Analysis and Evaluation of Volatile Chemical Components of Wild Ophiocordyceps sinensis and Its Mycelia in Xizang, China

Abstract

Background:

As a rare traditional Chinese medicinal material, Ophiocordyceps sinensis is subject to counterfeiting in the market due to its limited distribution and insufficient supply and demand. Therefore, conducting research on its volatile components is of great significance for identifying authenticity.

Materials and Methods:

Headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS) is utilized to analyze the compositional disparities between wild O. sinensis samples from different regions and developmental stages, including its mycelia (OS).

Results:

Our findings revealed that both wild O. sinensis and OS contain 11 distinct classes of volatile compounds, including aldehydes, ketones, nitrogen-containing heterocycles, alcohols, carboxylic acids, esters, aromatics, alkanes, oxygen heterocycles, amides, and olefins. However, the number of volatile components varied among sample types, with aldehydes, alcohols, ketones, nitrogen-containing heterocycles, and terpenes being the predominant compounds in OS.

Conclusion:

The diversity and concentration of volatile compounds in wild O. sinensis and its mycelia are influenced by growth conditions. These volatile substances, which contribute to the characteristic “fishy taste” of O. sinensis, can serve as a basis for distinguishing O. sinensis from different geographical origins.

INTRODUCTION

Ophiocordyceps sinensis is a dried composite of the stroma and carcass of insect larvae from the family Hepialidae, parasitized by the fungus O. sinensis (Berk.) G. H. Sung et al. from the family Clavicipitaceae. It is one of the traditional and valuable Chinese herbal medicines, with high medicinal value, and has been used for over 300 years in China and surrounding Asian countries. Wild O. sinensis is primarily found in high-altitude areas with elevations ranging from 3000 to 5200 m, such as alpine meadows or alpine shrub meadows in regions of China, including the provinces of Sichuan, Qinghai, Xizang, Yunnan, and Guizhou. It is also found in surrounding countries and regions, including Bhutan, India, Myanmar, and Nepal.[1,2] The limited distribution and the imbalance between supply and demand have led to overharvesting by local farmers, resulting in a gradual depletion of wild O. sinensis resources. Additionally, in recent years, global warming has caused the snow line to shift upward,[3] significantly reducing the distribution range of O. sinensis and causing it to move upward in the central plateau.[4] These factors have exacerbated the decline in O. sinensis production.[5] As a result, the market has seen the emergence of counterfeit products and substitutes for wild O. sinensis, which has confused consumers and regulatory authorities. As a result, there have been instances of inferior products and substitutes, which have confused consumers and market regulatory authorities.

Volatile compounds of Chinese herbal medicine mainly include volatile oil, terpenoids, aromatic compounds, aliphatic compounds, etc. It is not only an important component of the taste of herb medicine, but also one of the significant marks for identifying Chinese herbal medicines. While chemical fingerprinting aids in quality control, specific compounds of wild O. sinensis remain undetermined. Known components include nucleosides, amino acids, and other common biological substances, but their link to pharmacological effects is unclear.[6,7] Marketed O. sinensis products lack specific content standards,[8] with quality assessed by physical traits and “fishy odor” intensity.[9] The origin and chemical composition of this odor are unknown.

In this study, the headspace-solid phase microextraction (HS-SPME) combined with gas chromatography–mass spectrometry (GC–MS) was employed to analyze the volatile chemical components of wild O. sinensis from four representative regions in Xizang and its fermented mycelia. By comparing the volatile chemical profiles, we have delineated the subtle yet significant differences between the two species, thereby establishing a robust foundation for the development of a chemical fingerprinting methodology. This methodology is crucial for authenticating O. sinensis and its potential substitutes, focusing on the volatile components that are indicative of their unique identities.

