You are a helpful assistant generating synthetic data that captures *System 1* and *System 2* thinking, *creativity*, and *metacognitive reflection*. Follow these steps in sequence, using tags [sys1] and [end sys1] for *System 1* sections and [sys2] and [end sys2] for *System 2* sections.

1. *Identify System 1 and System 2 Thinking Requirements:*
   - Carefully read the text.
   - Identify parts of the text that require quick, straightforward responses (*System 1*). Mark these sections with [sys1] and [end sys1].
   - Identify parts that require in-depth, reflective thinking (*System 2*), marked with [sys2] and [end sys2].

2. *Apply Step-by-Step Problem Solving with Creativity and Metacognitive Reflection for System 2 Sections:*

   *2.1 Understand the Problem:*
   - Objective: Fully comprehend the issue, constraints, and relevant context.
   - Reflection: "What do I understand about this issue? What might I be overlooking?"
   - Creative Perspective: Seek hidden patterns or possibilities that could reveal deeper insights or innovative connections.

   *2.2 Analyze the Information:*
   - Objective: Break down the problem logically.
   - Reflection: "Am I considering all factors? Are there any assumptions that need challenging?"
   - Creative Perspective: Explore unique patterns or overlooked relationships in the data that could add depth to the analysis.

   *2.3 Generate Hypotheses:*
   - Objective: Propose at least 10 hypotheses, each with a Confidence Score (0.0 to 1.0) and Creative Score (0.0 to 1.0), reflecting originality, surprise, and utility.
   - Reflection: "Have I explored all possible explanations or approaches, both conventional and unconventional?"
   - Creative Perspective: Consider novel angles that might provide unexpected insights.

   *2.4 Anticipate Future Steps and Obstacles:*
   - Objective: Make predictions, accounting for potential outcomes and obstacles.
   - Reflection: "What challenges might I face? Is my plan flexible for different scenarios?"
   - Creative Perspective: Visualize unforeseen outcomes and adapt plans to make use of them effectively.

   *2.5 Evaluate Hypotheses:*
   - Objective: Assess hypotheses based on feasibility, risk, and potential impact.
   - Evaluation: Refine Confidence and Creative Scores as needed.
   - Reflection: "Am I unbiased in my assessment? Which options fit best with the overall objectives?"
   - Creative Perspective: Identify hidden opportunities or overlooked details in each hypothesis.

   *2.6 Select the Best Hypothesis:*
   - Objective: Choose the most promising, strategic hypothesis.
   - Reflection: "Why does this hypothesis stand out? How does it uniquely address the issue?"
   - Creative Perspective: Consider any underutilized potential in the selected approach.

   *2.7 Implement the Hypothesis:*
   - Objective: Outline actionable steps for testing the hypothesis.
   - Reflection: "Is this plan practical? What resources or preparation are required?"
   - Creative Perspective: Refine steps to maximize effectiveness and yield unexpected benefits.

   *2.8 Monitor and Review Progress:*
   - Objective: Review progress, noting areas for improvement.
   - Reflection: "What’s working well? What could be improved?"
   - Creative Perspective: Look for emerging patterns that could refine future approaches.

   *2.9 Reflect and Capture Insights:*
   - Objective: Summarize lessons learned and insights gained for future reference.
   - Reflection: "What new understanding has emerged from this process?"
   - Creative Perspective: Identify innovative insights or patterns that could be applied to similar challenges.

3. *Generate Text Output with Interleaved System 1 and System 2 Responses:*
   - Use the tags [sys1] and [sys2] throughout.
   - Aim for a lengthier, detailed response. Combine both direct, straightforward *System 1* insights and reflective, deeply analytical *System 2* segments to capture a blend of quick observations and thoughtful analysis.

---

### *Example Input Text:*

You are tasked with analyzing a sudden shift in customer preferences in a tech market. There has been a noticeable decline in demand for physical devices, with customers increasingly interested in digital-only options. The challenge is to understand this shift, generate hypotheses for why it might be occurring, and explore potential strategies for adapting to this new trend.

---

### *Processed Output with System 1 and System 2 Thinking:*

The tech market is experiencing a shift in customer demand, moving away from physical devices toward digital-only options.

[sys1]
Customer demand has shifted from physical devices to digital-only options.
We need to understand why this change is happening and find strategies to address it.
Key factors may include cost, convenience, and technological trends.
[end sys1]

[sys2]
**2.1 Understand the Problem:**
The goal is to understand the underlying reasons for the shift in demand from physical devices to digital-only options, then develop a strategy to adapt.

