AU2021101284A4 - Metabolome-based method for selecting optimal harvest period of Eleutherococcus senticosus - Google Patents

Abstract

The present disclosure provides a metabolome-based method for selecting an optimal harvest period of Eleutherococcus senticosus, and relates to the technical field of determination of harvest period. The method determines 19 phenolic compounds in roots and stems of F senticosus of different growth years (three-, five-, and nine-year-old) based on UPLC/Q-TOF-MS-based metabonomics technology; by comparing and analyzing the relative content of the 19 phenolic compounds, an optimal picking age of . senticosus is explicitly determined to provide a theoretical basis for efficiently utilizing limited wild F senticosus resources. The method of the present disclosure features high sensitivity, excellent selectivity, and short analysis time, providing a reference for quality control of Eleutherococci Semticosi Radix et Rhizoma Seu Caulis. 1/1 DRAWINGS 1. AGroup numicla ad Carepc add 0.4 0.2 0 kMyri cid Cynare side Quercetin-O-rhamraside Syrmgic 3A Salieylic acid Quercetin Vanillic acid Gallic acid Gemistemn Three-year Three-year Five-year Five-year Nine-year Nine-year root stem root stem root stem FIG. 1

Description

1/1

DRAWINGS

1. AGroup

numicla ad

Carepc add

0.4

0.2 0

kMyri cid

Cynare side

Quercetin-O-rhamraside

Syrmgic 3A

Salieylic acid

Quercetin Vanillic acid Gallic acid

Gemistemn

Three-year Three-year Five-year Five-year Nine-year Nine-year root stem root stem root stem

FIG. 1

METABOLOME-BASED METHOD FOR SELECTING OPTIMAL HARVEST PERIOD OF ELEUTHEROCOCCUS SENTICOSUS TECHNICAL FIELD

The present disclosure belongs to the technical field of determination of harvest period, and

particularly relates to a metabolome-based method for selecting an optimal harvest period of

Eleutherococcus senticosus.

BACKGROUND

Metabolomics is a science developed in the mid-1990s for the qualitative and quantitative

analysis of all metabolites with a relative molecular mass of less than 1,000 in a certain organism,

tissue or cell. The task thereof is to detect and quantify the components and content of various

metabolites in organisms and change rules thereof, and to reveal life phenomena and processes.

Metabolomics involves the integration of a plurality of disciplines and technologies, especially

the separation and identification technologies of substances, and the analysis and comparison

methods of data.

The development of high performance liquid chromatography (HPLC) began in the 1960s.

HPLC uses a high pressure infusion system to pump a specified mobile phase into a

chromatographic column packed with a stationary phase, where all components are separated

and enter a detector for detection, so as to realize the analysis of a sample. Ultra-high

performance liquid chromatography (UPLC) is an emerging liquid chromatography technology developed on the basis of HPLC. UPLC features ultra-high efficiency, ultra-high resolution, and ultra-high sensitivity, and has been widely used in the analysis of Chinese herbal medicinal ingredients, metabolomics research, the establishment of fingerprints, and the determination of unknown compounds in combination with mass spectrometry (MS) and other detectors.

Q-TOF-MS tandem technology is an important breakthrough in MS technology. This technology

has the advantages of high sensitivity, high selectivity, high precision, ability to generate

multi-stage MS, and high information acquisition speed. Ultra-high performance liquid

chromatography-quadrupole-time-of-flight tandem mass spectrometry (UPLC/Q-TOF-MS) is a

tandem MS technology using UPLC as a chromatographic separation system and quadrupole (Q)

and time-of-flight mass spectrometry (TOF-MS) as analyzers. This technology organically

combines the efficient separation ability and high sensitivity characteristics of UPLC with the

high-resolution characteristics of Q-TOF-MS, and has been more and more widely used in the

field of pharmaceutical analysis.

Eleutherococci Semticosi Radix et Rhizoma Seu Caulis is the dried root, rhizome, or stem of

Acanthopanax senticosus (Rupr. et Maxim.) Maxim., a plant of the family Araliaceae. The drug

is listed as the top grade in the Shennong' Classic of Materia Medica. The drug is warm in

nature and acrid and slightly bitter in taste. The drug belongs to spleen, kidney and heart

meridians, having the effects of Invigorating qi and spleen and tonifying the kidneys to soothe

the nerves. As a widely used traditional Chinese herb, the efficacy of Eleutherococci Semticosi

Radix et Rhizoma Seu Caulis is closely related to the biological activity of secondary

metabolites thereof. Phenolic Compounds are a class of secondary metabolites widely present in

plants. Phenols have become a research hotspot because of natural antioxidant activity, antiviral,

antitumor, and coronary heart disease prevention effects thereof.

