description

Technical Field

[0001] This invention relates to the field of Yunnan white pearl extraction technology, and in particular to a method for separating and purifying the effective components of Yunnan white pearl. Background Technology

[0002] *Gaultheria leucocarpa* Bl. var. *yunnanensis* (Franch.) TZHsuet RC Fang, belonging to the genus *Gaultheria* in the family Ericaceae, is an evergreen shrub and a medicinal plant, with the entire plant used medicinally. Other names for *Gaultheria leucocarpa* include Baizhushu, Touguxiang, Tougucao, Xiao Tougucao, Touguxiao, Manshanxiang, Mantianxiang, Wanlixiang, Jiulixiang, Fangxiangcao, Jiguxiang, Zuangufeng, Huotanzi, Meitanzi, Meitanguo, Zaozaoye, Soushanhu, Xiashanhuang, Xiashanhu, Donglvshu, Jianhuamu, Laohuoniao, Laohumian, Niutouyao, Tuobeiguniang, Naoshilala, Douzhengkong, and Donglian. *Gaultheria leucocarpa* was first recorded in *Diannan Bencao* and is currently documented in both the *Chinese Materia Medica* and *Chinese Ethnic Medicine Records*. Yunnan white pearl mainly contains salicylates, flavonoids, lignans, organic acids, coumarins, terpenes, sterols, and volatile oils. These active ingredients endow Yunnan white pearl with antibacterial, antioxidant, anti-inflammatory, analgesic, wind-dispelling, dampness-removing, heat-clearing, detoxifying, blood-activating, stasis-removing, qi-regulating, asthma-relieving, small intestine-improving, and diarrhea-relieving effects. Therefore, the efficient separation and extraction of the active ingredients of Yunnan white pearl is of great significance to the pharmaceutical field.

[0003] Currently, the extraction and separation of effective components from *Clematis chinensis* mainly employs methods such as water decoction, solvent extraction, or extraction. For example, patent CN102603811A discloses an extraction and separation process for apigenin-7-O-β-D-glucuronide from *Clematis chinensis*. This method involves first extracting with ethanol and then concentrating the extract, followed by solvent extraction with petroleum ether, ethyl acetate, and water-saturated n-butanol, respectively. Finally, the n-butanol extract is purified by column chromatography. This method only successfully separated apigenin-7-O-β-D-glucuronide and did not effectively separate other components from *Clematis chinensis*. Xing Haochun et al., in their study "Study on the Insecticidal Effect of Different Solvent Extracts of *Clematis chinensis* on Aphids," described using methanol cold-soaking of *Clematis chinensis*, followed by extraction with petroleum ether, ethyl acetate, and n-butanol, respectively, to evaluate the bioactivity of the extracts against aphids. This method achieved an extraction rate of only 12.46% for *Clematis chinensis*, with extraction rates ranging from 0.31% to 1.28%, indicating extremely low extraction rates and no clear identification of the specific components in the extract phase. The master's thesis from Northwest A&F University, titled "Extraction Methods and Separation Study of Insecticidal Components from Clematis chinensis," describes cold maceration, ultrasonic extraction, and sequential extraction methods, and studies the extraction of Clematis chinensis roots using different solvents. However, the extraction rates were all <10%. This indicates that existing extraction processes for the effective components of Clematis chinensis suffer from low extraction rates, inability to simultaneously separate multiple effective components, and cumbersome separation processes.

[0004] In summary, the proposed method for separating and purifying the effective components of *Phyllostachys yunnanensis* can effectively improve the extraction efficiency of *Phyllostachys yunnanensis* and achieve the separation and purification of multiple effective components, which is of great significance for the medical applications of *Phyllostachys yunnanensis*. Summary of the Invention

[0005] The purpose of this invention is to provide a method for separating and purifying the effective components of Dianbaizhu (a type of pearl) to address the shortcomings of existing technologies.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for separating and purifying the effective components of Dianbaizhu (a type of pearl), comprising the following steps: 1) Mix Yunnan white pearl powder with solvent and perform ultrasonic extraction to obtain the extract; 2) The extract was extracted using a mixed solvent to obtain the extract phase and the raffinate phase; 3) Remove the solvent from the raffinate to obtain the lignans, the active ingredients. The solvent is removed from the extract phase to obtain the salicylic acid ester active ingredients.

