A method for determining the volatile oil content of Amomum villosum

Abstract

The invention discloses a method for determining the content of volatile oil in Amomum villosum. The method comprises: 1) exploring the influence of five factors of Amomum villosum volatile oil extraction on the content of volatile oil through orthogonal design experiment, and finding the factors with significant influence on the determination of the content of volatile oil in Amomum villosum; 2) obtaining the best extraction method for determining the content of volatile oil in Amomum villosum by exploring the influence of the crushing degree of seed mass on the content of volatile oil; 3) establishing a method for determining the content of volatile oil in Amomum villosum; 4) determining the volatile oil of Amomum villosum from different origins according to the determination method of step 3), and calculating and comparing the difference of the content; 5) determining the water content of Amomum villosum from different origins, and combining it with the determination result of the determination method of step 3), calculating the actual volatile oil content of Amomum villosum (excluding water), and comparing the difference of the content; 6) comparing the difference between the volatile oil content of Amomum villosum from different origins and the actual volatile oil content (excluding water).

Description

Technical Field

[0001] The invention relates to the technical field of quality detection and control of Amomum villosum, and in particular to a method for measuring the content of volatile oil in Amomum villosum. Background Art

[0002] Amomum villosum Lour is the dried mature fruit of the ginger family plant Amomum villosum Lour. It has the effects of removing dampness and stimulating appetite, warming the spleen and stopping diarrhea, regulating qi and stabilizing pregnancy. It is native to Yangchun, Guangdong and surrounding areas, and was later introduced to Guangxi, Yunnan and other places. The one produced in Yangchun is the best.

[0003] Market research found that the prices of Amomum villosum from different origins vary greatly, and the quality is uneven. The price of Amomum villosum from the Yangchun original production area in Guangdong is 1,600 to 4,000 yuan / kg, while the price of Amomum villosum from Guangxi, Yunnan and other places is 150 to 300 yuan / kg. In addition, the appearance of Amomum villosum from different origins is similar and difficult to distinguish. The similarity of properties and price differences have led to mutual impersonation and inferior products being sold as good ones. This confusion has seriously restricted the quality evaluation, production of traditional Chinese medicines and clinical application of Amomum villosum.

[0004] The volatile oil content of Amomum villosum is one of the indicators for judging the quality of Amomum villosum. The determination of the volatile oil content of Amomum villosum is carried out according to the volatile oil determination method in Part IV of the 2020 edition of the Chinese Pharmacopoeia (General Rule 2204, Method A, hereinafter referred to as "Method A"), and the result shall not be less than 3.0% (mL / g).

[0005] However, the composition of plant volatile oils is complex and the content of volatile oils in different plants varies greatly. This method targets a large category of medicinal materials, and the relevant parameter regulations are relatively broad. Many experimental parameters are not clear enough, resulting in large deviations and poor repeatability in the actual production and measurement, which brings a lot of difficulties and inconveniences to the actual production and scientific research. At present, there is no special method for the determination of volatile oils in Amomum villosum; and although the pharmacopoeia stipulates that the moisture content of Amomum villosum shall not exceed 15.0%, no researchers have considered the impact of the difference in moisture content of Amomum villosum medicinal materials on the determination of volatile oil content.

[0006] In view of this, the present invention determines the optimal conditions for determining the volatile oil content of Amomum villosum, improves the stability of the determination method of the volatile oil content of Amomum villosum, can provide a reference for improving the determination method of the volatile oil content of Amomum villosum in the Chinese Pharmacopoeia, and provides a reliable method for related determination in production and research work. Summary of the invention

[0007] The invention aims to provide a method for determining the content of volatile oil in Amomum villosum, and solves the problem that the current determination of the content of volatile oil in Amomum villosum has large error and poor repeatability, and provides a method for determining the content of volatile oil in Amomum villosum.

[0008] The technical solution of the present invention is:

[0009] The present invention provides a method for determining the volatile oil content of Amomum villosum, the method comprising the following steps:

[0010] 1) Through orthogonal design experiments, the effects of five factors on the volatile oil content of Amomum villosum were explored, and the factors with significant influence on the determination of volatile oil content of Amomum villosum were found;

[0011] 2) The optimal extraction method for determining the volatile oil content of Amomum villosum was obtained by exploring the effect of the degree of seed crushing on the volatile oil content;

[0012] 3) Based on the principle of being close to the pharmacopoeia and convenient for conducting experiments, a method for determining the volatile oil content of Amomum villosum was established;

[0013] 4) Determine the volatile oil of Amomum villosum from different origins according to the method of step 3), and calculate and compare the differences in their contents;

[0014] 5) determining the water content of Amomum villosum from different origins, and then combining it with the determination result of the method of step 3), calculating the actual volatile oil content of Amomum villosum (excluding moisture), and comparing the difference in the content;

[0015] 6) Compare the differences in volatile oil content of Amomum villosum from different origins with the actual volatile oil content (excluding water).

