CN1869681A - Method for investigating Monacolin kind compound content in functional Monacolin - Google Patents

Abstract

The invention relates to a function red rice yeast Monacolin content detecting method, comprising the steps of: 1) sample preprocessing; 2) preparing acidic Monacolin K standard sample; 3) chromatographic sample analysis; 4) pattern analysis and calculating. And it excellently separates various Monacolin compounds from functional red rice yeast extract by HPLC, uses diode array detector to detect UV absorption spectrum of various Monacolin compounds, and finally uses external standard method to make qualitative analysis according to reserving time and UV absorption spectrum and makes quantitative analysis with peak areas. And the invention can detect total content of Monacolin compounds in the sample by detecting Monacolin compound-shared characteristic UV absorption spectrum on the condition of being shorting of Monacolin compound standard sample. And the method has good accuracy, precision and linearity and good detection results.

Description

The detection method of Monacolin kind compound content in a kind of functional red yeast rice

Technical field

The present invention relates to a kind of health food functional component detection method.

Background technology

Red colouring agent for food, also used as a Chinese medicine is widely used in food industry as natural colouring matter and food additives.Far rose report in 1979 and from the monascus fermentation liquor, separate and obtain a kind of material that falls the plasma cholesterol effect that has, be called Monacolin K, and find that it has the synthetic effect of cholesterol in human body that suppresses.The later class that monascus can produce in its growth later stage that studies confirm that has the material that suppresses active, both Monacolin compounds to animal and human's body 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase).They are gang's analogues, can be divided into: compositions such as Monacolin J, K, L, X, M, Dihydromevinolin and Dihydromonacolin L.Wherein maximum with Monacolin K content.

Because Monacolin class material inhibition cholesterol synthetic effect is remarkable, toxic and side effect is little, and better tolerance just has been applied to clinical since finding very soon.The Monacolin compound is applied to some as the health food functional component again and has the health food of hypolipemic function and the welcome that is subjected to consumers in general in recent years.In order to guarantee product quality, just seem very important for the detection of Monacolin class material in raw material and the product.

Since two thousand the method for measuring Monacolin K (claiming Lovastatin again) content in the red colouring agent for food, also used as a Chinese medicine with ultraviolet spectrophotometer and reversed-phase high-performance liquid chromatography-UV-detector has been reported in this laboratory successively on " Food Additives Used in China " and " Food Science " magazine.Because UV-detector can only obtain the chromatogram elution curve figure of each component under the single wavelength, and can not obtain each component abosrption spectrogram with the chromatographic component outflow; Because the standard model of present many Monacolin compounds does not have commodity selling, therefore give the qualitative difficulty of bringing of isolated each component again.So for the quantitative measurement of the hypolipemic function factor in the red colouring agent for food, also used as a Chinese medicine, in fact only measured the Monacolin K that content is maximum in the Monacolin compounds, and ignored other Monacolin compounds in the red colouring agent for food, also used as a Chinese medicine in the past.Obviously this is defective for suppressing the synthetic function factor of cholesterol in the accurate evaluation red colouring agent for food, also used as a Chinese medicine.

Summary of the invention

In order to address the above problem, the invention provides the detection method of Monacolin kind compound content in a kind of functional red yeast rice.The present invention is achieved in that

The detection method of Monacolin kind compound content in a kind of functional red yeast rice, it may further comprise the steps:

1) sample pre-treatment;

2) preparation of acid Monacolin K standard model;

3) the chromatogram sample introduction is analyzed;

4) atlas analysis and result calculate.

The detection method of Monacolin kind compound content in the described functional red yeast rice, its concrete steps can also for:

1) comprises in the sample pre-treatment step: the red colouring agent for food, also used as a Chinese medicine sample is pulverized (〉=80 order) also fully mix; Accurately take by weighing 400.0～600.0mg sample in the 50ml volumetric flask, add 30ml 75% ethanol (V/V), shake up ultrasonic 20min (frequency of operation 40KHz) under the room temperature.Add 75% ethanol extremely near scale, ultrasonic again 10min is cooled to room temperature afterwards, is settled to 50ml with 75% ethanol; With 3500 rev/mins of centrifugal 10min; Get supernatant and cross 0.45 μ m aperture miillpore filter, filtrate is treated sample introduction;

