Research and Application of TLC Identification Method for Homoisoflavanones in Polygonatum odoratum (Mill.) Druce

Abstract

We created a fast and effective thin-layer chromatography method to detect homoisoflavones in Polygonatum odoratum (Mill.) Druce (POD), demonstrating high sensitivity, specificity, and stability. Utilizing this method, we assessed the quality of 20 commercial batches of POD. Our results indicated that homoisoflavones (III, IV, and V) were present in 8 samples, absent in 7, and detected in 5. This method facilitates the effective evaluation of POD germplasm resources, laying a strong foundation for enhancing quality standards. This advancement aids in the standardization and quality control of POD, ensuring the consistency of its active ingredients and thereby supporting its therapeutic applications.

Keywords:
Polygonatum odoratum ; (Mill.) Druce; homoisoflavone; TLC; quality control

Introduction

Polygonatum odoratum (Mill.) Druce (POD) predominantly grows in mountainous and forestedge areas in China, Russia, and other regions, and is extensively used in traditional Chinese medicine. The primary components of POD include active ingredients like polysaccharides, saponins, and flavonoids,¹ which exhibit various pharmacological effects such as blood glucose reduction,² tumor prevention,³ and immune function regulation.⁴ In contemporary medical research, POD is gaining significant attention as a traditional herbal medicine. With deeper exploration of its pharmacological effects and clinical applications, POD is anticipated to play a larger role in health products and pharmaceutical development. However, ensuring its safe and effective use requires enhanced research on quality control and efficacy evaluation. The absence of standardized quality assessment methods impedes the sustainable growth and utilization of POD resources.⁵,⁶,⁷ Among the high-purity flavonoids in POD, three stand out for their high concentration and potent activity: 6,8-dimethyl-5,7,4'-trihydroxyisoflavone (III), 6-methyl-5,7,4'-trihydroxy-8-methoxyisoflavone (IV), and 6-methyl-5,7,4'-trihydroxyisoflavone (V). These components highlight the need for developing more specific and sensitive methods to evaluate the quality of POD, ensure efficacy, and support market stability.⁸

Recent studies have highlighted the therapeutic potential of plant-derived compounds, offering promising natural treatments for various health conditions. Guo et al.⁹ identified a dihydroflavonoid and several homoisoflavonoids in POD rhizomes. Their research showed that these compounds significantly activated adenosine monophosphate-activated protein kinase (AMPK) in rat liver epithelial cells, indicated by increased levels of phosphorylated AMPK and acetyl coenzyme A carboxylase. This suggests these compounds may positively influence metabolic pathways. Similarly, Ma et al.¹⁰ investigated the anti-hepatic injury properties of deoxyisoflavonoids from Cucumis bisexualis fruit. They isolated 12 compounds and analyzed their structures, then performed in vitro experiments to test their effectiveness in lowering aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in H₂O₂-induced HepG2 cells. Their research identified 4 compounds with significant anti-hepatic injury activity, indicating potential therapeutic applications for liver health. In ophthalmology, Amin et al.¹¹ discovered a new class of compounds, homoisoflavones, which inhibit retinal neovascularization, a critical factor in severe eye diseases like age, related to macular degeneration and retinopathy of prematurity. Their synthesis and evaluation of several homoisoflavones identified one compound that effectively inhibited this pathological process, laying the groundwork for new anti-angiogenic treatments. Furthermore, Tai et al.¹² examined the effects of extracts from POD, a traditional Chinese medicine, on breast cancer cells (MDA-MB-231). Their study found that these extracts inhibited cell proliferation and clone formation while promoting apoptosis. The extracts also affected mitochondrial membrane potential and regulated the expression of Bax and Bcl-2 proteins, key players in apoptosis. Collectively, these studies highlight the diverse therapeutic potential of plant-derived natural compounds, providing new insights and candidate compounds for developing treatments for liver injuries, cancer, diabetic complications, and ocular diseases.

