A Comparative Study on the Determination of Triterpene Content in Broken-Wall Ganoderma Lucidum Spor

A Comparative Study on the Determination of Triterpene Content in Broken-Wall Ganoderma Lucidum Spore Powder Using UV Spectrophotometry

Objective: This study aims to investigate the application of UV spectrophotometry based on different standards for determining the triterpene content in broken-cell-wall Ganoderma lucidum spore powder. Methods: Quantitative analysis of triterpenes in broken-cell-wall Ganoderma lucidum spore powder was performed using UV spectrophotometry in accordance with the Pharmacopoeia of the People’s Republic of China, industry standards, health food standards and in-house standards. Results: Among the four methods studied, the triterpene content results, from highest to lowest, were as follows: the in-house standard (17.9%), the industry standard (13.0%), the Pharmacopoeia of the People’s Republic of China (12.9%), and the health food standard (5.7%). Conclusion: This indicates that in practical applications, it is necessary to select an appropriate standard based on specific circumstances, or to establish a more accurate and standardised analytical method, to ensure the accuracy and consistency of triterpene content determination in broken-cell-wall Ganoderma lucidum spore powder.

Introduction

Ganoderma spores are the reproductive organs of Ganoderma lucidum and are rich in active compounds such as polysaccharides, triterpenes and steroids. Due to their hard chitinous outer shell, the active compounds within Ganoderma spores are difficult for the human gut to absorb and digest. Research indicates that the absorption rate of unbroken Ganoderma spore powder is only 12%, whereas this rises to 95% after cell wall disruption. Consequently, converting Ganoderma spores into broken-wall Ganoderma spore powder through cell wall disruption effectively releases the internal bioactive components, thereby enhancing their nutritional value. Triterpenes are key bioactive components of Ganoderma, exhibiting various pharmacological effects including anti-cancer, immune modulation, liver protection and blood glucose-lowering properties; their content serves as a crucial indicator for evaluating the medicinal value of Ganoderma. Currently, there is no standardised method for the determination of triterpenes. Methods are primarily based on the Pharmacopoeia of the People’s Republic of China (hereinafter referred to as the ‘Chinese Pharmacopoeia’), the group standard T/FJHX0001—2022, and relevant standards for health foods, all of which employ the vanillin -acetic acid-perchloric acid colourimetric method (using oleanolic acid or ursolic acid as reference standards), yet significant discrepancies exist in the results. This study compares different standards and internal corporate standards to analyse the factors influencing the determination of triterpenoid content in broken-cell wall Ganoderma lucidum spore powder, focusing on aspects such as standard solution preparation, extraction conditions and reaction parameters. The aim is to provide a reference basis for establishing more scientific quality standards and for authenticity verification.

1. Materials and Methods

1.1 Reagents

Raw material: Cell-broken Ganoderma lucidum spore powder

Methanol, ethanol, glacial acetic acid, ethyl acetate, chloroform, perchloric acid; vanillin; oleanolic acid standard; ursolic acid standard

1.2 Instruments and Equipment

UV-1700 UV-Vis spectrophotometer; analytical balance; programmable nitrogen evaporator; centrifuge; shaker; ultrasonic water bath; water bath.

1.3 Test Methods

1.3.1 Standards from the Chinese Pharmacopoeia

Weigh an appropriate amount of oleuropein reference standard and dissolve in methanol to prepare a 0.2 mg/mL solution. Dispense the solution in a gradient into capped test tubes, evaporate to dryness and allow to cool, then add 0.2 mL of freshly prepared vanillin-glacial acetic acid solution and 0.8 mL of perchloric acid, shake well, heat in a 70 °C water bath for 15 minutes, cool in an ice bath for 5 minutes, then add 4 mL of ethyl acetate and shake well; using the reagent blank as a reference, measure the absorbance at a wavelength of 546 nm and plot a standard curve. Weigh 0.15 g of cell-broken Ganoderma lucidum spore powder, add 50 mL of ethanol and sonicate for 45 minutes, filter, transfer the filtrate to a volumetric flask, make up to 100 mL with ethanol, measure the absorbance according to the method in the Chinese Pharmacopoeia, read the oleanolic acid content from the standard curve and calculate the triterpene content.

1.3.2 Group Standard

Weigh out the oleanolic acid reference standard, dissolve it in ethyl acetate and sonicate for 15 minutes to prepare a 0.1 mg/mL solution. Perform gradient sampling into test tubes; evaporate to dryness in a 100 °C water bath; add 0.2 mL of vanillin-glacial acetic acid solution and 0.8 mL of perchloric acid; heat in a 70 °C water bath for 15 minutes; cool in an ice bath for 5 minutes; then add 4 mL of ethyl acetate and shake well; measure the absorbance at a wavelength of 546 nm and plot a standard curve. Weigh 0.15 g of cell-broken Ganoderma lucidum spore powder, place it in a 100 mL volumetric flask, add 80 mL of anhydrous ethanol and sonicate for 30 minutes, then make up to volume and filter to obtain the test solution. Measure the absorbance according to the group standard method and calculate the triterpene content using the standard curve.

1.3.3 Health Food Standards

Weigh a quantity of bearberry acid reference standard, dissolve it in ethyl acetate, and prepare a standard solution in accordance with health food standards; simultaneously prepare a test solution of broken-cell wall Ganoderma lucidum spore powder. Dispense the standard solution in graduated volumes into test tubes; after evaporating to dryness in a 60 °C water bath, add 0.4 mL of a 5% vanillin-ice

acetic acid solution to dissolve the residue, followed by 1.0 mL of perchloric acid and mixed thoroughly. Heat in a 60 °C water bath for 15 minutes, cool in an ice bath, then add 5.0 mL of glacial acetic acid to develop the colour. Measure the absorbance at a wavelength of 548 nm to plot a standard curve. Measure 1.0 mL of the supernatant from the sample and treat it in the same manner; calculate the triterpene concentration based on the standard curve.