2) preparation process of acid Monacolin K standard model comprises: take by weighing Monacolin K (lactone) standard items 4mg, be settled to 100ml with the 0.2M sodium hydroxide solution, put under 50 ℃ of conditions ultrasonic conversion 1 hour, be cooled to room temperature after, lucifuge is placed after 3 hours and is used; Calculate conversion ratio according to transforming back acid Monacolin K and residual lactone Monacolin K chromatographic peak area; Utilize the acid Monacolin K of preparation, acid Monacolin K in the sample is carried out accurately qualitative and quantitative;

3) chromatogram sample introduction analytical procedure comprises: the filtrate of the filtering with microporous membrane of learning from else's experience enters liquid chromatograph, and described chromatographic chromatographic condition is: chromatographic column: C ₁₈Post, 4.6 * 250mm, 5 μ m; Column temperature: room temperature; Diode array detector: 200～350nm scanning, 238nm place chromatogram deal with data; Moving phase: methyl alcohol: water: phosphoric acid=385: 115: 0.14 (V: V); Flow velocity: 1.0ml/min; Sample size: 20 μ l;

4) atlas analysis comprises with calculation procedure as a result: find the component peaks that identical ultra-violet absorption spectrum is arranged with standard MonacolinK on chromatogram elution curve figure, be the Monacolin compounds, peak area addition with these components, compare Monacolin compounds total amount in the calculation sample with standard Monacolin K chromatographic peak area;

Result's computing formula:

$X=\frac{h_1\×c\×50}{h_2\×m}$

In the formula: Monacolin compounds total amount in the X-style, mg/g

h ₁-have a separated component chromatographic peak area sum of Monacolin compounds characteristic absorption spectrum

C-standard Monacolin K (lactone type or acid) solution concentration, mg/ml

50-sample constant volume, ml

h ₂-Monacolin K (lactone type or acid) peak area

The m-sample amount of taking by weighing, g.

The functional red yeast rice tunning contains various Monacolin compounds, based on lactone type and acid Monacolin K.In dehydrating the process of processing, acid Monacolin K changes lactone type gradually into.Because they all have physiologically active, so it is more meaningful to measure the Monacolin total amount of compound.

Diode array detector can provide all separated component chromatograms and flow out instant ultra-violet absorption spectrum, therefore can see clearly that on chromatogram elution curve figure those and Monacolin K have the separated situation of Monacolin compounds of identical ultra-violet absorption spectrum.If it is standard substance is arranged, qualitative one by one, quantitatively very convenient to them.For example because therefore acid Monacolin K instability does not have standard substance to sell at present.The condition of utilizing this method to provide can be converted into acid with standard lactone type Monacolin K easily.With the standard model sample introduction after transforming, just can be accurately qualitative and quantitative to the acid Monacolin K in the sample.

Those there is the peak area addition of the Monacolin compounds component of identical ultra-violet absorption spectrum with Monacolin K, quantitatively just can accesses the total amount of Monacolin compounds in the sample to them with standard lactone type Monacolin K.Although different Monacolin compounds has difference to the response of detecting device, the result who obtains has some errors, is lacking under the situation of Monacolin compounds reference material at present, and this method still is effective.Again since sample in various Monacolin compounds based on lactone type and acid Monacolin K, lactone type Monacolin K has commodity selling, this method has solved the preparation problem of acid Monacolin K standard model again, and other Monacolin kind compound contents seldom, and the Monacolin total amount of compound has higher accuracy in this method measurement function red colouring agent for food, also used as a Chinese medicine sample so adopt.

If active constituent content is low excessively in the sample, the supernatant that extracts after centrifugal suitably can be concentrated with the concentrating under reduced pressure method, but the result need be by the accurate volume calculation after concentrating.

The present invention is further illustrated below in conjunction with accompanying drawing.