Current pharmaceutical standards for POD are inadequate due to the lack of specific detection methods for its primary active ingredients, resulting in low-quality control. This lack of specificity hinders effective price management and impedes the rational development and use of POD resources. POD contains various active compounds, including polysaccharides, homoisoflavones, and steroidal saponins. Its sources range from southern to northern China, leading to significant environmental variations. These differences in germplasm resources can cause fluctuations in active ingredient concentrations, affecting the overall quality of POD. To overcome these challenges, it is essential to establish control methods tailored to the specific active ingredients important for the pharmaceutical, health food, and cosmetic industries. Implementing targeted control methods will provide a solid foundation for rigorous quality evaluation of POD, facilitating its rational development and use, and ensuring its efficacy and safety. Establishing precise quality control measures is crucial for maximizing the potential of POD, enhancing its value across various industries, and ensuring consistent and reliable product quality for consumers and patients.

This study aims to improve the understanding and use of homoisoflavones in POD by isolating and preparing three specific homoisoflavone controls. We will develop a thin-layer chromatography (TLC) method to identify these homoisoflavones in POD. Using this method, we will evaluate and analyze the quality of 20 POD batches from various sources. The results will provide a key reference for the rational development and use of germplasm resources of POD. Additionally, this study aims to enhance quality standards for POD. By accurately identifying and quantifying the homoisoflavones in POD, the research will ensure product consistency, efficacy, and safety, supporting their therapeutic applications and increasing their value in the pharmaceutical, health food, and cosmetic industries.

Experimental

General experimental procedures

Petroleum ether (60-90), ethyl acetate, acetone, methanol, ethanol and formic acid were purchased from commercial sources (Fuyu, Tianjin, China) and used without further purification. The above solutions were analytically pure (AR). Methanol (LC) were purchased from commercial sources (Merck, Berlin, Germany). All the information of herbs and tablets are provided in Table 1. Samples were prepared by ultra-performance liquid chromatography (UPLC, Waters 600, Waters, New York, USA). Preparation columns ZORBAX Eclipse XDB-C₁₈ (Agilent, San Francisco, USA) were employed to purify the samples. The extracts and fractions were concentrated by a rotary evaporator (SB-2000, Ailang, Shanghai, China). Digital display electronic thermostatic water bath HH-2 (Guohua, Jiangsu, China) was used for the heating treatment of samples. The UV-Vis information was recorded in an ultraviolet spectrophotometer (T6, PERSEE, Beijing, China). TLC images were recorded by dynamic thin layer chromatography (SC-5, Golden Sword, Beijing, China). In addition, C₁₈-H solid-phase extraction column (4000 mg/20 mL, Thermo Fisher, Beijing, China) and GF₂₅₄ aluminium thin-layer plate (10 cm x 10 cm, Merck, Berlin, Germany) were purchased commercially.

Table 1
The basic information of the samples used in this study

No.	Forma	Origin	Method (raw/ripe)b	Purchase date
1	tablet	Zhejiang	raw	2017
2	tablet	Hebei	ripe	2015
3	tablet	Hebei	raw	2017
4	tablet	Liaoning	raw	2019
5	tablet	Hunan	raw	2019
6	tablet	Anhui (Huangshan)	raw	2018
7	tablet	Anhui (Huangshan)	raw	2018
8	tablet	Anhui (Huangshan)	raw	2018
9	tablet	Hunan (Leiyang)	ripe	2014
10	tablet	Hunan	raw	2017
11	tablet	Hunan	raw	2015
12	tablet	Henan	ripe	2014
13	tablet	Heilongjiang	ripe	2014
14	tablet	Hebei	ripe	2014
15	tablet	Inner Mongolia	ripe	2018
16	herb	Heilongjiang (Shangzhi)	ripe	2018
17	herb	Heilongjiang (Shangzhi)	raw	2015
18	herb	Hebei (Chengde)	ripe	2018
19	herb	Inner Mongolia	ripe	2018
20	herb	Inner Mongolia	raw	2017

• ^aPresentation of samples: "tablet" refers to samples sliced and processed; "herb" refers to samples not processed. ^b"Ripe" refers to the process of maturing herbs through certain heating or treatment methods; "raw" refers to a process in which the herb is not made by heat or treatment, but is merely trimmed in shape.

Extraction and isolation

POD was extracted with 80% ethanol (40 L) for 6 h. The ethanol extract was concentrated using a rotary evaporator. Next, a large volume of distilled water was added and mixed thoroughly to produce a concentrated solution, which was then allowed to settle. The mixture was centrifuged at 9000 rpm for 15 min; the supernatant was removed, and the precipitate was collected in centrifuge bottles. The precipitate was dissolved in methanol, filtered to remove insoluble particles, and transferred to an evaporating dish. The methanol extract in the dish was evaporated to dryness to obtain the final alcohol extract.

