Advances in Extraction, Purification, and Analysis Techniques of the Main Components of Kudzu Root: A Comprehensive Review

Round 1

Reviewer 1 Report

I have some questioned regarding the articles 

1.      Page 1 line 43, it should be space between “naturalmaterial”

2.      The tittle of the manuscript is extraction, purification and analysis techniques of the main components of Kudzu root, but author focusing on isoflavone only without explained it as main components. No quantitative result of how much isoflavone content in Kudzu Root

3.      Figure 1 is not clear, author only write pharmacological activities with receptor that responsible for the activity or what?

4.      Table 2, better to add column about percentage of analytes obtain (recoveries) using extraction method that has been done

5.      Author should conclude which extraction method is better in obtaining isoflavone from Kudzu root rather than just reveal result of each extraction method before moving to next subchapter (purification techniques). The same suggestion apply to purification techniques and analytical method section

Author Response

In accordance with Reviewer 1’s suggestion:

Q1. Page 1 line 43, it should be space between “naturalmaterial”

A1: Thanks for your suggestion. The spelling mistake has been corrected now. The corrected sentence is as follows: Most importantly, as an excellent natural material for the development of new health foods, KR is extensively used in the food and health products industry and has great market potential.

Q2. The tittle of the manuscript is extraction, purification and analysis techniques of the main components of Kudzu root, but author focusing on isoflavone only without explained it as main components. No quantitative result of how much isoflavone content in Kudzu Root

A2: Thank you for the comment on this issue. We supplemented the relevant discussion on Pueraria isoflavones as the main active components in Kudzu root, and supplemented the specific content of various isoflavones in Table 1. The paragraph in manuscript appears as follows:

The common ingredients of KR include flavonoids, starch, cellulose fiber, protein, and etc. Among them, the best effect ingredients are isoflavones. Isoflavones are part of a large family of secondary plant metabolites called flavonoids, they are dietary phytoestrogens occurring naturally in legumes. A reliable source of isoflavones is Pueraria lobata, according to pertinent study studies. The mass fraction of puerarin can range from 1.58% to 7.68%, whereas the overall mass fraction of isoflavones in Pueraria lobata can range from 6.2% to 17.0%[7,8]. Currently, it has been revealed that more than 50 different types of puerarin isoflavone have unambiguous structures, Puerarin[9], Daidzein, Daidzin, Puerarinxyloside[10], 3'-Hydroxy Puerarin, Genistein, Genistin, Biochanin A, Formononetin [11], 6”-O-Malonylgenistin [12], 6″-O - α - d - Glucopyranosylpuerarin[13],Calycosin[14],4-Hydroxy-7-Hydroxymethyl-6-Methoxyisoflavone[15], 4′,6-Dimethoxy-8-Hydroxy-7-Hydroxymethyl Isoflavone [16], 3'-MethoxypuerarinA, 3'-MethoxypuerarinB[17], etc. The main isoflavones in KR was displayed in Table 1.

 Table 1. The main isoflavones in Kudzu Root.[42]

-: Not reported.

Q3. Figure 1 is not clear, author only write pharmacological activities with receptor that responsible for the activity or what?

A3: Thanks for your suggestion. We have replaced the images with improved quality. Figure 1 mainly describes the biological activity and related mechanism of Pueraria isoflavones.

Figure 1. Bioactivities of main isoflavones in KR and their underlying mechanisms.

Q4. Table 2, better to add column about percentage of analytes obtain (recoveries) using extraction method that has been done

A4: Thank you very much for this suggestion. We have modified it according to your suggestion. Increase the “Productive rate” column in the original table.

Q5. Author should conclude which extraction method is better in obtaining isoflavone from Kudzu root rather than just reveal result of each extraction method before moving to next subchapter (purification techniques). The same suggestion apply to purification techniques and analytical method section

A5: Thanks for your helpful comments. At the end of each summary of extraction, purification and analysis, we summarized each part.

2.1.5. Extraction summary

The conventional solvent extraction technique is ineffective, and using more sol-vents increases the possibility of environmental damage. In addition to being a popular topic in current research, the emergence of NADESs offers a new option for the extraction of Pueraria isoflavones and other plant active components. Technology for mod-ern instrument-assisted extraction has been continuously improved. Although Pueraria isoflavone extraction efficiency and time can be increased and decreased with the use of ultrasonic, microwave, and enzyme-assisted technologies, there are still certain drawbacks. For instance, some isoflavones that readily degrade and are not heat-resistant cannot be extracted with MAE. Ultrasonic blank area and high noise are drawbacks of UAE. The expense and easily influenced enzyme activity by the environment are the two drawbacks of EAE. As a result, the composite extraction approach is the main area of interest and development right now. A current issue is how to use it for the commercial extraction of Pueraria isoflavones.