[0007] Preferably, the solvent in step 1) comprises ethyl acetate and methanol.

[0008] Preferably, the mass ratio of ethyl acetate to methanol is 1:0.3~0.8.

[0009] Preferably, the ultrasonic extraction temperature in step 1) is 30~50℃, the ultrasonic extraction power is 30~50W, the time for a single ultrasonic extraction is 2~4h, and the number of ultrasonic extractions is 2~4 times.

[0010] Preferably, the solid-liquid ratio of the ultrasonic extraction in step 1) is 1:8~10.

[0011] Preferably, the mixed solvent in step 2) comprises n-butanol and dichloromethane.

[0012] Preferably, the mass ratio of n-butanol to dichloromethane is 1:1 to 3.

[0013] Preferably, the mass ratio of the mixed solvent in step 2) to the Yunnan white pearl powder in step 1) is 0.8~1.5:1.

[0014] Preferably, the solvent removal in step 3) is carried out by vacuum distillation.

[0015] The beneficial effects of this invention are: 1) This invention uses a mixed solvent of ethyl acetate and methanol to extract *Phyllostachys edulis* powder, which can simultaneously extract lignans and salicylates from *Phyllostachys edulis*. Combined with ultrasonic extraction, the temperature and power of ultrasonic extraction are optimized to achieve co-extraction of multiple components without damaging the active ingredients, and to achieve high-efficiency extraction. A mixed solvent of n-butanol and dichloromethane is used to extract the extract. The properties of the mixed solvent can simultaneously and efficiently separate lignans and salicylates, enabling the directional separation of the two active ingredients. The low-polarity salicylates are selectively extracted into the extract phase by dichloromethane, while the medium- and high-polarity lignans remain in the n-butanol phase as the raffinate. By reasonably setting the mass ratio of n-butanol to dichloromethane, two types of active ingredients with large polarity differences can be separated simultaneously in a single extraction, simplifying subsequent purification steps and improving separation efficiency.

[0016] 2) The present invention extracts the active ingredients in a mixed solvent of ethyl acetate and methanol, and then extracts them in a mixed solvent of n-butanol and dichloromethane, so that the extraction rate of lignan active ingredients reaches 4.5% and the extraction rate of salicylate active ingredients reaches 2.55%, which is significantly better than the separation and purification process of single alcohol extraction, single petroleum ether extraction, ethyl acetate extraction, etc. Detailed Implementation

[0017] This invention provides a method for separating and purifying the effective components of Dianbaizhu (a type of pearl), comprising the following steps: 1) Mix Yunnan white pearl powder with solvent and perform ultrasonic extraction to obtain the extract; 2) The extract was extracted using a mixed solvent to obtain the extract phase and the raffinate phase; 3) Remove the solvent from the raffinate to obtain the lignans, the active ingredients. The solvent is removed from the extract phase to obtain the salicylic acid ester active ingredients.

[0018] In this invention, the solvent in step 1) preferably includes ethyl acetate and methanol.

[0019] In this invention, the mass ratio of ethyl acetate to methanol is preferably 1:0.3~0.8, more preferably 1:0.4~0.7, and even more preferably 1:0.5~0.6.

[0020] In this invention, after mixing in step 1), it is preferable to let the mixture stand overnight before performing ultrasonic extraction.

[0021] In this invention, the temperature of the ultrasonic extraction in step 1) is preferably 30~50℃, more preferably 35~45℃, and even more preferably 40℃; the power of the ultrasonic extraction is preferably 30~50W, more preferably 35~45W, and even more preferably 40W; the time of a single ultrasonic extraction is preferably 2~4h, more preferably 2.5~3.5h, and even more preferably 3h; and the number of ultrasonic extractions is preferably 2~4 times, and even more preferably 3 times.

[0022] In this invention, the solid-liquid ratio of the ultrasonic extraction in step 1) is preferably 1:8~10, more preferably 1:8.5~9.5, and even more preferably 1:9.

[0023] In this invention, the extract in step 2) is preferably the extract after solvent removal.

