CN103808751B - A kind of method differentiating traditional Chinese medicine honeysuckle or spin-off - Google Patents

Abstract

The invention relates to a method for identifying honeysuckle medicinal materials or derivatives, comprising: extracting honeysuckle medicinal materials or derivatives to obtain a characteristic extract of honeysuckle organic acids or/and iridoid glycosides containing active ingredient groups; Carry out IGD carbon NMR fingerprint spectrum detection, according to the fingerprint spectrum to obtain the characteristic peak intensity of several active ingredients in the characteristic extract of organic acids or/and iridoid glycosides; and measure the corresponding standard reference product characteristics of each active ingredient in the same way peak intensity; determine the absolute content of the standard reference substance by means of quantitative analysis; use the ratio of the characteristic peak peak intensity and the absolute content of the standard reference substance to calculate the content of each active ingredient and the active ingredient group in honeysuckle medicinal materials or derivatives content. The invention can reflect which honeysuckle organic acids or/and iridoid glycoside compounds are contained in the honeysuckle and their ratios, so as to achieve the purpose of identifying the variety and quality of the honeysuckle medicinal material.

Description

A method for identifying medicinal materials or derivatives of honeysuckle

technical field

The invention belongs to the field of identification of natural medicinal plants, and in particular relates to a method for identifying medicinal materials or derivatives of honeysuckle.

Background technique

Honeysuckle (Loniceraejaponicaeflos) is the dry flower bud or the flower with the first bloom of Lonicera japonica Thunb., which is a famous Chinese medicinal material in my country. Honeysuckle belongs to the lung, heart, and stomach meridians. It is cold in nature and sweet in taste, and has the effects of clearing heat, detoxifying, and evacuating wind-heat [National Pharmacopoeia Committee. Pharmacopoeia of the People's Republic of China 2010 Edition Part One. China Medical Science and Technology Press, 205]. Its active ingredients are mainly organic acids, iridoid glycosides and flavonoids, which have the functions of antibacterial and antiviral, antipyretic and anti-inflammatory, liver protection and gallbladder, blood fat lowering, hemostasis, and antioxidant effects [①Zhang Shouping, et al. China Journal of Traditional Chinese Medicine Information 2007, 14(3):84. ②Wang Lichuan. Anhui Agricultural Sciences 2009, 37(5):2036.].

Qualitative identification of a certain type of active ingredient or quantitative determination of the content of a certain active ingredient has become an important method for evaluating the quality of honeysuckle and its preparations. However, this quality inspection mode for qualitative and quantitative analysis of a single component cannot effectively control the internal quality of Chinese medicinal materials, and cannot meet the current urgent requirements for objectively and effectively evaluating and controlling the quality of honeysuckle and its preparations.

Fingerprint technology is currently internationally recognized as the most effective method to control the quality of traditional Chinese medicine or natural medicine. People can not only confirm the authenticity of the product by virtue of the practical fingerprint, but also evaluate its quality. At present, the research work on the fingerprints of honeysuckle medicinal materials and their preparations is concentrated on HPLC fingerprints [①Bai Xuemei, et al. Chinese Patent Medicine 2004,26(7):521.②Xiong Yan, et al. ):1015.], capillary electrophoresis fingerprint (HPCE method) [Sun Guoxiang, et al. Chromatography 2007,25(1):96.], high performance liquid chromatography-mass spectrometry (LC-MS) [Zhang Qian, et al. Chinese Journal of Traditional Chinese Medicine, 2012,37(23):3564.] et al. Among them, the mainstream method HPLC fingerprint is affected by non-chromatographic conditions (such as chromatographic column inner diameter, length, stationary phase grade, carrier particle size, mobile phase flow rate, proportion of components in mixed mobile phase, column temperature, injection volume, detector sensitivity, etc.) There are many limitations in repeatability and feasibility due to the large influence and the need to use multiple standards for multi-component quantification; although the HPCE method is superior to the HPLC method because of its high efficiency, rapidity, simplicity, and less contamination of the column, but There are still deficiencies such as poor reproducibility, narrow linear range and low sensitivity; due to the complexity of traditional Chinese medicine compound and the lack of standard products, the single LC-MS analysis method also has problems such as ionization degree and matrix interference. Insufficient for accurate structural characterization of multiple components.

IGD 13 CNMR coupling (IGD ¹³ CNMR coupling) fingerprint technology, also known as anti-gated decoupling carbon NMR coupling fingerprint technology, this technology is based on the hydrogen nuclear magnetic resonance spectrum ( ¹ HNMR) fingerprint that has been studied for many years Map technology [Zhao Tianzeng, et al. ¹ HNMR fingerprint method to identify plant traditional Chinese medicine, Chinese herbal medicine 2000, 31 (11): 868-870] combined with other technologies (such as the most widely used high-performance liquid chromatography (HPLC) fingerprint technology [ Xie Peishan et al. Chromatographic Fingerprinting of Traditional Chinese Medicine, People's Medical Publishing House, 2005]) proposed a new non-single means comprehensive fingerprinting technology. So far, there are no relevant reports and disclosures of technical content using IGD carbon NMR coupled fingerprint technology to identify honeysuckle medicinal materials or derivatives.

The quality of honeysuckle does not lie in a single component, but its curative effect is the result of the synergistic ratio of multiple components. Therefore, it is imperative to establish an IGD carbon NMR coupled fingerprint that can not only control the active components of Lonicerae japonica, but also control the ratio of these active components. The research and application of honeysuckle medicinal materials or derivatives by C-NMR coupled fingerprints can not only solve the problem of identification and evaluation of honeysuckle medicinal materials or derivatives in my country, but also strengthen the systematization and standardization of research on the internal components of honeysuckle medicinal materials or derivatives, The realization of international standards provides a scientific guarantee. With the popularization and application of this technology in other Chinese medicinal materials or derivatives, the significant scientific value of this technology will become increasingly prominent.

Contents of the invention

In order to solve the problems in the prior art, the object of the present invention is to provide a method for identifying honeysuckle medicinal materials or derivatives, which utilizes IGD carbon NMR coupled fingerprint technology.

In order to achieve the above object, the method for identifying honeysuckle medicinal materials or derivatives provided by the present invention comprises the following steps:

1) Extracting honeysuckle medicinal materials or derivatives to obtain a characteristic extract of honeysuckle organic acids or/and iridoid glycosides containing active ingredient groups (organic acids or/and iridoid glycosides);

2) Perform IGD carbon NMR fingerprint detection on the characteristic extracts of honeysuckle organic acids or/and iridoid glycosides, and obtain the organic acids or/and iridoid glycosides characteristic extracts of honeysuckle according to the fingerprints. The characteristic peak intensity of several active ingredients; and measure the characteristic peak intensity of the corresponding standard reference product of each active ingredient in the same way (IGD carbon NMR fingerprint);

3) Determine the absolute content of the standard reference substance by means of quantitative analysis;

4) Using the ratio of the characteristic peak intensity (the characteristic peak intensity of each active ingredient and the characteristic peak intensity of the corresponding standard reference product) and the absolute content of the standard reference product, calculate the organic The content of active ingredients of acid or/and iridoid glycosides and the total content of main active ingredients, that is, the content of active ingredient groups.

Wherein, an extraction process with a clear IGD carbon NMR fingerprint is adopted as the extraction method of the honeysuckle organic acid or/and iridoid glycosides characteristic extract.

Wherein, in step 1), the preparation method of honeysuckle organic acids or/and iridoid glycosides characteristic extracts includes: weighing honeysuckle medicinal materials or decoction pieces and crushing, adding 20-25% ethanol for reflux extraction or ultrasonic extraction for 2-3 1-2 hours each time, filter and combine the filtrates, and concentrate under reduced pressure; take the concentrated filtrate, pack it into a column with a macroporous adsorption resin, wash the column with a water-ethanol system, collect different eluted parts, and evaporate under reduced pressure. dried to obtain the characteristic extract of honeysuckle organic acids or/and iridoid glycosides.