Description of drawings

Fig. 1 is Monacolin K (lactone formula) Monacolin K acid (open loop acid) standard colors spectrogram

Fig. 2 is certain red colouring agent for food, also used as a Chinese medicine sample chromatogram figure

Fig. 3 is the Monacolink typical curve

Embodiment

The detection of Monacolin kind compound content in a kind of functional red yeast rice

1. material

1.1 reagent

1.1 methyl alcohol chromatographically pure

1.2 it is pure that absolute ethyl alcohol is analyzed

1.3 phosphate analysis is pure

1.4 it is pure that NaOH is analyzed

1.5Monacolin the accurate storing solution of K accurately takes by weighing Monacolin K (lactone type) standard items 40.0mg, with moving phase constant volume 100ml.This solution concentration is 400 μ g/ml.

1.6Monacolin K standard operation liquid is accurately measured Monacolin K standard reserving solution 1ml, with moving phase constant volume 10ml.This solution concentration is 40 μ g/ml.

1.2 instrument and equipment

1.2.1 high performance liquid chromatograph (Waters 600E)

1.2.2 diode array detector (Photodiode Array Detecter, Waters 2996)

1.2.3 low speed centrifuge (Shanghai Surgical Operation Equipment Factory, 80-2)

1.2.4 ultrapure water system (U.S. PALL)

1.2.5 ultrasonic cleaner (Kunshan ultrasonic instrument company limited, KQ-100)

1.2.6 rigorous analysis balance (Mettler AE-100)

2. method

2.1 sample is handled

The functional red yeast rice sample is pulverized (〉=80 order) also fully to mix.Accurately take by weighing 400.0～600.0mg sample in the 50ml volumetric flask, add 30ml 75% ethanol (V/V), shake up ultrasonic 20min (frequency of operation 40KHz) under the room temperature.Add 75% ethanol extremely near scale, ultrasonic again 10min is cooled to room temperature afterwards, is settled to 50ml with 75% ethanol.With 3500 rev/mins of centrifugal 10min.Get supernatant and cross 0.45 μ m aperture miillpore filter, filtrate is treated sample introduction.

2.2 the preparation of acid Monacolin K standard items

Take by weighing Monacolin K (lactone) standard items 4mg, be settled to 100ml, put under 50 ℃ of conditions ultrasonic conversion 1 hour with the 0.2M sodium hydroxide solution, be cooled to room temperature after, lucifuge is placed after 3 hours and is used.Calculate conversion ratio according to transforming back acid Monacolin K and residual lactone Monacolin K chromatographic peak area.Utilize the acid Monacolin K of preparation, acid Monacolin K in the sample is carried out accurately qualitative and quantitative.

2.3 chromatographic condition

Chromatographic column: C ₁₈Post 4.6 * 250mm 5 μ m

Column temperature: room temperature

Diode array detector: 200～350nm scanning, 238nm place chromatogram deal with data

Moving phase: methyl alcohol: water: phosphoric acid=385: 115: 0.14 (V: V)

Flow velocity: 1.0ml/min

Sample size: 20 μ l

2.4 atlas analysis

Get the sample extract 20 μ l sample introductions of handling well, after the diode array detector detection obtains collection of illustrative plates, qualitative with standard Monacolin K (lactone and acid type) retention time and Monacolin compounds characteristic absorption spectrum.With the chromatogram under the 238nm wavelength is quantitative basis, and Monacolin component peaks area sum in the sample and standard Monacolin K (lactone or acid) chromatographic peak area is more quantitative.

2.5 result's computing formula

$X=\frac{h_1\×c\×50}{h_2\×m}$

In the formula: Monacolin compounds total amount in the X-style, mg/g

h ₁-have a separated component chromatographic peak of Monacolin compounds characteristic absorption spectrum

The area sum.

C-standard Monacolin K (lactone type or acid) solution concentration, mg/ml

50-sample constant volume, ml

h ₂-Monacolin K (lactone type or acid) peak area

The m-sample amount of taking by weighing, g

3. experimental result

3.1 chromatographic resolution effect expedition test

Under above-mentioned experiment condition standard Monacolin K (lactone and acid type) separation case as shown in Figure 1, certain red colouring agent for food, also used as a Chinese medicine sample Monacolin compounds separation case is as shown in Figure 2.

As seen from Figure 1, under above-mentioned test condition, lactone type Monacolin K and open loop acid Monacolin K obtain good separation.