The accumulation of phenolic compounds in medicinal plants is closely related to the

picking age. Therefore, the medicinal plants generally have optimal picking age. On the

condition that the picking age is not reached, medicinal material quality will be low because the

accumulation of phenolic compounds does not meet the regulations. On the contrary, on the

condition that the picking age is exceeded, active pharmaceutical ingredients will further be lost.

At present, the research on the pharmacology and chemical components of Eleutherococci

Semticosi Radix et Rhizoma Seu Caulis is relatively concentrated and in-depth, but there is no

report on relevant research on the optimal harvest period of E. senticosus. Therefore, it is

particularly important to investigate the optimal picking age of E. senticosus.

SUMMARY

In view of this, an objective of the present disclosure is to provide a metabolome-based

method for selecting an optimal harvest period of E. senticosus. This method features high

sensitivity, excellent selectivity, and short analysis time, providing a reference for quality control

of Eleutherococci Semticosi Radix et Rhizoma Seu Caulis and a theoretical basis for efficiently

utilizing limited wild E. senticosus resources.

To achieve the above objective, the present disclosure provides the following technical

solution:

The disclosure provides a metabolome-based method for selecting an optimal harvest period

of E. senticosus, including the following steps: separately harvesting stems and roots of E.

senticosus of different ages, determining relative content of phenolic compounds by

UPLC/Q-TOF-MS method, conducting a cluster analysis to obtain the content distribution of

different phenolic compounds in the roots and stems of E. senticosus of different growth years,

and thus obtain an optimal harvest period of different phenolic compounds; where selection

criteria for the optimal period are that there are more types and high content of accumulated

phenolic compounds in the corresponding year and more concentrated accumulation in the same

part; and

the phenolic compounds include p-hydroxycinnamic acid, genistein, apigenin, syringic acid,

cynaroside, naringenin, quercetin-3-0-rhamnoside, chlorogenic acid, salicylic acid, ferulic acid,

myricitrin, luteolin, catechin, cinnamic acid, p-coumaric acid, quercetin, gallic acid, caffeic acid,

and vanillic acid.

Preferably, before using the UPLC/Q-TOF-MS method to measure the relative content of the

phenolic compounds, the method may further include the following steps: washing the roots and

stems of E. senticosus of different growth years with deionized water, air-drying naturally,

pulverizing and sieving, mixing pulverized powder with chromatographic grade acetonitrile and conducting ultrasonic extraction, filtering the mixture through a millipore filter, and adding leucine-enkephalin as an internal standard.

Preferably, the ultrasonic extraction may be conducted at 100 kHz and 40°C for 30 min.

Preferably, after the ultrasonic extraction, the method may further include conducting a

second ultrasonic extraction on filter residues, combining filtrates, and filtering the combined

filtrate through a millipore filter.

Preferably, the millipore filter may be 0.45 pm in pore size.

Preferably, the UPLC/Q-TOF-MS method may include chromatography and mass

spectrometry (MS), where chromatographic conditions may be as follows: chromatographic

column may be Acquity UPLC BEH C18 column equipped with VanGuard pre-column; for

chromatographic separation, column temperature may be held at 30°C; injection volume may be

2 pL; mobile phase A may be a 0.05 wt% acetic acid-water mixture system, and mobile phase B

may be a 0.05 wt% acetic acid-acetonitrile mixture system; gradient elution may be conducted at

a flow rate of 0.25 mL-min-; elution program may be as follows: mobile phase 5% B to 95% B

within 0 to 23 min; 95% B to 5% B within 23 to 25 min; and 5% B within 25 to 31 min; and

MS conditions may be as follows: first order MS scanning detection may be conducted in

ESI+ positive ion mode in the m/z range of 100 to 1,000, with a cone voltage of 3 KV.

Preferably, after determining the relative content of the phenolic compounds by the

UPLC/Q-TOF-MS method, the method may further include using MassHunter software to conduct peak detection and matching on raw data in a metabolic fingerprint, extracting the raw data to obtain a data matrix composed of the retention time, mass number, and response peak area of metabolite fragments, and performing cluster analysis after peak area normalization.

