





































A comparative study of Salvia miltiorrhiza Radix &amp; Rhizoma raw material and granule products using chromatographic analysis and antioxidant activity


CTMJ | traditionalmedicinejournals.com Chinese Traditional Medicine Journal | 2023 | Vol 6 | Issue 6 

 

    ISSN : 2693 6356 

2023 | Vol 6 | Issue 6 
 

 
 

 
 
 
 
 

A comparative study of Salvia miltiorrhiza Radix & Rhizoma raw 

material and granule products using chromatographic analysis and 

antioxidant activity 

Zhou a, Valentina a, b, *, Mendoza a, John a, 
Kelvin a, c 
a NICM Health Research Institute, Western Sydney University, Westmead, 2145, Australia 
b South West Sydney Clinical Campuses, Faculty of Medicine & Health, University of New South Wales, Sydney, NSW, 2170, Australia 
c School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, Liverpool, L3 3AF, UK 

 

 

 

Introduction 

 

To make a therapeutic herbal decoction, 

practitioners of traditional Chinese medicine 

(TCM) choose certain raw plant components, dry 

them, and then either grind them into powder or 

chop them into smaller pieces. Inconsistent herbal 

ingredients, a bad aftertaste, and a lengthy 

preparation procedure are just a few of the 

numerous drawbacks of this method.1 Herbal 

extracts that are both concentrated and 

standardized, with excipients in a granular form, 

are thought to have reliability and effectiveness.2 

Over time, the granule form of herbal extracts 

surpassed the herbal decoction as the preferred 

method of giving herbal medications in clinical 

settings.3 On the other hand, some people are 

worried about the granule products' effectiveness, 

safety, and quality.There was a positive correlation 

between the amount of marker compounds and the 

extraction yields, marker compounds, and 

antioxidant capacities of the herb/decoction pieces 

 
 

Abstract— One common medicinal preparation used in traditional Chinese medicine is granules of botanical extracts. 

But there is no assessment of their quality or effectiveness. Dan Shen (Salvia miltiorrhiza Radix & Rhizoma) granule 

extracts and their herbal counterparts were the subjects of this comparative investigation on antioxidant activity and 

purity. Methods: A comparison was made between the herb's water extracts and 12 granule extracts using a 

chromatographic technique to ascertain the concentration of seven marker chemicals. Principal component analysis 

(PCA) and agglomerative hierarchical clustering (AHC) were used to differentiate the herbal and granule extracts 

according to the marker chemical content. This study used DPPH, ferric ion reducing antioxidant power (FRAP), and 

2, 20-azino-bis (3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS) tests to assess the antioxidant properties of herbal 

and granule extract The results revealed that salvianolic acid B, sodium danshensu, and cryptotanshinone levels were 

much greater in the herbal extracts group than in the granule group. The increased ABTS, DPPH, and FRAP activities 

of the herbal extracts were correlated with this (P <.05). Salvianolic acid B concentration was the primary 

differentiator between granule extracts and other herbal extracts in the AHC and PCA analyses. 

In order to better educate healthcare practitioners and customers about the quality and effectiveness of granule 

products, the findings reinforce the necessity for their evaluation.  

 



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compared to the granule products in a previous 

study that compared the raw and granular 

preparations of two popular medicinal plants 

(4,5).6,7 Our results are in line with those of a 

previous research by Liang et al., which found that 

the chemical profiles, antioxidant and anti-

inflammatory bioactivities of baical skullcap root 

(Scutellariae Radix) granules and traditional 

decoctions are only partially identical.8 A 

clustering study driven by bioactive markers 

1. taken 34 out of 39 samples of granules from 

the conventional decoction batches. In relation 

to the chemical fingerprint, 61.5% of the 

granule samples had anti-inflammatory 

activity that was bioequivalent to traditional 

decoction, while none of the granule samples 

had antioxidant activity that was bioequivalent 

to traditional decoction. Crucially, there is still 

no clear evidence that granules are safer and 

more effective than the traditional decoction in 

clinical practice.2 The significance and need 

of evaluating the biology and quality of 

granule products before they are used by both 

practitioners and consumers is highlighted by 

these findings. One of the most popular herbs 

in Asian therapeutic treatment, Dan Shen 

(Salvia miltiorrhiza Radix & Rhizoma) is a 

component in several formulas, including 

Danshen pian, Danshen injection, and 

compound Danshen dripping tablet.9, 10 S. 

miltiorrhiza is a versatile and important 

therapeutic plant, as shown by the 841 Chinese 

herbal remedies that include it, according to 

the Chinese National Medical remedies 

Administration database.11 While S. 