Further, the mass volume ratio of the honeysuckle medicinal material or decoction pieces to 20-25% ethanol is 1:(6-10) (g:mL).

Further, the weight of the macroporous adsorption resin is 1 to 2 times the weight of the concentrated filtrate.

Further, the temperature of the reflux extraction is 90-95°C, and the temperature of the ultrasonic extraction is 50-60°C.

Further, the honeysuckle medicinal material or decoction pieces are crushed and passed through a 24-65 mesh sieve.

Further, the model of the macroporous adsorption resin is HP-20, D-101, D-201, SP-825 or SP-70.

Further, the aspect ratio of the macroporous adsorption resin is 8-15:1, preferably 10-12:1.

Wherein, in step 2), the organic acid or/and iridoid glycosides characteristic extract of honeysuckle is subjected to IGD carbon-1-13 NMR fingerprint detection, and the solvent for dissolving the characteristic extract of honeysuckle organic acid or/and iridoid glycosides is Deuterated chloroform (CDCl ₃ ), deuterated methanol (CD ₃ COCD ₃ ) or deuterated dimethylsulfoxide (DMSO-d ₆ ), preferably deuterated methanol. The mass-to-volume ratio of the organic acid or/and iridoid glycosides characteristic extract of Lonicerae japonica to the corresponding solvent is 55:1-65:1 (mg:mL), preferably 60:1.

In addition to honeysuckle decoction pieces, other honeysuckle derivatives, such as honeysuckle organic acids or/and iridoid glycoside extracts, can be directly used as the characteristic extracts of honeysuckle organic acids or/and iridoid glycosides, and identified by the above method.

Wherein, in step 2), the characteristic peak of the active ingredient in the characteristic extract of honeysuckle organic acids or/and iridoid glycosides is the ester carbonyl absorption peak, and its chemical shift is δ _C 164.0-167.0 (with deuterated DMSO-d ₆ When dissolving honeysuckle organic acid or/and iridoid glycoside characteristic extract) or _δC 168.0～170.0 (when dissolving honeysuckle organic acid or/and iridoid glycoside characteristic extract with deuterated methanol).

Wherein, in step 2), several active ingredients in the honeysuckle organic acid or/and iridoid glycosides characteristic extract are sorted according to the size and position of the characteristic peak intensity.

Further, in step 2), according to the size and position of the characteristic peak intensity, the active ingredients in the honeysuckle organic acid or/and iridoid glycosides characteristic extract: chlorogenic acid, butyl chlorogenic acid, Vanillicacid7-O-β-D-(6-O-benzoylglucopyranoside), Butyl Chlorogenic Acid + 3,4-O-Dicaffeoyl Ethyl Quinate, Ferulic Acid, Ethyl Caffeate, α-Mono Glycoside + Secologanindimethylacetal, β-morroniside, swertiroside, ginjiside for sorting.

Wherein, in step 2), the peak intensity can be calculated by peak height method, area integration method or weight method.

Wherein, in step 3), the quantitative analysis means is: high performance liquid phase (HPLC) method.

Further, when detecting the characteristic extract of honeysuckle organic acid or/and iridoid glycosides, the conditions of the high performance liquid phase include: the mobile phase is acetonitrile: 0.4% phosphoric acid solution=(10～20):(80～95 ), preferably 13:87; the detection wavelength is 240nm.

Wherein, in step 3), the absolute content of the standard reference substance refers to: the mass percentage of the standard reference substance measured by quantitative analysis means.

Further, the standard reference substance of the active ingredients in the honeysuckle organic acids or/and iridoid glycosides characteristic extract is chlorogenic acid.

Wherein, in step 4), the coupling formula for calculating the content of each active ingredient is:

$W_{\mathrm{no}}=\frac{W_1{m}_{\mathrm{no}}{h}_{\mathrm{no}}}{m_1{h}_1};$ in:

W ₁ is step 3) the absolute content of the standard reference substance corresponding to a certain active ingredient in honeysuckle medicinal materials or derivatives determined by quantitative analysis means;

M ₁ is the molecular weight/quantitative peak corresponding to the carbon number of a standard reference product corresponding to a certain active ingredient in the honeysuckle medicinal material or derivative;

h1 is the characteristic peak intensity of a standard reference product corresponding to a certain active ingredient in honeysuckle medicinal materials or derivatives determined by IGD carbon- ₁₃ NMR fingerprint;

W _n is the mass percentage of a certain active ingredient in honeysuckle medicinal materials or derivatives;

M _n is the number of carbons corresponding to the molecular weight/quantitative peak of a certain active ingredient in honeysuckle medicinal materials or derivatives;

h _n is the characteristic peak intensity of a certain active ingredient in the honeysuckle medicinal material or derivatives determined by the _IGD carbon NMR fingerprint; That is, the content of the active ingredient group.

The derivation process of the above formula is:

$\begin{array}{c}\frac{{\mathrm{<span\; class="notranslate">\; W</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}}{{\mathrm{<span\; class="notranslate">\; m</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}}<span\; class="notranslate">\; :</span>\frac{{\mathrm{<span\; class="notranslate">\; W</span>}}_{\mathrm{<span\; class="notranslate">\; no</span>}}}{{\mathrm{<span\; class="notranslate">\; m</span>}}_{\mathrm{<span\; class="notranslate">\; no</span>}}}<span\; class="notranslate">\; =</span>\frac{{\mathrm{<span\; class="notranslate">\; h</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}}{{\mathrm{<span\; class="notranslate">\; h</span>}}_{\mathrm{<span\; class="notranslate">\; no</span>}}}\\ <span\; class="notranslate">\; \&DoubleRightArrow;</span>\frac{{\mathrm{<span\; class="notranslate">\; W</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}{\mathrm{<span\; class="notranslate">\; m</span>}}_{\mathrm{<span\; class="notranslate">\; no</span>}}}{{\mathrm{<span\; class="notranslate">\; m</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}{\mathrm{<span\; class="notranslate">\; W</span>}}_{\mathrm{<span\; class="notranslate">\; no</span>}}}<span\; class="notranslate">\; =</span>\frac{{\mathrm{<span\; class="notranslate">\; h</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}}{{\mathrm{<span\; class="notranslate">\; h</span>}}_{\mathrm{<span\; class="notranslate">\; no</span>}}}\\ <span\; class="notranslate">\; \&DoubleRightArrow;</span>{\mathrm{<span\; class="notranslate">\; W</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}{\mathrm{<span\; class="notranslate">\; m</span>}}_{\mathrm{<span\; class="notranslate">\; no</span>}}{\mathrm{<span\; class="notranslate">\; h</span>}}_{\mathrm{<span\; class="notranslate">\; no</span>}}<span\; class="notranslate">\; =</span>{\mathrm{<span\; class="notranslate">\; m</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}{\mathrm{<span\; class="notranslate">\; W</span>}}_{\mathrm{<span\; class="notranslate">\; no</span>}}{\mathrm{<span\; class="notranslate">\; h</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}\\ <span\; class="notranslate">\; \&DoubleRightArrow;</span>{\mathrm{<span\; class="notranslate">\; W</span>}}_{\mathrm{<span\; class="notranslate">\; no</span>}}<span\; class="notranslate">\; =</span>\frac{{\mathrm{<span\; class="notranslate">\; W</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}{\mathrm{<span\; class="notranslate">\; m</span>}}_{\mathrm{<span\; class="notranslate">\; no</span>}}{\mathrm{<span\; class="notranslate">\; h</span>}}_{\mathrm{<span\; class="notranslate">\; no</span>}}}{{\mathrm{<span\; class="notranslate">\; m</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}{\mathrm{<span\; class="notranslate">\; h</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}}\end{array}$

The active ingredient group described in the method of the present invention can be the active ingredient group in the honeysuckle single medicinal material, or the active ingredient group in the honeysuckle derivatives. The characteristic extract of honeysuckle organic acids or/and iridoid glycosides means that the extract contains active ingredients of honeysuckle organic acids or/and iridoid glycosides, and the active ingredient group refers to the sum of corresponding active ingredients of the same kind.