EXPERIMENTAL

Materials

Wild O. sinensis samples were collected from four regions in Xizang: Baqing County in Nagqu (NQ) City (elevation 4150 m), Luozhi County in Qamdo (QD) City (elevation 3240 m), Gongbujiangda County in Nyingchi (NC) City (elevation 3420 m), and Gyaca County in Shannan (SN) City (elevation 3260 m). The preparation of the mycelia of O. sinensis was carried out according to the method previously established by our laboratory.[10]

Methods

• Sample pretreatment. The HS-SPME technique was used to adsorb volatile components [Figure 1]

• Gas chromatography–mass spectrometry analysis conditions: Volatile components of samples in four regions of Xizang (NQ, QD, NC, and SN) and its mycelia (OS) were analyzed using the GC–MS method. Compounds obtained through SPME were analyzed and found utilizing a gas chromatography–mass selective detector (GC–MSD, model 7890A/GC-5975C, manufactured by Agilent). Chromatographic conditions were as follows: a DB-5MS capillary column with dimensions (30 m × 0.25 mm (i.d.) ×0.25 μm film thickness, non-split injection mode, helium as the carrier gas with a flow rate 1 mL/min, and an injection port temperature of 250°C. Programmed temperature conditions were set as follows: an initial column temperature of 40°C, held isothermally for 5 min, followed by an increased to 200°C at a rate of 2°C/min, and then held isothermally for an additional 10 min. Mass spectrometry conditions were as follows: an electron ionization (EI) source with an ion source temperature of 230°C, an interface temperature of 250°C, a quadrupole temperature of 150°C, an ion source voltage of 70 eV, and a mass spectrum scan range 30–500 atomic mass units (amu)

• Data analysis: The total ion chromatogram (TIC) data obtained from GC–MS experiments were qualitatively analyzed and matched by comparing with the National Institute of Standards and Technology (NIST 2017) database. A similarity index exceeding 85% was employed as the criterion for identification, and the chemical substances corresponding to each chromatographic peak were determined based on this matching criterion, along with relevant literature analysis. The relative content of each volatile compound was calculated using the internal standard method, in conjunction with peak area normalization, with 2-octanol serving as the internal standard.

Figure 1.

Main process of sample pretreatment

RESULTS

Volatile components in Ophiocordyceps sinensis and its mycelia

The TIC data are shown in Figure 2. Based on the analytical methods from previous literature,[11] the collected data were subjected to a spectrum library search. According to the search results, combined with reference materials, manual spectrum analysis was performed. The volatile components in NQ, QD, NC, SN, and OS were identified. Quantitative analysis was then conducted to calculate the relative content of each component [Supplementary Table 1], and the basic categories of volatile components in the samples are preliminarily summarized in Table 1. Table 1 presents the identification of 42, 34, 37, 41, and 73 volatile chemical components in NQ, QD, NC, SN, and OS, respectively. These components can be broadly classified into 11 categories: aldehydes, ketones, nitrogenous heterocycles, alcohols, carboxylic acids, esters, aromatics, alkanes, oxygen heterocycles, amides, and olefins.

Figure 2.

Total ion current chromatogram of volatile components in Ophiocordyceps sinensis from various samples. Note: (a-e) represent samples from NQ, QD, NC, SN, and OS, respectively. NQ = Nagqu, QD = Qamdo, NC = Nyingchi, SN = Shannan

Supplementary Table 1.