miltiorrhiza cultivation is common in most 

Chinese provinces, the provinces of Shanxi, 

Sichuan, Hebei, Henan, and Shandong have 

long been known as the main production 

locations for high-quality S. miltiorrhiza.12 Its 

chemical components include  

2. As early as the 1930s, S. miltiorrhiza was 

isolated. More than 70 structural components 

have been found and categorized according to 

whether they are hydrophilic or lipophilic.13 

Tanshinone I (TI), crypto-tanshinone (CT), 

dihydrotanshinone I (DT), and tanshinone IIA 

(TIIA) are among the over 30 lipophilic 

chemicals that have been isolated and 

identified. These molecules are mostly 

diterpene quinones. For example, phenolic 

acids, protocatechuic acid, sodium danshensu 

(DSS), and protocatechuic aldehyde are all 

hydrophilic substances. With more than 1% 

and 3%e5% of total dry weight, respectively, 

DSS and salvianolic acid B (SB) are the most 

abundant components.14, 15 Reference 

standards for S. miltiorrhiza in the People's 

Republic of China Pharmacopoeia (PPRC) are 

SB and TIIA at now. To represent the efficacy 

and purity of S. miltiorrhiza, the Korean 

Pharmacopoeia relied on SB, the only marker 

chemical.16, 17 The antioxi-dant, anti-

inflammatory, and angiogenesis 

characteristics of S. miltiorrhiza products 

make them a popular choice for the therapeutic 

treatment of cardiovascular illnesses, acute 

ischemic stroke, hyperlipidaemia, and 

cerebrovascular disorders in Asian 

nations.18e20  

The granule products of S. miltiorrhiza have 

not been evaluated for quality or efficacy, 

despite the fact that they are rather popular.13 

As part of their standard operating procedure 

for ensuring the highest quality of their 

Chinese herb products, the PPRC often 

employs thin layer chromatography (TLC), an 

analytical technique for the separation and 

identification of bioactive components in 

herbal combinations.16 Rapid and cost-

effective chemical quantification via TLC has 

also been achieved using the automated 

CAMAG Linomat system.21e23 Liquid 

chromatography methods, including HPLC 

and UPLC, are extensively used because they 

allow for the exact and accurate quantitative 

detection of biomarkers in Chinese herbs.24 It 

is unusual, nevertheless, to see herbal granules 

quality-controlled utilizing TLC and UPLC 

quantification in accordance with their 

original herbal decoction. Granule product 

efficacy is unclear, despite substantial in vitro 



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and in vivo investigation of S. miltiorrhiza 

antioxidant properties.15 In line with the 

general agreement that multiple methods 

should be used to determine the efficacy of 

herbal remedies, this study intends to compare 

the raw (unprocessed herbal 

material/decoction pieces) and manufactured 

granule samples of S. miltiorrhiza with respect 

to antioxidant activity, chemometrics, and 

TLC/UPLC chromatography. In addition to 

using statistical clustering techniques like 

agglomerative hierarchical clustering (AHC) 

and principal component analysis (PCA), this 

study also included a Pearson correlation of 

the chemical markers to antioxidant activity, 

which allowed for a thorough examination of 

the variations in product quality and ability.  

 

Part 2: Resources and Procedures  

     Part 2.1: Chemicals and botanicals  

Thermo Fisher Scientific of Scoresby, Australia, 

supplied the 85% phosphoric acid, 85% methanol, 

and HPLC grade acetonitrile. Ajax Finechem of 

Cheltenham, Australia, supplied the analytical 

grade ethyl acetate, toluene, and formic acid. 

Millipore, located in Burlington, MA, used its 

Milli-Q Reagent Water System to get the water. 

Sigma (Kemps Creek, Australia) supplied the 2, 

20-azino-bis (3-ethyl-benz-thiazoline-6-sulfonic 

acid) (ABTS) working solution, DPPH, Trolox, 

sodium acetate trihydrate, glacial acetic acid, 2, 4, 

6-tripyridyl-s-triazine (TPTZ), hydrochloric acid 

(HCl), ferric chloride hexahydrate, potassium 

persulfate, and hydrochloric acid (HCl) were all 

acquired from Sigma.  

Sources in China and Australia were consulted for 

the raw herbal materials of S. miltiorrhiza, which 

included both crude herbal materials (R2, R4eR6) 

and decoction components (R1, R3) 

(Supplemental Fig. 1). In accordance with the 

Hong Kong Materia Medica Standards and PPRC, 

all of the raw materials were certified by the 

Department of Applied Biology and Chemical 

Technology, Hong Kong Polytechnic University 

(Hong Kong, China) (China, 2015). The NICM 

Health Research Institute at Western Sydney 

University received a voucher specimen from 

every sample. The lack of permission for 

disclosure has resulted in the omission of product 

commercial names.  

To make it seem like a typical water decoction, 

the S. miltiorrhiza raw plant ingredients were 

soaked. The first step was to reflux 1 gram of R1–

R6 powdered S. miltiorrhiza herbal material in 30 

milliliters of boiling water. The liquid samples 

were then spun in a centrifuge at 672×g for 5 

minutes and dried using a rotary evaporator. To 

get rid of the water-soluble excipients, the granule 

products (1 g of G1-G12) and the water-based 

herbal extract were extracted three times with 10 

mL of methanol. Before being centrifuged at 

672×g for 5 minutes, the samples were sonicated 

for 30 minutes. After that, the liquid above the 

solid was collected and cooled using a rotary 

evaporator until it was completely dry. For 

immediate analysis, the dry residue was weighed 

and redissolved in methanol at a concentration of 

10 mg/mL. It was then kept at 4◦C, or at e20◦C, 

until it was needed again.  