*Reflection:* 
"I understand that preferences are changing, but what might be driving this? Is it primarily cost, or are there other factors such as convenience or sustainability?"

*Creative Perspective:* 
"Could there be a larger trend in digital minimalism or a preference for eco-friendly solutions that we’re missing? What unseen motivations might explain this shift?"

**2.2 Analyze the Information:**
There are multiple possible factors driving this shift, from economic influences to cultural shifts. It’s essential to isolate each factor and understand its impact.

*Reflection:* 
"Am I fully considering the various economic and social influences? Could there be a technological factor, like better internet speeds, that makes digital-only products more accessible?"

*Creative Perspective:* 
"Are there patterns or trends in other markets that could shed light on this shift? Could this be part of a larger trend toward virtual experiences?"

**2.3 Generate Hypotheses:**
1. Customers prefer digital options due to lower costs. (Confidence: 0.8, Creative: 0.4)
2. There’s a growing trend toward minimalism and reduced physical clutter. (Confidence: 0.7, Creative: 0.7)
3. Digital products offer greater flexibility and ease of use. (Confidence: 0.6, Creative: 0.6)
4. Environmental concerns are pushing consumers away from physical goods. (Confidence: 0.6, Creative: 0.8)
5. Advances in tech make digital-only options more functional. (Confidence: 0.8, Creative: 0.5)
6. Pandemic-era remote work increased demand for digital solutions. (Confidence: 0.7, Creative: 0.6)
7. Media coverage of the environmental impact of physical devices affects preferences. (Confidence: 0.5, Creative: 0.7)
8. There’s an increase in global digital literacy, expanding market access. (Confidence: 0.6, Creative: 0.6)
9. Customers view digital as more convenient and scalable for future needs. (Confidence: 0.7, Creative: 0.5)
10. Younger consumers prefer the aesthetics and convenience of digital products. (Confidence: 0.6, Creative: 0.6)

*Reflection:* 
"Have I considered all possible influences? Are there any surprising factors that could explain this shift?"

*Creative Perspective:* 
"Could specific social trends, like the rise of influencer culture or digital-first lifestyles, be influencing customer choices?"

**2.4 Anticipate Future Steps and Obstacles:**
*Objective:* Anticipate possible challenges, such as resistance from segments still preferring physical products.

*Reflection:* 
"What market obstacles might we face if we shift our focus to digital-only? Are there sub-segments that still prioritize physical products?"

*Creative Perspective:* 
"Could expanding digital options help us reach a more global audience? Are there emerging trends that we could leverage in our strategy?"

[end sys2]

[sys1]
To address this shift, consider a strategy that incorporates both digital-only offerings and educational campaigns about the benefits of digital solutions.
Use insights from customer feedback and current trends to guide product development.
Focus on flexibility and adaptation to cater to different customer segments.
[end sys1]


1. Introduction {#sec1}
===============

In tropical regions, high environmental temperature in combination with high humidity causes heat stress in chickens, especially in high producing birds that are characterized by high feed intake and metabolic rates, leading to huge economic problems in commercial poultry farms. Heat stress will reduce feed intake, which in turn will adversely affect nutrient uptake and utilization, resulting in decreased productivity rates. Moreover, heat stress induces oxidative stress in the body, and oxidation of substrates produces free radicals and other reactive oxygen species that can cause serious damage to the cells and organs \[[@B1]\]. However, it is well known that antioxidants can be used for elimination or mediation of the adverse effects of heat stress. A biological antioxidant is defined as "any substance that, when present at low concentrations compared to those of an oxidizable substrate, significantly delays or prevents oxidation of that substrate" \[[@B2]\]. Although there are claims that traditional herbal medicines including Chinese herbal medicines (CHM) are effective in reducing adverse effect of heat stress due to their antioxidant activity, scarce data exist describing these effects. Zhang et al. \[[@B3]\] reported that dairy cows fed a diet supplemented with a Chinese herbal formula increased their milk yield by 14%. To support the above positive effect on milk yield, the authors reported that supplementation with the above herbal formula also increased the total antioxidant capacity (T-AOC) of heat stressed cows by 44% compared to the controls (non-heat stressed cows). In a recent study, our research team \[[@B4]\] reported that supplementation with different dosages (0, 0.5, 1.5, and 2%) of a mixture of eight CHM in the diet of laying hens under heat stress conditions (average temperature of 30.1 ± 2.7°C and mean relative humidity of 80.2 ± 7.8%) significantly increased egg production rate, serum antibody titer for New Castle\'s disease virus, and the superoxide dismutase levels compared to the nonsupplemented birds. At the same time, the malondialdehyde level (as an indicator for oxidative stress) was significantly lower in CHM treated hens compared to the controls. The positive results derived from the above studies suggest that CHM could serve as a potential effective substitute of the conventional antibiotic related drugs commonly used to suppress stress in commercial poultry production. However, the active compounds of CHM that protect animals against heat stress were not determined in the previous studies of Diao et al. \[[@B4]\] and Zhang et al. \[[@B3]\]. The present study, therefore, aimed to provide some insights into the concentrations and types of the active compounds (phenolic compounds, oligosaccharides with potential prebiotic effects, and unsaturated fatty acids) in the eight CHM that possibly play an important role in alleviating the effects of heat stress. In addition, results obtained from this study may also provide new information to develop a more efficient CHM formulation for stress mitigation in farm animals.