Wherein, the honeysuckle derivatives include: honeysuckle decoction pieces, honeysuckle extract (such as honeysuckle organic acid extract, honeysuckle iridoid glycoside extract or honeysuckle flavonoid extract, or a combination of two or more of them) Or honeysuckle natural medicine.

The honeysuckle medical material of the present invention includes various parts of the honeysuckle plant, such as roots, bark, stems, leaves, flowers and fruits.

The calculation of the content of each active component and the total content of such active components in the present invention is to couple the IGD carbon nuclear magnetic resonance spectrum and analytical quantitative means through a coupling formula. Compared with the prior art, the present invention adopts the IGD ¹³ CNMR coupling fingerprint to have the following characteristics:

① Stability (repeatability): The chemical shift data obtained by IGD ¹³ CNMR is the second decimal place, with good resolution and repeatability; HPLC and GC non-chromatographic conditions (such as column inner diameter, length, stationary phase grade, Carrier particle size, mobile phase flow rate, component ratio of mixed mobile phase, column temperature, sample volume, detector sensitivity, etc.) changes, etc., the obtained retention time data changes greatly, which means that the overall chromatographic pattern varies and the repeatability is not good. it is good.

②Comprehensive (comprehensive): IGD ¹³ CNMR fingerprints contain the corresponding peaks of each active component carbon in the sample; HPLC, GC, UV, IR, and MS do not have this relationship.

③ Reliability (singleness): There is a strict one-to-one correspondence between IGD ¹³ CNMR peaks and carbons on different active ingredients and groups in the sample; HPLC, GC, UV, IR, and MS do not have such a relationship.

④ Feasibility (easiness of identification): IGD ¹³ CNMR fingerprints have strong regularity, and under normal circumstances, each carbon peak in the spectrum can be assigned; HPLC and GC need reference substances; IR is not easy to analyze; UV information is less; MS Then there are issues such as the degree of ionization and matrix interference.

The IGD carbon NMR fingerprint can only indicate which active ingredients are in the characteristic extract and the quantitative ratio between these active ingredients. obtained by combining the formula.

The advantages of the present invention are:

1. The four steps in the present invention are an integral whole, all of which are indispensable; the four steps have their own uniqueness; if they are separated or simply combined, the ratio and content of the complex components in the medicinal plants cannot be detected.

As we all know, although traditional Chinese medicine and botanical pesticides are drugs with a long history, their analysis and detection has always been a difficult problem. Therefore, there is a special need for a method that can identify and quantify various active components in plant varieties and their extracts, and then select the best extract components or their compositions so as to fully exert their effects. In addition, one of the obstacles for traditional Chinese medicine and botanical pesticides to go abroad is that there are no components and their quantitative labels. Limitations (see the content of the background technology description of this application for details). Facts have proved that IGD carbon NMR coupled fingerprint technology is the key technology to solve this problem, which has been reflected in the specification of this application.

2. Although the conventional extraction process is well known to ordinary technicians, no one has ever used the clear map and representative active ingredient group as a standard to select process parameters to obtain characteristic extracts. Utilizing this method will ultimately result in a better identification of the extract, which is one of the difficulties and one of the innovations of the present invention.

3. The IGD ¹³ CNMR coupling fingerprint is a mixed spectrum of multiple active ingredients, which will inevitably cause crowding and even overlapping of rod peaks. In order to make the calculation results accurate, it is necessary to select the carbon peaks of each active ingredient in the active ingredient group with large differences in chemical shifts to assign characteristic peaks. Since the carbon spectra of different types of compounds are very different, the selection of characteristic peaks can be varied; the difference in the carbon spectra of the same type of compounds is very small, and many spectral peaks overlap seriously. Select better characteristic peaks; If the characteristic peaks are not well selected, there is no way to carry out accurate quantitative analysis on the active ingredient group, which is the problem that the present invention needs to solve. According to the characteristics of chlorogenic acids and iridoid glycosides in Flos Lonicerae, the ester carbonyl peak was selected as the characteristic peak to distinguish the two types of components. Therefore, according to the characteristics of different active ingredients, it is necessary to select different active ingredient carbon peaks to designate characteristic peaks, which is another innovation point of the present application.

4. Due to the small difference in the chemical shifts of the characteristic peaks, in many cases, the difference is only in the first place after the decimal point, so the ordering of the characteristic peaks is the key to determining the main active components and their proportions. There is no deep knowledge of NMR spectroscopy and separation basis , it is difficult to determine the active ingredients and their proportions represented by the characteristic peaks, and it is impossible to conduct accurate qualitative and quantitative analysis on the active ingredient group. According to the characteristics of the carbon spectrum NMR data of chlorogenic acids and iridoid glycosides in honeysuckle, The characteristic peaks of the two types of components are accurately sorted; this is one of the difficulties and one of the innovations of the present invention.

5. The key to coupling calculation is the selection of standard products and quantitative analysis techniques. Analytical and quantitative methods can be selected from high performance liquid chromatography, gas chromatography, thin layer chromatography and weighing methods, etc. The standard reference product can be a certain active ingredient as the internal standard, or an external reference product as the external standard. According to the characteristics of chlorogenic acids and iridoid glycosides in Flos Lonicerae, liquid phase was selected as the analytical and quantitative means, and chlorogenic acid was selected as the standard reference product. This is one of the difficulties of the invention and also one of the innovation points of the present invention.

The research project involved in the present invention is funded by the special funds for scientific research in the public welfare industry of quality inspection (project number: 2012104019-1).

The present invention adopts IGD carbon nuclear magnetic resonance spectrum fingerprints to identify honeysuckle medicinal materials or derivatives, which can reflect the honeysuckle active ingredients contained in honeysuckle medicinal materials or derivative products and the ratio between them, so as to achieve the variety and quality identification of honeysuckle medicinal materials or derivative products the goal of. Wide linear range, high sensitivity, good repeatability and feasibility. Not only can it effectively control the inherent quality of honeysuckle medicinal materials or derivatives, but it can also meet the current urgent requirements for objectively and effectively evaluating and controlling the quality of honeysuckle. In general, it can not only solve the problem of identification and evaluation of honeysuckle medicinal materials or derivatives in my country, but also provide a scientific guarantee for strengthening the systematization and standardization of research on the internal components of honeysuckle medicinal materials or derivatives, and to achieve international standards.

Description of drawings

Figure 1-a is the IGD carbon NMR fingerprint of the commercially available honeysuckle medicinal material A organic acid or/and iridoid glycosides characteristic extract in Example 1.

Fig. 1-b is a partially widened and enlarged view of the characteristic peaks of the IGD carbon NMR fingerprint of the commercially available honeysuckle medicinal material A organic acid or/and iridoid glycosides characteristic extract in Example 1.

Fig. 2-a is the IGD carbon NMR fingerprint of the commercially available honeysuckle medicinal material B organic acid or/and iridoid glycosides characteristic extract in Example 2.

Fig. 2-b is a partially broadened and enlarged view of the characteristic peaks of the IGD carbon NMR fingerprint of the commercially available honeysuckle medicinal material B organic acid or/and iridoid glycosides characteristic extract in Example 2.

Figure 3-a is the IGD carbon NMR fingerprint of the commercially available chlorogenic acid 20% honeysuckle extract (Shanghe) in Example 4.

Fig. 3-b is a partially widened and enlarged view of the characteristic peaks of the IGD carbon NMR fingerprint of the commercially available chlorogenic acid 20% honeysuckle extract (Shanghe) in Example 4.

Figure 4-a is the IGD carbon NMR fingerprint of the self-made honeysuckle extract in Example 5.

