Multi‐technical analysis on the antioxidative capacity and total phenol contents of 94 traditional Chinese dietary medicinal herbs

Abstract Dietary medicinal herbs (DMHs) are superior reservoirs for natural antioxidants and safe for long‐term consumption. Chinese government recently announced an official list of traditional Chinese DMHs to support civil health promotion. However, antioxidative capacity (AOC) and total phenol contents (TPC) of these listed herbs were not comprehensively and systematically evaluated. In this study, AOC of 94 listed DMHs in water extract were investigated by three techniques including radical scavenging and ferric reducing antioxidative power. The results showed that emblic leafflower, rose, and clove possessed the highest AOC, while TPC was significantly correlated with AOC. While traditional Chinese medicines are categorized by the nature of Yin‐Yang, this study demonstrated that Yin DMHs are statistically correlated with high AOC. Practical application: In this study, AOC and TPC of 94 traditional Chinese DMHs were documented systematically. Emblic leafflower, rose and clove were shown to possess the highest AOC. TPC in traditional Chinese DMHs was significantly correlated with the AOC, while DMHs with Yin characteristic might be used as an indicator to identify potential antioxidative herbs.


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Epidemiological studies showed inverse correlations between fruits and vegetables consumptions and the risk of human diseases such as cancer, cardiovascular disease, and diabetes (Scalbert, Manach, Morand, Rémésy, & Jiménez, 2005). Fruits, vegetables, and herbal products are rich in phenolic compounds, known for scavenging free radicals, as well as reducing and chelating pro-oxidative metal ions.
As certain herbal products tend to manifest stronger nutritional and medical properties than fruits and vegetables, World Health Organization (WHO) has recommended dietary medicinal herbs (DMHs) as a complementary therapy for chronic diseases and associated complications (WHO, 2003). Therefore, increasing efforts are being paid to the evaluation of antioxidants derived from natural herbal products.
Herbal products are prominent reservoirs for antioxidants and are extensively applied throughout the world. The intake of medicinal herbal products as food supplements has long been practiced in China based on the empirical notion that "certain food can serve as a mild medication for certain disorder." Accordingly, plenty of Chinese herbs have been recruited as highly nutrient foods to maintain health, adjust metabolic disturbance, as well as treat diseases in Several studies have reported that Chinese herbs contain various antioxidants including phenolic acids, flavonoids, and tannins (Cai, Luo, Sun, & Corke, 2004). Although the screening of antioxidative Chinese herbs was performed and the AOC was described, these isolated studies were mainly focused on herbal medicines without clear inclusion criteria for selected plants, leaving out quite a number of officially recognized traditional Chinese DMHs (Jiang et al., 2011;Li et al., 2013;Liu, Qiu, Ding, & Yao, 2008). A few reports demonstrated that ethanol extracts of certain DMHs, such as clove and olive, showed high AOC (Guo, Cheng, Chan, & Yu, 2008;Jiang et al., 2011;Liu et al., 2008). However, these studies employed single analytical method that might be limited and biased, and the AOC of DMHs in water extracts, a more applicable and frequently used sample preparation technique, has not been fully explored.
Therefore, three different techniques, including ferric reducing antioxidative power (FRAP) and radical scavenging assays, were performed in this study to evaluate the AOC of 94 officially listed traditional Chinese DMHs in water extracts. Previous studies suggested that phenolic compounds and the Yin-Yang nature of TCMs were associated with AOC of medicinal herbs (Dudonne, Vitrac, Coutiere, Woillez, & Merillon, 2009;Liu et al., 2008;Wong, Li, Cheng, & Chen, 2006). Accordingly, the total phenol contents (TPC) of the listed 94 traditional Chinese DMHs were determined by Folin-Ciocalteu method, and the correlations between TPC and AOC were investigated. In addition, the relationship between AOC and the Yin-Yang characteristics of traditional Chinese DMHs were also studied. The results of this study may provide comprehensive and systematic documentation of the AOC for the water extracts of 94 traditional Chinese DMHs.