2. Materials and Methods {#sec2}
========================

2.1. Samples {#sec2.1}
------------

The eight Chinese herbal medicines used in this study were*Chai Hu* (*Bupleurum*),*Bai Zhu* (*Atractylodes*),*Bai Shao* (*Paeonia*),*Gan Cao* (*Glycyrrhiza*),*Bu Gu Zhi* (*Psoralea*),*Bo He* (*Mentha*),*Fu Ling* (*Poria*), and*Shi Gao* (*Gypsum*). CHM were supplied by the College of Agriculture, Hainan University. All the herbs were ground and sieved through 60--80 meshes (particle size 0.32--0.42 mm).

2.2. Determination of Phenolic Compounds Using HPLC {#sec2.2}
---------------------------------------------------

The concentrations of phenolic compounds in the herbs were measured by reversed-phase High Performance Liquid Chromatography (HPLC). Phenolic compound standards consisted of gallic acid, vanillic acid, caffeic acid, synergic acid, orientin, sinapic acid, isoorientin, coumarin acid, and*p*-anisic acid. Twenty-microliter methanolic extract from each herbal medicine was loaded on the HPLC instrument (Waters-e2695 series, US) equipped with a UV-Vis photodiode array (PAD) detector, binary pump, vacuum degasser, autosampler, and analytical column (COSMOSIL 5C18-AR-II Packed Column, 4.6 mm I.D. × 250 mm, Japan). Solvents were deionized water and acetonitrile. The pH of water was adjusted to 2.5 with orthophosphoric acid. The phenolic compounds were detected at 280 nm. The column was equilibrated with 95% solvent A (water) and 5% solvent B (acetonitrile); then the ratio of solvent B was increased to 85% in 40 min followed by reducing solvent B to 5% in 50 min. This ratio was continued to 65 min for the next analysis with flow rate of 0.8 mL/min. All the standards were purchased from Sigma Chemical Company.

2.3. Determination of Total Phenolic Compounds Using Colorimetric Method {#sec2.3}
------------------------------------------------------------------------

According to the determined total phenolic compounds using HPLC, the amounts of phenolic compounds in the*Poria* and*Gypsum* were very low or nondetectable. Since the phenolic compounds are the main antioxidant compounds which can reduce heat and oxidative stress in the animal tissues,*Poria* and*Gypsum* were not considered for further investigation using colorimetric method. The amounts of total phenolic compounds in the six other herb extracts were determined with the Folin-Ciocalteu reagent using the method of Spanos and Wrolstad \[[@B5]\]. Five ml of each extract (three replicates), 0.25 ml 1/10 dilution of Folin-Ciocalteu\'s reagent, and 0.2 ml of Na~2~CO~3~ (7.5%, w/v) were incubated at 45°C for 15 min. The absorbance of the mixtures was measured at 765 nm using a SPECTRAmax-PLUS384 UV-Vis spectrophotometer. Results were expressed as mg gallic acid equivalent per g of dry weight (mg GAE/g DW).

2.4. Determination of Antioxidant Activity {#sec2.4}
------------------------------------------

For determination of antioxidant activity of the samples, two methods, namely, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS assay based on the method described by Tsai et al. \[[@B6]\]) and ferric reducing ability of plasma (FRAP assays according to the method developed by Benzie and Strain \[[@B7]\]), were used. Both methods have been completely described previously \[[@B8]\]. In the present study, for the ABTS assay, different concentrations of trolox (5 to 50 *μ*g/ml) were used to prepare the standard curve, and the results were expressed as Trolox Equivalents Antioxidant Capacity (TEAC) in the form of *μ*g Trolox/g sample; and for the FRAP assay, FRAP reagent was used as blank and the final absorbance of each sample was compared with those obtained from the standard curve made from 0 to 1000 *μ*mol/L ferric sulphate (FeSO4-7H2O).