[0016] In the technical solution of the present invention, the degree of crushing (P < 0.05) is a factor that significantly affects the volatile oil content of Amomum villosum.

[0017] In the technical solution of the present invention, the extraction effect of the volatile oil of Amomum villosum is best when it is crushed for a certain time (5 to 40 seconds) and mixed and extracted for 5 hours.

[0018] In the technical scheme of the present invention, the method for determining the volatile oil content of Amomum villosum is as follows: the amount of water added is 500 mL; the crushing method is crushing for a certain time (5 to 40 seconds), without sieving, and mixing; the condensed water temperature is 28° C. (or room temperature); the micro-boiling time is 5 hours; and the weight of the test sample is 15 g.

[0019] In the technical scheme of the present invention, the volatile oil content of Amomum villosum from different origins is: Xishuangbanna>Chunwan, Huaiji, Wenshan II>Guangning Chikeng, Wenshan I>Guangning Hecang, Naruan, Panlong, Heshui, Shaoguan.

[0020] In the technical scheme of the present invention, the actual volatile oil content (excluding moisture) of Amomum villosum from different origins is: Xishuangbanna>Chunwan, Huaiji, Wenshan II>Guangning Chikeng, Wenshan I>Guangning Hecang, Heshui, Panlong, Naruan.

[0021] In the technical scheme of the present invention, the difference between the volatile oil content of Amomum villosum from different origins and the actual volatile oil content (excluding water) was compared. Except for the volatile oil content of Wenshan 1 (P>0.05) and Guangning Chikeng (P>0.05) which had no significant increase, the volatile oil content of Amomum villosum from other origins increased significantly. However, among the above origins, the final ranking results of the volatile oil content of the origin were all consistent.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] The present invention finds out the variables that significantly affect the content of volatile oil in Amomum villosum, greatly solves the problem that the results of the determination of the volatile oil content in Amomum villosum in actual production determination and scientific research are unstable and have poor repeatability, and improves the stability and repeatability of the determination in the actual production and scientific research process. At the same time, it provides a basis for the determination method of the volatile oil content in Amomum villosum to be improved in the Chinese Pharmacopoeia, and provides a reference for the standards of the determination methods of the volatile oil content in other medicinal materials to be scientific and specialized. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Here is a photo of Amomum villosum fruit;

[0025] Figure 2 The effect of different crushing time on the volatile oil content of each sieve group, from left to right: "not passing through No. 2 sieve", "passing through No. 2 sieve but not passing through No. 3 sieve", "passing through No. 3 sieve";

[0026] Figure 3 This is a picture of the powder after crushing and sieving; (from left to right: "not passing through No. 2 sieve", "passing through No. 2 sieve but not passing through No. 3 sieve", "passing through No. 3 sieve"). DETAILED DESCRIPTION

[0027] The present invention is further described in detail below through specific preferred embodiments combined with effect test examples, but the present invention is not limited to the following embodiments.

[0028] The invention discloses a method for measuring the content of volatile oil of Amomum villosum, and the influence of various factors of extracting Amomum villosum volatile oil on the content of volatile oil is explored by orthogonal design experiment, and the factors with significant influence are further studied to establish the optimal conditions for determining the content of volatile oil of Amomum villosum. Then, the volatile oil content of Amomum villosum from different origins and the influence of the moisture content of medicinal materials on the content of volatile oil are determined and compared by this method. The present invention determines the optimal conditions for determining the content of volatile oil of Amomum villosum, improves the stability of the determination method of the content of volatile oil of Amomum villosum, can provide reference for improving the determination method of the content of volatile oil of Amomum villosum in Chinese Pharmacopoeia, and provides a reliable method for relevant determination in production and research work.

[0029] A method for measuring the volatile oil content of Amomum villosum, comprising:

[0030] Example 1

[0031] We collected materials from 11 different places of origin, including Wenshan, Xishuangbanna, Yunnan, Yangchun, Shaoguan, Huaiji, Zhaoqing, Guangdong, etc. The specific sources are shown in Table 1 and the pictures are shown in Figure 2 . Using Yangchunsha from Wenshan No. 1 as the material, orthogonal experiments and further investigation experiments on variables with significant effects were carried out. All experimental materials were sealed and stored in a storage box in a cool and dry place. At the same time, water-absorbing silica gel was placed inside the storage box to prevent the medicinal materials from absorbing water and becoming moldy.

