CN105153082A - Large-scale production technology of andrographalide - Google Patents

Abstract

The invention provides a large-scale production process of andrographolide. The invention adopts the reflux method suitable for large-scale industrial production to extract andrographolide, and the extraction efficiency can reach more than 80%, which can save a large amount of raw medicinal materials and save costs; the extraction time is only 2 hours each time, the extraction time is short, and long-term heating is avoided Destroy andrographolide; extract with 4 times the amount of 95% ethanol, the amount of extraction solvent is small, and the extraction solvent can be reused after recovery, which saves a lot of cost.

Description

Large-scale production process of andrographolide

technical field

The invention relates to a large-scale production process of andrographolide.

Background technique

Andrographolide, molecular formula C20H30O5, is the main active ingredient of the natural plant Andrographis paniculata. It has the effects of dispelling heat and detoxification, reducing inflammation and relieving pain, and has special effects on bacterial and viral upper respiratory tract infections and dysentery. It is known as a natural antibiotic drug. This product is a diterpene lactone compound, which is insoluble in water and can only be administered orally.

The currently disclosed preparation methods of andrographolide include: Patent Application No. 200710142800.7, which discloses a method of separation with resin after alcohol extraction. The eluates with different concentrations of ethanol contained andrographolide to varying degrees when eluted by the macroporous resin method, and the eluate with 80% ethanol had a higher content, but the yield was lower. The literature of Lu Ping et al. (Study on Ultrasonic Extraction of Andrographolide, Journal of Chifeng University (Natural Science Edition) Volume 24, No. 2, 2008) reported that Andrographolide was extracted with ultrasonic-assisted extraction of Andrographolide stem powder. The results show that the most ideal extraction solvent is 75% ethanol, the ratio of material to material is 1:20, and the ultrasonic time is 40 minutes. The literature of Ma Zhiyu et al. (Microwave and Ultrasonic Assisted Extraction of Andrographolide, Fine Chemical Industry Volume 24, No. 8, 2007) reported that the stems and leaves of Andrographis paniculata were used as raw materials, and ultrasonic-assisted extraction and microwave-assisted extraction of andrographolide were used respectively. Results The optimal technological conditions of ultrasonic-assisted extraction were 75% ethanol as the extraction solvent, 40% ultrasonic efficiency, and 50 minutes of ultrasonic time; for 8 minutes. Ultrasonic-assisted extraction and microwave-assisted extraction have positive effects under certain conditions, but these two methods are difficult to apply to large-scale industrial production.

Contents of the invention

The technical scheme of the invention provides a large-scale production process of andrographolide.

The invention provides a kind of large-scale production technique of andrographolide, and it comprises the steps:

a. Take Andrographis paniculata coarse powder, add 95% ethanol or methanol to infiltrate, then add 95% ethanol or methanol, heat and reflux for extraction, filter, and combine the filtrates;

b. Add distilled water to the filtrate to adjust the ethanol concentration to 55%-85%, let it stand at room temperature, and filter to obtain the filtrate;

c. Add 0.4-0.8% activated carbon powder to the filtrate, heat to reflux for decolorization, and filter;

d. Recover the ethanol from the filtrate to 1-10g/ml (the amount of medicinal material/the volume of the concentrated solution), let it stand at 4°C, and filter to obtain the precipitation of andrographolide;

e, adding a small amount of 80% ethanol to the andrographolide precipitate, washing until it is off-white, and then washing with a small amount of water until the aqueous solution is colorless, to obtain a white powder of andrographolide;

f, the andrographolide white powder prepared by step e is added with chloroform to leave standstill, filter, and obtain refined andrographolide powder;

g, add 95% ethanol, heat to dissolve the refined andrographolide powder, filter, reclaim the ethanol to half the amount under reduced pressure, let it stand overnight at 4°C, make crystallization, filter, and obtain andrographolide crystallization; Half the amount, filtered, combined andrographolide crystals, dried under reduced pressure, that is.

Wherein, the extraction solvent described in step a is 95% ethanol. The extraction process described in step a is: add 4 times the amount of 95% ethanol each time, and reflux extraction for 3 times, each time for 2 hours.

Wherein, the ethanol concentration described in step b is up to 65%.

The standing time described in step b is 36 hours.

The consumption of the gac described in c step is 0.5%.

The activated carbon decolorization time described in step c is 0.5-3 hours.

The filtrate described in d step reclaims ethanol under reduced pressure to 1g/ml.

10 times the amount of chloroform described in step f was left standing for 1 hour, and left standing 3 times.

The vacuum drying condition described in step g is: drying under reduced pressure at 50° C. for 2 hours.

The invention adopts the reflux method suitable for large-scale industrial production to extract andrographolide, and the extraction efficiency can reach more than 80%, which can save a large amount of raw medicinal materials and save costs; the extraction time is only 2 hours each time, the extraction time is short, and long-term heating is avoided Destroy andrographolide; extract with 4 times the amount of 95% ethanol, the amount of extraction solvent is small, and the extraction solvent can be reused after recovery, which saves a lot of cost.