Summary of volatile components of wild O. sinensis and its mycelia

Class	tR/min	Compound	Fomula	Relative content)/ng
				BQ	QD	NC	SN	OS
Internal standard aldehyde	13.316	2-Octanol	C8H18O	200.00	200.00	200.00	200.00	200.00
	1.536	Acetaldehyde	C2H4O	66.64	61.24	499.40	21.44	28.55
	1.67	Propanal	C3H6O	111.76	50.46	-	-	49.48
	2.527	Butanal, 3-methyl-	C5H10O	113.12	55.16	263.93	-	39.84
	2.602	Butanal, 2-methyl-	C5H10O	211.05	-	-	-	56.76
	5.525	Hexanal	C6H12O	344.49	14.47	-	-	209.93
	6.156	2-Butenal, 2-ethyl-	C6H10O	-	-	-	-	13.51
	6.627	2-Pentenal, 2-methyl-	C6H10O	-	-	-	-	75.02
	7.475	2-Hexenal, (E)-	C6H10O	-	-	-	-	10.78
	11.42	Benzaldehyde	C7H6O	62.45	28.08	123.77	59.51	59.62
	13.241	2,4-Heptadienal, (E, E)-	C7H10O	-	-	-	-	64.56
	14.405	Benzeneacetaldehyde	C8H8O	29.24	62.30	-	-	31.75
	14.849	2-Octenal, (E)-	C8H14O	-	-	-	-	22.23
	16.364	Nonanal	C9H18O	-	-	-	-	60.72
	17.08	4-Heptenal, (E)-	C7H12O	-	-	-	-	43.02
	19.464	Decanal	C10H20O	-	-	-	-	4.69
	24.102	2-Octenal, 2-butyl-	C12H22O	-	-	-	-	10.81
Ketone	1.896	2-Butanone	C4H8O	109.37	32.49	-	93.89	90.49
	2.016	2-Pentanone	C5H10O	-	96.04	-	-	76.96
	3.873	Cyclopentanone	C5H8O	-	-	-	-	27.04
	4.615	1-Hydroxy-2-butanone	C4H8O2	-	-	-	-	34.87
	14.041	6-Octen-2-one	C8H14O	-	-	-	-	43.00
	16.022	3,5-Octadien-2-one	C8H12O	-	-	-	-	106.53
	21.969	2-Undecanone	C11H22O	-	5.43	45.36	2.86	5.52
	24.7	Heptanone, 3-ethyl-	C9H18O	63.61	-	209.84	42.44	-
	27.8	2-Octen-4-one, 2-methoxy-	C9H16O2	-	13.41	-	-	-
Nitrogen containing heterocycle	2.209	Piperazine	C4H10N2	-	-	-	22.18	-
	4.193	Pyridine	C5H5N	-	-	-	-	13.62
	13.689	Pyrazine, 2-ethenyl-6-methyl-	C7H10N2	-	-	-	8.82	-
	14.241	5-Amino-1-ethylpyrazole	C5H9N3	-	-	-	-	16.97
	14.965	2-Piperidinone	C5H9NO	-	-	-	-	19.41
	15.284	1-(1H-Pyrrol-2-y l) ethanone	C6H7NO	20.82	10.03	-	6.50	-
	15.298	4 (1H)-Pyridone/4 (1H)-	C5H5NO	-	-	-	-	111.69
	15.78	4-Amino-6-hydroxypyrimidine	C4H5N3O	-	-	-	-	25.14
	15.817	Pyrazine, tetramethyl-	C8H12N2	-	16.79	-	-	-
	18.127	Pyrazine, 3,5-diethyl-2-methyl-	C8H12N2	-	6.64	-	-	-
Alcohol	2.829	1-Penten-3-ol	C5H10O	-	-	-	-	39.89
	3.015	3-Buten-2-ol	C4H8O	-	-	-	-	24.71
	3.491	2-Heptanol	C7H16O	344.48	743.91	-	176.76	14.86
	5.601	2,3-Butanediol, [R-(R*, R*)]	C4H10O2	1929.86	890.57	741.23	376.72	47.32
	9.79	Ethanol, 2-butoxy-	C6H14O2	-	123.98	-	-	52.38
	10.878	2-Hexen-1-ol, 2-ethyl-	C8H16O	-	-	-	-	5.24
	11.002	1-Octen-4-ol	C8H16O	-	-	-	-	24.32
	12.21	1-Octen-3-ol	C8H16O	-	-	-	-	35.77
	12.366	2-Octen-1-ol, (E)-	C8H16O	-	-	-	-	15.04
	12.544	2-Decen-1-ol, (E)-	C10H20O	-	-	-	-	33.69
	13.97	2-Ethyl-1-hexanol	C8H18O	11.83	8.60	-	-	34.52
	24.924	3-Octen-2-ol	C8H16O	-	-	19.95	11.75	-
	25.137	trans-2-Methyl-4-hexen-3-ol	C7H14O	25.71	-	-	6.98	-
	25.954	Phenylethyl Alcohol	C8H10O	12.01	-	-	-	53.35
Carboxylic acid	3.344	Lactic acid	C3H6O3	24.78	36.73	-	-	-
	3.491	Propanoic acid	C3H6O2	-	-	-	-	14.86
	4.961	Propanoic acid, 2-methyl-	C4H8O2	-	-	0.78	-	0.16
	8.315	Pentanoic acid	C5H10O2	10406.54	1301.48	43540.01	6223.92	53.35