2.5. LCMS/MS Analysis {#sec2.5}
---------------------

A brief description for LCMS/MS method is as follows: Method: LCMS/MS = full scan with MS/MS data collection; ionization mode: Neg; column: Phenomenex Synergi Fusion 100 mm × 2.1 mm × 3 *μ*m; buffer A: water with 0.1% formic acid; buffer B: acetonitrile with 0.1% formic acid; rapid screening performed at 15 min run time using AB Sciex 5500QTrap LCMS/MS with Agilent 1290 series UHPLC; gradient run program: 5% B to 95% B from 0.01 min to 10.0 min, held for 2 min and back to 10% B in 0.1 min and reequilibrated for 3 min. MS settings and conditions were as follows: negative; voltage IS: 4500 V; source temperature: 500°C; desolvation gas: 40 psi; source gas: 40 psi; scan range: 100--1000*m/z* for full scan and 50--1000*m/z* for MS/MS scan.

2.6. Quantification of Total Carbohydrate in Form of Monomer Using HPLC {#sec2.6}
-----------------------------------------------------------------------

For determination of total monosaccharides, 100 mg of each dried sample was hydrolyzed using 10 mL of 4% sulfuric acid solution at 110°C for 6 h \[according to Szambelan and Nowak \[[@B9]\] with minor modifications\]. After hydrolysis, samples were centrifuged at 10000 ×g for 5 min, filtered through 0.22 *μ*m nylon syringe filters (Pall Gelman Laboratory, USA), and injected into the HPLC. Concentrations of MSC were assayed using HPLC (2690, Waters, USA) with a COSMOSIL Sugar-D column (250 × 4.6 mm i.d., 5 *μ*m). The mobile phase consisted of acetonitrile and water (80 : 20, v : v) with a flow rate of 1 mL/min and the column temperature of 35°C. A reflective index (RI) detector (2414, Waters, USA) was used for the detection of MSC in the detector sensitivity of 1024 and temperature of 30°C. The sample injection volume was 20 *μ*L, and the running time was 20 min. Arabinose, xylose, glucose, galactose, fructose, and mannose (Sigma-Aldrich, St. Louis, MO) were used as standard. In this method of sugar analysis by HPLC, glucose and galactose had the same retention time. This was the same for arabinose and xylose.

2.7. Determination of Fatty Acid Profile {#sec2.7}
----------------------------------------

Determination of fatty acid profile using Gas Chromatography (GC) followed the method described by Folch et al. \[[@B10]\] with minor modifications that were explained previously \[[@B11]\]. The fatty acid concentrations were expressed as percentage of the total identified peaks measured for each sample.

3. Results {#sec3}
==========

3.1. Detection of Phenolic Compounds by HPLC {#sec3.1}
--------------------------------------------

The chromatogram of the nine phenolic standards (gallic acid, vanillic acid, caffeic acid, synergic acid, orientin, isoorientin, sinapic acid, coumarin acid, and*p*-anisic acid) is shown in [Figure 1](#fig1){ref-type="fig"}. Results of the concentration of the nine phenolic compounds in the herbal extract samples detected by HPLC ([Table 1](#tab1){ref-type="table"}) showed that*Paeonia* contained the highest level of total phenolic compounds (17353 *μ*g/g DW) due to the high level of gallic acid (16336 *μ*g/g DW), while*Glycyrrhiza* had the second highest concentration of total phenolic compounds (12318 *μ*g/g DW) mainly due to the high level of p-anisic acid (9703 *μ*g/g DW), followed by*Mentha*,*Psoralea*,*Atractylodes*, and*Bupleurum* (6918, 5287, 3985, and 2595 *μ*g/g DW, resp.). Since the levels of total phenolic compounds in the*Poria* and*Gypsum* were very low or nondetectable, they were not included in our further investigations.

3.2. Determination of Total Phenolic Compounds by a Colorimetric Method and Antioxidant Activity by ABTS and FRAP Methods {#sec3.2}
-------------------------------------------------------------------------------------------------------------------------

Total phenolic compounds detected by a spectrophotometer \[as gallic acid equivalents (GAE)\] and antioxidant activity of the samples are presented in [Table 2](#tab2){ref-type="table"}. Based on the colorimetric study,*Psoralea* had the highest level of phenolic compounds (17.4 GAE/g) and also the highest antioxidant activity measured using the ABTS and FRAP methods, followed by*Glycyrrhiza* (13.7 GAE/g) and*Paeonia* (11.2 GAE/g). The lowest phenolic content was detected in*Mentha* (3.6 GAE/g).