As seen from Figure 2, under above-mentioned test condition, the various Monacolin compounds in the red colouring agent for food, also used as a Chinese medicine sample obtain good separation.Diode array detector has provided all separated component chromatograms and has flowed out instant ultra-violet absorption spectrum.Monacolin compound characteristic (230 will be had in these spectrum, 238, respectively there is an absorption peak at 246nm wavelength place, is maximum with the 238nm place) collection of illustrative plates pick out, the pairing chromatographic peak of these characteristic spectrums is each Monacolin compound in chromatogram elution curve figure.

3.2 the investigation of linear relationship and lowest detectable limit test

Configuration concentration is 0.1,1,10,30,75,150,300 μ g/ml Monacolin K (lactone type) standard solution, and sample introduction is measured under above-mentioned chromatographic condition, with peak area concentration is made typical curve, and the result as shown in Figure 3.Its equation of linear regression and related coefficient are respectively: Y=66084x-16840, R ²=0.9999.Show in this concentration lower linear relation good.

When Monacolin K concentration was 0.1 μ g/ml, the chromatographic peak that can see MonacolinK on chromatogram elution curve figure was a little more than 2 times of noises, so the limit of identification of this method is 0.1 μ g/ml (2ng).According to this typical curve as can be known the optimum linear scope of this method be 0.1～300 μ g/ml.

3.3 method collimation precision test

Same even sample is divided into 5 parts, begins parallel experimentizing from pre-treatment step, 5 duplicate samples measurement results see Table 1.

Table 1 collimation precision measurement result

The Monacolin compound is surveyed
				Sequence number	Decide result (g/100g)	Mean value	RSD
1 2 3	0.294 0.296 0.293	0.294	0.002

4 5

0.292 0.296

The result shows that this method has good collimation precision.

3.4 method repeatability precision test

Same even sample begins to measure once every day from sample pre-treatments under same experiment condition.Continuous coverage 5 days, it the results are shown in Table 2.

The repeated precision measurement result of table 2

The Monacolin compound determination
				Sequence number	Result (g/100g)	Mean value	RSD
1 2 3 4 5	0.41 0.43 0.42 0.42 0.44	0.424	2.69％

The result shows that this method repeatability precision is good.

3.5 method recovery test

Accurately take by weighing 10 parts in sample, wherein 5 parts respectively add standard Monacolin K.Begin to measure Monacolin K content 10 duplicate samples from sample pre-treatments.The result is as follows:

Table 3 method determination of recovery rates result

The N value

Monacolin K content mg in the sample

Addition mg

Detect Monacolin K content mg

Recovery %

Mean value

1 2 3 4 5

0.64 0.71 0.71 0.69 0.65

2.0 2.0 2.0 2.0 2.0

2.60 2.64 2.70 2.64 2.73

98.3 96.5 99.3 97.8 97.9

97.96％

Table 3 shows that the method has good accuracy.

3.6 the mensuration of Monacolin compounds content in 5 kinds of samples

Respectively get 5 kinds of red colouring agent for food, also used as a Chinese medicine samples, measure wherein Monacolin compounds content with above-mentioned detection method, the result is as follows:

Monacolin compounds content measurement result in table 4 sample

Sequence number	The sample title	The replication number of times	Monacolin compound determination result (g/100g)
				1 2 3 4 5	XX red colouring agent for food, also used as a Chinese medicine sample 1 XX red colouring agent for food, also used as a Chinese medicine sample 2 XX red colouring agent for food, also used as a Chinese medicine samples 3 XX red colouring agent for food, also used as a Chinese medicine samples 4 XX red colouring agent for food, also used as a Chinese medicine samples 5	6 6 6 6 6	1.02±0.02 0.80±0.01 0.99±0.02 1.20±0.11 0.45±0.10

As can be seen from Table 4, this method can obtain good result in actual applications.

4. discuss

4.1 the functional red yeast rice tunning contains various Monacolin compounds, based on lactone type and acid Monacolin K, diode array detector can provide all separated component chromatograms and flow out instant ultra-violet absorption spectrum, and this method can reflect clearly on chromatogram elution curve figure that those and Monacolin K have the separated situation (see figure 2) of Monacolin compounds of identical ultra-violet absorption spectrum.