Description of drawings

Figure 1 process flow chart

Figure 2 Andrographis paniculata TLC (A-andrographis paniculata TLC under ultraviolet lamp; B-andrographis paniculata TLC under sunlight; S1-andrographolide; S2-dehydroandrographolide; 1-mixed reference substance; 2-andrographis paniculata reference medicinal material; 3-CXL1; 4 -CXL2; 5-CXL3)

Figure 3254nm blank solvent HPLC chromatogram

Figure 4225nm condition blank solvent HPLC chromatogram

Mixed reference substance HPLC chromatogram (1-andrographolide; 2-dehydroandrographolide) under the condition of figure 5254nm

Mixed reference substance HPLC chromatogram (1-andrographolide; 2-dehydroandrographolide) under the condition of figure 6225nm

Sample HPLC chromatogram (1-andrographolide; 2-dehydroandrographolide) under the condition of figure 7254nm

Sample HPLC chromatogram (1-andrographolide; 2-dehydroandrographolide) under the condition of Fig. 8225nm

Fig. 9 dehydroandrographolide standard curve

Figure 10 andrographolide standard curve

Fig. 11 Investigation of concentrated volume of Andrographis paniculata

Figure 12 TLC of andrographolide (1-J1; 2-J2; 3-J3)

Figure 13 andrographolide reference substance HPLC chromatogram (1-andrographolide)

Figure 14 Crystalline HPLC chromatogram (1-andrographolide)

Detailed ways

The extraction process of embodiment 1 andrographolide of the present invention (process flow chart sees Fig. 1)

1 Experimental instruments and materials

1.1 Experimental Instruments

Shimadzu LC-20AT high performance liquid chromatography (Shimadzu, Japan), SIL-20A autosampler, SPD-20A detector, CTO-20A column thermostat, LCsolution chromatographic workstation software, BP211D electronic analytical balance (Germany Sartouris Co., Ltd. company), rotary evaporator RE-2000B (Shanghai Yarong Biochemical Instrument Factory); UPT-I-10T Youpu series ultrapure water machine (Chengdu Ultrapure Technology Co., Ltd.); BP211D electronic analytical balance (1/100,000 ) (German Sartorius Company), BP121S (1/10,000) (German Sartorius Company), SB25-12D Ultrasonic Cleaner (Ningbo Xinyi Ultrasonic Equipment Co., Ltd.); Electric Blast Drying Oven (Model: DB-211SD) .

1.2 Experimental materials

1.2.1 Medicinal materials

A total of 3 batches of Andrographis paniculata were collected, one batch was purchased from Chengdu Hehuachi medicinal materials professional market, the origin is unknown, the other batch was purchased from the Affiliated Hospital of Chengdu University of Traditional Chinese Medicine, the origin is unknown, and the other batch was purchased from Qingyuan Shengzhitang Modern Chinese Medicine Co., Ltd. The place of origin is Qingyuan, Guangdong. See Table 1 for medicinal materials collection.

Table 1 Collection of Andrographis paniculata

"-" means unclear

1.2.2 Reagents and Reagents

Andrographis paniculata reference medicinal material (China Food and Drug Control Institute, batch number 121082-200603), andrographolide (China Food and Drug Control Institute, for content determination, 110797-201108), dehydroandrographolide (China Food and Drug Control Institute, for content determination (batch number 110854-201308), water was ultrapure water, methanol was chromatographically pure (Sinopharm Chemical Reagent Co., Ltd.), and other reagents were analytically pure.

2 Quality inspection of Andrographis paniculata

21 TLC identification

2.1.1 Preparation of the test solution

Take about 0.5g of the powder of this product (passed through a No. 4 sieve), accurately weigh it, put it in a stoppered Erlenmeyer flask, add 25ml of 40% methanol accurately, weigh it, soak for 1 hour, and perform ultrasonic treatment (power 250W, frequency 33kHz) After 30 minutes, let it cool down, weigh it again, make up the lost weight with 40% methanol, shake well, and filter. Precisely measure 10ml of the continued filtrate, put it on a neutral alumina column (200-300 mesh, 5g, inner diameter of 1.5cm), elute with 15ml of methanol, collect the eluate, put it in a 50ml measuring bottle, add methanol to the mark, Shake well and serve.

2.1.2 Preparation of reference medicinal solution

Take Andrographis paniculata reference drug 0.5g, add ethanol 30ml, ultrasonic treatment for 30 minutes, filter, and the filtrate is concentrated to 5ml, as the reference drug solution.

2.1.3 Preparation of reference solution

Then take the dehydrated andrographolide reference substance and the andrographolide reference substance, add absolute ethanol to make a mixed solution each containing 1mg per 1ml, as the reference substance solution.