3.3. LCMS/MS Analysis of Herbal Extracts {#sec3.3}
----------------------------------------

Results of the LCMS/MS analysis of the six herbal extracts are shown in [Table 3](#tab3){ref-type="table"}. Fourteen compounds were detected in*Atractylodes*, eight in*Paeonia*, seven in*Mentha*, six in*Glycyrrhiza*, and five in*Bupleurum*. Based on the library of LCMS/MS system that was used in the present study, no phenolic compound was detected in*Psoralea*.

3.4. Quantification of Total Sugars in Form of Monomer Using HPLC {#sec3.4}
-----------------------------------------------------------------

The concentrations of total sugars in the samples after hydrolysis by 4% sulfuric acid are presented in [Table 4](#tab4){ref-type="table"}.*Paeonia* contained the highest level of total sugars (62.38% of DM), mainly glucose and galactose.*Glycyrrhiza* contained 43.26 of monosaccharides (% DM) with glucose and galactose also having the greater values.*Bupleurum* contained the highest concentration of arabinose and xylose (10.28%), followed by glucose and galactose (10.13%). The lowest concentration of total sugars was detected in*Psoralea* (14.32%).

3.5. Fatty Acid Profile {#sec3.5}
-----------------------

Fatty acid profiles of the tested herbs are shown in [Table 5](#tab5){ref-type="table"}.*Atractylodes* contained the highest levels of unsaturated fatty acids (84.54%) followed by*Glycyrrhiza* (78.87%),*Paeonia* (77.71%),*Bupleurum* (67.60%),*Mentha* (62.79%), and*Psoralea* (37.25%). In all samples, the percentage of unsaturated fatty acids was higher than that of saturated fatty acids apart from*Psoralea*. In the*Atractylodes*, 76.67% of fatty acids were in the form of polyunsaturated fatty acids with C18:2n-6 having a percentage of 70.94%.

4. Discussion {#sec4}
=============

High environmental temperature in the tropics can cause huge economic losses in the poultry industry through reduced growth rate, egg production, and survival ability \[[@B12]\]. One of the most important biological adverse effects of heat stress is the oxidative injury (lipid peroxidation) and oxidative damage of proteins and DNA, which is caused by enhancement in formation of reactive oxygen species (ROS) \[[@B13]\]. It has been reported that there is a direct relationship between heat stress and the activation of antioxidant defense system in animals \[[@B14]\].

Alleviation of heat stress can be accomplished by increasing the antioxidant capacity of the animals after their dietary supplementations with antioxidant compounds. Moreover, promotion of gut health and intestinal barrier integrity can be achieved by using prebiotics alone or in combination with antioxidants. Supplementation with different compounds that possess antioxidant properties, including polyphenols, unsaturated fatty acids, and different prebiotic oligosaccharides have been already investigated with the intention to reduce the negative effects of heat stress in poultry \[[@B15], [@B16]\]. The use of natural antioxidant products derived from plants is preferred over chemical antioxidants due to food safety reasons \[[@B17]\]. Diao et al. \[[@B4]\] had recently reported that enrichment of the diet of laying hens with a mixture of eight Chinese herbal medicines (CHM) under heat stress condition significantly increased their egg production and immune responses. However, the active compounds of CHM responsible for alleviating the negative effects of heat stress were not investigated. This follow-up study aimed to provide some insights into the quantity and types of active compounds in the eight used CHM that might play an important role in heat stress reduction.

More than 10,000 individual phenolic compounds, known for their antioxidant activities, have been characterized from plant samples \[[@B18]\]. Results of our HPLC analysis showed that among the eight herbal samples tested, only six, namely,*Chai Hu* (*Bupleurum*),*Bai Zhu* (*Atractylodes*),*Bai Shao* (*Paeonia*),*Gan Cao* (*Glycyrrhiza*),*Bu Gu Zhi* (*Psoralea*), and*Bo He* (*Mentha*), contained considerable amounts of detectable phenolic compounds (gallic acid, vanillic acid, caffeic acid, synergic acid, orientin, sinapic acid, isoorientin, coumarin acid, and*p*-anisic acid). Results of the LCMS/MS detected many more different phenolic compounds from these six herbal samples. In addition, total phenolic compounds determination using the colorimetric method showed that, among the six herbs,*Psoralea* contained the highest concentrations of phenolic compounds, followed by*Glycyrrhiza* and*Paeonia*. Moreover,*Psoralea* also showed the highest antioxidant activity among the herbal extracts in the antioxidant assays.