[0024] In this invention, the mixed solvent in step 2) preferably comprises n-butanol and dichloromethane. Extraction with the mixed solvent improves the extraction rate of the active ingredients and enables the simultaneous separation of multiple active ingredients.

[0025] In this invention, the mass ratio of n-butanol to dichloromethane is preferably 1:1 to 3, more preferably 1:1.5 to 2.5, and even more preferably 1:2.

[0026] In this invention, the mass ratio of the mixed solvent in step 2) to the Yunnan white pearl powder in step 1) is preferably 0.8~1.5:1, more preferably 0.9~1.3:1, and even more preferably 1~1.2:1.

[0027] In this invention, the raffinate phase in step 3) is preferably the n-butanol phase, and the extract phase is preferably the dichloromethane phase.

[0028] In this invention, the solvent removal in step 3) is preferably carried out by vacuum distillation.

[0029] In this invention, during the solvent removal process of the extract phase in step 3), the temperature of vacuum distillation is preferably 50~60℃, more preferably 55℃; the pressure of vacuum distillation is preferably -0.1~-0.05MPa, more preferably -0.09~-0.07MPa, and more preferably -0.08MPa; and the vacuum distillation is preferably carried out until the liquid volume is 1 / 3~1 / 5 of the initial volume of the extract phase, more preferably 1 / 4. In step 3), the temperature of vacuum distillation in the solvent removal of the raffinate phase is preferably 30~40℃, more preferably 35℃; the pressure of vacuum distillation is preferably -0.1~-0.05MPa, more preferably -0.09~-0.07MPa, and more preferably -0.08MPa; and the vacuum distillation is preferably carried out until the liquid volume is 1 / 3~1 / 5 of the initial volume of the extract phase, more preferably 1 / 4.

[0030] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0031] The Yunnan white pearl powder used in the embodiments and comparative examples of the present invention was obtained by pulverizing Yunnan white pearl after drying at 80°C for 8 hours, and the particle size range of Yunnan white pearl powder was 1~3cm.

[0032] Example 1

[0033] Yunnan white pearl powder was added to a mixed solvent of ethyl acetate and methanol (mass ratio of ethyl acetate to methanol 1:0.5), with a solid-liquid ratio of 1:8. After mixing thoroughly, the mixture was allowed to stand overnight at room temperature. Subsequently, the mixture was ultrasonically extracted at 30℃ and 40W for 4 hours, and the ultrasonic extraction was repeated 3 times under the same conditions. After solid-liquid separation, the extract was obtained. The extract was concentrated and evaporated to dryness to remove the solvent, yielding a crude extract, which was then dispersed in distilled water to obtain a mixture. The mixture was extracted using a mixed solvent of n-butanol and dichloromethane in a mass ratio of 1:2. The extract phase (dichloromethane phase) and the raffinate phase (n-butanol phase) were then separated, with the mass ratio of the n-butanol and dichloromethane mixed solvent to the Yunnan white pearl powder being 1.5:1. The raffinate phase was distilled under reduced pressure at 55℃ and -0.08MPa until the volume was reduced to 1 / 5 of the initial volume, yielding the lignans as the effective components. The extract phase was subjected to vacuum distillation at 35℃ and -0.08MPa until the volume was reduced to 1 / 5 of the initial volume, thus obtaining the salicylic acid ester active ingredients.

[0034] Example 2

[0035] Yunnan white pearl powder was added to a mixed solvent of ethyl acetate and methanol (mass ratio of ethyl acetate to methanol 1:0.3), with a solid-liquid ratio of 1:9. After mixing thoroughly, the mixture was allowed to stand overnight at room temperature. Subsequently, the mixture was ultrasonically extracted at 40℃ and 30W for 2 hours, and the ultrasonic extraction was repeated once under the same conditions. After solid-liquid separation, the extract was obtained. The extract was concentrated and evaporated to dryness to remove the solvent, yielding a crude extract, which was then dispersed in distilled water to obtain a mixture. The mixture was extracted using a mixed solvent of n-butanol and dichloromethane in a mass ratio of 1:3. The raffinate phase (n-butanol phase) and the extract phase (dichloromethane phase) were then separated, with the mass ratio of the n-butanol and dichloromethane mixed solvent to the Yunnan white pearl powder being 1:1. The raffinate phase was distilled under reduced pressure at 55℃ and -0.08MPa until the volume was reduced to 1 / 5 of the initial volume, yielding the lignans as the effective components. The extract phase was subjected to vacuum distillation at 35℃ and -0.08MPa until the volume was reduced to 1 / 5 of the initial volume, thus obtaining the salicylic acid ester active ingredients.