Using the Herbal Chemical Marker Ranking 

System (Herb MaRS), the primary chemical 

components of S. miltiorrhiza were chosen for this 

investigation.Twenty-five marker chemicals were 

chosen for bioassay testing and content 

measurement; all seven of them scored higher 

than 1 on this scale. Salvianolic acid A (SA), DSS, 

SB, DT, CT, TI, and TIIA are the chosen 

compounds. Chengdu Biopurify Phytochemicals 

Ltd of Chengdu, China, supplied the reference 

standards (with a purity level of >98%), and we 

used liquid chromatography-mass spectrometry 

(LC-MS) to confirm their accuracy.  

The reference compounds' standard stock 

solutions were made in methanol at a 

concentration of 2 mg/mL and kept at 4◦C for 

quick analysis or at —20◦C for later usage. For the 

UPLC calibration curve, a range of concentrations 

was produced, ranging from 0.1 to 2000 mg/mL 

for DSS, DT, CT, and TIIA, and from 1 to 1500 

mg/mL for SA, SB, and TI.  

Instrumentation and chromatographic conditions 



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2.1.1. Thin layer liquid chromatography 

3. Preparation for usage included cutting the 

silica gel 60 F254 TLC plates (20 x 20 cm) 

from Merck KGaA in Darmstadt, Germany, 

into 10 × 10 cm squares. Camag Chemie-

Linomat 5 automated applicator was used to 

apply the S. miltiorrhiza samples and 

standards (6 mL) on the plate. 

(Erzeugnisse & Adsorptionstechnik AG, 

Muttenz, Switzerland) using 100 mL syringes 

that were adjusted as follows: With an 8-

millimeter band width, 2-millimeter track 

spacing, and 8 tracks per plate. Ten 

millimeters below the TLC plate's bottom 

border was where the application was made. 

After that, the prepared mobile phase solution 

consisting of ethyl acetate, toluene, formic 

acid, and methanol (15:20:10:10:1) was added 

to a stainless steel lid on top of a CAMAG 

Twin Trough chamber (10 × 10 cm) that held 
around 6 mL of the plate.16 For a minimum of 

30 minutes at 20 ◦C, the mobile phase was 

applied vertically to the plate, starting from the 

bottom border and continuing up to 80 mm. 

The plate was allowed to air dry for 10 minutes 

after development prior to imaging. The TLC 

plates were examined using a Canon PSG × 

digital camera and a CAMAG Scanner 3 with 

366 nm light from CAMAG in Muttenz, 

Switzerland. Applying the winCATs ver.1.3.0 

system (CAMAG, Muttenz, Switzerland), the 

data and images were analyzed.  

3.1.1. Ultra-performance liquid chromatography 

4. The analysis was carried out utilizing a Waters 

ACQUITY UPLC system, manufactured by 

Waters in Milford, MA, which stands for ultra-

performance liquid chromatography 

combined with photodiode array. UPLC 

separations were carried out with the use of a 

pre-column (2.1 × 5 mm, 1.7 mm) from 

Waters in Milford, MA, connected to an 

ACQUITY UPLC BEH C18 column (150 × 

2.1 mm, 1.7 mm). Temperatures of 20 ◦C and 

4 ◦C were maintained for the column and 

sample, correspondingly. S. miltiorrhiza was 

cultured in mobile phase solutions that 

included 0.1 percent phosphoric acid.  

 

mixture of (A)-acetonitrile and (B). The 

gradient elution conditions were derived from 

a previously disclosed approach with the 

following modifications: 0–5 minutes, A 

90%–75%; 5–10 minutes, A 75%–50%; 10–

16 minutes, A 50%–20%; and 16–18 minutes, 

A 20%–90%.26 For a further two minutes, the 

column was isocratically reconditioned with 

90% A. A volume of 10 mL was injected at a 

flow rate of 0.3 mL/min. A wavelength of 280 

nm was used for detection.  

Using linearity and repeatability, the UPLC 

techniques were partly validated in accordance 

with the International Guidelines for Single 

Laboratory Validation of Chemical techniques 

for Dietary Supplements and Botanicals 

(AOAC) as outlined in the aforementioned 

publication.27 For the purpose of validating 

the standard curve calibration, we created over 

four concentrations of each standard 

component and evaluated the compound in 

triplicate at each concentration.  

The calibration regression was determined by 

plugging in the values of the reference 

samples' concentration (x) and the peak area 

(y), which were y ¼ ax + b. Using the formulas 

as follows: LOD = 3.33 × (standard deviation 

[SD] of y-intercept/mean of slope) and LOQ = 

10 × (SD of y intercept/mean of slope) from 

the repeated analyses, the UPLC techniques' 

limit of detection (LOD) and limit of 

quantification (LOQ) were determined.27 One 

way to quantify consistency was by looking at 

the relative standard deviation-RSD. Each 

marker compound's intra-day accuracy was 

tested by measuring four different 

concentrations three times in a single day, 

while the inter-day repeatability was tested 

over the course of three days in a row.  

 
4.1. Antioxidant activity assays 

 
4.1.1. ABTS antioxidant assay 

5. We used a standard protocol to determine the 

ABTS radical scavenging capabilities of the 



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raw and granule extracts of S. miltiorrhiza.28 

After 7 mmol/L of ABTS and 2.45 mmol/L of 

potassium persulfate were mixed in equal 

volumes, they were let to sit at room 

temperature for 12–16 hours in the dark to 

create the ABTS radical working solution. An 

initial absorbance value of 0.4 at 410 nm was 

achieved by diluting the stock solution with 

phosphate-buffered saline (PBS, pH 7.4) prior 

to analysis. After that, 20 mL of either the 

sample or the Trolox standard (0.045e0.330 

mmol/L) was mixed with 200 mL of diluted 

ABTS. Five minutes after mixing, the 

absorbance was measured at 410 nm using a 

microplate reader (BMG).  