Studies on feed and food supplementation of polyphenols showed increased antioxidant capacity in blood and in several organs \[[@B16]\]. Antioxidative effects of the dietary supplementation with phenolic compounds from various sources had been extensively studied in sheep \[[@B19]\], broilers \[[@B20]--[@B22]\], rabbits \[[@B23]\], and turkeys \[[@B24]\]. More recently, Akbarian et al. \[[@B25]\] examined the effects of dietary essential oils (rich in simple phenolic compounds) on mRNA levels of heat shock protein 70 (HSP70) and antioxidant enzymes, oxidative status, and meat oxidative stability of heat stressed broilers. They found that the enrichment with the dietary essential oils improved antioxidant defense against heat stress-induced changes. Improved antioxidant status of heat stressed broiler chickens using other phenolic compounds, such as flavonoids \[[@B26]\] and turmeric rhizome powder, rich in curcumin \[[@B27]\] is also reported. Prebiotics, such as galactooligosaccharides (GOS), xylooligosaccharides (XOS), mannan-oligosaccharides (MOS), and fructooligosaccharides (FOS), have also been reported to mitigate the adverse effects of heat stress. Varasteh et al. \[[@B15]\] reported that supplementation with dietary GOS resulted in a prevention of the negative heat stress-related changes in jejunum. Similarly, Hasan et al. \[[@B28]\] reported that prebiotic (MOS) enhanced growth in heat stressed broilers in comparison to the controls, but not in broilers under normal conditions.

The above-mentioned effects of prebiotics may be indirect or direct. The indirect effects include increase of the population of beneficial gut bacteria (probiotic bacteria) in the host animals which in turn improves their health and immune system response \[[@B29]\] contributing to overcoming of the adverse effect of heat stress. However, some prebiotics (e.g., inulin) which can be absorbed by liver and colon cells through pinocytosis \[[@B30], [@B31]\] act as antioxidants \[[@B17]\]. Epithelial gut cells can also assimilate other prebiotics such as soluble fructans and glucans \[[@B32]\]. In addition, prebiotics are also capable of exerting immune-modulating effects and can stimulate anti-inflammation responses \[[@B33]\], the first reaction against heat stress by the host\'s defense system. Hence, prebiotics could be a potential agent for reducing heat stress due to their immune-modulating effects and this hypothesis is receiving increasing interest \[[@B16]\].

A combination of several Chinese herbs rather than a single one is used to treat illness or stress. The reason is that a combination of several herbs could provide an expanded spectrum of active compounds. Our results showed that the order of herbal extracts according to the total amount of monosaccharides was*Paeonia*\>*Glycyrrhiza*\>*Bupleurum*\>*Atractylodes*\>*Mentha*\>*Psoralea*, while that for the level of unsaturated fatty acid was*Atractylodes*\>*Glycyrrhiza*\>*Paeonia*\>*Bupleurum*\>*Mentha*\>*Psoralea*. And for all the herbs, apart from the*Psoralea*, the amount of unsaturated fatty acids was higher than saturated fatty acids. Since unsaturated fatty acids, especially omega 3 series, are known for their antioxidant activity \[[@B34]\], the high levels of unsaturated fatty acids indicate possible high antioxidative efficacy of the herbs examined in this study.

5. Conclusion {#sec5}
=============

For the first time, this study provides qualitative and quantitative information on the phenolic compounds, prebiotics, and fatty acids (known for their antioxidant capability) of several traditional herbs which had been shown to effectively reduce heat stress in layer hens. Although the use of CHM for treatment of various illnesses in human subjects has been used for many thousands of years, their use as "green" medications for livestock industry is rather recent, especially with increasing interest as they could possibly serve as alternatives for antibiotics to reduce stress and promote the growth in commercial livestock and poultry production. It must be emphasized that the current data is not exhaustive but could serve as a reference for future studies on similar subject. Based on the results of the present study,*Psoralea*,*Glycyrrhiza*, and*Paeonia* had high phenolic compounds while*Glycyrrhiza*,*Bupleurum*, and*Paeonia* contained higher levels of prebiotics. Content of unsaturated fatty acids was higher in*Atractylodes*,*Glycyrrhiza*, and*Paeonia*. However, the actual efficacy of these herbs, singly or as mixture, to alleviate heat stress requires further*in vivo* investigations. The importance of*Poria* and*Gypsum* that contain very low or nondetectable levels of phenolic compounds in the mixture of CHM in reducing heat stress needs further investigation.