[0036] Example 3

[0037] Yunnan white pearl powder was added to a mixed solvent of ethyl acetate and methanol (mass ratio of ethyl acetate to methanol 1:0.8), with a solid-liquid ratio of 1:10. After mixing thoroughly, the mixture was allowed to stand overnight at room temperature. Subsequently, the mixture was ultrasonically extracted at 50℃ and 50W for 3 hours, and the ultrasonic extraction was repeated twice under the same conditions. After solid-liquid separation, the extract was obtained. The extract was concentrated and evaporated to dryness to remove the solvent, yielding a crude extract, which was then dispersed in distilled water to obtain a mixture. The mixture was extracted using a mixed solvent of n-butanol and dichloromethane in a mass ratio of 1:1. The raffinate phase (n-butanol phase) and the extract phase (dichloromethane phase) were then separated, with the mass ratio of the n-butanol and dichloromethane mixed solvent to the Yunnan white pearl powder being 0.8:1. The raffinate phase was distilled under reduced pressure at 55℃ and -0.08MPa until the volume was reduced to 1 / 5 of the initial volume, yielding the lignans as the effective components. The extract phase was subjected to vacuum distillation at 35℃ and -0.08MPa until the volume was reduced to 1 / 5 of the initial volume, thus obtaining the salicylic acid ester active ingredients.

[0038] Comparative Example 1

[0039] The ethyl acetate-methanol mixed solvent in Example 1 was replaced with a 60% (w / w) aqueous ethanol solution, and everything else was the same as in Example 1.

[0040] Comparative Example 2

[0041] The ethyl acetate-methanol mixed solvent in Example 1 was replaced with a 90% (w / w) methanol aqueous solution, as in other examples of Example 1.

[0042] Comparative Example 3

[0043] The mixed solvent of n-butanol and dichloromethane in Example 1 was replaced with petroleum ether, and everything else was the same as in Example 1.

[0044] Comparative Example 4

[0045] Yunnan white pearl powder was added to a mixed solvent of ethyl acetate and methanol (mass ratio of ethyl acetate to methanol 1:0.5), with a solid-liquid ratio of 1:8. After mixing thoroughly, the mixture was allowed to stand overnight at room temperature. Subsequently, the mixture was ultrasonically extracted at 30℃ and 40W for 4 hours, and the ultrasonic extraction was repeated 3 times under the same conditions. After solid-liquid separation, the extract was obtained. The extract was concentrated and evaporated to dryness to remove the solvent, yielding a crude extract, which was then dispersed in distilled water to obtain a mixture. The mixture was extracted sequentially with n-butanol and dichloromethane to obtain n-butanol extract phase and dichloromethane extract phase. The n-butanol extract phase was distilled under reduced pressure at 55℃ and -0.08MPa until the volume was reduced to 1 / 5 of the initial volume, yielding the lignan active ingredients. The dichloromethane extract phase was distilled under reduced pressure at 35℃ and -0.08MPa until the volume was reduced to 1 / 5 of the initial volume, yielding the salicylate active ingredients.

[0046] Comparative Example 5

[0047] The mass ratio of ethyl acetate to methanol in Example 1 was modified to 1:1.5, and all other aspects remained the same as in Example 1.

[0048] Comparative Example 6

[0049] The mass ratio of ethyl acetate to methanol in Example 1 was modified to 1:0.1, the solid-liquid ratio was modified to 1:15, and the rest were the same as in Example 1.

[0050] Comparative Example 7

[0051] In Example 1, the mass ratio of n-butanol to dichloromethane was modified to 1:5, and the mass ratio of the mixed solvent of n-butanol and dichloromethane to the mass of Yunnan white pearl powder was modified to 0.5:1. Other aspects were the same as in Example 1.

[0052] Comparative Example 8

[0053] The mass ratio of n-butanol to dichloromethane in Example 1 was modified to 1:0.5, and all other aspects remained the same as in Example 1.