6. Victoria, Australia is home to CLARIOstar. 

Measured in milligrams per gram of dry 

weight (mg/g of DW), the ABTS antioxidant 

activity was calculated.29  

 

 
6.1.1. DPPH antioxidant assay 

7. The DPPH test followed the protocol that had 

been previously detailed.30 Combine each 

sample with the same volume of DPPH radical 

solution (0.24 mg/mL DPPH in methanol), 

incubate for 30 minutes in the dark, and then 

measure absorbance at 515 nm. The 

experiment was conducted on a 96-well plate. 

As for the calibration curve, Trolox was used. 

Following the same procedure as for ABTS, 

we were able to acquire the data and the 

standard curve. 
 

7.1.1. FRAP antioxidant assay 

8. The FRAP (ferric ion reducing antioxidant 

power) test was carried out according to the 

methods previously detailed.30 The FRAP 

working solution was made by combining 10 

volumes of 300 mM acetate buffer (pH 3.6), 1 

volume of 20 mM ferric chloride 

(FeCl3$6H2O), and 1 volume of 10 mM 2,4,6-

tris(2-pyridyl)-s-triazine (TPTZ) in 40 mM 

HCl. After that, every sample was combined 

with the pre-heated FRAP solution on a 96-

well plate and let to sit at 37 ◦C for half an 

hour. Microplate readers (BMG CLARIOstar, 

Victoria, Australia) were used to measure the 

absorbance at 595 nm. The methodology used 

to generate the data and standard curve was 

same to that which was detailed for ABTS.  

 

 
8.1. Statistical analyses 

 

To compare the granule samples to the original 

herbal material, we divided their values by their 

concentrated ratio. This included yield, TLC and 

UPLC quantification, and antioxidant capacities. 

For example, a ratio of 1:5 would mean that 1 g of 

granule is equivalent to 5 g of the original raw 

material (refer to Supplemental Table 1), allowing 

us to draw comparisons.  

We used non-parametric tests in GraphPad Prism 8 

(GraphPad, San Diego, CA) or SPSS 20.0 (IBM 

Corp., Armonk, NY) to look for statistically 

significant differences in the data between the raw 

herbs and the granules. For AHC and PCA, the 

compounds that demonstrated significant 

differences were designated as variables. Ward's 

approach and Euclidean distances formed the basis 

of AHC analysis, which yielded data shown as 

dendrograms. The degree of similarity between 

analytes was indicated by the length of the 

branches. The original variables, which consisted 
of seven marker compounds, were transformed into 

a new set of linearly uncorrelated factors (PCs) that 

matched the original variables' maximum potential 

variance via the use of principal component 

analysis (PCA) in XLSTAT (Addinsoft, New 

York, NY). The PCA-generated biplot (scoring 

plot and loading plot) displayed the sample 

distribution according to the variables' and PCs' 

correlations.31 

Antioxidant capabilities vary significantly, and 

their Graph-Pad Prism 8 was used for non-

parametric analysis, whereas Pearson correlation 

was used for content correlation analysis with the 

marker molecules. The strength of the connection 

was shown by the Pearson correlation coefficients 



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(r), and the statistical significance was suggested 

by the P-value, which was fixed at P <.05.  

9. Results 

 
9.1. Quantification of the marker compounds 

 

The seven marker chemicals were identified and 

quantified using TLC and UPLC-PDA on both the 

raw and granule samples of S. miltiorrhiza. The 

seven marker compounds' molecular blueprints are 

shown forth in Fig. 1. The raw herb extract (R6) 

and granules of S. miltiorrhiza are shown in 

Supplemental Figs. 1e3, which exhibit 

representative TLC and UPLC fingerprints. 

Validation of the TLC and UPLC methods using 

linear regression equations, R2, LOQ, and LOD 

 

 

 

 

 
 

Fig. 1. Chemical structures of seven standard compounds in S. miltiorrhiza extracts using ACD/ChemSketch (Canada). 

Notes: The hydrophilic compounds include (A) DSS, (B) SB and (C) SA. Lipophilic compounds include (D) DT, (E) CT, (F) TI, (G)  TIIA. Their contents in the S. miltiorrhiza raw herb and 

granule samples were determined by TLC and UPLC analysis. In particular, SB and TIIA were used as marker compounds for the quality control of S. miltiorrhiza raw herb extract 

in the PPRC. DSS:sodium danshensu; SB: salvianolic acid B; SA: salvianolic acid; DT: dihydrotanshinone I; CT: cryptotanshinone; TI: tanshinone I; TIIA: tanshinone IIA. 

Table 1 displays them. Good linearity of the 

experimental data for the analytical procedures 

was shown by R2 values better than 0.988 (TLC) 

and 0.994 (UPLC) for all analytes. In terms of 

linear optical density (LOD), the seven marker 

chemicals were detected using UPLC with a 

range of 0.001–0.215 mg/mL and TLC with a 

range of 0.001–0.019 mg/mL. The 7 marker 

compounds have LOQs ranging from 0.003 to 

0.647 mg/mL for TLC and 0.002 to 0.056 



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mg/mL for UPLC. When compared to TLC, 

UPLC often had lower LOD and LOQ values, 

indicating that UPLC was more sensitive when it 

came to identifying and quantifying chemicals. 