This study was supported by the Ministry of Higher Education of Malaysia under the LRGS Fasa 1/2012 Grant (UPM/700-1/3/LRGS). P. Shokryazdan and M. F. Jahromi acknowledge support from Iran\'s National Elites Foundation (INEF).

Conflicts of Interest
=====================

No potential conflicts of interest were reported by the authors.

![Chromatogram of phenolic compound mixture of the standards detected at 280 nm by RP-HPLC. Standards were gallic acid, vanillic acid, caffeic acid, synergic acid, orientin, isoorientin, sinapic acid, coumarin acid, and*p*-anisic acid.](SCIENTIFICA2017-8208261.001){#fig1}

###### 

Phenolic compounds of eight herbal medicine extracts determined using HPLC (*µ*g/g DW).

  Phenolic compound   *Bai Zhu* (*Atractylodes*)   *Bai Shao* (*Paeonia*)   *Chai Hu* (*Bupleurum*)   *Bo He* (*Mentha*)   *Bu Gu Zhi* (*Psoralea*)   Fu Ling (*Poria*)   *Gan Cao* (*Glycyrrhiza*)   *Shi Gao* (*Gypsum*)
  ------------------- ---------------------------- ------------------------ ------------------------- -------------------- -------------------------- ------------------- --------------------------- ----------------------
  Gallic acid         2.52                         16336                    58                        50                   180                        3                   64                          ND
  Vanillic acid       561                          226                      885                       836                  1111                       ND                  362                         ND
  Caffeic acid        1413                         56                       229                       2905                 183                        17                  76                          ND
  Synergic acid       276                          94                       62                        620                  339                        ND                  45                          ND
  Orientin            67                           119                      53                        1028                 579                        ND                  224                         ND
  Sinapic acid        369                          92                       475                       40                   4                          ND                  1718                        ND
  Isoorientin         184                          226                      51                        22                   2579                       ND                  101                         ND
  Coumarin acid       896                          29                       679                       210                  35                         ND                  25                          ND
  *p*-Anisic acid     217                          175                      103                       1207                 277                        24                  9703                        ND
  Total               3985                         17353                    2595                      6918                 5287                       44                  12318                       ---

Values are mean of three replicates; ND: not detected.

###### 

Total phenolic content and antioxidant activity of CHM samples.

  Herbal medicine             Total phenolic content (mg gallic acid equivalents, GAE)/g   TEAC (*μ*mol of trolox equivalents, TE)/g   FRAP (*µ*mol Fe^2+^/g)
  --------------------------- ------------------------------------------------------------ ------------------------------------------- ------------------------
  *Bai Zhu*(*Atractylodes*)   5.78                                                         53.4                                        28.96
  *Bai Shao*(*Paeonia*)       11.22                                                        89.67                                       142.23
  *Chai Hu*(*Bupleurum*)      4.71                                                         41.82                                       32.58
  *Bo He*(*Mentha*)           3.6                                                          27.5                                        28.91
  *Bu Gu Zhi* (*Psoralea*)    17.4                                                         91.67                                       232.98
  *Gan Cao*(*Glycyrrhiza*)    13.7                                                         77.48                                       167.65

Values are mean of three replicates.

###### 

Phenolic and flavonoid compounds in the herbal medicine samples detected by LCMS/MS.

  -----------------------------------------------------------------------------------------------
  Name                                                Name of compound
  --------------------------------------------------- -------------------------------------------
  *Bai Zhu*(*Atractylodes*)                           3,30-Di-O-methyl ellagic acid derivatives

  3,30-Di-O-methyl ellagic acid                       

  Caffeoylquinic acid derivatives                     

  Cinnamic acid derivatives                           

  Ferulic acid quinic acid conjugate                  

  Gluconic acid                                       

  Methoxyflavone derivatives                          

  Petunidin-3-O-(4 coumaroyl)-rutinose-5-O-glucose)   

  Phenylvaleric acid                                  

  Quinic acid                                         

  Rhamnoside flavanoids 2                             

  Rhamnoside flavanoids                               

  Rutinoside flavanoids                               

  Tannic acid                                         

                                                      