[0032] Table 1 Collection information of Amomum villosum fruit

[0033]

[0034] Example 2

[0035] Volatile oil determination method:

[0036] According to the provisions of Method A, the relevant operating regulations during the orthogonal test of volatile oil content are as follows:

[0037] 1) Fruit preservation: Take the Amomum villosum fruit from Wenshan, Yunnan (Wenshan 1), store it in a sealed and dry storage box (with a small amount of water-absorbing silica gel inside), and store the storage box in a cool and dry place, avoiding direct sunlight;

[0038] 2) Shelling and weighing: Because the weight of the fruits of Wenshanyi varies greatly, before the experiment, the fruits of Amomum villosum were shelled and the seed clusters were weighed, and the seed clusters with a single grain weight of 0.8 to 1.3 g were taken;

[0039] 3) Crushing: Put the seed mass into the powder grinder and crush it according to the "10+5s" crushing method (crush for 10s, sieve, crush for 5s, and sieve again) to minimize the degree of heating of the instrument;

[0040] 4) Screening: Screening is carried out according to the requirements of orthogonal test;

[0041] 5) Extraction: Weigh the sieved powder, weigh a certain amount of powder, pour it into a 1000 mL round-bottom flask, add 10 glass beads, add a certain amount of distilled water, and put it into an electric heating mantle; install a volatile oil extractor and a condenser, turn on the condensation water circulation, and extract it at a certain temperature of condensation water for a certain time with slight boiling;

[0042] 6) Standing: After the extraction is completed, stop heating and keep the condensed water in a circulating state;

[0043] 7) Collection: After standing for 1 hour, read and collect the volatile oil;

[0044] 8) Calculation: Calculate the volatile oil content (%, mL / g).

[0045] Example 3

[0046] Orthogonal experimental design method:

[0047] Orthogonal experiments can understand the situation of comprehensive experiments by selecting representative test points for testing and analyzing the results. This can greatly reduce the number of tests, achieve efficient and economical testing, and optimize the process.

[0048] To this end, this study screened out five factors with greater influence from the many factors that affect the volatile oil content in Amomum villosum, and set three levels for each factor, as shown in Table 2.

[0049] Afterwards, SPSS software (version 21) was used to carry out orthogonal experimental design, and the design results are shown in Table 3. After deleting the repeated groups, there were 15 groups in this orthogonal experiment.

[0050] Table 2 Variable factors and levels of the volatile oil extraction test of Amomum villosum

[0051]

[0052] Table 3 Orthogonal design table of orthogonal test of volatile oil extraction factors L16 (35)

[0053]

[0054] Example 4

[0055] Method for determining moisture content of Amomum villosum:

[0056] Based on the water content determination method of the fourth volume of the 2020 Chinese Pharmacopoeia (General Rule 0832, Method 4), and according to the results of the preliminary experiment, the following operations are taken for water content determination:

[0057] 1) Take the shelled Amomum villosum seeds, place them in a grinder, grind them and pass them through a No. 2 sieve;

[0058] 2) Weigh 25.00 g of powder and place it in a 500 mL dry distillation flask;

[0059] 3) Add a few dry glass beads to the distillation flask, add 200 mL of toluene solution, and place the distillation flask on an electric heating mantle in a fume hood;

[0060] 4) Connect the moisture meter, condenser tube, condensate water pipe, and open the condensate water circulation;

[0061] 5) Use a small funnel or a long rubber-tipped dropper to inject toluene along the top of the condenser tube to fill the moisture analyzer with toluene;

[0062] 6) Turn on the power of the electric heating mantle until turbidity appears in the moisture analyzer, start timing, and continue to maintain a slight boiling state;

[0063] 7) Extract for 5 hours until no new water droplets appear in the measuring device, and wash off the water droplets attached to the condenser with toluene solution;

[0064] 8) When the solution in the moisture meter cools to room temperature, read the moisture content.

[0065] Results and Analysis

[0066] 1 Orthogonal test of factors for volatile oil extraction

[0067] After conducting the orthogonal test according to Table 3, the test data (Table 4) were statistically analyzed to obtain the orthogonal test significance analysis results (Table 5) and the crushing degree pairwise comparison results (Table 6).

[0068] Table 4 Results of orthogonal test of factors for volatile oil extraction

[0069]

[0070] The results of orthogonal test significance analysis (Table 5) showed that the amount of water added A (P>0.05), condensed water temperature C (P>0.05), micro-boiling time D (P>0.05) and test sample weight E (P>0.05) were not factors that significantly affected the volatile oil content of Amomum villosum. The degree of crushing B (P<0.05) was a factor that significantly affected the volatile oil content of Amomum villosum.