According to the thin-layer chromatography (Appendix VIB) test, draw the above-mentioned need testing solution, 6 μl of the reference medicinal material solution and 4 μl of the reference solution, respectively spot on the same silica gel GF ₂₅₄ thin-layer plate, and use chloroform-ethyl acetate- Methanol (4:3:0.4) was used as the developer, developed, taken out, dried in the air, and inspected under ultraviolet light (254nm). In the chromatogram of the test product, on the positions corresponding to the chromatogram of the contrast medicinal material and the chromatogram of the reference substance, spots of the same color are shown respectively; spray with 2% 3,5-dinitrobenzoic acid ethanol solution-2mol/L potassium hydroxide solution (1:1) mixed solution (pre-use preparation), immediately inspected under sunlight. In the chromatogram of the test product, spots of the same color are displayed at the positions corresponding to the chromatogram of the reference medicinal material and the chromatogram of the reference product. (See Figure 2)

2.2 Moisture determination

Measure according to "Chinese Pharmacopoeia" in 2010 (Appendix IX H first method). The results are shown in Table 2.

Table 2. Moisture determination of Andrographis paniculata (n=3)

2.3 Content determination

Measure according to "Chinese Pharmacopoeia" 2010 edition one high performance liquid chromatography (appendix VI D), chromatographic conditions, reference substance solution preparation, need testing solution preparation are with " Chinese pharmacopoeia " 2010 edition one one Andrographis paniculata content determination item. The HPLC chromatograms of reference substances and samples are shown in Figures 3-8.

2.3.1 Standard curve

Take 2 μl, 5 μl, 10 μl, 15 μl, and 20 μl of the above-mentioned mixed reference substance solution and inject them into the liquid chromatograph, measure the peak area according to the method under "2.4.1", and take the peak area as the ordinate, and the injection volume as the abscissa, Draw a standard curve, the regression equation is shown in Table 3, Figure 9-10.

Table 3. Standard curve

It can be known from Table 3 that the dehydroandrographolide has a good linear relationship in the range of 0.1848-1.848 μg, and the andrographolide has a good linear relationship in the range of 0.1964-1.946 μg.

2.3.2 Measurement results

See Table 4 for the results of HPLC determination of Andrographis paniculata medicinal material.

Table 4. Andrographis paniculata content determination (n=3)

The HPLC content determination results show that the theoretical plate number of dehydroandrographolide is 6084, and the theoretical plate number of andrographolide is 4305, which is in line with the regulations under the content determination item of Andrographis paniculata in the 2010 edition of "Chinese Pharmacopoeia".

As can be seen from Table 4, the content of andrographolide and dehydroandrographolide in CXL1 samples is unqualified, and the contents of andrographolide and dehydroandrographolide in samples CXL2 and CXL3 all meet the regulations under the determination item of Andrographis paniculata in the 2010 edition of the Chinese Pharmacopoeia, but due to The origin of CXL2 is not clear, and it does not comply with the relevant provisions of the "Letter on Soliciting Opinions on Strengthening the Supervision and Management of Chinese Medicine Extraction and Extracts", and its andrographolide content is lower than that of the CXL3 sample with a clear origin, so the CXL3 sample was selected for experimental research.

3 andrographolide preparation

In the experiment, the key processes such as extraction and purification of andrographolide were systematically studied. The result is as follows.

3.1 Pretreatment of Andrographis paniculata

Crush the Andrographis paniculata into coarse powder.

3.2 Extraction

3.1.1 Investigation of extraction methods

The experimental research compared and studied the effects of three methods, which are suitable for industrial production, immersion method, percolation method and reflux method, on the extraction efficiency of andrographolide. The methods and results are as follows.

Immersion method: take 3 parts of Andrographis paniculata crude powder, each 25g, add 1.5 times the amount of 95% ethanol to soak for 30 minutes. Then add 5 times the amount of 95% ethanol and soak in a 55°C water bath for 2 times, each time for 12 hours, filter, and combine the filtrates. Get the soaking extract.

Percolation method: take 3 parts of Andrographis paniculata crude powder, each 25g, add 1.5 times the amount of 95% ethanol to infiltrate for 30 minutes. Slowly collect 250ml of percolate at a speed of 1-3ml/min (add 95% ethanol while percolating). Obtain percolation extract.

Reflux method: take 3 parts of Andrographis paniculata crude powder, 25g each, add 1.5 times the amount of 95% ethanol to infiltrate for 30 minutes. Add 5 times the amount of 95% ethanol to reflux for extraction twice, each time for 3 hours, filter, and combine the filtrates. The reflux extract was obtained.

Take the above-mentioned soaking extract, percolation extract, and reflux extract 1ml respectively and put them in a 10ml volumetric flask, add 95% ethanol to make the volume up to the mark, take the subsequent filtrate, measure the concentration of andrographolide in each extract, and calculate the concentration of andrographolide. quantity. The transfer rate of andrographolide was used as an index to investigate the extraction method. The results are shown in Table 5.