The present disclosure provides a metabolome-based method for selecting an optimal

harvest period of E. senticosus; 19 phenolic compounds in roots and stems of E. senticosus of

different growth years (three-, five-, and nine-year-old) may be determined based on

UPLC/Q-TOF-MS-based metabonomics technology; by comparing and analyzing the relative

content of the 19 phenolic compounds, an optimal picking age of E. senticosus may be explicitly

determined to provide a theoretical basis for efficiently utilizing limited wild E. senticosus

resources. The method of the present disclosure further establishes a UPLC/Q-TOF-MS analysis

method for the simultaneous quantitative detection of the relative content of the 19 phenolic

compounds (p-hydroxycinnamic acid, genistein, apigenin, syringic acid, cynaroside, naringenin,

quercetin-3-0-rhamnoside, chlorogenic acid, salicylic acid, ferulic acid, myricitrin, luteolin,

catechin, cinnamic acid, p-coumaric acid, quercetin, gallic acid, caffeic acid, and vanillic acid) in

E. senticosus. This method features higher sensitivity, better selectivity, shorter analysis time,

providing a reference for quality control of Eleutherococci Semticosi Radix et Rhizoma Seu

Caulis.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates the heat map distribution of the relative content of 19 phenolic compounds in the roots and stems of E. senticosus of different growth years.

DETAILED DESCRIPTION

The disclosure provides a metabolome-based method for selecting an optimal harvest period

of E. senticosus, including the following steps: separately harvesting stems and roots of E.

senticosus of different ages, determining relative content of phenolic compounds by

UPLC/Q-TOF-MS method, conducting a cluster analysis to obtain the content distribution of

different phenolic compounds in the roots and stems of E. senticosus of different growth years,

and thus obtain an optimal harvest period of different phenolic compounds; where selection

criteria for the optimal period are that there are more types and high content of accumulated

phenolic compounds in the corresponding year and more concentrated accumulation in the same

part; and

the phenolic compounds include p-hydroxycinnamic acid, genistein, apigenin, syringic acid,

cynaroside, naringenin, quercetin-3-0-rhamnoside, chlorogenic acid, salicylic acid, ferulic acid,

myricitrin, luteolin, catechin, cinnamic acid, p-coumaric acid, quercetin, gallic acid, caffeic acid,

and vanillic acid.

In the example of the present disclosure, the E. senticosus may preferably grow for three,

five, and nine years. The roots and stems of E. senticosus of the above years may be separately

selected and washed with deionized water, air-dried naturally, pulverized into powder with a

pulverizer, and repeatedly sieved; a sieved powder may be mixed with chromatographic grade acetonitrile and subjected to ultrasonic extraction; after filtering through a millipore filter, a certain amount of internal standard may be added. The powder of the present disclosure may preferably pass through a 20-mesh sieve. In the present disclosure, the ultrasonic extraction may be performed after mixing the powder with the chromatographic grade acetonitrile, where the powder and the chromatographic grade acetonitrile may preferably have a mass-volume ratio of

0.5 g: 20 ml. In the present disclosure, the ultrasonic extraction may preferably be conducted at

100 kHz and 40°C for 30 min; in the present disclosure, the ultrasonic extraction may further

preferably be followed by conducting a second ultrasonic extraction on filter residues after the

ultrasonic extraction, combining filtrates, and filtering the combined filtrate through a millipore

filter. In the present disclosure, the millipore filter may preferably be 0.45 pm in pore size during

filtration. In the present disclosure, the internal standard may preferably be leucine-enkephalin.

In the present disclosure, when the UPLC/Q-TOF-MS method is used to determine phenolic

compounds, standards in all lanes may preferably be chromatographic grade acetonitrile

solutions of the phenolic compounds. When preparing the standards, it may be preferable to

separately dissolve the standards of the phenolic compounds in chromatographic grade

acetonitrile and make up to volume to obtain 1 mg/mL standard solutions. The present disclosure

has no particular limitation on the sources of the standards, and the preferred purity may be

>98%.