[0071] Table 5 Results of significance analysis of orthogonal test

[0072]

[0073] Note: The significant analysis results of water addition amount A are the comparison results between A1 (group 3, 4, 6, 8, 9, 10, 15, 16) and A2 (group 1, 5, 12, 13), A3 (2, 7, 11, 14); the significant analysis results of crushing degree B are the comparison results between B1 (group 3, 4, 6, 8, 11, 12, 13, 14) and B2 (group 1, 7, 9, 16), B3 (group 2, 5, 10, 15); the significant analysis results of condensation water temperature C are the comparison results between C1 (3, 5, 7, 8, The significance analysis results of the micro-boiling time D are the comparison results between D1 (groups 3, 5, 7, 8, 9, 10, 13, 14) and D2 (groups 1, 6, 11, 15), D3 (groups 2, 4, 12, 16); the significance analysis results of the test sample weight E are the comparison results between E1 (groups 1-8) and E2 (groups 9-12), E3 (groups 13-16).

[0074] The results of the paired comparison of the degree of pulverization (Table 6) show that the volatile oil content is: passing through the No. 3 sieve > passing through the No. 2 sieve > passing through the No. 2 sieve but not through the No. 3 sieve. That is, the finer the powder, the higher the volatile oil content.

[0075] Table 6 Paired comparison results of crushing degree

[0076]

[0077] Note: The same letters indicate no significant difference at the 0.05 level, no or different letters indicate significant difference at the 0.05 level, the same below. The significance analysis results of crushing degree B are the comparison results between B1 (groups 3, 4, 6, 8, 11, 12, 13, 14) and B2 (groups 1, 7, 9, 16), B3 (groups 2, 5, 10, 15).

[0078] In summary, there are significant differences in the volatile oil content of three different degrees of crushing, namely, passing through No. 2 sieve, passing through No. 3 sieve, and passing through No. 2 sieve but not passing through No. 3 sieve (the results of the determination are very different). Therefore, the sentence "crushed to pass through No. 2 to No. 3 sieves and mixed evenly" written in the fourth general chapter of the Chinese Pharmacopoeia is inappropriate for the determination of the volatile oil content of Amomum villosum. In line with the principle of being close to the pharmacopoeia and convenient for conducting experiments, the amount of water added in the extraction method of the volatile oil of Amomum villosum can be set to 500mL, the condensed water temperature can be set to 28℃ (or room temperature), the micro-boiling time can be set to 5h, and the weight of the test sample can be set to 15g. The results of the orthogonal test show that the degree of crushing has a very significant effect on the volatile oil content of Amomum villosum, but when designing the orthogonal experimental table, only three levels of "passing through No. 2 sieve", "passing through No. 2 sieve but not passing through No. 3 sieve", and "passing through No. 3 sieve" were selected. It is not clear whether the best method for extracting volatile oil from Amomum villosum is among them. Therefore, it is necessary to conduct further exploration and research on the degree of crushing of Amomum villosum seed mass.

[0079] Effect of 2 seed pellet crushing degree on volatile oil content

[0080] 2.1 Effect of different crushing degrees on the volatile oil content of different screening fractions of Amomum villosum volatile oil

[0081] It is clear that the finer the powder, the higher the volatile oil content. The factors that affect the fineness of powder particles mainly include the power of the crushing instrument and the crushing time. Under the premise of fixing the crushing instrument, by setting different crushing times, we examine whether the conclusion is still valid. With this in mind, four groups were set: crushing for 5s (the average rate of K1 that can pass the No. 2 sieve after crushing is 51.4%), crushing for 10s (K2 is 72.5% on average), crushing for 20s (K3 is 85.2% on average), and crushing for 40s (K4 is 97.2% on average).

[0082] Depend on Figure 2 It can be seen that the grinding time does not affect the volatile oil content: the rule of "passing through the No. 3 sieve > passing through the No. 2 sieve > passing through the No. 2 sieve but not through the No. 3 sieve" is true. That is, the finer the powder, the higher the volatile oil content.

[0083] As shown in Table 7, the volatile oil content of the powder that passed through the No. 2 sieve but not the No. 3 sieve, passed through the No. 2 sieve, and passed through the No. 3 sieve all showed a downward trend with the extension of the crushing time. As the crushing time increases, the proportion of powder that can pass through the No. 3 sieve increases, but the volatile oil content of the powder that passes through the No. 3 sieve decreases. The possible reason is that the seed coat ( Figure 3 The beige part in the middle) was crushed and entered the No. 3 sieve as the crushing time increased. However, due to the difficulty in separating seeds and embryos, it was difficult to separate them completely and they were not successfully separated for determination.

[0084] Table 8 Effect of different crushing time on the volatile oil of Amomum villosum (`X±S)

[0085]

[0086] Note: The letters in this table are the results of longitudinal comparison (5, 10, 20, 40s).