Table 5 andrographolide extraction method investigation

It can be seen from Table 5 that the extraction effects of the dipping method and the percolation method are similar, and the extraction efficiency of the reflux method is the highest, and the reflux method is used for the test.

3.2.2 Investigation of extraction solvent

Take 2 portions of Andrographis paniculata crude powder, 25g each, add 1.5 times the amount of 95% ethanol and methanol to infiltrate for 30 minutes. Add 5 times the amount of 95% ethanol and methanol to reflux extraction twice, each time for 3 hours, filter, combine the filtrate, and dilute the filtrate to 500ml. The reflux extract was obtained. The results are shown in Table 6.

Table 6 Extraction Solvent Investigation

As can be seen from Table 6, the use of 95% ethanol and methanol as the extraction solvent has no significant difference in the extraction rate of andrographolide. Considering that the cost of using methanol as the extraction solvent is high, and methanol residues have certain potential safety hazards in clinical use, Therefore, 95% ethanol was chosen as the extraction solvent.

3.2.3 Orthogonal design to optimize key process parameters

The concentration of ethanol, the amount of ethanol, the extraction time, and the number of extractions have a great influence on the extraction efficiency of andrographolide. Therefore, the above four factors are selected to set three levels respectively to investigate their influence on the extraction efficiency of andrographolide. For the design of factor levels, see Table 7.

Get the Andrographis paniculata medicinal material, each about 25g, use 95% ethanol as the extraction solvent, test according to the orthogonal test table, combine the filtrate, and the filtrate measures the andrographolide content according to the Andrographis paniculata medicinal material content determination method. Orthogonal design and results are shown in Table 8.

Table 7 Test factor level table

Table 8 Orthogonal Experimental Results (n=2)

The variance analysis was carried out on the results of the orthogonal test. Since the SS value of factor A was less than the error, it was attributed to the error and then analyzed again. The results are shown in Table 9.

Table 9 Orthogonal test analysis of variance results

Carrying out visual analysis and analysis of variance to orthogonal experiment, we can know that the influence order of each factor on the extraction rate of Andrographis paniculata is C>D>B>A, that is, the number of extractions is the main factor, the extraction time is the important factor, and the concentration of ethanol and the ratio of solid to liquid are secondary. Important factors, considering that low-concentration ethanol brings out more water-soluble impurities, so the ethanol concentration is selected as 95% ethanol. Thus the optimal extraction process D ₃ C ₂ B ₂ A ₁ was obtained, that is, adding 4 times the amount of 95% ethanol each time, and refluxing extraction 3 times, 2 hours each time.

3.2.4 Extraction process verification test

Take Andrographis paniculata medicinal powder 200g, add 4 times the amount of 95% ethanol, reflux extraction 3 times, each time 2h. The results are shown in Table 10.

Table 10 Extraction process verification test

It can be seen from Table 10 that the average extraction rate of andrographolide after the process optimization is 83.28, and the RSD is 0.80%, indicating that the extraction rate of the optimized process is high and stable.

3.3 Investigation of purification process

The purification of andrographolide adopts the method of water precipitation and reflux with activated carbon to remove impurities such as chlorophyll, uses chloroform to stand still to remove dehydrated andrographolide, and finally adopts the method of ethanol recrystallization to obtain andrographolide crystals.

3.3.1 Adjustment of ethanol concentration investigation

Take 100ml of Andrographis paniculata extract in 3.2.4 (equivalent to 9g of crude drug), add appropriate amount of distilled water to adjust the ethanol concentration to 55%, 65%, 75%, and 85% respectively, observe the precipitation and filtration after standing overnight, and determine The content of andrographolide in the solution was used to calculate the loss of andrographolide. The results are shown in Table 11.

Table 11 regulates ethanol concentration investigation (n=3)

"+" means precipitation, "+++" means the largest amount of precipitation, "—" means no precipitation

"**" means filterable, "***" means easy to filter

As can be seen from Table 11, the content of andrographolide has a small decrease after adding water to adjust the ethanol concentration to 55%, but there is no significant change at 65% to 95%, and there is a large amount of precipitation when the ethanol concentration is adjusted to 65%, and the filtration is rapid, so Choose to adjust the ethanol concentration to 65%.

3.3.2 Investigation of the standing time of water sinking

Get Andrographis paniculata medicinal material powder 500g, press the andrographolide extract solution extracted by the extraction method in 3.2.4 and measure the andrographolide content in the extract solution, add the extract solution and be divided into 3 parts, add the distilled water equivalent to 6 times the amount of crude drug respectively and let it stand, Samples were taken at 3, 6, 12, 24, and 36 hours to measure the andrographolide content, and the loss of andrographolide was calculated, and the precipitation was observed. The results are shown in Table 12.

Table 12 adds water precipitation time investigation (n=3)

"+" means precipitation, "+++" means the largest amount of precipitation, "—" means no precipitation

"**" means filterable, "***" means easy to filter

It can be seen from Table 12 that after adding water and standing still for 36 hours, the precipitation is complete and the content of andrographolide has no obvious change, that is, after adding water, let stand at room temperature for 36 hours.