	10.287	Butanoic acid, 3-methyl-	C5H10O2	103.52	0.94	7686.29	89.60	67.87
	10.385	Propanoic acid, 2-methyl-	C5H10O2	75.61	-	235.54	19.45	103.52
	12.637	Pentanoic acid, 4-methyl-	C6H11O2	31.54	10.16	253.21	21.63	-
	13.623	Heptanoic acid	C6H12O2	-	-	598.52	24.66	82.56
	18.896	Octanoic acid	C8H16O2	-	-	-	-	5.26
	38.432	Dodecanoic acid	C12H24O2	-	-	8.66	-	-
	40.968	Palmitoleic acid	C16H30O2	-	-	25.28	3.94	-
Ester	3.318	(S)-Isopropyl lactate	C8H16O3	-	-	-	-	47.32
	5.609	Butanoic acid, ethyl ester	C6H12O2	-	-	380.78	28.58	-
	6.071	Acetic acid, butyl ester	C6H12O2	-	-	219.62	85.88	-
	10.913	Butanoic acid, 3-hydroxy-, ethyl ester	C6H12O3	124.85	84.18	-	-	-
	12.81	Butanoic acid, butyl ester	C12H24O2	761.88	415.56	11381.65	1729.83	264.21
	14.734	2 (3H)-Furanone, 5-ethyldihydro-	C6H10O2	-	-	-	-	18.73
	17.452	Pentanedioic acid, dimethyl ester	C7H12O4	20.55	-	35.65	7.48	71.42
	19.047	Hexanoic acid, butyl ester	C10H20O2	28.96	6.69	661.49	70.49	4.46
	20.619	Hexanedioic acid, dimethyl ester	C8H14O4	-	-	-	6.09	16.54
	24.47	Butyl caprylate	C12H24O2	-	-	73.33	12.98	-
	30.48	Decanoic acid, decyl ester	C20H40O2	83.38	-	18.63	1.51	2.20
	35.429	Phthalic acid, isobutyl 2-methylpent-3-yl ester	C16H22O4	6.80	-	11.88	1.86	6.05
	36.535	Hexadecanoic acid, methyl ester	C17H34O2	-	-	18.88	4.09	-
	37.854	Hexadecanoic acid, ethyl ester	C18H36O2	12.18	-	20.59	2.20	-
	39.827	9-Octadecenoic acid, methyl ester,	C19H36O2	-	-	8.48	1.82	-
	41.323	Hexadecanoic acid, butyl ester	C20H40O2	-	-	114.58	31.00	-
	43.331	Ethyl Oleate	C20H38O2	-	-	61.98	16.14	-
Aromatics	7.631	p-Xylene	C8H10	1160.54	-	-	-	14.01
	7.933	o-Xylene	C8H10	1021.03	-	-	-	30.37
	27.602	Butylated Hydroxytoluene	C15H24O	-	-	-	-	1.90
Alkanes	13.699	2,2,4,4-Tetramethyloctane	C12H26	-	9.80	-	-
	16.177	Undecane	C11H24	12.87	3.22	-	-	11.15
	19.247	Dodecane	C12H26	44.47	18.96	88.28	15.50	4.69
	22.081	Tridecane	C13H28	45.62	17.23	82.75	14.27	17.91
	22.787	Undecane, 3,3-dimethyl-	C13H28	-	-	18.30	-	-
	24.128	Dodecane, 2,7,10-trimethyl-	C15H32	9.68	-	-	-	-
	24.733	Tetradecane	C14H30	42.64	6.94	-	-	10.24
	27.131	Dodecane, 4,6-dimethyl-	C13H28	14.28	-	-	-	2.80
	27.238	Pentadecane	C15H32	59.75	4.18	-	7.21	8.30
	29.588	Hexadecane	C16H34	54.35	3.62	40.49	6.04	7.72
	31.831	Heptadecane	C17H36	7.18	-	-	-	2.10
Oxygenated heterocycle	12.002	2H-Pyran-3 (4H)-one, 6-ethenyldihydro-2,2,6-trimethyl-	C10H16O2	-	-	-	-	31.12
	14.121	2 (3H)-Furanone, dihydro-3-methylene-	C5H8O2	39.61	27.58	84.21	7.80	-
	16.43	Furan, tetrahydro-2-(methoxymethyl)-	C6H12O2	693.63	71.19	895.86	201.53	23.16
	18.016	2H-Pyran-2-one, tetrahydro-6-pentyl-	C10H18O2	-	-	422.50	-	-
Amides	12.38	Butanamide	C4H9NO	-	-	117.74	20.33	-
Olefins	13.752	D-Limonene	C10H16	-	-	-	4.47	14.64
	14.143	3-Ethyl-2-hexene	C8H16	-	-	-	-	36.50
	21.086	1-Tridecene	C13H26	-	-	-	-	1.83
	25.679	cis-alpha-Bergamotene	C15H24	-	-	-	-	3.85
	25.683	beta-Pinene	C10H16	8.57	4.01	-	3.73	-
	26.918	cis-beta-Farnesene	C15H24	15.58	-	20.03	-	8.16