The UPLC technique and instruments both 

produced very accurate results. The intra-day 

precision of TLC varied between 4.656% and 

21.251% and that of UPLC between 1.334% and 

3.732%, as indicated in Table 1. For TLC, the 

RSD for inter-day accuracy was 7.257% to 

27.536%, whereas for UPLC, it was 3.040% to 

7.065%. Accordingly, when compared with 

TLC, the UPLC technique and apparatus seem to 

have superior reproducibility for  

S. miltiorrhiza chemical analysis.  

Table 2 displays the contents of the marker 

chemicals that were found in the samples using 

TLC. Marker chemicals DSS and tanshinones 

(CT, DT, TI, and TIIA) were not adequately 

quantified independently for certain samples due 

to insufficient TLC resolution (Supplemental 

Fig. 1). The amounts of SA and SB in the 

granules were much lower than what was found 

in the samples of raw herbs. SB and SA were the 

two most abundant compounds in the raw and 

granule samples, respectively. The raw herb 

samples often showed negligible or non-existent 

levels of tanshinones, including CT, DT, TI, and 

TIIA. The amounts of all the chemicals 

examined did not differ substantially between 

the raw and granule samples, according to the 

non-parametric t-test (P >.05). The raw sample 

group had a considerably greater SB than the 

granule sample group (P <.0001).  

See Table 2 for a list of the marker chemicals 

found in the UPLC-analyzed raw and granule 

samples. Repeatability in the quantification was 

shown by the low standard deviations of the 

seven standards in the raw herbs and granules. 

The S. miltiorrhiza samples showed that SB and 

SA were the two most abundant compounds, in 

agreement with the TLC quantification findings. 

The amounts of all the chemicals examined did 

not differ substantially between the raw and 

granule samples, according to the non-

parametric t-test (P >.05). Still, a non-parametric 

independent t-test revealed that, when 

comparing the two groups, the raw samples 

included considerably more DSS, SB, and CT 

than the granule samples (P =.010, 0.000, and 

0.000, respectively).  

8.2 AHC and PCA multivariate analysis  

The quantification findings from UPLC were 

submitted to AHC utilizing all 7 marker 

chemicals as variables as UPLC demonstrated 

improved sensitivity and capacity to quantify the 

marker compounds in S. miltiorrhiza samples. 

The most striking contrast, as illustrated in 

Figure 2A, was found between the cluster 

containing all twelve granules and two raw herbs 

(R2 and R4) and the cluster containing four raw 

samples of S. miltiorrhiza [R1, R3 (both 

decoction pieces), R5 and R6]. This is because 

the four raw samples contained a substantially 

higher concentration of the seven marker 

compounds when contrasted with the twelve 

granule samples. The granule samples clustered 

with R2 and R4 because their concentrations of 

the seven chemicals were similar. From most to 

least, the clusters might be further subdivided 

into several groups based on  



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Fig. 2. AHC dendrograms and PCA biplot of S. miltiorrhiza extracts analyzed by UPLC. 

Notes: A: AHC dendrograms for the UPLC results divided the raw herb and granule extracts into 2 main clusters, with raw herb samples R1, R3, R5 and R6 classified into one cluster, 

whereas R2 and R4 were grouped with the granules. B: PCA biplot (loading and score plot) of S. miltiorrhiza raw herb and granule extracts as analyzed by UPLC. From the UPLC 

biplot, raw samples (R5 and R6) demonstrated relatively higher amount of DT, TI, TI IA, SB, CT and DSS (cluster 1), especially R6 (highest amount). The content of these compounds in 

R1 and R3 were slightly lower. In contrast, all the granule samples plus R2 and R4 were closely distributed due to lower amou nt of these marker compounds (cluster 2). F1 

represents 77.95% of the total variance from the 7 marker compounds and F2 (17.11%) cumulatively explains up to 95.06% of tot al variance. AHC: agglomerative hierarchical 

clustering; PCA: principal component analysis; UPLC: ultra-performance liquid chromatography. 

57.74 mg/g DW, which were much higher than 

that of granule samples (4.37e9.57 mg/g DW). 

R6 exhibited the highest antioxi- dant activity 

among all the samples (87.33 and 57.74 mg/g 

DW) in both the ABTS and DPPH assays. In the 

FRAP assay (Fig. 3C), the optical density (O.D.) 

at 590 nm of the raw herbs/decoction pieces 

and granules ranged from 1.06 to 1.69 and 0.08 

to 0.20, respectively. The non-parametric 

analysis in Fig. 3D showed that the antioxidant 

capacities of the raw herbs (as one group) were 

significantly higher than that of the granule 

group (P < .0001) in all 3 assays. 
Pearson correlation examined the relationship between the 

content of the marker compounds and radical scavenging 

capac- ities. There were significant correlations between the 

UPLC results for the marker contents of DSS, SB and CT to the 

3 antioxidant ac- tivities (Table 3). R6 possessed the highest 

amount of these marker compounds and thus showed the 

highest ABTS and DPPH scav- 

enging capacities. In contrast, the amount of DT was negatively 

correlated with ABTS, DPPH and FRAP assays (—0.343, —

0.283 and —0.293) without any significance (P > .05). The results 
showed 
that the compounds SA, TI and TIIA did not contribute to the 

antioxidant activities of the raw and granule samples. 