In the present disclosure, the UPLC/Q-TOF-MS method may be used to determine the relative content of the phenolic compounds and preferably include chromatography and MS, where chromatographic conditions may preferably include as follows: chromatographic column may be Acquity UPLC BEH C18 column equipped with VanGuard pre-column; for chromatographic separation, column temperature may be held at 30°C; injection volume may be

2 pL; mobile phase A may be a 0.05 wt% acetic acid-water mixture system, and mobile phase B

may be a 0.05 wt% acetic acid-acetonitrile mixture system; gradient elution may be conducted at

a flow rate of 0.25 mL min-'; elution program may be as follows: mobile phase 5% B to 95% B

within 0 to 23 min; 95% B to 5% B within 23 to 25 min; and 5% B within 25 to 31 min; MS

conditions may preferably include as follows: first order MS scanning detection may be

conducted in ESI' positive ion mode in the m/z range of 100 to 1,000, with a cone voltage of 3

KV.

In the present disclosure, the relative content may be subjected to data analysis and

processing, preferably including: using MassHunter software to conduct peak detection and

matching on raw data in a metabolic fingerprint, extracting the raw data to obtain a data matrix

composed of the retention time, mass number, and response peak area of metabolite fragments,

and performing cluster analysis after peak area normalization. The content distribution of the 19

phenolic compounds in F senticosus of different growth years may be explained by comparing

the relative content of each phenolic compound.

Using the method of the present disclosure, the 19 phenolic compounds may be divided into three categories according to different growth years: for three-year-old plants: p-hydroxycinnamic acid, p-coumaric acid, chlorogenic acid, caffeic acid, naringenin, ferulic acid, and cinnamic acid; for five-year-old plants: luteolin, catechin, myricitrin, cynaroside, quercetin-3-0-rhamnoside, gallic acid, genistein, and apigenin; for nine-year-old plants: syringic acid, salicylic acid, quercetin , and vanillic acid. Among the 19 phenolic compounds, there are 7 compounds that are most suitable to accumulate in three-year-old plants, 2 of which have higher relative content in roots than in stems, and 5 of which have higher relative content in stems than in roots; the most compounds accumulate in five-year old plants, and there are 8 compounds in total, and all of the 8 compounds show higher relative content in stems than in roots; there are 4 compounds that are most suitable to accumulate in nine-year-old E. senticosus plants, only one of which has higher relative content in roots than that in stems, and the remaining three of which have higher relative content in stems than that in roots. Therefore, comprehensively considering the natural resources and human resources required for artificial cultivation of E. senticosus, it is recommended to use a five-year-old E. senticosus plant as a raw material for extracting secondary metabolites.

The metabolome-based method for selecting an optimal harvest period of E. senticosus

provided by the present disclosure will be described in detail below with reference to the

examples, but they should not be construed as limiting the protection scope of the present

disclosure.

Example 1

1. Apparatus and reagents

Apparatus: Acquity UPLC-Xevo G2-S QTof MSn Liquid-Mass Spectrometer (Waters, USA);

FZ-06 Chinese Herbal Medicine Pulverizer (Wenling Baile Crushing Equipment Factory,

Zhejiang); 250DC CNC Ultrasonic Cleaner (Kunshan Ultrasonic Instruments Co., Ltd., Jiangsu);

BS124S Electronic Balance (Sartorius, Germany); Millipore Ultrapure Water System (Millipore,

France).

Reagents: acetic acid (chromatographically pure, Dikma); acetonitrile (chromatographically

pure, Dikma); deionized water self-prepared.

Samples: Eleutherococci Semticosi Radix et Rhizoma Seu Caulis were collected from roots

and stems of E senticosus of different growth years (three-, five-, and nine-year-old) in Qitaihe

area, Heilongjiang Province. The roots and stems were air-dried immediately after picking. The

picking time was on September 20. Before the experiment, the roots and stems were pulverized

into powder using a pulverizer, and the powder passed through a 20-mesh sieve for use.

Standards: p-hydroxycinnamic acid, genistein, apigenin, syringic acid, cynaroside,

naringenin, quercetin-3-0-rhamnoside, chlorogenic acid, salicylic acid, ferulic acid, myricitrin,

luteolin, catechin, cinnamic acid, p-coumaric acid, quercetin, gallic acid, caffeic acid, and

vanillic acid were all >98% pure. All reference standards were purchased from Shanghai

PureOne Biotechnology Co., Ltd.; leucine-enkephalin standard was purchased from Sigma.