Fig. 4-b is a partially widened and enlarged view of the characteristic peaks of the IGD carbon NMR fingerprint of the self-made honeysuckle extract in Example 5.

detailed description

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

Unless otherwise specified, the materials used in the present invention are conventional materials in this field, and the operation methods not mentioned are also conventional methods in this field.

In the present invention, the concentrations of methanol and ethanol are volume percentages.

1. Study on IGD carbon NMR fingerprints of honeysuckle medicinal materials or derivatives

(1) IGD carbon NMR fingerprints of honeysuckle organic acids or/and iridoid glycosides characteristic extracts extracted from honeysuckle medicinal materials or derivatives

1) Obtaining the characteristic extracts of honeysuckle organic acids or/and iridoid glycosides

① Obtained from honeysuckle medicinal materials or decoction pieces

Weigh honeysuckle medicinal materials or decoction pieces and grind them (through a 24-65 mesh sieve), add 6-10 times the volume of ethanol at 20%-25% (volume ratio), reflux at 90-95°C or sonicate at 50-60°C Extract 2 to 3 times, 1 to 2 hours each time, filter and combine the filtrate, concentrate under reduced pressure; take the concentrated filtrate, pack it into a column with 1 to 2 times its weight of macroporous adsorption resin, and mix with water-ethanol Punch the column with the system, and collect different eluted fractions to obtain the characteristic extract of honeysuckle organic acids or/and iridoid glycosides (CET).

The model of the macroporous adsorbent resin is HP-20, D-101, D-201, SP-825 or SP-70; the aspect ratio of the macroporous adsorbent resin is 8～15:1, preferably 10～12: 1.

② Obtained from other honeysuckle derivatives

Directly take other honeysuckle derivatives (honeysuckle derivatives except honeysuckle decoction pieces, such as honeysuckle organic acid or/and iridoid glycoside extract) as the characteristic extract of honeysuckle organic acid or/and iridoid glycoside.

2) IGD carbon NMR fingerprint detection of honeysuckle organic acids or/and iridoid glycosides characteristic extracts

Take honeysuckle organic acids or/and iridoid glycosides characteristic extracts, dissolve them in deuterated chloroform, DMSO-d ₆ or deuterated methanol, and perform IGD carbon NMR fingerprint detection to obtain honeysuckle organic acids or/and Carbon NMR fingerprint of iridoid glycoside IGD.

The mass-to-volume ratio of the organic acid or/and iridoid glycosides characteristic extract of Lonicerae japonica to the corresponding solvent is 55:1-65:1 (mg:mL), preferably 60:1.

3) IGD carbon NMR fingerprint analysis of honeysuckle organic acids or/and iridoid glycosides characteristic extracts

① Identification

The characteristic signals of organic acids or/and iridoid glycosides of honeysuckle organic acids or/and iridoid glycosides are clearly displayed in the fingerprints of IGD carbon NMR spectrum fingerprints of honeysuckle organic acids or/and iridoid glycosides.

②Selection of the characteristic peaks of each active ingredient in the characteristic extract of honeysuckle organic acids or/and iridoid glycosides

According to the characteristics of different active ingredients, it is necessary to select different active ingredient carbon peaks to specify the characteristic peaks. The selection principles are as follows: ①The designated characteristic peaks of the same compound should preferably be carbon-carbon peaks with the same positions for each compound; ②The chemical shift difference between the designated characteristic peaks of each compound and other carbon peaks is relatively large; The difference in chemical shift is large; ④The chemical shift effect affecting the specified characteristic peaks of each compound is quite different.

Since the characteristic extracts of honeysuckle organic acids or/and iridoid glycosides contain a series of such compounds, the carbon peaks cross more, in order to determine the proportion of each active ingredient, the corresponding peaks with large chemical shift differences must be selected as the characteristic peaks . For this reason, after actual investigation, the carbon-carbon peak of ester carbonyl was selected as the designated characteristic peak of the carbon peaks of the two types of active ingredients. There are also some differences in chemical shifts.

③ Selection of standard reference products

Chlorogenic acid is one of the main active components in the characteristic extract of honeysuckle organic acids or/and iridoid glycosides, and its carbonyl chemical shift does not overlap with other main components; therefore, chlorogenic acid was selected as the standard reference substance.

4) Determination of chlorogenic acid content in honeysuckle medicinal materials or derivatives by HPLC method

i) Chromatographic conditions (refer to the 2010 Pharmacopoeia)

Mobile phase: acetonitrile: 0.4% phosphoric acid solution = 13:87;

Chromatographic column: C18 (250*4.6mm, 5um);

Detection wavelength: 240nm.

ii) Preparation of chlorogenic acid standard reference solution

Accurately weigh 5mg of chlorogenic acid, put it in a 50mL volumetric flask, dissolve and dilute to the mark with methanol with a mass concentration of 50%, and shake well to obtain a chlorogenic acid standard reference solution.

iii) Preparation of test solution containing active ingredients of honeysuckle organic acid and/or iridoid glycosides

Accurately weigh the test product: 0.5g of powder of honeysuckle medicinal material/decoction pieces, accurately weighed, put in a stoppered Erlenmeyer flask, accurately add 50mL of 50% methanol, weigh, heat and reflux for 1 hour, let cool, and use 50% Make up the weight with methanol, filter, and take the continued filtrate as the test solution of honeysuckle medicinal material/decoction pieces. Put 30-50mg of other honeysuckle derivatives into a 50mL volumetric flask, dissolve and dilute to the mark with methanol with a mass concentration of 50%, and shake well to obtain the test solution of other honeysuckle derivatives.

iv) Assay

Accurately draw 20 μl each of the standard reference solution of chlorogenic acid and the test solution of honeysuckle medicinal material/decoction pieces or the test solution of derivatives, inject it into the liquid chromatograph, and measure it to obtain the content of chlorogenic acid.

②Calculation of absolute content of chlorogenic acid (calculation of absolute content of chlorogenic acid in honeysuckle medicinal materials/decoction pieces or derivatives)

i) Calculate the mass concentration of chlorogenic acid in the test solution of honeysuckle herbal medicine/decoction pieces or derivatives by the following formula:

${\mathrm{<span\; class="notranslate">\; C</span>}}_{\mathrm{<span\; class="notranslate">\; x</span>}}<span\; class="notranslate">\; =</span>{\mathrm{<span\; class="notranslate">\; C</span>}}_{\mathrm{<span\; class="notranslate">\; R</span>}}<span\; class="notranslate">\; \&times;</span>\frac{{\mathrm{<span\; class="notranslate">\; A</span>}}_{\mathrm{<span\; class="notranslate">\; x</span>}}}{{\mathrm{<span\; class="notranslate">\; A</span>}}_{\mathrm{<span\; class="notranslate">\; R</span>}}}$

C _X : Mass concentration of chlorogenic acid in the test solution of honeysuckle herbal medicine/decoction pieces or derivatives (ug/mL);

C _R : mass concentration of chlorogenic acid standard reference solution (ug/mL);

A _X : the peak area of honeysuckle medicinal material/decoction pieces or derivatives for the test solution;

A _R : the peak area of the chlorogenic acid standard reference solution.

ii) Calculate the mass percentage of chlorogenic acid in honeysuckle medicinal materials/decoction pieces or derivatives by the following formula

${\mathrm{<span\; class="notranslate">\; W</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}<span\; class="notranslate">\; (</span><span\; class="notranslate">\; \%</span><span\; class="notranslate">\; )</span><span\; class="notranslate">\; =</span>\frac{{\mathrm{<span\; class="notranslate">\; C</span>}}_{\mathrm{<span\; class="notranslate">\; x</span>}}<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; ug</span>}<span\; class="notranslate">\; /</span>\mathrm{<span\; class="notranslate">\; mL</span>}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; \&times;</span>\mathrm{<span\; class="notranslate">\; 100</span>}<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; mL</span>}<span\; class="notranslate">\; )</span>}{\mathrm{<span\; class="notranslate">\; m</span>}<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; mg</span>}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; \&times;</span>\mathrm{<span\; class="notranslate">\; 1000</span>}}<span\; class="notranslate">\; \&times;</span>\mathrm{<span\; class="notranslate">\; 100</span>}<span\; class="notranslate">\; \%</span>$

W ₁ (%): the mass percentage of chlorogenic acid in honeysuckle medicinal materials/decoction pieces or derivatives;

C _X : Chlorogenic acid mass concentration (ug/mL) in honeysuckle medicinal material/decoction piece or derivative product for test solution;

m: the mass (mg) of the honeysuckle medicinal material or derivatives weighed.