[0087] 2.2 Effect of extraction of intact seed mass without crushing on volatile oil content

[0088] According to the results of 2.1, the volatile oil content of different sieves is significantly different, and the volatile oil content of multiple components shows a downward trend with the extension of the crushing time. In the actual production and scientific research process, the crushing instrument itself has differences in power, and the crushing time will have a significant impact on the volatile oil content. Therefore, it is unreasonable to select a certain component for the determination of volatile oil content. So is it possible to not crush it, that is, to use the whole seed mass to determine the volatile oil content? If the whole seed mass is used, is it necessary to extend the distillation time? With this in mind, two groups of "whole seed mass, extraction 5h" and "whole seed mass, extraction 10h" were set up, and compared with the total volatile oil content of each group in 2.1, see Table 8 for details.

[0089] The results show that:

[0090] 1) There was no significant difference in the total content of volatile oil between the intact seed mass, extracted for 5 h (group ①) and the volatile oil content of the seed mass, which was sieved after crushing for 5, 10, 20, and 40 seconds and then extracted for 5 h (groups ③, ④, ⑤, and ⑥);

[0091] 2) The volatile oil content of the intact seed mass extracted for 10 h (Group ②) was significantly higher than that of the intact seed mass extracted for 5 h (Group 1). It can be seen that for the intact seed mass, extending the distillation time can significantly increase its volatile oil content.

[0092] 3) The volatile oil content of the intact seed mass extracted for 10 h (Group ②) was significantly higher than the sum of the volatile oil extracted separately for 5 h after being crushed for 5s, 10s, 20s, and 40s and sieved (Groups ③, ④, ⑤, and ⑥).

[0093] Table 8 Effect of different crushing procedures on volatile oil content (`X±S)

[0094]

[0095] In summary, compared with the method of crushing and then sieving to extract volatile oil, the method of directly extracting from the intact seed mass is more effective.

[0096] 2.3 Effect of crushing for a certain time and mixed extraction on the volatile oil content

[0097] According to the results of 2.2, extending the extraction time can increase the volatile oil content of the complete seed mass, but for actual production and scientific research, 10 hours of extraction is too long. If appropriate crushing or manual crushing (hammering or pounding) is adopted to increase the crushing degree of the seed mass, and then mixed extraction is performed, can the volatile oil content of "complete seed mass, extraction for 10 hours" be achieved or even exceeded? In addition, mixed extraction can also reduce the influence of factors such as screening and reduce the subjectivity of the experiment.

[0098] Based on this consideration, we set ⑦ pulverizing for 5 seconds and mixing and extracting for 5 hours; ⑧ pulverizing for 10 seconds and mixing and extracting for 5 hours; ⑨ pulverizing for 20 seconds and mixing and extracting for 5 hours; ⑩ pulverizing for 40 seconds and mixing and extracting for 5 hours; The seeds were hammered open manually (an iron hammer wrapped in tin foil or plastic wrap was used to hammer open the seeds), mixed and extracted for 5 hours, and the Shapiro-Wilk test was used to test the normality of each group of data. All the data conformed to the normal distribution, as shown in Table 9 for details.

[0099] The results show that:

[0100] 1) The volatile oil content of the seeds crushed for 5, 10, 20, 40 seconds and mixed extracted for 5 hours (⑦-⑩ groups) was higher than that of the intact seed mass, the total volatile oil of the seeds extracted for 5 hours (① group), crushed for 5, 10, 20, 40 seconds, and extracted separately for 5 hours (③-⑥ groups) and manually hammered, mixed extracted for 5 hours ( Group), but the content was similar to that of the intact seed mass, which was extracted for 10 hours (Group ②). That is, the volatile oil content of crushing for 5, 10, 20, and 40 seconds, and mixed extraction for 5 hours; the volatile oil content of the intact seed mass, which was extracted for 10 hours, was greater than the sum of the volatile oil of manual hammering; mixed extraction for 5 hours; intact seed mass, extraction for 5 hours; crushing for 5, 10, 20, and 40 seconds, and separate extraction for 5 hours.

[0101] 2) For the crushing time of 5, 10, 20, and 40 seconds and mixed extraction of 5 hours (7-10 groups), there was no significant difference in the volatile oil content between the two groups. This shows that the crushing time does not affect the determination results of the volatile oil content when mixed extraction is used for 5 hours.

[0102] 3) Manual hammering and mixed extraction for 5h ( The comparison results of the two groups (group 7-10) with the groups 7-11 and 7-12 showed that manual hammering resulted in a large loss. In addition, manual hammering is subjective in actual production, such as the strength of force and the degree of crushing, so it is not applicable.