3.3.3 Activated carbon decolorization investigation

3.3.3.1 Investigation on the dosage of activated carbon

Take 3 parts of Andrographis paniculata extract in 3.3.2, each 500ml (equivalent to 30g of crude drug), add 2.0g, 2.5g, 3g, 3.5g, 4.0g of activated carbon powder, equivalent to 0.4%, 0.5%, 0.6% of the solution %, 0.7%, 0.8%, reflux decolorization for 1 hour, observe the color of the decolorization solution after cooling and measure the content of andrographolide, and calculate the loss of andrographolide. The results are shown in Table 13.

Table 13 Investigation of the amount of activated carbon added (n=3)

"+" indicates the lightest color, ""++" indicates the lighter color, "+++" indicates the darker color, and "++++" indicates the darkest color

It can be seen from Table 13 that the content of andrographis paniculata was not significantly reduced by adding 0.4% to 0.5% of the solution to reflux for decolorization for 1 hour, and there was no obvious green color in the decolorization solution. When the amount of activated carbon added was greater than 0.5% of the solution, the content of andrographolide was obvious Reduce, so the active carbon consumption gets final product 0.5% of medicinal material amount.

3.3.3.1 Inspection of decolorization time

Take 4 parts of Andrographis paniculata extract in 3.3.2, each 500ml (equivalent to 30g of crude drug), add 2.5g of activated carbon powder, reflux for decolorization for 0, 0.5, 1, 2, 3 hours, filter, observe the color of the decolorization solution after cooling and Determination of andrographolide content, calculation of andrographolide loss. The results are shown in Table 14.

Table 14 Investigation of the amount of activated carbon added (n=3)

"++" is the darkest color; "+" is lighter

As can be seen from Table 14, there is a small decrease in andrographolide as the reflux time prolongs, and if the temperature is too high, the andrographolide will be destroyed. If the decolorization time is too short, the decolorization purpose cannot be achieved, so the reflux decolorization time can be selected for 1 hour.

3.3.4 Concentration volume inspection

Get Andrographis paniculata medicinal material powder 500g, get Andrographis paniculata decolorization liquid after the above-mentioned operation, get Andrographis paniculata decolorization 5 parts, every part of 500ml (equivalent to 31g of crude drug), concentrate under reduced pressure to 1ml/g, 2ml/g, 3ml/g, 5ml/g , 8ml/g, 10ml/g, measure the andrographolide content in the filtrate after standing overnight. The results are shown in Figure 11.

As can be seen from Figure 11, when concentrated to 1ml/g, andrographolide has a large amount of precipitation, and when the Andrographis paniculata decolorization solution is concentrated to 1ml/g, it gets final product.

3.3.5 Investigation of Alcohol Washing and Precipitation

In the previous prediction test, it was found that the precipitate in 3.3.4 was dark yellow, and the crystals of andrographolide obtained through subsequent purification and crystallization operations were yellow flake or needle crystals, and the purity could only reach 87%. According to the prediction, it is found that after washing the precipitate with a small amount of ethanol, the color of the precipitate becomes off-white, and after purification and crystallization, the crystallization is in the shape of flakes or needles, with a purity of more than 95%, which reaches Part 1 of andrographolide in the "Chinese Pharmacopoeia" 2010 edition. Relevant regulations on purity under this item.

Therefore, the effects of ethanol concentration and ethanol dosage on the washing effect were investigated. The specific test methods and results are as follows.

3.3.5.1 Ethanol consumption

Take 4 parts of the precipitate in 3.3.4, each 1g, put them in the Buchner funnel respectively, add 0, 3, 5, 8ml (0, 3, 5, 8 times) 95% ethanol to wash, dry under reduced pressure at 50°C and observe Precipitate color, and measure andrographolide content, calculate andrographolide loss amount, the results are shown in Table 15.

Table 15 Investigation of Ethanol Consumption

Note: "+" indicates that the precipitate color is yellow, "-" indicates that the precipitate color is off-white

It can be seen from Table 15 that when the amount of ethanol added is less than 5 times the amount of precipitation, the color of the precipitate is yellow, and when the amount of ethanol added is greater than 5 times, the color of the precipitate is off-white, so choose to use 5 times the amount of ethanol for washing.

3.3.5.2 Investigation of ethanol concentration

Take 4 parts of the precipitate in 3.3.4, each 1g, put them in the Buchner funnel respectively, add 5ml of 65%, 80%, and 95% ethanol to wash, and wash the other part without ethanol, and observe the color of the precipitate after drying under reduced pressure at 50°C. And measure andrographolide content, calculate andrographolide loss amount, the results are shown in Table 16.

Table 16 Investigation of ethanol concentration

Note: "+" indicates that the precipitate color is yellow, "-" indicates that the precipitate color is off-white

As can be seen from Table 16, when the ethanol concentration is less than 80%, although the loss of andrographolide is less, the precipitate color is yellow. When the ethanol concentration is greater than 80%, the precipitate color is off-white, so it is selected to use 80% ethanol for washing. Can.

