Application of fluorescence spectrophotometry in the field of medicinal materials research

2024-01-24

In today's society, with the change of people's thinking, traditional Chinese medicine has gradually become a hot topic among the public. At present, the Chinese medicine market is a mixed bag, with fake Chinese medicines and inferior medicines emerging one after another. Quality evaluation and quality control of traditional Chinese medicines in the traditional Chinese medicine market are the means to ensure the effectiveness and safety of traditional Chinese medicines. They are also the bottleneck that has always plagued the modern development of traditional Chinese medicines. First of all, the quality of traditional Chinese medicine is affected by multiple links and factors. There are many types of Chinese herbal medicines, their origins are scattered, and factors such as the growth environment, harvest season, and processing techniques lead to differences in the chemical composition

With the continuous development of traditional Chinese medicine, the advantages of fluorescence analysis in the identification of Chinese medicinal materials have gradually emerged. Chinese medicinal materials contain a variety of chemical substances with fluorescent properties, such as cyclic compounds, aromatic compounds, etc., and leaf medicinal materials are easy to shrink and break during the process of collection, processing, transportation and storage, making it difficult to identify properties; and because leaf medicinal materials contain more tannins, chlorophyll and other ingredients, general physical and chemical identification is also difficult. It can be easily and quickly identified by fluorescence method.

Identification of senna medicinal materials and Indigowoad leaf medicinal materials by fluorescence spectrophotometry

Senna is a common laxative and is widely used in various tea drinks. Most of their sources are imported, and domestic production is small, resulting in the proliferation of counterfeit and inferior products. The current application of Indigowoad leaf is confusing, and the quality on the market is mixed. And as Indigowoad leaf was listed as a drug to prevent H1N1 by the Beijing Municipal Food and Drug Administration, the dosage of Indigowoad leaf increased sharply, and a large number of counterfeit products appeared on the market for confusing use.

Xinrui Wang and others crushed senna leaves and Daqing leaves, added water, dilute ethanol, methanol, dilute hydrochloric acid, and sodium hydroxide solution, and obtained the leachate after ultrasonic and centrifugation. After consulting the information, set the excitation wavelength to 254nm and 365nm.

Figure 1-2 Fluorescence spectra of Indigowoad leaf with different solvents and ultrasonic excitation wavelength of 254nm.

Fluorescence spectra of senna leaves with different solvents and ultrasonic excitation wavelength of 254nm.

Figure 3-4 Fluorescence spectra of Indigowoad leaf with different solvents and ultrasonic excitation wavelength of 365nm.

Fluorescence spectra of senna leaves with different solvents and ultrasonic excitation wavelength of 365nm.

This study found that different spectra can be obtained from the leaching solutions of the two medicinal materials using a fluorescence spectrophotometer. When the excitation wavelength is 254nm, there are two fluorescence peaks in water and hydrochloric acid leachates of Indigowoad leaf, and only one fluorescence peak in Senna leaves. When the excitation wavelength is 365nm, there are two fluorescence peaks in methanol, sodium hydroxide, and ethanol leachates of Senna leaves. Indigowoad leaf has only one fluorescence peak. And the fluorescence peak intensity of Indigowoad leaf was significantly stronger than that of Senna.

Determination of riboflavin concentration in wolfberry by fluorescence spectrophotometry

Riboflavin, also known as vitamin B2, was first discovered by British chemist Bruce from whey. Riboflavin is an essential nutrient for maintaining the normal body. It has a variety of physiological functions. It can promote the metabolism of various organic substances and promote the growth and development of the body. It is involved in the metabolism of proteins, fats and carbohydrates in the body, and is also related to animal vision. It is essential nutrients for animals. The most widely used method to measure riboflavin concentration in today's society is fluorescence spectrophotometry. The fluorescence method uses riboflavin to produce yellow-green fluorescence at an excitation wavelength in the range of 440 to 500 nm in an environment of pH 4 to 9, and the fluorescence intensity of riboflavin is measured based on the principle that the fluorescence intensity is proportional to the concentration of riboflavin. The method is simple and easy to operate.

Jiying Song and others added hydrochloric acid solution to wolfberry, centrifuged and filtered the supernatant. Pour the filtrate into the cuvette and set the parameters:

Wavelength	Scan range	Slit width	Scan speed	Delay	Repeat times
EX-490.0nm, EM-512.0nm	EX-300~600nm, EM-300~600nm	EX-2.5nm, EM-2.5nm	300nm/min	0s	1 time

Figure 5 Fluorescence intensity at excitation wavelengths of samples extracted with different hydrochloric acid solutions.