[0103] Table 9 Effect of different crushing procedures on volatile oil content (`X±S)

[0104]

[0105] In summary, the best effect is achieved by crushing for a certain period of time (5 to 40 seconds) and mixing and extracting for 5 hours.

[0106] 2.4 Effect of pulverization for a certain period of time and mixed extraction on the actual volatile oil content of the powder

[0107] Crushing or manual hammering and pounding can speed up the extraction of volatile oils and improve the determination efficiency to a certain extent. However, no matter what kind of crushing method is used, it will cause the loss of powder. Excluding this part of the lost powder, the volatile oil content is calculated based on the actual weight of the powder after crushing, which is determined as the actual powder volatile oil content.

[0108] Calculation method: actual powder volatile oil content % (mL / g) = volatile oil volume (mL) / actual weight of powder after crushing (g) × 100%.

[0109] The Shapiro-Wilk test was used to test the normality of each group of data after calculation. All groups of data were in line with the normal distribution. The statistical analysis results are shown in Table 10.

[0110] The results show that:

[0111] 1) In order to consider the powder loss caused by crushing, the actual powder weight (the weight of the powder weighed again after the seed mass was crushed) was substituted into Formula 3 to calculate the actual powder volatile oil content. Except for Group ① and Group ②, the remaining groups all needed to undergo this crushing operation. Therefore, except for Group ① and Group ②, the volatile oil content of the remaining groups increased.

[0112] 2) Volatile oil content of Amomum villosum powder: Crushing for 5, 10, 20, 40 seconds, and extracting for 5 hours of volatile oil content (⑦-⑩ groups); intact seed mass, extracting for 10 hours (② group) > Manual hammering, mixed extraction, extracting for 5 hours of volatile oil content; crushing for 5, 10, 20, 40 seconds (③-⑥, The volatile oil content of the whole seed mass, extracted for 5 hours (Group ①), was greater than that of the volatile oil content of the whole seed mass, extracted for 5 hours (Group ①).

[0113] Table 10 Effect of different pulverization procedures on the actual powder volatile oil content (`X±S)

[0114]

[0115] In summary, the comparison results of volatile oil content under different pulverization procedures and the comparison results of volatile oil content of actual powder can be obtained:

[0116] 1) Comparison of volatile oil content: volatile oil content of crushing for 5, 10, 20, and 40 seconds and mixed extraction for 5 hours; volatile oil content of intact seed mass and extraction for 10 hours > volatile oil content of manual hammering; mixed extraction for 5 hours; volatile oil content of intact seed mass and extraction for 5 hours; the sum of volatile oil of crushing for 5, 10, 20, and 40 seconds and separate extraction for 5 hours.

[0117] 2) Comparison of actual powder volatile oil content: volatile oil content after crushing for 5, 10, 20, and 40 seconds and extracting for 5 hours; volatile oil content after intact seed mass and extracting for 10 hours> volatile oil content after manual hammering and mixed extraction for 5 hours; volatile oil content after crushing for 5, 10, 20, and 40 seconds> volatile oil content after intact seed mass and extracting for 5 hours. Whether the volatile oil content is calculated by the weight of the seed mass or the actual weight of the powder after crushing, it can be concluded that the crushing procedure with a certain crushing time (5 to 40 seconds) and mixed extraction for 5 hours has the best effect.

[0118] 3. Establishment of determination method of volatile oil in Amomum villosum

[0119] The orthogonal test results show that:

[0120] The amount of water added A (P>0.05), condensed water temperature C (P>0.05), micro-boiling time D (P>0.05) and test sample weight E (P>0.05) were not factors that significantly affected the volatile oil content of Amomum villosum. However, the degree of crushing B (P<0.05) was a factor that significantly affected the volatile oil content of Amomum villosum. Further investigation of the degree of crushing B showed that the best effect was achieved when the crushing time (5-40s) was fixed and the mixed extraction was performed for 5h.

[0121] Based on the principle of being close to the pharmacopoeia and facilitating the experiment, the amount of water added was determined to be 500 mL; the crushing method was to crush for a certain time (5 to 40 seconds) without sieving and mixing; the condensed water temperature was 28°C (or room temperature); the micro-boiling time was 5 hours; and the weight of the test sample was 15 g.