[0054] The extraction rates of lignans and salicylates were calculated using the formula: Extraction rate = (Mass of extract / Mass of Dianbaizhu powder) × 100%. The results are shown in Table 1.

[0055] Table 1 Extraction rate of active ingredients

[0056] As shown in Table 1, the extraction rates of lignans and salicylates in Examples 1-3 were significantly higher than those in Comparative Examples 1-8. Comparative Examples 1 and 2 used conventional single alcohol solvents as extraction solvents, which showed some extraction effect on lignans, but the extraction rate of salicylates was low, resulting in extremely low extraction rates and making simultaneous separation and purification of both active ingredients impossible. Comparative Example 3 used petroleum ether for extraction after mixed solvent extraction, resulting in poor extraction of both lignans and salicylates, failing to achieve efficient separation and purification. Comparative Example 4 used a mixed solvent… After extraction, the samples were sequentially extracted with n-butanol and dichloromethane to obtain n-butanol and dichloromethane extract phases. Although sequential extraction can separate lignans and salicylates, the multi-step extraction process is complex and results in the loss of active ingredients, thus reducing the extraction rate. Comparative Examples 5-8 involved adjusting the mass ratio of ethyl acetate and methanol, and the mass ratio of n-butanol and dichloromethane, in the mixed solvents used for extraction. Changes in the solvent mass ratio altered the properties of the mixed solvents, thereby reducing the extraction efficiency and effectiveness of the active ingredients.

[0057] As can be seen from the above embodiments, the present invention provides a method for separating and purifying the effective components of *Phyllostachys edulis*. *Phyllostachys edulis* powder is extracted using a mixed solvent of ethyl acetate and methanol, combined with ultrasonic extraction to achieve efficient extraction of lignans and salicylates. The extract is further extracted using a mixed solvent of n-butanol and dichloromethane, achieving efficient separation of lignans and salicylates in a single extraction step. By rationally controlling the mass ratio of the components in the mixed solvent used for extraction, efficient extraction and separation of lignans and salicylates are achieved.

[0058] 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 without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for separating and purifying the effective components of Dianbaizhu (a type of pearl), characterized in that, It includes the following steps: 1) Mix Yunnan white pearl powder with solvent and perform ultrasonic extraction to obtain the extract; 2) The extract was extracted using a mixed solvent to obtain the extract phase and the raffinate phase; 3) Remove the solvent from the raffinate to obtain the lignans, the active ingredients. The solvent is removed from the extract phase to obtain the salicylic acid ester active ingredients.

2. The method for separating and purifying the effective components of Dianbaizhu (a type of pearl) according to claim 1, characterized in that, Step 1) The solvent comprises ethyl acetate and methanol.

3. The method for separating and purifying the effective components of Dianbaizhu (a type of pearl) according to claim 2, characterized in that, The mass ratio of ethyl acetate to methanol is 1:0.3~0.

8.

4. The method for separating and purifying the effective components of Dianbaizhu according to any one of claims 1 to 3, characterized in that, Step 1) The ultrasonic extraction temperature is 30~50℃, the ultrasonic extraction power is 30~50W, the time for a single ultrasonic extraction is 2~4h, and the number of ultrasonic extractions is 2~4 times.

5. The method for separating and purifying the effective components of Dianbaizhu (a type of pearl) according to claim 4, characterized in that, Step 1) The solid-liquid ratio of the ultrasonic extraction is 1:8~10.

6. The method for separating and purifying the effective components of Dianbaizhu (a type of pearl) according to claim 1 or 5, characterized in that, Step 2) The mixed solvent comprises n-butanol and dichloromethane.

7. The method for separating and purifying the effective components of Dianbaizhu (a type of pearl) according to claim 6, characterized in that, The mass ratio of n-butanol to dichloromethane is 1:1~3.

8. The method for separating and purifying the effective components of *Phyllostachys edulis* according to claim 1 or 7, characterized in that, The mass ratio of the mixed solvent in step 2) to the Yunnan white pearl powder in step 1) is 0.8~1.5:

1.

9. The method for separating and purifying the effective components of Dianbaizhu (a type of pearl) according to claim 8, characterized in that, Step 3) The solvent removal is performed by vacuum distillation.

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