In the follow-up verification test, it was found that when the precipitate was washed with 5 times the amount of 80% ethanol, the loss of andrographolide was relatively large, which could reach more than 20%. Therefore, when washing with ethanol, wash until the color of the precipitate is off-white, then add a small amount of water to wash until the aqueous solution is colorless.

3.3.6 Purification investigation of chloroform

References ^[9-10] , using the property that dehydroandrographolide is easily soluble in chloroform, but andrographolide is insoluble in chloroform, the dehydroandrographolide in the above precipitate was removed by standing in chloroform. Considering the influence of the amount of chloroform, standing time and standing times on the impurity removal effect, the following three single factor experiments were designed to select the best test plan. The specific test methods and results are as follows.

3.3.6.1 Investigation on the amount of chloroform

Get 200g of Andrographis paniculata medicinal material powder, obtain andrographolide coarse crystal after the above-mentioned operation, get 6 parts of andrographolide coarse crystal after drying, every part of 1g, add 5, 10, 20, 30, 40, 50ml trichloromethane (equivalent to Let stand for 1 hour at 5, 10, 20, 30, 40, 50 times the amount of sedimentation), filter, and dry to obtain refined andrographolide powder, respectively measure the dehydroandrographolide content, and calculate the loss of dehydroandrographolide. The results are shown in Table 17.

Table 17 Chloroform dosage investigation

It can be seen from Table 17 that when the amount of chloroform used is 10 times greater than the amount of precipitation, the content of dehydroandrographolide does not decrease significantly. Considering the residual chloroform and the cost, it is sufficient to select 10 times the amount of chloroform and let it stand.

3.3.6.1 Investigation on the standing time of chloroform

Take 200g of Andrographis paniculata medicinal material powder, obtain andrographolide precipitation after the above operation, take 4 parts of andrographolide precipitation after drying, each part of 1g, add 10ml of chloroform, let stand for 0.5, 1, 2, 3 hours respectively, filter, Dry to obtain refined andrographolide powder, measure the content of dehydroandrographolide respectively, and calculate the loss of dehydroandrographolide. The results are shown in Table 18.

Table 18 Chloroform dosage investigation

It can be seen from Table 18 that the loss ratio of dehydroandrographolide is relatively small after standing for 0.5 hours, and there is no significant change in the loss ratio of dehydroandrographolide after standing for 1, 2, and 3 hours. Can.

3.3.6.1 Investigation on the standing times of chloroform

Take 200g of Andrographis paniculata medicinal material powder, and obtain andrographolide precipitation after the above operation, after drying, take 3 parts of andrographolide precipitation, each 1g, add 10ml of chloroform, let stand 1, 2, 3 times respectively, 1 hour each time , filtered, and dried to obtain refined andrographolide powder, and the content of dehydroandrographolide was determined respectively. The results are shown in Table 19.

Table 19 Chloroform dosage investigation

It can be seen from Table 19 that the number of times of standing has a great influence on the refining effect, and the purpose of removing a large amount of dehydroandrographolide can be achieved after standing for 3 times, and it is sufficient to choose to stand for 3 times.

3.4 Crystallization

References ^[9-10] , the method of recrystallization of coarse crystals of andrographolide is mainly to purify andrographolide by heating and dissolving with ethanol, recovering the solvent, and standing for crystallization.

Take 4g of refined andrographolide powder, add 200ml of 95% and heat to dissolve, filter, recover the solvent to half the amount under reduced pressure, stand overnight at 4°C, filter to obtain andrographolide crystals. Concentrate the mother liquor to half the amount again, repeat the above operation to obtain andrographolide crystals, combine the two andrographolide crystals, dry under reduced pressure at 50°C, and obtain andrographolide crystals. The results are shown in Table 20.

Table 20 Crystallization Results

It can be seen from Table 20 that according to the above crystallization method, the crystal purity of andrographolide obtained through twice concentrated crystallization is greater than 95%, which meets the requirements of the "Chinese Pharmacopoeia".

3.5 Process verification

Take Andrographis paniculata crude powder 200g, operate according to the technological process, the results are shown in Table 21.

Table 21 Process Validation

The results showed that the average yield of andrographolide was 1.09, the average purity was 97.88%, and the RSD of yield and purity were both less than 5%, indicating that the method was stable and feasible.

