Ganoderma spore powder contains little triterpenoids (2024)

Background

Ganoderma spore is a minuscule germ cell ejected from Ganoderma gills during its growth maturity period, it has been considered with high exploitable potential in health-care products manufacture.

Methods

After testing sporoderm-broken rate, the triterpenoids in 12 batches of broken and unbroken Ganoderma spore powder (GSP) samples were compared with Ganoderma lucidum fruiting body (GL) by high performance thin-layer chromatography (HPTLC) and further verified by liquid chromatography coupled with electrospray ionization quadrupole-time-of-flight mass spectrometry (LC–QTOF–MS); meanwhile, the dissolution of triterpenoids after bionic extraction was also investigated by HPTLC.

Results

The sporoderm-broken rate of all the broken GSP samples was over 85%. The relative peak area of triterpenoids in GSP samples were lower than 50% of that in fruiting body, and the dissolution of triterpenoids in artificial gastrointestinal fluid was lower than in methanol.

Conclusions

This study demonstrated that there were little triterpenoids in GSP. Triterpenoids in GSP also seldom be dissolved in artificial gastrointestinal fluid.

Keywords: Ganoderma spore powder, Triterpenoids, Bionic extraction, Artificial gastroenteric fluid, HPTLC, LC–QTOF–MS

Ganoderma samples and chemicals

All Ganoderma samples were listed in Table 1, including one batch of Ganoderma lucidum fruiting body (GL) was collected from Zhejiang Province of China, 12 batches of Ganoderma spore powder (GSP-01 to GSP-12) were collected from different regions of China. The voucher specimens were stored at the Institute of Chinese Medical Sciences, University of Macau, Macao, China. Methanol, dichloromethane, ethyl acetate, n-hexane and formic acid were highest available purity (Merck, Darmstadt, Germany), deionized water was produced by a Millipore Milli-Q Plus system (Millipore, Bedford, USA).

Sporoderm-broken rate of GSP

Powder of each sample (2.0mg) was soaked in 1.0mL of 30% ethanol. Before observing, each mixture was fully oscillated, taken 10μL to observed under 40-fold objective lens (Olympus IX73 Inverted Microscope System, Tokyo, Japan). Each mixture was observed three times and averaged, samples from a same origin were compared, sporoderm-broken rate of broken GSP samples was calculated by following equation:

$$Sporoderm-brokenrate\left(\%\right)=100-N_2/N_1\ast 100$$

(N₁ = the average of spore from unbroken GSP sample; N₂ = the average of unbroken spore from broken GSP sample).

Extraction of Ganoderma samples

Each sample (1.0g) was soaked in 10.0mL of methanol for 1h and extracted by the ultrasonic extractor (Bransonic Branson 8510, Danbury, USA) for 45min (room temperature). Then the extract was centrifuged at 4500× rcf for 10min (ThermoFisher Heraeus Multifuge X3R Centrifuge, Osterodeam Harz, Germany). Supernatant was evaporated to desiccation under vacuum by rotary evaporator (Büchi Rotavapor R-205, Flawil, Switzerland), and residue was re-dissolved in 1.0mL of methanol. After centrifugation at 16,000× rcf for 5min (Eppendorf AG Centrifuge 5415D, Hamburg, Germany) and filtration through a 0.45μm membrane filter, the supernatant was used for HPTLC analysis subsequently.

Bionic extraction

Artificial gastrointestinal fluid was prepared by the 2015 Chinese Pharmacopoeia Part IV general principles as followed: Artificial gastric fluid: 16.4mL of dilute hydrochloric acid was added about 800mL of water and 10g of pepsin, and the mixture was diluted with water to 1000mL. Artificial intestinal fluid: 6.8g of potassium dihydrogen phosphate was dissolved with 500mL of water, and the pH was adjusted to 6.8 with 0.1mol/L sodium hydroxide solution; meanwhile, 10g of trypsin was dissolved with an appropriate amount of water, then the mixture was diluted with water to 1000mL.

Each sample (1.0g) was soaked in 10.0mL of artificial gastric fluid and incubated by the thermostatic oscillation incubator (Eppendorf AG Thermomixer comfort, Hamburg, Germany) for 3h (37℃, 150× rpm). Then the extract was centrifuged at 4500× rcf for 10min. The supernatant was evaporated to desiccation under vacuum using rotary evaporator, and the residue was re-dissolved in 1.0mL of methanol. After centrifugation at 16,000× rcf for 5min and filtration through a 0.45μm membrane filter, the supernatant was used for HPTLC analysis subsequently.

Then the residues were immersed in 10.0mL of artificial intestinal fluid and incubated by the thermostatic oscillation incubator for 6h (37℃, 150× rpm). Then the extract was centrifuged at 4500× rcf for 10min. The supernatant was evaporated to desiccation under vacuum using rotary evaporator, and the residue was re-dissolved in 1.0mL of methanol. After centrifugation at 16,000× rcf for 5min and filtration through a 0.45μm membrane filter, the supernatant was used for HPTLC analysis subsequently. Finally, the residues were extracted by methanol according to the section of “Extraction of Ganoderma samples”.

HPTLC analysis of triterpenoids

All samples (7μL) were formed into band shape on a 20 × 10cm silica-gel 60 plate (Merck, Darmstadt, Germany) with an automatic HPTLC sampler (CAMAG ATS4, Muttenz, Switzerland). Bands were 8mm wide, 6mm apart and at 10mm from the bottom edge. The plate was developed to 90mm with dichloromethane/ethyl acetate/methanol/formic acid, 30:20:1:1 (v/v/v/v) as developing solvent at room temperature and 88% relative humidity, after pre-saturation in developing solvent vapor for 15min. Then the developed plates were colorized by spraying 10% (v/v) sulfuric acid in ethanol and heated at 105℃ for 5min on a plate heater (KEZHE-SP-III, Shanghai, China). The plate was photographed under daylight and UV 365nm respectively, and the figure results were integrated by grayscale scanning (GelAnalyzer2010a, Debrecen, Hungary).