For further purification, the samples were analyzed using preparative UPLC at a flow rate of 1 mL min^-1 and an injection volume of 50 µL. Gradient elution was carried out with solvent A (CH₃OH) and solvent B (H₂O) according to the following profile: 0-5 min (65% A), 5-8 min (65-70% A), 8-10 min (70-73% A), 10-12 min (73-75% A), 12-39 min (75% A), 39-40 min (75-77% A), 40-45 min (77-82% A), 45-70 min (82% A). The temperature was set at 21ºC, with a wavelength of 297 nm and a pressure of 1600 psi. The eluate corresponding to the main peak was collected, concentrated, and dried to yield high-purity Flavonoids III, IV, and V. The structural formulas of these Flavonoids are shown in Figure 1.

Figure 1
Structural formulas of homoisoflavanones III, IV and V.

Preparation of the test sample solutions

The reference standards for homoisoflavones III, IV, and V were prepared separately as well as solutions of sample 1 and sample 16. Additionally, mixed solutions of sample 16 with the reference standard of homoisoflavone III, and sample 16 with the reference standard of homoisoflavone IV were prepared. Each solution had a concentration of 1 mL min^-1 for future use.

Results and Discussion

Appearance of POD

Figure 2 illustrates the appearance and morphology of 20 batches of POD, divided into two main categories: medicinal slices and herbs. The medicinal slices are labeled from 1 to 15 and include both thin and thick slices. Batches 5,9, 10, and 12 are thin slices, with a thickness of 0.08 to 0.2 cm and a light-yellow color, originating from Hunan and Henan provinces. In contrast, batches 1 to 4, 6 to 8, 11, and 13 to 15 are thick slices, with a thickness of 0.2 to 0.4 cm, sourced from Zhejiang, Hebei, Liaoning, Anhui, Hunan, Northeast China, and Inner Mongolia. They exhibit an elongated cylindrical shape, noticeably curved, slightly flattened, and with few branches. Their diameters range from 0.4 to 1.1 cm, and lengths vary from 5 to 18 cm. These crude drugs appear yellow-brown, with longitudinal wrinkles and slightly raised fissures on the surface. They range in texture from hard and brittle to slightly soft, and are easily breakable. Descriptively, these crude drugs present an angular or granular cross-section. Originating from Heilongjiang, Hebei, and Inner Mongolia, they demonstrate the geographical diversity of POD's native habitats. This comprehensive classification and description provide valuable information for understanding the physical characteristics of POD, aiding in better comprehension of its quality and potential applications.

Figure 2
The images of the appearance of PODs.

Methodological validation

Positive and simulated positive sample tests

Figure 3 presents the TLC analysis results for homoisoflavone reference standards (III, IV, and V), positive samples, simulated positive samples, and a negative sample, before and after 1 h of dark incubation under UV light at 254 and 365 nm. Initially, the main spots of the homoisoflavone reference standards and two positive samples appeared at the same positions and colors, with no corresponding spots in the negative sample. This result confirms the specificity of the method, indicating that other POD components did not interfere with the TLC of homoisoflavones III, IV, and V. However, due to the structural similarity between homoisoflavones III and IV, their initial resolution was poor. To address this, the TLC plates were incubated in darkness for 1 h to observe changes in spot characteristics. Post-incubation observations showed no significant changes at 254 nm but revealed significant differences at 365 nm. The fluorescence spot color of reference group IV remained unchanged, while that of reference group III changed from dark blue to light blue, showing a noticeable difference. The color of spot S2 remained unchanged, indicating it contained only homoisoflavone IV. The deep blue fluorescence spots of the positive samples became lighter, yet still predominantly blue, indicating the presence of both III and IV. These findings demonstrate the effectiveness and specificity of the method in distinguishing closely related homoisoflavones, thereby enhancing the analytical capability for assessing the active ingredients of POD. This method provides a proprietary technique for identifying and distinguishing homoisoflavones in POD, offering valuable insights for quality control and research applications.

Figure 3
TLC of positive and simulated positive samples. ("V" represents homoisoflavone V, "IV" represents homoisoflavone IV, "III" represents homoisoflavone III, "P" represents positive sample, "S1" represents simulated positive sample 1, "S2" represents simulated positive sample 2, "N" represents negative sample).