3.6 Summary

Final determination of andrographolide extraction, purification process is: get Andrographis paniculata coarse powder 200g, add 1.5 times the amount of 95% ethanol, infiltrate for 30 minutes, then add 4 times the amount of 95% ethanol, heat and reflux extraction 3 times, each time for 2 hours, filter , combined 3 filtrates. Add distilled water to the filtrate to adjust the ethanol concentration to 65%, let it stand at room temperature for 36 hours, filter, add 0.5% activated carbon powder to the filtrate, heat and reflux for 1 hour to decolorize, filter, and recover ethanol from the filtrate to 1g/ml (medicinal material amount/concentrate volume) under reduced pressure. Stand at 4°C for 36 hours, filter to obtain andrographolide precipitate. The andrographolide precipitate is washed with a small amount of 80% ethanol (washed and precipitated to off-white), and then washed with a small amount of water (washed until the aqueous solution is colorless), and the white powder of andrographolide is obtained, and the white powder of andrographolide is added with 10 times the amount of chloroform Let it stand for 3 times, each time for 1 hour, filter to obtain refined andrographolide powder. Add 1 times the amount of crude drug 95% ethanol, heat slightly to dissolve the refined andrographolide powder, filter, recover the ethanol to half the amount under reduced pressure, stand overnight at 4°C to crystallize, filter to obtain andrographolide crystals. The mother liquor was recovered under reduced pressure to reduce the solvent to half, filtered, combined two crystals of andrographolide, dried under reduced pressure at 50°C for 2 hours, and obtained.

3.7 Process flow

3.7.1 Description of key process parameters

Note: The color of the solution, the volume of the solution, and the density of the solution in each step are all observed or measured after the solution is cooled.

[1] After reflux extraction, the filtered filtrate is green, with a volume of about 2300-2600ml and a density of about 0.790-0.810g/ml.

[2] Add water and let it stand. After filtering, the filtrate should be yellow-green, with a volume of about 3300-3700ml and a density of about 0.890-0.910g/ml.

[3] Add 0.5% activated carbon to reflux for decolorization. After filtration, the filtrate is yellow, with a volume of about 2900-3300ml and a density of about 0.890-0.910g/ml.

[4] When the decolorization solution is concentrated under reduced pressure, in order to avoid the loss of andrographolide, the temperature should not be too high. This study chooses 55°C.

[5] After the decolorization solution is concentrated, the solution is yellow, and a large amount of yellow precipitates are precipitated at the bottom of the solution, with a volume of about 200-300ml and a density of about 1.05-1.25g/ml. After standing at 4°C for 36 hours, a large amount of off-white precipitate precipitated at the bottom.

[6] After standing still at 4°C for the second time, the amount of precipitation is less, so the second standing precipitation precipitation step is omitted.

[7] Add a small amount of 80% ethanol to wash the precipitate and wash the precipitate until off-white, then add a small amount of water to wash.

[8] When recovering ethanol under reduced pressure after crystallization, in order to avoid the loss of andrographolide, the temperature should not be too high, 50°C was selected in this study.

[9] During crystallization drying, in order to avoid excessive temperature and quickly achieve the purpose of drying, this study chooses the method of decompression drying and drying in the field at 50°C for 2 hours.

4 Andrographolide Quality Check

4.1 Other lactones

Take this product, add absolute ethanol to make a solution containing 2mg per 1ml as the test solution. According to the thin-layer chromatography (Appendix VIB) test, draw 10 μl of the above solution, spot it on the silica gel G thin-layer plate, use chloroform-methanol (19:1) as the developer, develop, take it out, dry it, and spray it with 2 % 3,5-dinitrobenzoic acid ethanol solution and 5% potassium hydroxide ethanol solution in equal volume (preparation). In the chromatogram of the test product, except for the main spot, other spots shall not appear (as shown in Figure 12).

4.2 Loss on drying

Take this product and dry it to constant weight at 105°C, and the weight loss should not exceed 1.0% (Appendix IXG). The results are shown in Table 22.

Table 22 andrographolide loss on drying (n=3)

It can be seen from Table 22 that the weight loss of the three groups of crystals is less than 1.0%, which meets the requirements of the Pharmacopoeia.

4.2 Content determination

4.2.1 Chromatographic conditions and system suitability test Octadecylsilane bonded silica gel was used as filler; methanol-water (60:40) was used as mobile phase; detection wavelength was 225nm. The number of theoretical plates should not be less than 5000 based on the peak of andrographolide.

4.2.2 Preparation of reference substance solution Take an appropriate amount of andrographolide reference substance, weigh it accurately, add methanol to make a solution containing 0.101mg per 1ml, and obtain it.

4.2.3 Preparation of the test solution Take about 4.91, 4.94, and 5.07 mg of this product, weigh them accurately, put them in a 50ml measuring bottle, add methanol to dissolve and dilute to the mark, shake well, and you get it.

4.2.4 Determination of samples Accurately draw 10 μl each of the reference substance solution and the test solution, inject it into the liquid chromatograph, and measure it. The results are shown in Figures 13-14.