1.3.4 Internal Laboratory Standards

Weigh a quantity of oleuropein reference standard; after titration, dissolve in methanol and dispense in graduated volumes into capped test tubes. Evaporate to dryness and allow to cool, then add 0.2 mL of freshly prepared vanillin-glacial acetic acid solution and 0.8 mL of perchloric acid; heat in a 70 °C water bath for 30 minutes, cool in an ice bath for 5 minutes, add 4 mL of ethyl acetate and shake well; using the reagent blank as a reference, measure the absorbance at a wavelength of 546 nm and plot a standard curve. Weigh 0.15 g of cell-broken Ganoderma lucidum spore powder, add 50 mL of ethanol, extract by sonication and filter; make up the filtrate to 100 mL, measure the absorbance according to the company standard method, and calculate the triterpene content using the standard curve.

1.3.5 Methodological Validation

Compare the differences between the four triterpene determination methods and investigate the influence of key factors—such as standard solutions (selection of reference standards), extraction conditions (solvent type, extraction time) and reaction parameters (colour development temperature, time)—on the results.

2. Results and Analysis

2.1 Comparison of Triterpene Results from Different Analytical Methods

The triterpene content was determined using four methods: the Chinese Pharmacopoeia, industry standards, health food standards and in-house standards. The results were 12.9%, 13.0%, 5.7% and 17.9% respectively. Among these, the in-house standard yielded the highest value, whilst the health food standard yielded the lowest. The colour reaction conditions for each method are as follows: both the Chinese Pharmacopoeia and the industry standard involved a 15-minute water bath at 70°C; the health food standard involved a 15-minute water bath at 60°C; and the in-house standard involved a 30-minute water bath at 70°C. The results indicate that, for the same reaction time, the higher the reaction temperature, the higher the triterpene content; and for the same reaction temperature, the longer the reaction time, the higher the triterpene content.

2.2 Analysis of Differences Among Triterpene Determination Methods

All four methods are based on UV spectrophotometry, utilising the principle of chemical colourimetric reactions to quantitatively analyse the triterpene content of Ganoderma lucidum via absorbance at specific wavelengths. However, there are differences in the operational details of each standard (Table 1), primarily reflected in the use of different reference standards, different solvent evaporation methods, and variations in the temperature and duration of the colourimetric reaction.

2.3 Effect of Different Filtration Methods on Results

2.3.1 Effect of Filter Paper and Centrifugation on Results

Following the standard method for health foods described in 1.3.3, the sample extracts were subjected to filtration through a filter membrane (pore size 0.22 μm) and centrifugation at 6000 r/min for 10 minutes, respectively. The extract filtered through the membrane was clear and transparent, consistent with the standard solution, with a triterpene content of 3.57%; the supernatant after centrifugation was turbid, interfering with the colourimetric reaction, and the triterpene content was 5.70%.

2.3.2 Effect of Different Filter Papers on Results

Using the in-house standard method described in 1.3.4, the extract was filtered using GF/A filter paper, double-ring quantitative filter paper and double-ring qualitative filter paper. The results showed that the filtrate from GF/A filter paper was clear and transparent, whilst the filtrates from the other two types of filter paper were light brown (with precipitation appearing after standing overnight).

The The triterpene content of the three filtrates was 17.9%, 17.8% and 18.9% respectively, with minimal variation; however, after colour development, the colour tones of the filtrates did not match those of the standard. It is surmised that non-terpenoid substances may be present in the extract following filtration, leading to inflated measurement values.

2.4 Analysis of Results for Different Reference Materials Using the Same Determination Method

Using the standard method for health foods described in 1.3.3, standard curves were plotted using bearberry acid and oleanolic acid as reference standards for the determination. The results (Table 2) show that the mean triterpene content across the 10 data sets was 3.8%, with a relative standard deviation of 4.90%. A one-way analysis of variance (ANOVA) yielded a P-value of <0.5, indicating that the two reference standards had no significant effect on the triterpene determination results.

2.5 Analysis of Combined Tests of Health Food Standards and In-House Standards

Combined tests were conducted using health food standards and in-house standards that differed in terms of reference material, solvent volatilisation method and colour development conditions (Table 3). The results showed that when the standard solution and sample extraction steps were consistent, differences in colour development conditions (30 min in a 70°C water bath vs. 15 min in a 60°C water bath) led to significant differences in triterpene results (Scheme 1: 15.3% vs. Scheme 2: 5.1%, a difference of 10.2%; Scheme 3: 16.0% vs. Scheme 4: 4.0%, difference of 12%). When the colour development conditions were consistent with those of the reference standard, the sample extraction method had a minor impact on triterpene results (difference of 0.7% between Scheme 1 and Scheme 3; difference of 1.1% between Scheme 2 and Scheme 4). This indicates that the temperature and duration of the colour development reaction are positively correlated with triterpene results.

3. Discussion and Conclusions

This study employed four UV-spectrophotometric methods to determine the triterpene content in broken-cell wall Ganoderma lucidum spore powder. The results indicated significant variations between the different methods, with the triterpene content ranging from highest to lowest as follows: in-house standard (17.9%), industry standard (13.0%), Chinese Pharmacopoeia (12.9%) and health food standard (5.7%). In practical applications, it is recommended that an appropriate analytical method be selected based on the research objectives and practical conditions. Where necessary, results should be validated using other technical methods, such as high-performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-MS), to eliminate discrepancies in the results.