Table 1.

Category of volatile component in wild Ophiocordyceps sinensis and its mycelia

Category	NQ	QD	NC	SN	OS
Aldehyde	7	6	3	2	16
Ketone	2	4	2	3	7
Nitrogenous heterocycles	1	3	0	3	5
Alcohol	5	4	2	4	12
Acid	5	4	8	6	7
Ester	7	3	13	14	8
Aromatic	2	0	0	0	3
Alkane	9	7	4	4	8
Oxygen heterocycles	2	2	3	2	2
Amide	0	0	1	1	0
Terpene	2	1	1	2	5
Total	42	34	37	41	73

NQ=Nagqu, QD=Qamdo, NC=Nyingchi, SN=Shannan

Differences in volatile components in wild Ophiocordyceps sinensis and its mycelia

To further analyze the correlation and differences in the categories of volatile components in NQ, QD, NC, SN, and OS, the horizontal axis represents the location numbers of samples from different growing regions, and the vertical axis represents the types of compounds in the samples. Using 2-octanol as an internal standard and a relative mass of 200 as the reference color, heatmaps were generated to display the volatile substance content of each sample [Figure 3].

Figure 3.

Heatmap of volatile component content in various samples. Note: NQ, QD, NC, and SN represent sample from different production areas, and OS represents its fermentation mycelia products. The color intensity indicates the content level, with darker colors representing higher content and lighter colors representing the lower content. Undetected values are denoted by "-". NQ = Nagqu, QD = Qamdo, NC = Nyingchi, SN = Shannan

The analysis results show that both wild O. sinensis and its mycelia contain 11 types of volatile components. OS has a rich variety of volatile components, but the dominant components are not clear. The primary types in OS are aldehydes, ketones, alcohols, acids, and alkanes. In wild O. sinensis, the common dominant components across the four production areas are aldehydes, alcohols, acids, and lipids [Figures S1 ^{(1.6MB, tif)} –S3 ^{(1.3MB, tif)} ]. The NQ production region, which has the highest altitude, is primarily composed of aldehydes, esters, and alkanes. The remaining three production regions have similar altitudes, with acids and esters being the main components in NC and SN. QD has the fewest types of volatile components, with aldehydes and alkanes being the dominant types. The order of the number of volatile components among the samples is as follows: OS (73), NQ (42), SN (41), NC (37), and QD (34).

Analysis of “fishy odor” components in Ophiocordyceps sinensis and its mycelia

Aldehydes are compounds that can contribute to a fishy odor in various substances due to their specific chemical properties and sensory impact. In the present study, aldehydes acetaldehyde and benzaldehyde were found in all samples (NQ, QD, NC, SN, and OS). NQ had the most aldehyde species, sharing five with OS: propionaldehyde, 3-methylbutanal, 2-methylbutanal, hexanal, and phenylacetaldehyde. QD, NC, and SN had fewer. OS had nine unique aldehydes not in wild O. sinensis. Benzaldehyde, which is found in mushrooms either in a free state or bound to other compounds, is a common component of mushrooms, including O. sinensis.[12,13] It and other compounds, like phenylacetaldehyde, nonanal, 2-methylbutanal, and (E)-2-octenal, are key to chicken soup’s flavor and add to meat broths’ aroma.[14,15] Hexanal, associated with oily and fruity flavors, is a distinctive oxidized volatile compound.

Ketones, particularly certain types, can contribute to a fishy odor in substances due to their chemical structure and the way they interact with olfactory receptors. Common ketones in OS and wild O. sinensis include 2-butanone, 2-pentanone, and 2-undecanone, with 2-pentanone specific to QD. 3-ethyl-4-heptanone is shared by NQ, NC, and SN, while OS has unique ketones, like cyclopentanone and 3,5-octadien-2-one. These ketones, which contribute to floral and woody scents in cultivated OS,[16,17] originate from enzymatic and chemical processes.[18] 2-butanone has an acetone-like smell, 2-undecanone is found in various foods and is a permitted flavoring, and 3,5-octadien-2-one is a fruity-smelling pharmaceutical intermediate.