 
10. Discussion 

 

There have been many attempts worldwide at developing 

rapid techniques and methods for analyzing the chemical 

variability of herbal granule products using TLC, high-

performance liquid chro- matography, UPLC, and using new 

techniques such as FT-NIR spectroscopy.32e34 However, it is 

important to note that industry requires simple, accessible and 

inexpensive methods to examine the quality control of their 

manufactured products. TLC has been used extensively as an 

initial tool for the identification and semi- quantitative analysis 

of herbal products due to its simple and inexpensive set up.35 

In the PPRC and herbal monographs, TLC analysis is highly 

recommended for the identification and quality control of S. 

miltiorrhiza samples.16,36 HPLC is the standard chro- 

matography methods used by industry for the quality control 

ofherbal products. However, UPLC is preferred over HPLC for 

the accurate quantification of marker compounds in herbal 

products due to its higher sensitivity and resolution.35 In our 

previous studies, we have shown that the content of the marker 

compounds and antioxidant capacities using UPLC and/or TLC 

were generally lower in the granule form compared to that of 

the raw herbs in Sanchi (Notoginseng Radix et Rhizoma) and 

Chinese Angelica (An- gelica Sinensis Radix) which raises 

concern for the quality control and efficacy of herbal granule 

products.6,7 In 2009, Song et al compared the amount of 8 major 

components in S. miltiorrhiza granules with raw extracts using 

a HPLC system, and their results showed that the average 

content of the total components were similar to that in aqueous 

extracts of S. miltiorrhiza.37 However, the equivalent ratio of 

granule was not taken into account for the calculation, and the 

bioactivities were not conducted. The present study assesses 

the quality and efficacy of granule formulations of another 

valuable Chinese herbal medicine, S. miltiorrhiza. By using 

advanced UPLC system, antioxidant assays and multi-variant 

analysis, our data suggests that granules presented with a lower 

quality and antioxidant activity compared with their herbal 

coun- terparts when compared with the dried weight of the 



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herb. 
Twelve S. miltiorrhiza. granules and 6 raw samples 
were 

collected from various sources and examined in this study. The 

raw samples were decocted with water (to reflect traditional 

con- sumption) and then extracted with methanol, whilst the 

granule samples (which were manufactured extracts) were 

extracted with methanol only to remove the water-soluble 

excipients. This is because herbal granule products are 

assumed to be formed from a concentrated water decoction. 

Based on the standard manufacturing process, granules are 

marketed as the concentrated herbal extract with added 

excipients. The labelled ratio (e.g. 1 g of the granule is 

equivalent to 5 g of the crude material etc.) is essential for 

correct dosing. Thus, the results generated from granule 

samples were divided by their concentration ratio as indicated 

on the product label so that the granule was compared with 

the raw sample at the same baseline. By comparing the 

 
 

Fig. 3. ABTS, DPPH and FRAP activities of S. miltiorrhiza raw herb and granule extracts. 

Notes: A: Raw herb samples (25.95e87.33 mg/g DW) showed a higher ABTS radical scavenging activity compared with granule samples (5.38e15.67 mg/g DW). R6 exhibited 

the highest ABTS antioxidant activity among all the samples (87.33 ± 9.97 mg/g DW). B: Raw herb samples (24.71e57.74 mg/g DW) showed a higher DPPH radical scavenging 

activity compared with granule samples (4.37e9.57 mg/g DW). R6 exhibited the highest DPPH antioxidant activity among all the samples (57.74 ± 0.09 mg/g DW). C: At 590 nm, 

raw herb samples (1.06e1.69) showed a higher FRAP radical scavenging activity than granule samples (0.08e0.20). R1 exhibited the highest FRAP antioxidant activity among all 

the samples (1.69 ± 0.04). D: Group comparison between raw and granule samples in ABTS, DPPH and FRAP assays. ****P < .0001 vs. raw group. n = 3. ABTS: 2,2'-azino-bis 

(3- ethylbenzothiazoline-6-sulfonic acid; DPPH: 2,2-diphenyl-1-picrylhydrazyl; FRAP: ferric ion reducing antioxidant power. 



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extraction yield between groups, the raw herbs were generally 

higher than the granules group. The different extraction yield in 

the granule products could be caused by the different 

manufacturing processes used to produce the granules. The 

excipients added to the granules during manufacturing may be 

soluble in methanol and this may affect the final calculation of 

the content of the granule product. Upon a closer inspection, 

granulation was generally inconsistent for each product 

examined and thus, smaller particles 

 

 
Table 3 

Pearson correlation between the 3 chemical markers and antioxidant activities. 
 