5) Calculate the content and total amount of each main active ingredient of organic acids or/and iridoid glycosides in honeysuckle medicinal materials or derivatives through the coupling formula, that is, the active ingredient group of organic acids or/and iridoid glycosides content

${\mathrm{<span\; class="notranslate">\; W</span>}}_{\mathrm{<span\; class="notranslate">\; no</span>}}<span\; class="notranslate">\; =</span>\frac{{\mathrm{<span\; class="notranslate">\; W</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}{\mathrm{<span\; class="notranslate">\; m</span>}}_{\mathrm{<span\; class="notranslate">\; no</span>}}{\mathrm{<span\; class="notranslate">\; h</span>}}_{\mathrm{<span\; class="notranslate">\; no</span>}}}{{\mathrm{<span\; class="notranslate">\; m</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}{\mathrm{<span\; class="notranslate">\; h</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}}$

W ₁ : the mass percentage of chlorogenic acid in the standard reference product of honeysuckle medicinal materials/decoction pieces or derivatives;

M ₁ : the molecular weight of the standard reference product chlorogenic acid/the number of carbons corresponding to the quantitative peak;

h ₁ : the characteristic peak intensity (peak height) of the standard reference product chlorogenic acid;

W _n : mass percentage content of a certain organic acid or/and iridoid glycoside active ingredient in honeysuckle medicinal material/decoction pieces or derivatives;

M _n : the number of carbons corresponding to the molecular weight/quantitative peak of a certain organic acid or/and iridoid glycoside active ingredient in honeysuckle medicinal materials/decoction pieces or derivatives;

h _n : characteristic peak intensity (peak height) of an organic acid or/and iridoid glycoside active ingredient in honeysuckle medicinal materials or derivatives.

2. Instruments, reagents and materials

Main instruments and equipment

NMR spectrometer Bruker DPX400.

Mass spectrometer: WatersMicromass company Q-TofMicroTM type.

Semi-preparative high-performance liquid chromatography: Waters600 type.

High performance liquid chromatography: Agilent1200 type.

2000mL distillation flask, 5000mL distillation flask, spherical condenser, 2000mL separatory funnel.

DE-52AA rotary evaporator: Shanghai Yarong Biochemical Instrument Factory.

DEF-6020 vacuum drying oven: Shanghai Jinghong Experimental Equipment Co., Ltd.

Column chromatography silica gel G and thin layer chromatography silica gel H: Qingdao Ocean Chemical Factory.

Silica gel chromatography column 6cm×70cm (diameter×height).

Commercially available honeysuckle medicinal material A (Zhongyuan Zhengxin Company, purchased from Anhui Bozhou medicinal material market in June 2013, origin Henan), honeysuckle (Zhongyuan Zhengxin Company, purchased from Anhui Bozhou medicinal material market in July 2013, origin Henan), all passed through Henan Province Identification by Professor Zhu Changshan of Agricultural University; chlorogenic acid, chemical reference substance, purchased from China Institute for the Control of Pharmaceutical and Biological Products.

Reagents: chromatographically pure (methanol, Tianjin Siyou Fine Chemicals Co., Ltd.) and analytically pure (Tianjin No. 1 Chemical Reagent Factory).

3. The structure and carbon NMR data of the main active ingredients in honeysuckle medicinal materials or derivatives

Butyl Chlorogenic Acid

protocatechin caffeic acid

Ethyl Caffeate Ferulic Acid

Vanillicacid7-O-β-D-(6-O-benzoylglucopyranoside)

Methylchlorogenate (3-caffeoylquinic acid methylester)

3,4-O-Dicaffeoyl quinic acid ethyl ester

Loganin 7-dehydrologanin

Morroniside Swertiin

7-O-Methyl-morroniside Secologanindimethylacetal

Kingiside 8-epi-KINGISIDE

Chlorogenic acid (C ₁₆ H ₁₈ O ₉ )

¹³ CNMR (75MHz, DMSO-d ₆ )δ: 73.6(C-1), 36.4(C-2), 70.5(C-3), 70.9(C-4), 68.3(C-5), 37.3(C -6), 175.1(C-7), 125.6(C-1′), 114.3(C-2′), 145.0(C-3′), 146.0(C-4′), 115.8(C-5′) , 121.4 (C-6′), 148.4 (C-7′), 114.8 (C-8′), 165.8 (C-9′).

¹³ CNMR (125MHz, CD ₃ OD) δ: 127.8(C-1), 115.2(C-2), 146.8(C-3), 149.6(C-4), 116.0(C-5), 123.0(C- 6), 147.1(C-7), 115.3(C-8), 168.7(C-9); B: 76.1(C-1), 38.8(C-2), 72.0(C-3), 73.4(C -4),71.3(C-5),38.2(C-6),177.0(C-7)

Butyl chlorogenic acid

13C-NMR (MHz, DMSO-d6) δ: 73.2(C-1), 37.3(C-2), 69.4(C-3), 71.1(C-4), 66.9(C-5), 35.2(C -6), 173.2(C-7), 64.2(C-8), 30.1(C-9), 18.6(C-10), 13.6(C-11), 125.4(C-1′), 114.6(C -2′), 145.2(C-3′), 148.8(C-4′), 115.9(C-5′), 121.4(C-6′), 145.7(C-7′), 113.9(C-8 '), 165.5 (C-9').

3,4-O-Dicaffeoyl quinic acid ethyl ester

13C-NMR (MHz, CD ₃ OD) δ: 75.1(C-1), 36.8(C-2), 75.1(C-3), 69.8(C-4), 66.0(C-5), 41.3(C -6),175.7(C-7),62.5,14.2(-7-OCH ₂ CH ₃ ),127.7,127.6(C-1′,1′′),115.2,115.1(C-2′,2′′ ), 146.8, 146.8 (C-3′, 3′′), 149.0, 149.0 (C-4′, 4′′), 116.5, 116.5 (C-5′, 5′′), 123.2, 123.1 (C- 6', 6''), 147.4, 147.4 (C-7', 7''), 115.0, 115.0 (C-8'), 168.5, 168.4 (C-9').

Caffeic acid (C ₉ H ₈ O ₄ )

¹³ CNMR (CD ₃ OD, 75MHz) δ: 127.8(C-1), 115.1(C-2), 147.0(C-3), 149.4(C-4), 115.5(C-5), 122.8(C- 6), 146.8(C-7), 116.8(C-8), 170.9(C-9)

Ethyl Caffeate (C ₁₁ H ₁₂ O ₄ )

¹³ CNMR (CD ₃ OD, 75MHz) δ: 127.7(C-1), 115.0(C-2), 146.8(C-3), 149.5(C-4), 115.2(C-5), 122.9(C- 6), 146.7(C-7), 116.5(C-8), 169.3(C-9), 61.4, 14.6(-OCH ₂ CH ₃ )

Ferulic acid (C ₁₀ H ₁₀ O ₄ )

¹³ CNMR (125MHz, CD ₃ OD): δC 51.9(q, OCH ₃ -3), 114.8(d, _C -8), 115.1(d, C-2), 116.4(d, C-5), 122.7 (d,C-6),127.6(s,C-1),146.6(s,C-4),146.7(s,C-7),149.3(s,C-3),169.5(s,C-3) 9).

protocatechuic acid

¹³ CNMR (CD ₃ OD, 125MHz) δ _C : 123.2(C-1), 115.8(C-2), 146.1(C-3), 151.5(C-4), 117.8(C-5), 123.9(C -6), 170.3(C-7);

¹³ CNMR(DMSO-d ₆ ,100MHz)δ _C :121.7(C-1),116.7(C-2),144.8(C-3),149.8(C-4),115.1(C-5),122.5( s, C-6), 167.7 (C-7).