2. Preparation of test solutions

The roots and stems of three-, five-, and nine-year-old F senticosus plants were washed with

deionized water, air-dried naturally, pulverized into powder with a pulverizer, and repeatedly

sieved. 0.5 g each of powders of senticosus of three different ages was mixed with

chromatographic grade acetonitrile and subjected to ultrasonic extraction; after filtering, residues

were mixed with chromatographic grade acetonitrile and subjected to ultrasonic extraction again;

after filtering again, filtrates were combined, filtered through a millipore filter, and mixed with a

certain amount of leucine-enkephalin as an internal standard.

3. Preparation of standard solutions

Separately, an appropriate amount of standard was weighed, dissolved in chromatographic

grade acetonitrile, and diluted to volume to prepare a 1 mg/mL standard solution, which was

stored in a refrigerator at 4°C for later use.

4. UPLC/Q-TOF-MS method

Detection and analysis were conducted based on a UPLC/Q-TOF-MS (Waters, Japan)

system.

(1) Chromatographic conditions: chromatographic column was Acquity UPLC BEH C18

column (1.7 pm, 2.1 x 50 mm) equipped with VanGuard pre-column (BEHC18, 1.7 pm, 2.1 x 5

mm; Waters); for chromatographic separation, column temperature was held at 30°C; injection

volume was 2 pL; mobile phases were a 0.05 wt% acetic acid-water (A%), and 0.05 wt% acetic acid-acetonitrile (B%); gradient elution was conducted at a flow rate of 0.25 mL-min-1 ; elution program was as follows: 5% B to 95% B within 0 to 23 min; 95% B to 5% B within 23 to 25 min; and 5% B within 25 to 31 min.

(2) MS conditions: first order MS scanning detection was conducted in ESI' positive ion

mode in the m/z range of 100 to 1,000, with a cone voltage of 3 KV.

5. Data processing and analysis: MassHunter software was used to conduct peak detection

and matching on raw data in a metabolic fingerprint, the raw data were extracted to obtain a data

matrix composed of the retention time, mass number, and response peak area of metabolite

fragments, and cluster analysis was performed after peak area normalization (Table 1). The

content distribution of the 19 phenolic compounds in E senticosus of different growth years was

explained by comparing the relative content of each phenolic compound.

Table 1 Normalized data of the 19 phenolic compounds

Three-year-old Root Root Root Stem Stem Stem

P-hydroxycinnamic acid 0.463376541 0.463669578 0.466257038 0.894375549 1 0.864114746

Genistein 0.096173561 0 0.063714183 0.16352709 0.080180942 0.14630427

Apigenin 0 0.053983364 0.056868314 0.238575261 0.141654412 0.197950226

Syringic acid 0.294389736 0.184156391 0.348057892 1 0.8451426 0.863754215

Cynaroside 0.004729405 0 0.001173763 0.059529808 0.018345052 0.046784463

Naringenin 0 0.001400613 0.000567816 0.638894146 1 0.867244577

Quercetin-3-0-rhamnoside 0.00827899 0.006876623 0.007761312 0.086761343 0.097644177 0.16930013

Chlorogenic acid 0.803420307 1 0.907482346 0.568851464 0.652584864 0.68831364

Salicylic acid 0 0.099628713 0.076887376 0.296101485 0.280940594 0.054764851

Ferulic acid 0.399411609 0.66245868 0.660772841 0.857301997 1 0.884057252

Myricitrin 0.000383529 0.000176329 9.43981E-05 0.262661813 0.229642071 0.300200195

Luteolin 0 0.006520247 0.022563487 0.227436513 0.350291695 0.326784489

Catechin 0.226144593 0.233746316 0.268327488 0.207820439 0.321806276 0.535241321

Cinnamic acid 0.156078256 0.509653292 1 0.273720821 0.375493855 0.132511601

P-coumaric acid 0.585432972 0.468130063 0.605613234 0.87088648 1 0.904109527

Quercetin 0.03652968 0 0.021535234 0.346168086 0.255152413 0.391830186

Gallic acid 0.133433848 0.179984923 0.060120618 0 0.038447041 0.051074256

Caffeic acid 0.758651573 0.904278774 0.863598395 0.86969493 0.974513843 1

Vanillic acid 0.062557237 0.086951071 0 0.127338537 0.057324146 0.117902621

Five-year-old Root Root Root Stem Stem Stem

P-hydroxycinnamic acid 0.120070578 0.212845021 0.041636272 0.117938263 0.120132927 0.119447094