Assay Markers  

 DSS SB SA DT CT TI TIIA  

ABTS 0.774** 0.965** 0.202 —0.343 0.886** 0.340 0.231  

DPPH 0.730** 0.934** 0.184 —0.283 0.936** 0.347 0.272  

FRAP 0.665** 0.844** 0.083 —0.293 0.890** 0.320 0.267  

Notes: The r value (between —1 and +1) as analyzed by the Pearson correlation 

represents the correlation coefficient, where an r value < 0 refers to a negative 

correlation, and an r value > 0 refers to a positive correlation. **P < .01 refers 
to a 

significant correlation (either positive or negative). ABTS: 2,2'-azino-bis 

(3- 

ethylbenzothiazoline-6-sulfonic acid; DPPH: 2,2-diphenyl-1-picrylhydrazyl; 

FRAP: ferric ion reducing antioxidant power; DSS: sodium danshensu; SB: 

salvianolic acid B; SA: salvianolic acid; DT: dihydrotanshinone I; CT: 

cryptotanshinone; TI: tan- shinone I; TIIA: tanshinone IIA. 

(which have greater surface area for 

extraction) may find them- selves at the 

bottom of the container. Manufacturing 

differences such as extraction with solvents 

other than water, temperature/ pressure 

conditions, the type and quantity of excipients 

used and adulteration can affect the quality of 

the finished product. 
It is well-recognized that the quality control of medicinal 

plant products is the foundation for their development and 

acceptance in integrative medicine.38 The complex nature of 

plants, environ- mental influences and poor-quality control 

processes bring great challenge  for  the  quality  control  

of  the  herbal  products. 

S. miltiorrhiza has a complex chemical composition which 

includes 2 major groups of chemicals: salvianolic acids 

(hydrophilic) and tanshinones (hydrophobic). Based on the 

TLC method documented in the PPRC, we have further 

optimized the condition and mobile phase, and identified 6 

compounds presented in the S. miltiorrhiza raw and granule 

samples. Unfortunately, DSS was not detected due to its 

extremely hydrophilic property and thus, could not move with 

the mobile phase. This may limit the single TLC method (as 

used in this study) for establishing the herb's complete 

profile.39 In addition, the optimized TLC method was partially 

validated in terms of linearity, LOD, LOQ and precision and 

compared to UPLC. In agreement with our previous studies, the 

relatively large RSD value for precision reflected the drawbacks 

of using TLC for quantification purposes as the resolution of 

TLC is easily affected by the 

 
temperature, humidity, mobile phase condition, etc.6,7 

Moreover, we noticed that TLC failed to quantify the lipid-

soluble compounds of tanshonines which may be due to their 

minor amounts in the extracts and the sensitivity limit of the 

TLC method. Here, a vali- dated UPLC method was employed to 

quantify S. miltiorrhiza marker compounds. The significantly 

lower values of LOD, LOQ and RSD suggested good instrumental 

and method precision for UPLC compared with TLC. In 

agreement with literature, SB was found to be the most 

abundant compound in S. miltiorrhiza raw and granule 

samples.40 The amounts of SB were significantly higher in the 

raw samples compared to that of the granule sample. However, 

the PPRC marker compound, TIIA, did not show significant 

difference between the 2 main sample groups which is 

consistent with its hydrophobicity in water. Although TLC and 

UPLC had differing quantification results, the trend was similar, 

with SB significantly higher in the raw herb groups compared 

with that of the granule group. Thus, our results showed that 

TLC is not as accurate and efficient as UPLC for quantitative 

purposes. However, it can be used as a qualitative tool for a 

quick scan of the bioactive compounds in the herbal extract. 
We then examined the similarities of the products 
using 

multivariate analysis. Using the 7 marker compounds as 

variables, AHC analysis showed that most of the raw samples 

were grouped in the same cluster, except for R2 and R4 (raw 

herb samples) which were classified into the granule cluster 

due to its lower amount of marker compounds. This reflects the 

lower content of components in the starting raw material 

which may influence the granule quality.41,42 This could be a 

primary reason for S. miltiorrhiza's inconsistency which would 

subsequently affect the quality of the manufactured granule, 

especially if the raw materials are collected from different 

sources (i.e not the recommended growing areas as per PPRC) 

or not at the optimal time of the year. These results are in line 

with our previous comparative studies on Notoginseng and 

Angelica Sinensis which showed comparable amounts of marker 

compounds in the raw herbal samples, whereas the contents of 

marker compounds in the granule samples were significantly 

lower and possessed large variance.6,7 It is interesting to note 

that the 
S. miltiorrhiza decoction pieces (R1 and R3) showed 
comparable 

amounts of all the marker compounds as in most raw herb 

mate- rials. This corresponded to our previous study where the 

Angelica Sinensis decoction pieces were consistent in 

composition and showed comparable amounts of the marker 

compounds to the raw herbs.7 Decoction pieces are processed 

raw materials (i.e., washed, fried, cut, dried as per TCM 

practice), and can be applied directly to clinical treatment. 

Thus, they should present a similar quality as for the raw 

material. However, it is reiterated that the quality man- 

agement of decoction pieces needs to be strengthened, as the 

processing mechanism remains unclear and the quality control 

not standardized.43 

The AHC analysis showed that SB and TIIA are the key com- 

pounds that differentiate the raw and granule samples which 

support the PPRC. Moreover, SB, the most abundant compound, 

played a predominant role amongst the tested compounds. 