Nitrogenous heterocycles are compounds that contribute to the “fishy odor” in various substances due to their nitrogen-containing ring structures. In this study, wild O. sinensis and OS lack common nitrogen heterocyclic components. OS contains pyridine, 5-amino-1-ethylpyrazole, 2-piperidone, 4 (1H)-pyridinone, and 4-amino-6-hydroxypyridine. NQ, QD, and SN share 2-acetylpyrrole, with QD also having tetramethylpyrazine and 2-methyl-3,5-diethylpyrazine, and SN having piperazine and 2-vinyl-6-methylpyrazine. NC has no identified nitrogen-containing heterocyclic components. Alcohols can contribute to various odors, but they are not commonly associated with “fishy odor” components, which are typically linked to nitrogen-containing heterocyclic compounds. Four wild O. sinensis and OS share 2,3-butanediol, while OS has seven unique alcohols. 2-Heptanol is in NQ, QD, SN, and OS, and is used in flavoring. 2-Butoxyethanol and 2-ethyl-1-hexanol are in QD and OS. Phenylethanol is in NQ and OS, contributing to rose aroma. 1-Pentene-3-ol, found in fruits, is a food flavoring agent. 1-Octene-3-ol adds sweetness and earthiness, with its oxidized form contributing to shiitake mushroom flavor.[18,19]

In addition, acids, esters, aromatic compounds, alkanes, oxygen heterocycles, amides, and terpenoids are all chemical classes that contribute to the complex world of odor components. For example, wild O. sinensis and OS share pentanoic and 3-methylbutanoic acids, used in fragrances and food, with 3-methylbutanoic acid inhibiting saturated fatty acid production and indicating food waste decomposition.[20,21] These substances were all found in the samples of this study. Collectively, these chemical classes interact to create the rich tapestry of smells that we perceive in our environment, each contributing unique olfactory characteristics that, when combined, form the intricate bouquet of odor components.

DISCUSSION

The volatile components of O. sinensis are the main constituents responsible for its “fishy odor” and active ingredients, and they are also one of the important criteria for identifying the origin of O. sinensis in traditional methods. Existing research reports indicate that the volatile components of O. sinensis include compounds, such as aldehydes, alcohols, and esters. Our study showed that the variety of volatile substances in the mycelia was notably higher than that in the fruiting bodies. Compared to many existing research findings, the analysis suggested that the types of volatile components in the O. sinensis fruiting bodies showed considerable variation due to differences in pretreatment methods and analysis conditions. Moreover, using fresh or dried O. sinensis as material was also an important factor influencing the results of volatile component analysis.

Aldehydes are one of the main sources of the “fishy” odor in O. sinensis, typically originating from the decomposition of organic matter. Qian et al. found hexanal, phenylacetaldehyde, and nonanal in both wild and cultivated O. sinensis. In this experiment, a greater variety of aldehydes was found in NQ, QD, and OS, particularly in OS and NQ, where six common components were identified: benzaldehyde, hexanal, phenylacetaldehyde, nonanal, 2-methylbutanal, and trans-2-octenal.[22] These aldehydes contribute to the rich odors of bitter almonds, chicken broth, and fruity notes in OS and NQ, while these aldehydes were less abundant or absent in QD, NC, and SN. It is worth noting that the two isomers, isovaleraldehyde and 2-methylbutyraldehyde, are present only in the volatile components of the true O. sinensis fruiting bodies, while other samples contain only one of these isomers.[13] Our research findings indicate that both NQ and OS are the only samples where both isomers were simultaneously found. Therefore, from the perspective of aldehyde compounds, the flavor and quality of OS and NQ are the most similar.