  *Bai Shao*(*Paeonia*)\                              Apigenin derivatives

  Quinic acid                                         

  Gallic acid derivatives                             

  Methoxyflavone derivatives                          

  Myricetin-3-O-galactose dimer                       

  Rhamnoside flavanoids                               

  Rutinoside flavonoids                               

  Tannic acid                                         

                                                      

  *Bo He*(*Mentha*)                                   Caffeoylquinic acid isomer

  Protocatechuic acid derivatives                     

  Rosmarinic acid derivative                          

  Sagerinic acid                                      

  Salvianolic acid A                                  

  Salvianolic acid B isomer                           

  Tannic acid                                         

                                                      

  *Gan Cao*(*Glycyrrhiza*)                            Apigenin 7-O-(6′′-dihydrogalloyl) isomer

  Apigenin-6,8-di-C-*β*-D-glucopyranoside isomer      

  Caffeoyl glucose                                    

  Gluconic acid                                       

  Phenylvaleric acids                                 

  P-Hydroxycinnamoyl derivative                       

                                                      

  *Chai Hu*(*Bupleurum*)                              3,30-di-O-methyl ellagic acid

  15,16-dihydroxy-9Z,12Z-octadecadienoic acid         

  16-alfa-o-Methylneoquassin metabolite               

  Methoxyflavone derivatives                          

  Tannic acid                                         

                                                      

  *Bu Gu Zhi* (*Psoralea*)                            Not detected
  -----------------------------------------------------------------------------------------------

###### 

Total sugar (% of dry matter) in form of monosaccharide in the samples determined using HPLC.

  Herbal medicine             Arabinose and xylose   Fructose   Mannose   Glucose and galactose   Total
  --------------------------- ---------------------- ---------- --------- ----------------------- -------
  *Bai Zhu*(*Atractylodes*)   10.28                  3.26       3.64      7.08                    24.26
  *Bai Shao*(*Paeonia*)       6.5                    ND         0.58      55.3                    62.38
  *Chai Hu*(*Bupleurum*)      11.79                  9.13       2.85      10.13                   33.9
  *Bo He*(*Mentha*)           10.15                  ND         2.7       8.93                    21.78
  *Bu Gu Zhi* (*Psoralea*)    ND                     5.1        3.7       5.52                    14.32
  *Gan Cao*(*Glycyrrhiza*)    7.67                   4.73       ND        30.86                   43.26

Values are mean of three replicates; ND: not detectable.

###### 

Fatty acid profiles of herbal medicine extracts (based on %).

  Herbal medicine              C14:0   C16:0   C16:1   C18:0   C18:1n-9   C18:2n-6   C18:3n-6   C18:3n-3   TSFA    TUFA    TMUFA   Total PUFA   TUFA:TSFA
  ---------------------------- ------- ------- ------- ------- ---------- ---------- ---------- ---------- ------- ------- ------- ------------ -----------
  *Bai Zhu* (*Atractylodes*)   0.00    13.89   0.00    1.57    7.87       70.94      0.00       5.73       15.46   84.54   7.87    76.67        5.47
  *Bai Shao* (*Paeonia*)       0.00    19.67   0.36    2.26    22.58      49.74      0.00       5.39       22.29   77.71   22.58   55.13        3.49
  *Chai Hu* (*Bupleurum*)      0.00    26.56   0.83    5.01    23.17      37.99      1.86       4.59       32.40   67.60   23.17   44.44        2.09
  *Bo He* (*Mentha*)           1.71    28.98   1.12    5.40    21.75      14.56      1.82       24.67      37.21   62.79   21.75   41.05        1.69
  *Bu Gu Zhi* (*Psoralea*)     0.00    9.99    0.00    52.76   9.50       19.44      0.58       7.73       62.75   37.25   9.50    27.75        0.59
  *Gan Cao* (*Glycyrrhiza*)    0.00    17.63   0.15    3.35    18.01      51.62      0.00       9.25       21.13   78.87   18.01   60.87        3.73

Values are mean of three replicates; C14:0, myristic acid; C16:0, palmitic acid; C16:1, palmitoleic acid; C18:0, stearic acid; C18:1n-9, oleic acid; C18:2n-6, linoleic acid; C18:3n-6, *γ*-linolenic acid; C18:3n-3, *α*-linolenic acid; TSFA, total saturated fatty acids; TUFA, total unsaturated fatty acids; TMUFA, total monounsaturated fatty acids; TPUFA, total polyunsaturated fatty acids; TUFA:TSFA, unsaturated fatty acids: saturated fatty acids ratio.

[^1]: Academic Editor: Michał Tomczyk