Examination of limits of detection

Table 2 presents the limits of detection (LODs) of homoisoflavones III, IV, and V. As shown in Figure 4, the LODs for homoisoflavones III, IV, and V at 254 and 365 nm are 1.0, 4.0, and 6.0 µg, respectively. These results highlight the excellent sensitivity of the method in detecting homoisoflavones in POD. The differing LODs for each homoisoflavone demonstrate the method's ability to detect these compounds at very low concentrations, with homoisoflavone III showing the highest sensitivity, followed by IV and V. This high sensitivity is essential for accurate quality control, confirming the effectiveness of the method in analyzing these active components in POD. It supports the standardization and quality assessment of POD, ensuring its efficacy and reliability.

Table 2
Dosage of homoisoflavones (III, IV, and V) solution used for LOD analysis

No.	1	2	3	4	5
Dosagea /µg	0.5	1.0	2.0	4.0	6.0

• ^aAmount of sample solution used for limit of detection (LOD) analysis.

Figure 4
Images of the LODs of homoisoflavanones (III, IV, V) in TLC with 254 and 365 nm.

Examination of stability

Figure 5 illustrates the results of a time-course analysis of a positive sample using TLC, conducted in a light-protected environment and assessed at 0, 0.5, 1, 2, 4, and 8 h. Observations at 254 and 365 nm showed consistent results. The main TLC spot remained unchanged at 254 nm throughout the time-course. The spots for homoisoflavones IV and V also showed no change at 365 nm, indicating their stability under the testing conditions. In contrast, the spot for homoisoflavone III changed from dark blue to light blue fluorescence at 365 nm. This fluorescence remained stable from 1 to 6 h, offering a reliable timeframe for differentiating between homoisoflavone III and IV. This stability demonstrates the reliability and robustness of the method for detecting and distinguishing these homoisoflavones in POD. Such consistency is essential for analytical methods, ensuring accurate and confident detection over time. This characteristic underscores the method's suitability for quality control and research, where precise and stable identification of active ingredients is crucial.

Figure 5
Stability of TLC for positive and simulated positive samples. Samples in order: V, IV, III, positive sample, negative sample.

Detection of samples

Figure 6 displays TLC images of homoisoflavone controls (III, IV, and V) alongside extraction solutions from 20 batches of POD, all analyzed under the same chromatographic conditions. By reviewing these images and the batch information detailed in Table 1, the commercial POD batches can be classified into three categories based on the presence or absence of homoisoflavones III, IV, and V:

Figure 6
The TLC images of the samples compared to the homoisoflavones reference standards (III, IV, and V).

Category (i): this category comprises eight POD batches (Nos. 1 to 6, 8, 9), identified as drinking slices from Zhejiang, Hebei, Liaoning, Hunan, and Anhui. These batches were found to contain homoisoflavones III, IV, and V, suggesting higher quality due to the presence of these active components.

Category (ii): this category includes seven POD batches (Nos. 11, 15 to 20) where homoisoflavones III, IV, and V were not detected. These batches consisted of five herb samples and two tablet samples, primarily from wild POD in Inner Mongolia and cultivated POD in Heilongjiang Province. Four of these batches (Nos. 15, 16, 18, 19) were boiled, potentially leading to oxidative degradation of homoisoflavones. The remaining three batches (Nos. 11, 17, 20) were raw wild POD, indicating that differences in growing conditions or harvest timing might have led to reduced or absent homoisoflavone levels.

Category (iii): this category includes five POD batches (Nos. 7, 10, 12 to 14) where homoisoflavones III and IV were detected, but homoisoflavone V was not. These batches came from Anhui, Hunan, Henan, Heilongjiang, and Hebei. The lack of homoisoflavone V in these samples may be due to a higher limit of detection or reduced sensitivity for this compound.

The results in Figure 6 and the classification into three categories reveal significant quality differences among commercially available POD batches. Understanding these variations is essential for assessing the efficacy and safety of POD products, aiding both consumers and manufacturers in making informed choices. Furthermore, these findings underline the importance of standardized practices in harvesting, processing, and storage to maintain consistency and preserve valuable homoisoflavones in POD products.