How- ever, PCA analysis suggested that the other 3 tanshinones 

also contributed to the differentiation of raw and granule 

samples. Moreover, the AHC display of samples using individual 



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2023 | Vol 6 | Issue 6 
 

 
 

compounds showed distinct dendrogram (excluding SB to 

TIIA), suggesting that more compounds (i.e. CT, DT and TI) may 

need to be considered for the quality control of S. miltiorrhiza 

raw and granule samples. 

Although TIIA and SB are listed as the key marker compound 

for the quality control of S. miltiorrhiza in the PPRC, it is noted that 

the quantity of SB and TIIA in granules were all below the standard 

in PPRC.16 For S. miltiorrhiza, methanol is used as the solvent in 

the PPRC  and  this  would  favor  higher  extraction yields  

of  the 

compounds. However, it is suggested that the quality control 

standard for the herbal water decoction should be considered 

for inclusion in the PPRC or relevant regulation standard to 

honor the tradition method of preparing herbs for medicinal 

use. 

SB is the most abundant and bioactive compound found 
in 

S. miltiorrhiza and is known for its potent antioxidative and 

reactive oxygen species scavenging activity which is 

attributed to its poly- phenolic structure.40 Thus, S. miltiorrhiza 

has been extensively used clinically as a principal herb in TCM 

for cardiovascular diseases.44 Tanshinones are a hydrophobic 

group of compounds isolated from S. miltiorrhiza, and 

emerging experimental and clinical in- vestigations have 

supported their pharmacological activities in preventing or 

slowing the progression of a wide variety of diseases due to 

their potent antioxidant, anti-inflammatory and anti-cancer 

activities.45 Our study assessed the antioxidant activity of 

the 

S. miltiorrhiza raw and granule products and correlated their 

ac- tivity to the content of the key bioactive compounds. The 

results show that the significantly lower amounts of SB, DSS 

and CT (relatively more polar compounds) in the granule 

samples not only contributed to differentiating the granule 

samples from raw sam- ples in the PCA analysis, but also lead 

to significantly lower ABTS, DPPH and FRAP scavenging 

activity. This was evidenced by the positive and significant 

correlation. It was observed that marker compound TIIA also 

played a minor role in discerning the antioxi- dant activities of 

the S. miltiorrhiza raw and granule samples but was not 

significantly correlated to the overall antioxidant activity of the 

extract (P > .05). Isolated TIIA has been recognized as a phar- 

macological active compound with various promising health 

ben- efits including antioxidant, anti-inflammatory and anti-

cancer. Here, TIIA did not contribute to the antioxidant activity 

which was attributed to its minimal presence in the S. 

miltiorrhiza water extract.46 The different antioxidant 

activities in the raw and granule samples can also be attributed 

to other minor bioactives that were not included in this study 

such as rosmarinic acid, rosmarinic acid methyl ester and 

rosmarinic acid ethyl ester.47 

 
11. Conclusions 

 
The present study assessed the quality and bioactivity 

differ- ences between S. miltiorrhiza raw and granule products 

using TLC and UPLC coupled with multivariate analysis, and 

antioxidant as- says. UPLC proved to be a better differentiator 

of the marker com- pounds between samples. It was revealed 

that the content of the marker compounds (SB, DSS, CT) was 

significantly lower in the granule samples compared with that 

of the raw samples which led to the lower antioxidant activity 

of the granule. It has been recog- nized that the inconsistent 

amount of bioactive compounds in herbal products is likely to 

induce fluctuated levels of therapeutic effects, which 

represent one of the major concerns for the clinical 

effectiveness of herbal products, including granule 

formulations.1 With the gaining popularity of herbal medicinal 

granules around the world, this study provides important 

scientific evidence for standardization committees, industry, 

practitioners and consumers on the quality control and 

efficacy assessment of herbs and its related granule products. 

It is vital for healthcare professionals to be aware of granule 

quality differences and know the correct dos- ages to match 

the contents of traditional decoctions. We believe this study 

provides a rational argument for the continued investi- gation 

of the quality and efficacy assessment of the granular form of 

medicinal plants. More rigorous pharmacological, toxicological 

and clinical studies using granules compared to herbal extract 

water decoctions are warranted to confirm these findings and 

to advocate high-standard, safe and efficacious herbal 

preparations to the consumers. 
 

Funding 

 
The project was supported by the Joint Chair in Traditional 

Chinese Medicine (JCTCM) Program, funded by the Office of 

Science and Research in NSW, the University of Sydney and 

Western Sydney University, Australia. The labor cost of Xian 

Zhou was supported by the International Postgraduate 

Research Scholarship, Western Sydney University and the 

Linkage Project from the Australian Research Council (ARC) grant 

(LP160101594). Her salary is currently supported by Research 

Support Program Fellowship, Western Sydney University. The 

general consumables needed for this project was supported by 

Western Sydney University Research Grant Scheme 

(P00021202). 

 
CRediT authorship contribution statement 

 
Xian Zhou: Methodology, validation, formal analysis, investi- 

gation, resources, writing ‒ original draft, writing ‒ review & 

editing.  Valentina  Razmovski-Naumovski:  

Conceptualization, 

supervision, methodology, validation, resources, writing ‒ 

original draft, writing ‒ review & editing, project 
administration. Raynold Mendoza: Methodology, investigation. 
John Truong: Methodology, 

formal analysis. Kelvin Chan: Conceptualization, supervision, 

methodology, resources, writing ‒ review & editing, project 
administration. 

 
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