Vanillicacid7-O-β-D-(6-O-benzoylglucopyranoside) (vanillic acid 7-o-β-D–(6-o-phenylglucopyranoside)

¹³ C-NMR (MHz, CD ₃ OD) δ: 126.1 (C-1), 114.3 (C-2), 150.4 (C-3), 151.6 (C-4), 116.5 (C-5), 124.6 ( C-6), 169.5(C-7), 56.5(-OMe), 131.2(C-1′), 130.6(C-2′), 129.6(C-3′), 134.4(C-4′), 129.6 (C-5'), 130.6 (C-6'), 167.7 (C-7').

Methylchlorogenate(3-caffeoylquinicacidmethylester)[chlorogenic acid methyl ester ( ₃ -caffeoylquinic acid methylester)]

¹³ C-NMR (MHz, MeOD) δ: 75.8 (C-1), 38.0 (C-2), 70.3 (C-3), 72.1 (C-4), 72.1 (C-5), 37.7 (C- 6), 175.4(C-7), 53.0(OMe), 127.6(C-1′), 115.1(C-2′), 146.9(C-3′), 149.7(C-4′), 116.5(C -5'), 123.0 (C-6'), 145.7 (C-7'), 115.0 (C-8'), 168.2 (C-9').

Loganin

¹³ CNMR (100MHz, MeOD) δC: 97.71( _C -1), 152.10(C-3), 114.03(C-4), 32.15(C-5), 42.70(C-6), 75.04(C-7 ), 42.16 (C-8), 46.50 (C-9), 13.41 (C-10), 169.52 (C-11), 51.62 (C-12), 100.05 (C-1′), 74.72 (C-2 ′),78.02(C-3′),71.59(C-4′),78.35(C-5′),62.76(C-6′) ¹³ CNMR(100MHz,DMSO-d ₆ )δ _C :96.15(C -1), 150.71(C-3), 112.21(C-4), 30.88(C-5), 41.74(C-6), 73.11(C-7), 40.58(C-8), 44.80(C- 9), 13.62(C-10), 167.14(C-11), 51.13(C-12), 98.63(C-1′), 72.18(C-2′), 76.64(C-3′), 70.05( C-4'),77.31(C-5'),61.12(C-6')

7-dehydrologanin

¹³ CNMR (100MHz, D ₂ O) δ _C :95.04(C-1), 152.60(C-3), 110.35(C-4), 26.74(C-5), 42.52(C-6), 225.04(C -7), 44.08(C-8), 44.87(C-9), 12.61(C-10), 169.78(C-11), 52.26(C-12), 99.07(C-1′), 73.00(C -2'),75.95(C-3'),69.94(C-4'),76.72(C-5'),61.07(C-6')

Swertiin

¹³ CNMR (100MHz, DMSO-d ₆ )δ _C :95.75(C-1), 151.60(C-3), 105.01(C-4), 26.94(C-5), 24.45(C-6), 67.86( C-7), 132.54(C-8), 41.69(C-9), 120.48(C-10), 164.83(C-11), 98.27(Glc-1), 73.30(Glc-2), 76.57(Glc -3), 70.22 (Glc-4), 77.52 (Glc-5), 61.21 (Glc-6)

¹³ CNMR (100MHz, MeOD) δC: 98.2( _C -1), 153.9(C-3), 106.0(C-4), 28.5(C-5), 26.0(C-6), 69.7(C-7 ), 133.5 (C-8), 43.8 (C-9), 120.1 (C-10), 168.5 (C-11), 99.7 (C-1′), 74.7 (C-2′), 77.8 (C- 3'),71.5(C-4'),78.4(C-5'),62.7(C-6')

α-morroniside

¹³ CNMR (100MHz, DMSO-d ₆ )δ _C :95.38(C-1), 152.95(C-3), 109.92(C-4), 30.30(C-5), 36.39(C-6), 94.37( C-7), 73.74(C-8), 39.99(C-9), 19.56(C-10), 166.51(C-11), 51.34(C-12), 98.41(C-1′), 71.60( C-2'),76.82(C-3'),70.35(C-4'),77.65(C-5'),61.24(C-6')

¹³ CNMR (CD ₃ OD) 97.2(C-1), 154.6(C-3), 111.0(C-4), 34.7(C-5), 37.4(C-6), 96.1(C-7).75.1 (C-8),40.0(C-9),20.0(C-10),168.8(C-11),100.2(C-1′).74.2(C-2′),78.0(C-3′) ,71.7(C-4′),78.5(C-5′),62.9(C-6′).

β-morroniside

¹³ CNMR (100MHz, DMSO-d ₆ )δ _C :93.98(C-1), 153.00(C-3), 110.70(C-4), 25.74(C-5), 33.64(C-6), 90.01( C-7), 40.12(C-8), 40.43(C-9), 19.52(C-10), 166.47(C-11), 51.28(C-12), 98.37(C-1′), 73.74( C-2'),76.82(C-3'),70.35(C-4'),77.70(C-5'),78.46.(C-5'),61.39(C-6')

¹³ CNMR(CD ₃ OD)δC:95.8( _C -1),154.6(C-3),111.8(C-4),27.6(C-5),34.7(C-6).92.5(C-7 ), 66.0(C-8), 40.7(C-9), 20.0(C-IO), 168.8(C-11), 100.2(C-1′), 74.2(C-2′), 78.0(C- 3'),71.7(C-4'),78.5(C-5'),62.9(C-6').

7-O-Methyl-morroniside

¹³ CNMR (100MHz, CD ₃ Cl ₃ ) δ _C :95.1(C-1), 152.0(C-3), 110.7(C-4), 29.9(C-5), 33.8(C-6), 102.4( C-7), 71.6(C-8), 38.9(C-9), 18.4(C-10), 166.1(C-11), 50.9(C-12), 96.7(C-1′), 70.7( C-2'),72.0(C-3'),68.6(C-4'),72.5(C-5'),61.6(C-6')

¹³ CNMR (100MHzCD _{3 Cl 3} ,) δC: 94.7(C-1), 152.1(C-3), 111.7(C-4), 26.1(C-5), 32.6(C-6), 97.7(C -7), 64.1(C-8), 39.4(C-9), 18.6(C-10), 166.5(C-11), 51.0(C-12), 96.7(C-1′), 71.0(C -2'),72.0(C-3'),68.6(C-4'),72.5(C-5'),61.7(C-6')

Secologanindimethylacetal (Secologanindimethylacetal)

¹³ CNMR (100MHz, DMSO-d ₆ )δ _C :95.7(C-1), 151.5(C-3), 109.7(C-4), 25.2(C-5), 31.7(C-6), 102.1( C-7), 134.5 (C-8), 43.1 (C-9), 119.3 (C-10), 166.6 (C-11), 98.7 (C-1′), 73.0 (C-2′), 77.4 (C-3'),69.9(C-4'),76.7(C-5'),61.0(C-6')

Jinjiside

¹³ CNMR(MeOD)δC: 94.4( _C -1), 154.3(C-3), 111.4(C-4), 28.0(C-5), 34.3(C-6), 174.3(C-7), 76.8(C-8), 39.9(C-9), 18.4(C-10), 168.2(C-11), 100.1(C-1′), 74.8(C-2′), 78.0(C-3′ ),71.6(C-4′),78.5(C-5′),62.8(C-6′)

8-epi-KINGISIDE (8-epijinjiside)

¹³ CNMR(MeOD)δC: 96.3(Cl), 154.4( _C -3), 109.6(C-4), 28.1(C-5), 34.6(C-6), 174.7(C-7), 75.8( C-8), 41.9 (C-9), 21.7 (C-lo), 168.3 (C-11), 52.0 (OMe), 100.7 (C-l'), 74.7 (C-2'), 78.5 (C -3'),71.7(C-4'),77.9(C-5'),62.9(C-6').