Figure 6 Fluorescence intensity at excitation wavelength of samples extracted with different material-to-liquid ratios.

Figure 7 Fluorescence intensity at excitation wavelength of samples extracted at different stirring times.

Through experiments with different control groups, the research team obtained the best experimental conditions. The research team accurately weighed 1.000g of riboflavin standard and diluted it to 1000ml with 0.5% acetic acid as the mother liquor. The mother liquor was removed to prepare riboflavin standard solution of 0.2, 0.4, 0.6, 0.8 and 1μg/ml respectively. With the riboflavin concentration as the abscissa and the excitation wavelength fluorescence intensity as the ordinate, make a standard working curve. The resulting standard curve equation F=15.998c+0.7684, F is the fluorescence intensity, c is the concentration of riboflavin, and the linear correlation coefficient r²=0.9993.

Products Recommended

Figure 8 ATF4500 Fluorescence Spectrophotometer.

1) Comprehensive overview: A fluorescence spectrophotometer is an instrument used to scan the fluorescence spectrum emitted by liquid phase fluorescent markers. It can provide many physical parameters including excitation spectrum, emission spectrum, fluorescence intensity, quantum yield, fluorescence lifetime, fluorescence polarization, etc., reflecting the bonding and structure of molecules from all angles. By measuring these parameters, not only general quantitative analysis can be done, but also the conformational changes of molecules under various environments can be inferred, thereby clarifying the relationship between molecular structure and function. Can be used for scanning liquid and solid samples.

In order to solve the problem of instrument light source performance declining with the increase of service life, ATF4500 uses a xenon lamp with superior performance and improves the lamp power supply. The brightness of the light source is improved and the service life of the xenon lamp is effectively extended to 1000 hours. Extending the lamp replacement cycle can reduce consumable costs and reduce the burden of lamp replacement and other operations. Coupled with the scientifically designed optical path system that embodies the long-term development of Optosky's superb technology and new technology, it ensures the superior precision and outstanding measurement accuracy pursued by fluorescence analysis. While ensuring accurate results, high-speed three-dimensional fluorescence collection can also ensure high-speed scanning speed, not only that, but also a variety of accessories to choose from to meet users' various measurement needs.

This product is equipped with Optosky's unique simple and intuitive operation page. From the user's perspective, the intelligent integrated system allows users to get started quickly with only a little training. In terms of data processing, data can be better organized through spectral processing online.

2) Product selection

Model	Wavelength range
ATF4500	185-900nm
ATF4500-17	185-1700nm
ATF4500-TM	185-900nm, with time resolution function, can measure fluorescence lifetime 150ps~100ns
ATF4500-17-TM	185-1700nm, with time resolution function, can measure fluorescence lifetime 150ps~100ns

3) Product parameters

Model	ATF4500	ATF4500-17
Wavelength range	185~900nm	185~1700nm
Scan speed	30、60、240、 1,200、2,400、 12,000、 30,000、60,000nm/min	30、60、240、 1,200、2,400、 12,000、 30,000、60,000nm/min
Sensitivity	Noise at water Raman peak: S/N>800(RMS) Minimum background noise: S/N>7500(RMS) Noise at water Raman peak: S/N>7500(RMS)	Noise at water Raman peak: S/N>800(RMS) Minimum background noise: S/N>7500(RMS) Noise at water Raman peak: S/N>7500(RMS)
Wavelength accuracy	±1.0nm	±1.0nm
Light source	150W xenon lamp	150W xenon lamp
Spectral Bandwidth	Excitation： 1.0/2.5/5/10nm Adjustable Emission：1.0/2.5/5/10/20.0nm Adjustable	Excitation： 1.0/2.5/5/10nm Adjustable Emission：1.0/2.5/5/10/20.0nm Adjustable
Resolution	1.0nm	1.0nm
Average sampling time	Fluorescence: 0.0125-999sec Phosphorescence: 1usec-10sec Bio/chemiluminescence: 40usec-10sec	Fluorescence: 0.0125-999sec Phosphorescence: 1usec-10sec Bio/chemiluminescence: 40usec-10sec
Detector	PMT	PMT & InGaAs
Communication interface	USB2.0	USB2.0
Wavelength Repeatability	±0.2nm	±0.2nm
Working humidity	45-80%	45-80%
Working temperature	10-35℃	10-35℃