[0122] Therefore, it is recommended that the detailed determination method of the volatile oil content of Amomum villosum be established as follows:

[0123] 1) Fruit preservation: Take the Amomum villosum fruit and store it in a sealed and dry storage box (with a small amount of water-absorbing silica gel inside). The storage box should be placed in a cool and dry place, away from direct sunlight;

[0124] 2) Shelling and weighing: Before the experiment, shell the Amomum villosum fruit, weigh about 15 g of seed mass (accurate to 0.01 g), and record the balance reading;

[0125] 3) Crushing: Put it into the powder grinder and crush it for a certain time (5 to 40 seconds can be selected according to the condition of the crushing instrument, and try to avoid the crushing instrument from getting hot);

[0126] 4) Weigh again: pour out as much of the crushed powder as possible, weigh the powder, and record the balance reading to calculate the powder recovery rate, which should be as high as possible;

[0127] 5) Extraction: Pour the powder into a 1000 mL round-bottom flask, add a few glass beads, add 500 mL of distilled water, and place it in an electric heating mantle; install a volatile oil extractor and a condenser, turn on the condenser water circulation, and extract at a low boiling temperature for 5 hours;

[0128] 6) Standing: After the extraction is completed, stop heating and keep the condensed water in a circulating state;

[0129] 7) Collection: After standing for 1 hour, read the reading and collect the volatile oil.

[0130] 8) Calculation: Calculate the volatile oil content (%, mL / g).

[0131] 4. Comparison of the volatile oil content of Amomum villosum from different origins

[0132] The new method for determining the volatile oil content of Amomum villosum was used to determine the volatile oil content of Amomum villosum from Wenshan (WS1, WS2), Xishuangbanna (XSBN), Yunnan, Yangchun (PL, HS, CW, NR), Huaiji (HJ), Shaoguan (SG), Guangning (GNHC, GNCK) and other places in Guangdong. The Shapiro-Wilk method was used to test the normality of the measured data from each production area, and each group of data conformed to the normal distribution. The detailed results are shown in Table 11.

[0133] The results show that:

[0134] 1) In this test, the volatile oil content is: Xishuangbanna>Chunwan, Huaiji, Wenshan II>Guangning Chikeng, Wenshan I>Guangning Hecang, Naruan, Panlong, Heshui, Shaoguan. Among them, the volatile oil content of Amomum villosum in Xishuangbanna area is the highest, reaching more than 5%; the volatile oil content of Chunwan, Huaiji, Wenshan area (Wenshan I, Wenshan II), Guangning Chikeng is not much different; the volatile oil content of Guangning Chikeng, Guangning Hecang, Naruan, Panlong, Heshui, Shaoguan is not much different.

[0135] 2) For Amomum villosum in Yunnan, the volatile oil content is: Xishuangbanna > Wenshan 1 and Wenshan 2.

[0136] 3) For the Amomum villosum in Guangdong area, the volatile oil content is: Chunwan, Huaiji > Guangning Chikeng, Guangning Hecang, Naruan, Panlong, Heshui, Shaoguan.

[0137] Table 11 Determination results of volatile oil content of Amomum villosum from different origins (`X±S)

[0138]

[0139] 5. Comparison of the actual volatile oil content (excluding water) of Amomum villosum from different origins

[0140] The water content of Amomum villosum from Wenshan (WS1, WS2), Xishuangbanna (XSBN), Yunnan, Yangchun (PL, HS, CW, NR), Huaiji (HJ), Guangning (GNHC, GNCK) in Guangdong was measured, and then combined with the results of 3.4 to calculate the actual volatile oil content of Amomum villosum (excluding water). The Shapiro-Wilk test was used to test the normality of each group of data, and each group of data conformed to the normal distribution. The detailed results are shown in Table 12.

[0141] The results show that: from the results of moisture content determination: Guangning Chikeng, Wenshan No. 1, Heshui> Guangning Hecang, Panlong> Chunwan, Wenshan No. 2, Xishuangbanna, Huaiji> Naruan. The highest moisture content is Guangning Chikeng, which is 11.60±0.40(%, mL / g); the lowest moisture content is Naruan, which is 8.00±0.00(%, mL / g).

[0142] From the actual volatile oil content (excluding water):

[0143] 1) Xishuangbanna> Chunwan, Huaiji, Wenshan II> Guangning Chikeng, Wenshan I> Guangning Hecang, Heshui, Panlong, Naruan. In this test, Amomum villosum in Xishuangbanna has the highest volatile oil content, reaching more than 6%.

[0144] 2) For Amomum villosum in Yunnan, the actual volatile oil content (excluding water): Xishuangbanna > Wenshan 1, Wenshan 2. For Amomum villosum in Guangdong, the volatile oil content: Chunwan, Huaiji > Guangning Chikeng, Guangning Hecang, Heshui, Panlong, Naruan.

[0145] Table 12 Volatile oil content of Amomum villosum from different origins (`X±S)

[0146]

[0147] 6 Comparison of the difference between the volatile oil content of Amomum villosum from different origins and the actual volatile oil content (excluding water)

[0148] The independent sample T test in SPSS was used to examine whether there was a significant difference between the determination results of the volatile oil content and the actual volatile oil content (excluding moisture) of Amomum villosum in various regions. The results are shown in Table 13.