LC–QTOF–MS analysis of triterpenoids

LC–QTOF–MS analysis was performed according to our previous study [21]. Briefly, an Ultimate 3000 UHPLC system equipped with Ultimate 3000 degasser, pump, RS autosampler, and RS column compartment, coupled with diode-array detector (Thermo Fisher, Osterodeam Harz, Germany). Waters Acquity BEH C₁₈ column (2.1 × 150mm i.d., 1.7μm, Waters, Milford, MA, USA) was used for sample separation. The mobile phase was consisted of 0.2% acetic acid in water (A) and acetonitrile (B) with gradient elution: 0–8min, 3% B-22% B; 8–25min, 22% B-25% B; 25–40min, 25% B-35% B; 40–65min, 35% B-100% B, 65–70min, 100% B. The flow rate was 0.3mL/min, injection volume was 1μL, column temperature was 25℃ and detection wavelength was set at 257nm.

A high-resolution impact HD QTOF mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany) equipped with an electrospray ionization (ESI) source was operated in the negative ion mode. The mass range was set at m/z 100–1500 in the full scan mode. The capillary voltage was set at 3500V. The fragmentation mode was CID. The source temperature was set at 250℃. Nitrogen was used as the drying gas. The gas flow rate was set at 8L/min. MS² data analysis of the three highest intensive ion fragments was intelligently performed in real time.

Sporoderm-broken rate of GSP

As shown by objective lens, broken spore had brown translucent fragmented contents, and unbroken spore had double intact plump walls. The sporoderm-broken rate of all the broken GSP samples was over 85% (Table 1), thus, it could be considered that all the broken GSP samples were broken completely. Furthermore, filiform mycelia could not be observed in the samples, which meant that there was no fruiting body powder mixed into GSP samples.

HPTLC analysis of triterpenoids in GSP

HPTLC fingerprints of triterpenoids in 12 batches of GSP at UV 365nm was shown in Fig.1a. GL was taken as reference and demonstrated that it supplied yellow or green bands at triterpenoids area (red dotted line part, Rf = 0.27–0.60). However, most of GSP samples just had some trace of bands at this part and the colors were different from those of fruiting body (except GSP-01 and GSP-02), which revealed that these components corresponding to the bands was not as same as the triterpenoids in fruiting body, although their Rf value were similar. As HPTLC fingerprints at UV 365nm is more sensitive and clearer than under UV 254nm and daylight [22], it was chosen for image grayscale analysis. Triterpenoids in GSP samples were compared with GL, specifically, by setting the triterpenoids peak area of GL as standard and calculating the relative peak area between GL and each GSP sample. Grayscale scanning integral results of GL and GSP-05 were shown in Fig.1b. As demonstrated in Table 2, whether the spore walls were broken or not, the triterpenoids peak areas of all GSP samples were lower than 50% of that in fruiting body. Spore wall-breaking process was helpful to dissolve internal ingredients, however, p value of triterpenoids peak area was 0.096 (p>0.05), which suggested that there was no significant difference between broken and unbroken GSP samples in triterpenoids. This result might attribute to the GSP samples contained little triterpenes.

LC–QTOF–MS further verification of triterpenoids

As the sensitivity of HPTLC was not high enough, LC–QTOF–MS was used to further verify the results. In previous studies [21, 23], oxygenated tetracyclic triterpenoid was the main type of triterpenoids in Ganoderma, and major specific fragment ions for identification of oxygenated tetracyclic triterpenoid were m/z 301.1809 (d1 and d1′ type, Fig.2a) and m/z 303.1966 (d2 type, Fig.2a). P1, P2 and P3 were three main peaks of these type triterpenoid, which detected in GL and representative broken GSP samples, so P1–P3 were representative for profiling the triterpenoids in GSP samples. As listed in Table 3, the main ions in P1 was m/z 515.3004 (d1 type), P2 was m/z 513.2858 (d1′ type) and the main ions in P3 was m/z 515.2990 (d2 type). Furthermore, the proposed fragmentation pathway was referred to our previous study [21], the possible structures of P1, P2 and P3 was shown in Fig.2b, respectively. The extraction ion chromatograms (MS²) of GL and GSP-05 were shown in Fig.2c, respectively. Relative total peak areas of d1, d1′ and d2 type were shown in Table 4, accounted for 0.43% to 0.27% of fruiting body. And relative peak area of P1–P3 were shown in Table 4, accounted for 15.62% to 1.57% of fruiting body. Thus, there were little triterpenoids in GSP samples compared with fruiting body, which was matched up with the above results of HPTLC.

HPTLC analysis of triterpenoids by bionic extraction

As demonstrated in Fig.3a, b, respectively, there were no obvious band of triterpenoids in artificial gastric fluid, and only some faint bands at triterpenoids area in artificial intestinal fluid. The residues after bionic extraction were extracted in methanol again, as Fig.3c shown, its dissolution of triterpenoids was higher than that in artificial gastrointestinal fluid. However, there were just a few bands at triterpenoids area, which were almost as same as Fig.1a. Grayscale scanning integral was still used to profile the triterpenoids, and the relative peak area results demonstrated in Table 2. As Table 2 indicated, triterpenoids in GSP samples could not be dissolved in artificial gastrointestinal fluid, and most of triterpenoids were still in the residues.

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Data Availability Statement

The data in this study is available from the corresponding author upon reasonable request.