Example 1: Commercially available honeysuckle medicinal material A (Zhongyuan Zhengxin) organic acid and iridoid glycosides characteristic extract IGD C-NMR coupled fingerprint

(1) Preparation of characteristic extracts

Weigh 25 grams of commercially available honeysuckle medicinal material A after crushing (passing through a 65 mesh sieve), add 6, 6, and 10 times the volume (150 mL, 150 mL, 250 mL), 20% (volume ratio) of ethanol, and reflux extraction at 90°C 3 times, extract for 1 hour each time, filter and combine the filtrates, concentrate under reduced pressure until there is no alcohol smell, about 500mL, adjust pH=1 with concentrated hydrochloric acid; take the filtrate after acid adjustment, use a macroporous Adsorptive resin (model: HP-20, diameter-to-height ratio 12:1) was packed into a column, washed with water-ethanol system, and 30% of the eluted part was collected to obtain the characteristic extract of honeysuckle A organic acid and iridoid glycosides ( CET).

(2) Feature extraction IGD carbon NMR fingerprint detection

Take commercially available honeysuckle A (Zhongyuan Zhengxin) organic acid and iridoid glycoside characteristic extract 30mg, dissolve it in 0.5mL DMSO-d ₆ , and do IGD carbon NMR fingerprint spectrum detection to obtain honeysuckle A organic acid and cycloalkene Carbon NMR fingerprint of iridoid glycoside IGD.

(3) Commercially available honeysuckle A (Zhongyuan Zhengxin) organic acid and iridoid glycosides characteristic extract IGD carbon NMR fingerprint

1) IGD carbon NMR fingerprint identification

In the IGD carbon NMR fingerprint of honeysuckle organic acids and iridoid glycosides characteristic extract (CET) of honeysuckle A (Zhongyuan Zhengxin) medical material, it clearly shows that the organic acids and iridoid glycosides of honeysuckle are characteristic signal. Honeysuckle organic acids and iridoid glycosides: chlorogenic acid, butyl chlorogenic acid, ethyl caffeate, Vanillicacid7-O-β-D-(6-O-benzoylglucopyranoside), α-morroniside, Secologanindimethylacetal, β-morroniside, swertiside, etc. have corresponding NMR signals in the IGD carbon NMR fingerprint. See attached drawing 1-a for the carbon NMR fingerprint of IGD, and see attached drawing 1-b for the partially broadened and enlarged view of its characteristic peaks.

2) The results of the determination of the proportions of the active ingredients in the characteristic extracts of organic acids and iridoid glycosides from the commercially available honeysuckle A (Zhongyuan Zhengxin) medicinal material are as follows:

(4) The results of the determination of the mass percentage of chlorogenic acid in the commercially available honeysuckle A (Zhongyuan Zhengxin) medicinal material are as follows:

(5) The results of the mass percent determination of organic acids and iridoid glycosides active ingredients in commercially available honeysuckle A (Zhongyuan Zhengxin) medicinal materials are as follows:

The molecular formula of α-morroniside + Secologanindimethylacetal in the table below is the molecular formula represented by α-morroniside (the following examples are the same as Example 1).

Example 2: Commercially available honeysuckle medicinal material B (Zhongyuan Zhengxin) organic acid and iridoid glycosides characteristic extract IGD C-NMR coupled fingerprint

(1) Preparation of characteristic extracts

Weigh 25g of commercially available honeysuckle B (Zhongyuan Zhengxin) decoction pieces after being crushed (passed through a 24 mesh sieve), add 10 or 10 times the volume (250mL, 250mL), 25% (volume ratio) of ethanol, and ultrasonically extract at 50°C 2 times, extracting for 2 hours each time, after filtering, combine the filtrates, concentrate under reduced pressure until there is no alcohol smell, about 500mL, adjust the pH=2 with concentrated hydrochloric acid; take the filtrate after acid adjustment; take the concentrated filtrate, and use its weight as 1 times the amount of macroporous adsorption resin (model: D-101, diameter-to-height ratio 12:1) was packed into a column, washed with water-ethanol system, and 35% of the eluted fraction was collected to obtain honeysuckle B organic acid and cycloalkene ether Terpene Characteristic Extract (CET).

(2) Feature extraction IGD carbon NMR fingerprint detection

Take commercially available honeysuckle B (Zhongyuan Zhengxin) organic acid and iridoid glycosides characteristic extract 30mg, dissolve it in 0.5mL deuterated DMSO-d ₆ , and do IGD carbon NMR fingerprint detection to obtain honeysuckle B organic acid and iridoid glycosides IGD carbon NMR fingerprints.

(3) Commercially available honeysuckle B (Zhongyuan Zhengxin) organic acids and iridoid glycosides characteristic extract IGD carbon NMR fingerprint

1) IGD carbon NMR fingerprint identification

The organic acids and iridoid glycosides of honeysuckle B (Zhongyuan Zhengxin) medicinal material commercially available in the IGD carbon NMR fingerprint of the characteristic extract (CET) of honeysuckle B (Zhongyuan Zhengxin) clearly show the organic acids and iridoid glycosides of honeysuckle characteristic signal. Honeysuckle Organic Acids and Iridoid Glycosides: Chlorogenic Acid, Butyl Chlorogenic Acid, Ethyl Caffeate, Vanillicacid7-O-β-D-(6-O-benzoylglucopyranoside), α-Morroniside, Secologanindimethylacetal, β-morroniside, swertiside, etc. have corresponding NMR signals in the IGD carbon NMR fingerprint. See attached drawing 2-a for the carbon NMR fingerprint of IGD, and see attached drawing 2-b for the partially broadened and enlarged view of its characteristic peaks.

2) The results of the determination of the proportions of the active ingredients in the characteristic extracts of organic acids and iridoid glycosides from the commercially available honeysuckle B (Zhongyuan Zhengxin) medicinal material are as follows:

(4) The results of the determination of the mass percentage of chlorogenic acid in the commercially available honeysuckle B (Zhongyuan Zhengxin) medicinal material are as follows:

(5) The results of the mass percent determination of organic acids and iridoid glycosides active ingredients in commercially available honeysuckle B (Zhongyuan Zhengxin) medicinal materials are as follows:

Example 3: Commercially available honeysuckle extract IGD carbon nuclear magnetic resonance coupled fingerprint

(1) Feature extraction selection

The commercially available chlorogenic acid 20% honeysuckle extract (Hunan Changsha Shanghe Biotechnology Co., Ltd.) was directly selected as the characteristic extract.

(2) Feature extraction IGD carbon NMR fingerprint detection

Take 30mg of chlorogenic acid 20% honeysuckle extract, dissolve it in 0.5mL deuterated DMSO-d ₆ , and perform IGD carbon NMR fingerprint detection to get chlorogenic acid 20% honeysuckle extract IGD nuclear magnetic resonance Carbon spectrum fingerprint.

(3) IGD carbon NMR fingerprint of chlorogenic acid 20% honeysuckle extract

1) IGD carbon NMR fingerprint identification

In the IGD carbon NMR fingerprint of chlorogenic acid 20% honeysuckle extract, the characteristic signals of organic acid compounds of honeysuckle are clearly displayed. Organic acids of honeysuckle: chlorogenic acid, butyl chlorogenic acid, ethyl 3,4-O-biscaffeoylquinic acid, ethyl caffeic acid, ferulic acid, etc. are all present in the IGD carbon NMR fingerprint Corresponding NMR signal. See attached drawing 3-a for the carbon NMR fingerprint of IGD, and see attached drawing 3-b for the partially broadened and enlarged view of its characteristic peaks.