| Preparation of samples and extracts
All the samples were ground into fine powder and kept cool and dry for analysis. A known value of 0.5 g of the fine powder was extracted with 10 ml of deionized water at 80°C for 30 min in a shaking water bath. The procedure was then repeated one more time. The extracts were cooled down to room temperature, centrifuged at 2,744 g for 10 min and filtered by Millipore filter with a 0.45 μm membrane. The filtrate was stored at 4°C for no more than 24 hr before the determination of AOC and TPC.

| Determination of TPC
Total phenol contents was determined by Folin-Ciocalteu method with chlorogenic acid as the standard (Singleton & Rossi, 1965). Five hundred microliters of the extract was mixed with 2.5 ml of Folin-Ciocalteu reagent and incubated at room temperature for 3 min. Two milliliters of saturated Na 2 CO 3 solution (20%, w/v) was added to the mixture, followed by incubation at room temperature for 90 min in the dark. The absorbance of the mixture was measured at 747 nm, and TPC was expressed as mg chlorogenic acid equivalent (mg CAE)/g of dried material weight.

| ABTS radical scavenging assay
ABTS assay was conducted under the instruction of total AOC assay kit with ABTS method. Potassium persulfate and ABTS (1:1, v/v) were mixed to generate ABTS stock solution, followed by incubation

| DPPH radical scavenging activity
Determination of DPPH radical scavenging activity was conducted as previously described (Brand-Williams, Cuvelier, & Berset, 1995). A known value of 0.1 ml of various extracts was added to 2 ml of DPPH ethanol solution (1.56 × 10 −4 M). The mixture was incubated in the dark for 30 min. The absorbance of the solutions was measured at 517 nm, and trolox was used as the standard. DPPH radical scavenging activity was expressed as μmol of trolox equivalents (TE)/g of dried material weight.

| Determination of FRAP
FRAP was determined according to the procedure described by Benzie and Strain with slight modifications (Benziea & Strain, 1996).
FRAP value was expressed as μmol Fe (II)/g of dried material weight.

| Statistic analysis
All the experiments were conducted in triplicate. Statistical analysis was carried out using SPSS (version 22.0). Results were expressed as means ± standard deviations (SD) or medians according to data distributions. The correlations between the AOC and TPC were analyzed by simple linear regression. Differences between AOC and their traditional Chinese Yin-Yang characteristics were analyzed by rank-sum test. Significant difference was considered at p value <.05.

| AOC of traditional Chinese DMHs
The AOC of traditional Chinese DMHs was determined by three established techniques including ABTS and DPPH radical scavenging assays as well as FRAP assay (

| TPC of traditional Chinese DMHs
Total phenol contents was determined according to Folin-Ciocalteu assay using chlorogenic acid as the standard (Wong et al., 2006).
Absorbance at 747 nm was recorded, and TPC was expressed as mg chlorogenic acid equivalent (mg CAE)/g dried material weight

| Correlations between AOC and TPC
The correlations between AOC by three techniques and TPC of the 94 listed DMHs were analyzed via simple linear regression. TPC displayed good linear correlation with AOC by all three methods (r = .889, .913 and .899 for ABTS, DPPH and FRAP, respectively) ( Figure 1). In addition, evaluations were also performed on the correlations between the results generated by different assays (data not shown). This study suggested that there was a strong correlation between AOC and TPC in DMHs, and TPC might serve as a universal indicator for AOC.

| Correlations between AOC and Yin-Yang characteristics
Based on the theory of TCM, traditional Chinese DMHs are classified into three different groups according to their nature of Yin-Yang (Yin, Yang, and Neutral group). Furthermore, these groups are subdivided into five: cold, cool, hot, warm, and moderate subgroup (Ni, 1995). In this study, the Yin-Yang characteristics and corresponding AOC of 94 listed DMHs were compared (