The method for preparing andrographolide of the present invention has the following characteristics: the reflux method suitable for industrial large-scale production is used to extract andrographolide, the extraction efficiency is high, the time consumption is short, the organic solvent consumption is small, and the cost is low; the present invention uses The combination of alcohol extraction, water sedimentation and activated carbon reflux decolorization removes impurities such as chlorophyll, which has the advantages of less activated carbon consumption and short antipyretic time, avoiding the adsorption of andrographolide by activated carbon and the decomposition of andrographolide by prolonged heating; this study The method of washing the coarse crystals of andrographolide with ethanol to remove impurities avoids the decomposition of andrographolide by repeated use of activated carbon to decolorize and remove impurities in the traditional process. No macroporous resin is used in the preparation process; the reaction time is short, saving energy; the product is safe and effective, convenient for "three wastes" treatment, and beneficial to environmental protection; the product has high purity, and the andrographolide content can reach more than 97%; the process is simple to operate, suitable for Industrial production.

references:

[1] Lu Ping, Ding Teng, Sheng Minli. Study on Ultrasonic Extraction of Andrographolide. Journal of Chifeng University, 2008, 24(2): 28-29

[2] Wang Hongyan, Liu Xuefei. Extraction process of andrographolide. Traditional Chinese Medicine, 2011, 24(3): 170

[3] Wu Aiqun, Zhang Guoqiang. Research on extraction process of andrographolide. Journal of Hechi University, 2009, (5): 66-68

[4] Dai Ping, Wei Hanyan, Huang Guixiang, etc. Research on the extraction process of andrographolide. Science and Technology Information, 2010, (1): 305-307

[5] Zhang Jianqiang, Zhang Ling, Wang Huan, etc. Research on the extraction process of Andrographis paniculata. Food and Drugs, 2005, 7, (1): 37-40

[6] Liu Lei, Xu Xiaoxiao, Han Guang. Optimizing the Extraction Process of Andrographolide by Uniform Design Method, Journal of Henan University, 2006, 25(4): 33-35

[7] Ma Zhiyu, Lin Cuiwu, Liao Sen, etc. Microwave and Ultrasonic Assisted Extraction of Andrographolide, Fine Chemical Industry, 2007, 24(8): 758-760

[8] Yang Tao, Sheng Huanhuan, Li Yan, et al. Optimizing the Extraction Process of Andrographis paniculata by Star Point Design-Response Surface Method, Chinese Journal of Pharmaceutical Sciences, 2011, 46(3): 208-213

[9] Zhuo Ju, Wang Xiaogen. Orthogonal design study on the extraction process of dehydroandrographolide from Andrographis paniculata. Zhongnan Pharmacy, 2005, 3(3): 155-157

Claims (10)

1. the large production technique of rographolide, it comprises the steps:

A, get Herba Andrographis meal, add 95% ethanol or methyl alcohol infiltrates, then add 95% ethanol or methyl alcohol, heating and refluxing extraction, filter, merging filtrate;

B, filtrate adding distil water regulate alcohol concn to 55%-85%, and room temperature leaves standstill, and filter, obtain filtrate;

C, filtrate add 0.4-0.8% active carbon powder, and reflux is decoloured, and filter;

D, decompression filtrate recycling ethanol are to 1-10g/ml (medicinal material amount/concentrated solution volume), and 4 DEG C leave standstill, and filter, and obtain rographolide precipitation;

E, rographolide precipitation adds a small amount of 80% washing with alcohol, wash to off-white color, then it is colourless to the aqueous solution to add a small amount of water washing, obtains rographolide white powder;

F, rographolide white powder step e prepared add trichloromethane and leave standstill, and filter, obtain refining rographolide powder;

G, add 95% ethanol, heating makes refining rographolide powder dissolution, filters, and decompression recycling ethanol is to half amount, and 4 DEG C of hold over night, make crystallization, filters, obtains rographolide crystallization; Mother liquor again decompression and solvent recovery, to half amount, filters, and merge rographolide crystallization, drying under reduced pressure, to obtain final product.

2. the large production technique of rographolide according to claim 1, is characterized in that: the Extraction solvent described in a step is 95% ethanol.

3. the large production technique of rographolide according to claim 1, is characterized in that: the extraction process described in a step is: add 95% ethanol 4 times amount, refluxing extraction 3 times, each 2h at every turn.

4. the large production technique of rographolide according to claim 1, is characterized in that: the alcohol concn to 65% described in b step.

5. the large production technique of rographolide according to claim 1, is characterized in that: the time of repose described in b step is 36 hours.

6. the large production technique of rographolide according to claim 1, is characterized in that: the consumption of the gac described in step c is 0.5%.

7. the large production technique of rographolide according to claim 1, is characterized in that: the activated carbon decolorizing time 0.5-3 hour described in step c.

8. the large production technique of rographolide according to claim 1, is characterized in that: the decompression filtrate recycling ethanol described in Step d is to 1g/ml.

9. the large production technique of rographolide according to claim 1, is characterized in that: 10 times amount trichloromethanes described in f step leave standstill 1 hour, leaves standstill 3 times.

10. the large production technique of rographolide according to claim 1, is characterized in that: the drying under reduced pressure condition described in g step is: 50 DEG C of drying under reduced pressure 2 hours.