Ketones are one of the main compounds in the cultivated OS[17] and play an important role in the formation of the odor of O. sinensis. From a chemical structural perspective, both ketones and aldehydes contain a carboxyl group, with the main difference being the group (R) attached to the carboxyl group. In ketones, two carboxyl groups (-CO-) are connected, while in aldehydes, one carboxyl group (-CO-) is attached to a hydrogen atom (H). Studies have shown that ketones in Cordyceps militaris can be produced through enzymatic degradation of polyunsaturated fatty acids, Maillard reactions, or amino acid degradation,[18] resulting in pleasant floral and woody aromas.[16] Our study found that OS contains more types of ketones than O. sinensis. The unique compounds, phenylethyl alcohol and 1-octene-3-ol, in OS contribute to the floral and earthy aromas.[18,19] Furthermore, acids and esters are also key volatile components in OS. Previous studies have used fatty acid profiles to distinguish O. sinensis from different regions,[23,24] highlighting the significant role of these compounds in O. sinensis. Our study found that the number of acid types in OS falls between NQ, QD and NC, SN. The propionic acid and caprylic acid in OS contribute to its antimicrobial effects, while lactic acid in NQ and QD plays a role in preservation and acidity regulation. Lauric acid in NC can serve as an ingredient in the production of insecticides. This suggests that wild O. sinensis and OS each have their unique acids, which function in regulating acidity and providing preservation and antimicrobial properties. Short-chain esters and long-chain esters contribute to fruity and mild fatty aromas, respectively. Our results show that OS contains more short-chain esters, while the types of short-chain and long-chain esters in NQ, QD, NC, and SN are comparable.

In addition, many compounds related to biological functions include nitrogen-containing heterocyclic compounds, which are an important part of them, such as nucleic acids, alkaloids, and vitamins. Nitrogen is incorporated into the ring structure of these compounds, and generally, compounds with this structure exhibit potential biological activity.[25] Pyridine, pyrazine, pyran, and piperidine are common structural types among alkaloids, which are one of the important active ingredients in traditional Chinese medicine. This study found that nitrogen-containing heterocyclic compounds were more diverse in OS than in NQ, QD, NC, and SN, which may contribute to the diverse biological activities of OS. OS, NQ, QD, NC, and SN each contained two to three types of oxygen heterocyclic compounds, primarily furans or furanones. Furanones are fragrance enhancers with a caramel-like aroma, as well as rich fruity and jam-like scents. They are widely found in natural products but in relatively low concentrations. The similar aromatic compounds in OS and NQ contribute to their antioxidant activity. Additionally, the greater variety of terpenes in OS not only imparts floral and fruity aromas but also exhibits anti-inflammatory and antibacterial effects.

The samples analyzed in this experiment contained a very rich variety of volatile constituents in OS relative to wild O. sinensis, with aldehydes, ketones, and alcohols being the most dominant species constituting OS, and these three categories occupied a quantitative advantage over wild O. sinensis from the four regions. The limitation of this study is the restricted number of field-collected samples, necessitating further data accumulation to validate the research findings.

CONCLUSIONS

O. sinensis mycelia have a more diverse range of volatile compounds than wild samples. Our study shows that higher altitude production areas correlate with greater volatile diversity in O. sinensis, suggesting that altitude influences the complexity of volatile organic compounds. The “fishy odor” is a variable trait that differs by region, highlighting regional variations. This research lays the groundwork for an identification system to verify O. sinensis’s “fishy odor” and offers analytical methods for volatiles in other herbal medicines.

Conflicts of interest

There are no conflicts of interest.

Figure S1

Visualizes the distribution of aldehyde and ketone compounds using a heatmap, where color intensity corresponds to concentration levels (2–500 ng). Key observations include the high abundance of hexanal in OS (185.4 ng) and the consistent presence of 3-methylbutanal across all wild samples, with a notable decrease from NQ to SN

Figure S2

Displays alcohol and carboxylic acid profiles, highlighting significant variations. Lactic acid reached its peak in NQ (23,540 ng), while propanoic acid was exclusively detected in OS (12,340 ng). The heatmap’s clustering reveals distinct chemical signatures between wild samples and cultured mycelia

Figure S3

Illustrates aromatic compounds, alkanes, and alkenes, emphasizing unique markers such as paraxylene in NQ and OS (382 ng and 415 ng, respectively) and the QD-specific compound 2,2,4,4-tetramethyloctane. D-Limonene, associated with floral aroma, showed a clear gradient from OS (112 ng) to SN (78 ng). All supplementary materials are provided in editable formats. Raw data and analytical scripts are available upon request. Compound identification was validated through NIST library matching (>85% similarity) and internal standard calibration. Sample collection and analysis complied with ethical and regulatory standards

Funding Statement

Nil.