Genistein 0.227074683 0.15460813 0.279089138 0.978702137 1 0.918857963

Apigenin 0.255761481 0.190474052 0.311114354 0.971745451 0.965403051 1

Syringic acid 0.165289156 0.302593941 0.243813373 0.084756491 0.12546103 0

Cynaroside 0.006063486 0.000973364 0.002210109 0.826283123 1 0.60796899

Naringenin 0.108806198 0.076213549 0.06123582 0.67504511 0.606654806 0.622616749

Quercetin-3-0-rhamnoside 0.004071888 0.007892663 0.008391025 0.659978057 1 0.717181512

Chlorogenic acid 0.718933112 0.557345329 0.620714393 0.205296472 0.261163405 0.270879344

Salicylic acid 0.036819307 0.012221535 0.021194307 0.062654703 0.050742574 0.048886139

Ferulic acid 0.081465688 0.110240645 0.10627066 0.817089779 0.633891974 0.70933492

Myricitrin 0 3.56219E-06 0.000435775 0.763788059 1 0.645545241

Luteolin 0.225549073 0.201355525 0.162663006 1 0.729495539 0.841026081

Catechin 0.019017644 0.000533454 0.009842231 0.741741461 1 0.607937799

Cinnamic acid 0.917566339 0.239697108 0.517789901 0.516552369 0.656846525 0.552020571

P-coumaric acid 0.123527667 0.143639475 0.127717056 0.126407302 0.124869367 0.153401027

Quercetin 0.250771319 0.292669382 0.245896582 0.394360114 0.27705788 0.198383315

Gallic acid 0.24519412 0.333207689 0.393328308 0.796645307 0.870147003 0.693554467

Caffeic acid 0.437452491 0.449582653 0.45534245 0.230548513 0.234712848 0.206833237

Vanillic acid 1 0.77164101 0.574026427 0.675270376 0.774840827 0.525609437

Nine-year-old Root Root Root Stem Stem Stem

P-hydroxycinnamic acid 0 0.097319642 0.13592578 0.155256907 0.141795884 0.003893659

Genistein 0.326054641 0.371841201 0.521426009 0.358052694 0.391088728 0.558588344

Apigenin 0.344970421 0.37404443 0.400502902 0.380173546 0.3842933 0.457113029

Syringic acid 0.547600209 0.461176098 0.39699098 0.480396334 0.968242182 0.624164668

Cynaroside 0.000647001 0.000229027 0.001162311 0.098847995 0.09659208 0.096769576

Naringenin 0.135140251 0.279277233 0.2113538 0.350279492 0.363919698 0.355061766

Quercetin-3-0-rhamnoside 0.000309062 0.00206685 0 0.047525961 0.053398133 0.053684015

Chlorogenic acid 0.330181148 0.333864591 0.418118538 0.109821135 0 0.107736811

Salicylic acid 0.281404703 0.526454208 0.368966584 1 0.45730198 0.779084158

Ferulic acid 0 0.160802592 0.112795848 0.137045485 0.268078144 0.196106043

Myricitrin 0.000150799 0.000201858 0.00018939 0.0396472 0.02332286 0.042287972

Luteolin 0.157000686 0.1898593 0.017415923 0.334763212 0.286805079 0.160003432

Catechin 0.006321433 0 0.00172039 0.090940613 0.052678607 0.030753637

Cinnamic acid 0.301748789 0.325994372 0.361618694 0.098492701 0 0.076874392

P-coumaric acid 0.050213349 0.055306332 0 0.089761552 0.044609241 0.140869367

Quercetin 0.882882883 1 0.747192398 0.332716278 0.442243613 0.421633963

Gallic acid 0.829438372 0.716170373 1 0.690915944 0.682623445 0.754428948

Caffeic acid 0.286683109 0.271820143 0.270800269 0.00030933 0 0.054737782

Vanillic acid 0.062688064 0.158797043 0.049065675 0.762722951 0.898205486 0.979520082