[0149] The results show that:

[0150] 1) Except for Wenshan No.1 (P>0.05) and Guangning Chikeng (P>0.05), the volatile oil content of Amomum villosum from other production areas increased significantly.

[0151] 2) Based on the average value, the volatile oil content of the samples from Xishuangbanna area increased the most, by 0.60% mL / g; the volatile oil content of the samples from Naruan area increased the least, by only 0.29% mL / g.

[0152] Table 13 Volatile oil content of Amomum villosum from different origins (`X±S)

[0153]

[0154] The present invention finds out the variables that significantly affect the content of volatile oil in Amomum villosum, greatly solves the problem that the results of the determination of the volatile oil content in Amomum villosum in actual production determination and scientific research are unstable and have poor repeatability, and improves the stability and repeatability of the determination in the actual production and scientific research process. At the same time, it provides a basis for the determination method of the volatile oil content in Amomum villosum to be improved in the Chinese Pharmacopoeia, and provides a reference for the standards of the determination methods of the volatile oil content in other medicinal materials to be scientific and specialized.

[0155] The present invention is not limited to the above-mentioned embodiments. If various changes or modifications to the present invention do not depart from the spirit and scope of the present invention, and if these changes and modifications fall within the scope of the claims and equivalent technologies of the present invention, the present invention is also intended to include these changes and modifications.

Claims

1. A method for measuring the volatile oil content of Amomum villosum, characterized in that: The method comprises the following steps: 1) Through orthogonal design experiments, the effects of five factors on the content of volatile oil in Amomum villosum, namely, the amount of water added for extraction of volatile oil from Amomum villosum, degree of crushing, condensed water temperature, micro-boiling time, and test sample, were explored to find the factors that have a significant impact on the determination of volatile oil content in Amomum villosum; 2) The optimal extraction method for determining the volatile oil content of Amomum villosum was obtained by exploring the effect of the degree of seed mass crushing on the volatile oil content; the degree of seed mass crushing was P < 0.05; the amount of water added was 500 mL; the crushing method was crushing for a certain time of 5 to 40 seconds, without sieving, and mixing; the condensed water temperature was 28°C or room temperature; the micro-boiling time was 5 hours; the weight of the test sample was 15 g; 3) Based on the principle of being close to the pharmacopoeia and convenient for conducting experiments, a method for determining the volatile oil content of Amomum villosum was established; 4) Determine the volatile oil of Amomum villosum from different origins according to the method of step 3), and calculate and compare the differences in their contents; 5) determining the water content of Amomum villosum from different origins, and then combining it with the determination result of the method of step 3), calculating the actual volatile oil content of Amomum villosum after removing the water, and comparing the difference in the content; 6) Compare the differences in volatile oil content of Amomum villosum from different origins with the actual volatile oil content excluding water.

2. The method for measuring the volatile oil content of Amomum villosum as claimed in claim 1, wherein: The volatile oil content of Amomum villosum from different origins: Xishuangbanna > Chunwan, Huaiji, Wenshan II > Guangning Chikeng, Wenshan I > Guangning Hecang, Naruan, Panlong, Heshui, Shaoguan.

3. The method for measuring the volatile oil content of Amomum villosum as claimed in claim 1, wherein: The actual volatile oil content of Amomum villosum from different origins after removing water is: Xishuangbanna > Chunwan, Huaiji, Wenshan II > Guangning Chikeng, Wenshan I > Guangning Hecang, Heshui, Panlong, Naruan.

4. The method for measuring the volatile oil content of Amomum villosum as claimed in claim 1, wherein: The determination method is as follows: 1) Fruit preservation: Take the Amomum villosum fruit and store it in a sealed and dry storage box. The storage box should be stored in a cool and dry place, away from direct sunlight; 2) Shelling and weighing: Before the experiment, shell the Amomum villosum fruit, weigh 15 g of the seed mass, and record the balance reading; 3) Crushing: Put it into the powder grinder and crush it for a certain time, choose 5 to 40 seconds, and try to avoid the crushing instrument getting hot; 4) Weigh again: pour out as much of the crushed powder as possible, weigh the powder, record the balance reading, and use it to calculate the powder recovery rate, which should be as high as possible; 5) Extraction: pour the powder into a 1000mL round-bottom flask, add a few glass beads, add 500mL of distilled water, and put it into an electric heating mantle; install a volatile oil extractor and a condenser, turn on the condensing water circulation, and extract at a low boiling temperature for 5 hours; 6) Standing: After the extraction is completed, stop heating and keep the condensed water in a circulating state; 7) Collection: After standing for 1 hour, read and collect the volatile oil; 8) Calculation: Calculate the volatile oil content.

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