4.2 lactone type Monacolin K has commodity selling.Because therefore acid Monacolin K instability does not have standard substance to sell at present.The condition of utilizing this method to provide can be converted into acid with standard lactone type Monacolin K easily.Ratio with transforming back acid and lactone type Monacolin K chromatographic peak area just can accurately obtain conversion ratio, and can be accurately quantitative to lactone type in the sample and acid Monacolin K.

Flow out instant ultra-violet absorption spectrum scanning 4.3 adopt diode array detector to carry out chromatogram to all separated components.Therefore, this method can utilize the characteristic absorption spectrum of Monacolin K qualitative except the retention time that adopts Monacolin K is qualitative simultaneously.This just makes the qualitative analysis of this method can only utilize retention time qualitative more reliable than UV-detector in the past.

4.4Monacolin compounds all has the physiologically active of similar Monacolin K, the peak area addition of these components, quantitatively just can draw the total amount of Monacolin compounds in the sample with standard lactone type Monacolin K to them, although because the problem of detector response, have some errors, but lacking under the situation of Monacolin compounds standard substance at present, this method still is effective.Again owing to various Monacolin compounds in the sample are main (see figure 2) with lactone type and acid Monacolin K, utilize this method can be accurately quantitative to these two kinds of compounds, and other Monacolin compounds contents seldom, and the Monacolin total amount of compound has higher accuracy in this method measurement function red colouring agent for food, also used as a Chinese medicine sample so adopt.

Claims (2)

1. the detection method of Monacolin kind compound content in the functional red yeast rice, it may further comprise the steps:

1) sample pre-treatment;

2) preparation of acid Monacolin K standard model;

3) the chromatogram sample introduction is analyzed;

4) atlas analysis and result calculate.

2, the detection method of Monacolin kind compound content in the functional red yeast rice according to claim 1 is characterized in that:

1) comprises in the sample pre-treatment step: the red colouring agent for food, also used as a Chinese medicine sample is pulverized (〉=80 order) also fully mix; Accurately take by weighing 400.0～600.0mg sample in the 50ml volumetric flask, add 30ml 75% ethanol (V/V), shake up ultrasonic 20min (frequency of operation 40KHz) under the room temperature.Add 75% ethanol extremely near scale, ultrasonic again 10min is cooled to room temperature afterwards, is settled to 50ml with 75% ethanol; With 3500 rev/mins of centrifugal 10min; Get supernatant and cross 0.45 μ m aperture miillpore filter, filtrate is treated sample introduction;

2) preparation process of acid Monacolin K standard model comprises: take by weighing Monacolin K (lactone) standard items 4mg, be settled to 100ml with the 0.2M sodium hydroxide solution, put under 50 ℃ of conditions ultrasonic conversion 1 hour, be cooled to room temperature after, lucifuge is placed after 3 hours and is used; Calculate conversion ratio according to transforming back acid Monacolin K and residual lactone Monacolin K chromatographic peak area; Utilize the acid Monacolin K of preparation, acid Monacolin K in the sample is carried out accurately qualitative and quantitative;

3) chromatogram sample introduction analytical procedure comprises: the filtrate of the filtering with microporous membrane of learning from else's experience enters liquid chromatograph, and described chromatographic chromatographic condition is: chromatographic column: C ₁₈Post, 4.6 * 250mm, 5 μ m; Column temperature: room temperature; Diode array detector: 200～350nm scanning, 238nm place chromatogram deal with data; Moving phase: methyl alcohol: water: phosphoric acid=385: 115: 0.14 (V: V); Flow velocity: 1.0ml/min; Sample size: 20 μ l;

4) atlas analysis comprises with calculation procedure as a result: find the component peaks that identical ultra-violet absorption spectrum is arranged with standard MonacolinK on chromatogram elution curve figure, be the Monacolin compounds, peak area addition with these components, compare Monacolin compounds total amount in the calculation sample with standard Monacolin K chromatographic peak area;

Result's computing formula:

$X=\frac{h_1\×c\×50}{h_2\×m}$

In the formula: Monacolin compounds total amount in the X-style, mg/g

h ₁-have a separated component chromatographic peak area sum of Monacolin compounds characteristic absorption spectrum

C-standard Monacolin K (lactone type or acid) solution concentration, mg/ml

50-sample constant volume, ml

h ₂-Monacolin K (lactone type or acid) peak area

The m-sample amount of taking by weighing, g.

Images