Establishment of TLC

The UV absorption spectra of homoisoflavones III, IV, and V, measured using a UV-Vis spectrophotometer, showed maximum absorption wavelengths at 297 nm for III and IV, and 294 nm for V (see Figure 7). Absorption was also noted at 254 and 365 nm. Therefore, GF₂₅₄ thin-layer plates were used for TLC analysis, where homoisoflavones III, IV, and V showed fluorescence under 254 nm and appeared dark blue under 365 nm. Selecting 254 and 365 nm for detection, even though they are not the peak absorption wavelengths, was a deliberate choice. The lower absorption coefficients at these wavelengths lead to a higher LOD for the TLC spots. While this makes the method semi-quantitative, it is still highly valuable for quality control of active ingredients in POD. This technique allows for qualitative assessment of homoisoflavones in POD, aiding in the standardization and validation of POD's pharmacological potential. By detecting these compounds through their distinctive fluorescence and color responses under UV light, researchers and quality control professionals can ensure the consistency and effectiveness of POD products, demonstrating the practical application of the method in the herbal and pharmaceutical fields.

Figure 7
UV-Vis spectra of the homoisoflavones reference standards (III, IV, and V).

Homoisoflavones III and IV differ structurally in their 8-position substituents: III has a methyl group, while IV has a methoxy group. This similarity in polarity makes them indistinguishable under standard TLC conditions, as shown in Figure 8 at 0 h, where their spots overlap and cannot be separated. However, these substituents react differently to oxidation, which affects adjacent phenolic hydroxyl groups and provides a method for distinguishing them. The rate of color change in TLC spots can indicate the presence of either III or IV, as the methyl group, an electron-donating substituent, increases the electron density on the neighboring hydroxyl hydrogen, making it more prone to oxidation compared to the methoxy group. Consequently, the TLC spot for compound III changes color within 1 h (see Figure 8-1 h), indicating a faster oxidation rate. In contrast, the methoxy group influences the benzene ring through two mechanisms: an electron-withdrawing inductive effect, which decreases the electron density around the neighboring hydroxyl hydrogen, making it less likely to oxidize, and an electron-donating resonance effect. This resonance involves the lone pair of electrons on the methoxy group's oxygen interacting with the benzene ring and the adjacent hydroxyl group to form a π-π conjugation system, thereby stabilizing the structure. As a result, the TLC spot for compound IV changes color only after 72 h (shown in Figure 8-72 h), reflecting a slower oxidation rate. This variation in oxidation rates, driven by the electronic effects of the substituent groups, provides a reliable method for distinguishing between homoisoflavones III and IV. It also highlights the complex interplay between molecular structure and chemical reactivity. Utilizing these properties for identification improves analytical techniques for studying and quality-controlling substances with similar molecular structures, such as homoisoflavones in POD.

Figure 8
Stability of TLC for homoisoflavanones III, IV and V. Samples in order: V, IV, III, positive sample, negative sample.

Conclusions

The TLC method developed in this study for identifying homoisoflavones in POD is praised for its simplicity, speed, sensitivity, specificity, and stability. When applied to assess the quality of 20 commercially available POD batches, the method provided clear results: homoisoflavones (III, IV, and V) were present in 8 batches, indicating high-quality material. In contrast, 7 batches lacked these homoisoflavones, suggesting potential degradation or initial absence, which might be due to factors like harvest timing, processing methods, or storage conditions. Additionally, 5 batches contained only homoisoflavones (III and IV), reflecting variability in their concentration or preservation. This method enables a thorough evaluation of POD quality, offering a reliable tool for assessing the consistency and effectiveness of its active ingredients. By distinguishing between batches based on the presence of specific homoisoflavones, it serves as a crucial reference for evaluating POD's germplasm resources and guiding quality improvements. Accurate identification and quantification of homoisoflavones enhance quality control processes and support the rational development and use of POD resources. Consequently, this TLC method is valuable for advancing research, ensuring product quality, and supporting the therapeutic applications of POD in the pharmaceutical, health food, and cosmetics industries.

Acknowledgments

We gratefully acknowledge the financial support by Fundamental Scientific Research Business Expenses of Colleges and Universities in Heilongjiang Province (2022-KYYWF-0819), Qiqihar Science and Technology Program Joint Guidance Project (LSFGG-2022038) and Qiqihar Institute of Medical Sciences Project (QMSI2020M-09).

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Edited by

• Editor handled this article:
  Paulo Cezar Vieira

Publication Dates

• Publication in this collection
  11 Oct 2024
• Date of issue
  2025

History

• Received
  12 Mar 2024
• Accepted
  17 Sept 2024