2) The results of the determination of the proportions of active ingredients in 20% honeysuckle extract of chlorogenic acid are as follows:

(4) The results of the determination of the mass percentage of chlorogenic acid in 20% honeysuckle extract of Shanghe chlorogenic acid are as follows:

(5) The results of the determination of the mass percentage of the active ingredients in the 20% honeysuckle extract of chlorogenic acid are as follows:

Embodiment 4: Self-made honeysuckle extract IGD carbon nuclear magnetic resonance spectrum fingerprint

(1) Feature extraction selection

Directly select self-made honeysuckle extract [Weigh 50 grams of commercially available honeysuckle medicinal material (Zhongyuan Zhengxin Company, sourced from Anhui Bozhou medicinal material market, place of origin Henan) after crushing (through a 65-mesh sieve), and add volumes of 6, 6, and 10 times , 20% (volume ratio) ethanol was refluxed at 90°C for 3 times, each time for 1 hour, filtered and combined the filtrate, concentrated under reduced pressure until no alcohol smell, about 500mL, adjusted pH = 2 with concentrated hydrochloric acid; Ethyl extraction was carried out 4 times, and the combined ethyl acetate solution was evaporated to dryness to obtain 2.22 g of homemade honeysuckle extract] as the characteristic extract.

(2) Feature extraction IGD carbon NMR fingerprint detection

Take 30mg of self-made honeysuckle extract, dissolve it in 0.5mL of deuterated methanol, and perform IGD carbon NMR fingerprint detection to obtain the IGD carbon NMR fingerprint of 25% honeysuckle extract of chlorogenic acid.

(3) Self-made honeysuckle extract IGD carbon NMR fingerprint

1) IGD carbon NMR fingerprint identification

In the IGD carbon NMR fingerprint of the self-made honeysuckle extract, the characteristic signals of honeysuckle organic acids and iridoid glycosides are clearly displayed. Honeysuckle organic acids and iridoid glycosides: chlorogenic acid, chlorogenic butyl ester, caffeic acid ethyl ester, morroniside, swertiroside, ginji glycoside, etc. have corresponding in IGD carbon NMR fingerprint NMR signal. See Figure 4-a for the IGD carbon NMR fingerprint, and Figure 4-b for the partially broadened and enlarged view of its characteristic peaks.

2) The results of the ratio determination of the active ingredients in the self-made honeysuckle extract are as follows:

(4) The results of the determination of the mass percentage of chlorogenic acid in the self-made honeysuckle extract are as follows:

(5) The results of the determination of the mass percentage of active ingredients in the self-made honeysuckle extract are as follows:

Claims (9)

1. differentiate a method for traditional Chinese medicine honeysuckle or spin-off, comprise the following steps:

1) traditional Chinese medicine honeysuckle or spin-off are extracted, obtain honeysuckle organic acid containing active component group or/and iridoid glycoside feature extraction thing;

Honeysuckle organic acid is or/and the preparation method of iridoid glycoside feature extraction thing, comprise: take traditional Chinese medicine honeysuckle or medicine materical crude slice is pulverized, add 20 ~ 25% alcohol reflux and extract or ultrasonic extraction 2 ~ 3 times, extract 1 ~ 2 hour at every turn, merging filtrate after filtering, reduced pressure concentration; Get the filtrate after concentrating, fill post with macroporous absorbent resin, rush post with water-ethanol system, collect different elution fraction, evaporated under reduced pressure, obtain honeysuckle organic acid or/and iridoid glycoside feature extraction thing;

The mass volume ratio of described traditional Chinese medicine honeysuckle or medicine materical crude slice and 20 ~ 25% ethanol is 1:(6 ~ 10);

The weight of described macroporous absorbent resin is 1 ~ 2 times of the filtrate weight after concentrating;

The temperature of described refluxing extraction is 90 ~ 95 DEG C, and the temperature of ultrasonic extraction is 50 ~ 60 DEG C;

2) to described honeysuckle organic acid or/and iridoid glycoside feature extraction thing carries out IGD carbon-13 nmr spectra finger-print respectively detects, obtain described honeysuckle organic acid according to finger-print or/and several active component characteristic peak peak intensities in iridoid glycoside feature extraction thing; And determine the characteristic peak peak intensity of described each active component respective standard with reference to product by same way;

Wherein, honeysuckle organic acid is or/and the active component characteristic peak in iridoid glycoside feature extraction thing is ester carbonyl group absorption peak, and its chemical shift is δ _c164.0 ~ 167.0 or δ _c168.0 ~ 170.0;

Honeysuckle organic acid is or/and the standard of active component in iridoid glycoside feature extraction thing is chlorogenic acid with reference to product;

3) absolute content obtaining described standard reference product is measured by efficient liquid phase means;

The condition of described efficient liquid phase comprises: mobile phase is acetonitrile: 0.4% phosphoric acid solution=(10 ~ 20): (80 ~ 95), and determined wavelength is 240nm;

4) utilize the ratio of the characteristic peak peak intensity of each active component characteristic peak peak intensity and respective standard reference product and the absolute content of described standard reference product, calculate each organic acid in traditional Chinese medicine honeysuckle or spin-off or/and the content of iridoid glycoside active component and the content of two active component groups.

2. method according to claim 1, is characterized in that, adopt have obtain clear IGD carbon-13 nmr spectra finger-print extraction process as described honeysuckle organic acid or/and the extracting mode of iridoid glycoside feature extraction thing.

3. the method according to claim 1 ~ 2 any one, is characterized in that, the model of described macroporous absorbent resin is HP-20, D-101, D-201, SP-825 or SP-70; The blade diameter length ratio of described macroporous absorbent resin is 8 ~ 15:1.

4. method according to claim 3, it is characterized in that, step 2) in, to honeysuckle organic acid or/and iridoid glycoside feature extraction thing carries out the detection of IGD carbon-13 nmr spectra finger-print, dissolve honeysuckle organic acid or/and the solvent of iridoid glycoside feature extraction thing is deuterochloroform, deuterated methanol or deuterated dimethyl sulfoxide.

5. the method according to claim 1 or 2 or 4, is characterized in that, step 2) in, according to size and the position of characteristic peak peak intensity, to described honeysuckle organic acid or/and in iridoid glycoside feature extraction thing several active components sort.

6. method according to claim 5, is characterized in that, the condition of described efficient liquid phase comprises: mobile phase is acetonitrile: 0.4% phosphoric acid solution=13:87.

7. the method according to claim 1 or 2 or 4 or 6, is characterized in that, step 3) in, described standard refers to reference to the absolute content of product: the standard measured by quantitative test means is with reference to the mass percentage of product.

8. method according to claim 7, is characterized in that, step 4) in, the coupling formula calculating the content of each active component is:

$W_n=\frac{W_1{M}_n{h}_n}{M_1{h}_1};$ Wherein:

W ₁for step 3) a certain active component is corresponding in the traditional Chinese medicine honeysuckle that measures by quantitative test means or spin-off standard is with reference to the absolute content of product;

M ₁for standard corresponding to active component a certain in described traditional Chinese medicine honeysuckle or spin-off is with reference to the carbon number that molecular weight/quantitatively peak is corresponding of product;

H ₁for by the characteristic peak peak intensity of standard corresponding to a certain active component in the traditional Chinese medicine honeysuckle of IGD carbon-13 nmr spectra determining fingerprint pattern or spin-off with reference to product;

W _nfor the mass percentage of active component a certain in traditional Chinese medicine honeysuckle or spin-off;

M _nfor the carbon number that molecular weight/quantitatively peak is corresponding of active component a certain in traditional Chinese medicine honeysuckle or spin-off;

H _nfor the characteristic peak peak intensity by a certain active component in the traditional Chinese medicine honeysuckle of IGD carbon-13 nmr spectra determining fingerprint pattern or spin-off.

9. the method according to claim 1 or 2 or 4 or 6 or 8, it is characterized in that, honeysuckle spin-off comprises: honeysuckle medicine materical crude slice, Honegsukle flower P.E or honeysuckle natural drug.