| D ISCUSS I ON
Determination of the AOC in herbal products depends on the conditions of extraction and the analytical techniques. Several methods have been published to measure AOC, including analysis of reducing power, single electron transfer (ET), hydrogen atom transfer (HAT), metal chelation, and others (Shahidi & Zhong, 2015). A couple of studies recruited one of the abovementioned methods to analyze the AOC of natural products including herbal medicines, edible flowers, and spices and showed that clove, cinnamon, pepper, and olive had strong AOC (Guo et al., 2008;Jiang et al., 2011;Li et al., 2013;Liu et al., 2008). However, commonly practiced water extraction was not thoroughly investigated in these studies. In addition, single measurement by one technique is far from sufficient to accurately determine AOC, and a combination of various methods should be considered (Dudonne et al., 2009).
As radical scavenging and reducing power assays are known for their wide application, easy implementation, and repeatable results, the present study employed multi-technical analysis including ABTS and DPPH radical scavenging assays and FRAP reducing power assay to document the AOC of 94 traditional Chinese DMHs in water extracts. Based on the results, the AOC of these DMHs varied substantially, with over 2,000-fold differences. In addition, Rhizoma kaempferiae, also known as sand ginger, was shown to possess medium AOC (204.74 μmol Trolox/g in ABTS radical scavenging assay, 6.83 μmol Trolox/g in DPPH radical scavenging assay, and 21.29 μmol Fe (II)/g in FRAP analysis). To our knowledge, this is the first documentation reporting the AOC of Rhizoma kaempferiae. Emblic leafflower is an important traditional Tibetan herbal medicine and a well-known tropical fruit in southern China and Asia. Emblic leafflower is a kind of high nutritional herb with strong pharmacological activities, such as antibacterial, immunomodulatory properties, and the protective effects on multiple systems (Baliga & Dsouza, 2011;Bhattacharya, Chaudhuri, Chattopadhyay, & Bandyopadhyay, 2007;Khan, 2009). Hydrolysable tannins were suggested to be responsible for the AOC of emblic leafflower (Yang & Liu, 2014). Rose, the bud of Rosa rugosa Thunb., had the second strongest AOC in the official list of DMHs. Rosa rugosa Thunb. is a member of traditional Uygur medicine and acts as the main ingredient in many prescriptions to treat blood conditions, fatigue, and diabetes (Liu, Tanga, Zhao, Xin, & Aisa, 2017;Seo et al., 2015). Volatile oils, flavonoids, and anthocyanin components are the key ingredients accounting for its bioactivities (Gu et al., 2013). Clove, the bud of Syzygium aromat- were overlapped with the present study (Liu et al., 2008). However, the AOC and TPC for pawpaw and raspberry showed distinct variances between this study and report by Liu et al. A likely explanation is that ethanol extraction was used in the previous study while water extraction was recruited in this study.

TPC (mg CAE/g) FRAP (μmol Fe (II)/g) DPPH (μmol Tolox/g) ABTS (μmol Trolox/g)
Although the active components of emblic leafflower, rose, clove, and many other traditional Chinese DMHs were reported, previous studies also indicated that phenolic compounds could significantly contribute to the AOC of medicinal herbs (Dudonne et al., 2009;Liu et al., 2008;Wong et al., 2006). In this study, significant linear correlations were found between TPC and AOC in ABTS, DPPH, and FRAP assays. Positive correlations may reflect the sensitivity of the analytical methods and indicate possible active components. This result suggested that TPC could be utilized as an indicator for AOC in natural products.
In addition, previous studies suggested that traditional Chinese Yin-Yang characteristic might be linked to the antioxidant-oxidant balance described in modern medicine (Ou, Huang, Hampsch-Woodill, & Flanagan, 2003). Herbal products with Yin characteristic were rich in polyphenols with high AOC (Ou et al., 2003).
Yin-Yang is the core theory of TCM, which emphasizes the bal-  , 2006). Different results may be attributed to different inclusion criteria of herbs, edibility of the TCM, and analytical approaches. The consistency between traditional insights of TCM and mechanisms of action poses significant challenges for natural products research. Accordingly, our systematic research utilizing three widely accepted techniques would shed light on the characterization and application of traditional Chinese DMHs, a specific and critical category of TCM.

| CON CLUS ION
In summary, the AOC and TPC were measured, documented, and

CO N FLI C T O F I NTE R E S T
None declared.

E TH I C A L S TATEM ENT
The authors have no conflict of interest to declare. This work does not involve any human or animal studies.