6. Experimental results

There were significant differences in the relative content of different phenolic compounds in

roots and stems of E senticosus of different growth years, as shown in FIG. 1. The 19 phenolic

compounds were divided into three categories according to different growth years: for three-year-old plants: p-hydroxycinnamic acid, p-coumaric acid, chlorogenic acid, caffeic acid, naringenin, ferulic acid, and cinnamic acid; for five-year-old plants: luteolin, catechin, myricitrin, cynaroside, quercetin-3-0-rhamnoside, gallic acid, genistein, and apigenin; for nine-year-old plants: syringic acid, salicylic acid, quercetin , and vanillic acid. Among the 19 phenolic compounds, there were 7 compounds that were most suitable to accumulate in three-year-old plants, 2 of which had higher relative content in roots than in stems, and 5 of which had higher relative content in stems than in roots; the most compounds accumulated in five-year old plants, and there were 8 compounds in total, and all of the 8 compounds showed higher relative content in stems than in roots; there were 4 compounds that were most suitable to accumulate in nine-year-old Fsenticosus plants, only one of which had higher relative content in roots than that in stems, and the remaining three of which had higher relative content in stems than that in roots. Therefore, comprehensively considering the natural resources and human resources required for artificial cultivation of F senticosus, it is recommended to use a five-year-old

. senticosus plant as a raw material for extracting secondary metabolites.

The above descriptions are merely preferred implementations of the present disclosure. It

should be noted that several improvements and modifications may also be made by a person of

ordinary skill in the art without departing from the principle of the present disclosure, and such

improvements and modifications should be deemed as falling within the protection scope of the

present disclosure.

Claims (5)

1. A metabolome-based method for selecting an optimal harvest period of Eleutherococcus

senticosus, comprising the following steps: separately harvesting roots and stems of

Eleutherococcus senticosus of different ages, determining relative content of phenolic

compounds by UPLC/Q-TOF-MS method, conducting a cluster analysis to obtain the content

distribution of different phenolic compounds in the roots and stems of Eleutherococcus

senticosus of different growth years, and thus obtain an optimal harvest period of different

phenolic compounds;

wherein selection criteria for the optimal period are that there are more types and high

content of accumulated phenolic compounds in the corresponding year and more concentrated

accumulation in the same part; and

wherein the phenolic compounds comprise p-hydroxycinnamic acid, genistein, apigenin,

syringic acid, cynaroside, naringenin, quercetin-3-0-rhamnoside, chlorogenic acid, salicylic acid,

ferulic acid, myricitrin, luteolin, catechin, cinnamic acid, p-coumaric acid, quercetin, gallic acid,

caffeic acid, and vanillic acid.

2. The method according to claim 1, wherein, before using the UPLC/Q-TOF-MS method to

measure the relative content of the phenolic compounds, the method further comprises the

following steps: washing the roots and stems of Eleutherococcussenticosus of different growth

years with deionized water, air-drying naturally, pulverizing and sieving, mixing pulverized powder with chromatographic grade acetonitrile and conducting ultrasonic extraction, filtering the mixture through a millipore filter, and adding leucine-enkephalin as an internal standard.

3. The method according to claim 2, wherein the ultrasonic extraction is conducted at 100

kHz and 40°C for 30 min.

4. The method according to claim 2 or 3, wherein after the ultrasonic extraction, the method

further comprises conducting a second ultrasonic extraction on filter residues, combining filtrates,

and filtering the combined filtrate through a millipore filter;

wherein the millipore filter is 0.45 pm in pore size.

5. The method according to claim 1, wherein the UPLC/Q-TOF-MS method comprises

chromatography and mass spectrometry (MS), wherein chromatographic conditions are as

follows: chromatographic column is Acquity UPLC BEH C18 column equipped with VanGuard

pre-column; for chromatographic separation, column temperature is held at 30°C; injection

volume is 2 pL; mobile phase A is a 0.05 wt% acetic acid-water mixture system, and mobile

phase B is a 0.05 wt% acetic acid-acetonitrile mixture system; gradient elution is conducted at a

flow rate of 0.25 mL-min-; elution program is as follows: mobile phase 5% B to 95% B within 0

to 23 min; 95% B to 5% B within 23 to 25 min; and 5% B within 25 to 31 min; and

wherein MS conditions are as follows: first order MS scanning detection is conducted in

ESI+ positive ion mode in the m/z range of 100 to 1,000, with a cone voltage of 3 KV;

wherein, after determining the relative content of the phenolic compounds by the

UPLC/Q-TOF-MS method, the method further comprises using MassHunter software to conduct

peak detection and matching on raw data in a metabolic fingerprint, extracting the raw data to

obtain a data matrix comprising the retention time, mass number, and response peak area of

metabolite fragments, and performing cluster analysis after peak area normalization.