2.2.5 Purification techniques summary

Because it is inexpensive and simple to use, column chromatography can be widely employed in industrial production, but it has a drawback—low purifying effective-ness. SPE and QuEChERS have developed quickly in recent years, with one of the main development paths being the study and use of novel materials for the effective extraction of KR. HSCCC has effective separation skills. And can decrease the usage of organic solvents, but the price of the necessary equipment is too costly and makes it un-suitable for promotion. Although the CPE approach does not require the use of big or complicated apparatus and can reduce the amount of organic solvents used, its range of applications is limited. Therefore, it is important to think about ways to increase separation efficiency while lowering extraction and separation costs. This supports the expansion of the use of Pueraria isoflavones in the food and pharmaceutical industries.

3.4. Analytical methods summary

In the current detection method, TLC has little personnel and equipment needs, it is inaccurate and requires lengthy experimental processes. The sensitivity and speed of ELISA are advantages, but the cross-contamination reaction is a disadvantage. A novel method known as pCEC combines the benefits of capillary electrophoresis with the great selectivity of HPLC. In recent years, it has become one of the chromatography's hot sites. However, it is still in the development stage, with few applications, and there are problems such as small sample load and difficult cleaning. LC-UV / Vis and LC-MS are widely used in the detection and analysis of Pueraria isoflavones. The sensitivity and selectivity of MS have been improved as a result of the development of HRMS, TOF MS, Ion Trap MS, and orbital trap. Improved detection techniques can also help with standardization, reliability, and food quality control, as well as chemical analysis of natural goods

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript submitted for evaluation is a review of advances in kudzu root phytochemical research. This applies primarily to methodological issues related to typical analytical procedures such as extraction and identification of bioactive compounds. This is a valuable study summarizing the achievements in this field over the last several years, when a clear progress in research on this valuable plant was observed. Observable through the development of analytical techniques related primarily to the development of chromatography, in particular detection techniques and the development of unconventional processing methods.

The present manuscript in this respect has been properly compiled with a thorough review of the available literature which includes 131 cited items. Nevertheless, I believe that some points could be added to the content of the current manuscript for the benefit of its upgrading.

First of all, kudzu is a plant that contains many valuable bioactive substances, the consumption of which has a positive effect on human health and has a preventive effect in many chronic diseases. The authors could include an extended description of many valuable biological activities, which in the current form of the manuscript have been described quite casually. I suggest adding a dedicated paragraph describing examples of biological activity. In particular, the recently discovered aid in addiction treatment.

I also believe that paragraph 2. Sample pretreatment methods can be extended. The authors mention the possible use of pre-treatment of the plant matrix for better extraction of analytes. It is possible to specify here, for example, procedures related to enzymatic treatment or hydrolysis. In paragraph 2.1.1 I would like more details on the use of deep eutectic fluids. It is quite popular today and fast growing sector of green chemistry. Including an additional table with NADES examples would be appreciated. Paragraphs 3.1 and 3.2 should be supplemented with a list of spectral properties such as UV-Vis maxima and m/z and MS/MS characteristic fragments.

I believe that after a positive response to my comments, the whole manuscript will be significantly improved and will be a valuable source material to help other scientists dealing with this subject.

Author Response

In accordance with Reviewer 2’s suggestion:

Q1: First of all, kudzu is a plant that contains many valuable bioactive substances, the consumption of which has a positive effect on human health and has a preventive effect in many chronic diseases. The authors could include an extended description of many valuable biological activities, which in the current form of the manuscript have been described quite casually. I suggest adding a dedicated paragraph describing examples of biological activity. In particular, the recently discovered aid in addiction treatment.

A1: Thanks for your suggestion. We added a dedicated paragraph describing examples of biological activity. The paragraph in manuscript appears as follows:

Research has revealed that puerarin isoflavone has positive effects on common health issues like diabetes, hypertension, and hyperlipidemia. It has been established that puerarin, genistein, daidzein and daidzin are efficient treatments for hypertension[33–38]. Pueraria extract, according to Li et al., can lower blood pressure in mice fed a high-salt diet through modulating gut flora[37]. According to Yang et al., pueraria is effective in treating physical illness-es that co-occur with depression[38]. It covers post-stroke depression as well as de-pression caused by coexisting conditions such diabetes, coronary heart disease, migraines, Parkinson's disease, and others. KR have potential in an alcohol intake reduction[39], and thus in the long-term prevention of alcohol addiction[40]. Zhou et al. gave alcoholic male Wistar rats puerarin, daidzein, and puerarin[41]. The findings demonstrated that daidzin and puerarin were able to dramatically raise the levels of two types of ghrelin, which in turn decreased alcohol consumption.

Q2: I also believe that paragraph 2. Sample pretreatment methods can be extended. The authors mention the possible use of pre-treatment of the plant matrix for better extraction of analytes. It is possible to specify here, for example, procedures related to enzymatic treatment or hydrolysis.

A2: Thanks for your helpful comments. We expanded the paragraph 2. And briefly described the procedures related to enzyme treatment and hydrolysis in the extraction process. The paragraph in manuscript appears as follows:

Sample pretreatment refers to the separation of components from complex systems by enzymatic hydrolysis, extraction, enrichment, purification and concentration. It is a process of removing impurity interference, increasing the concentration of the component to be tested, and facilitating the qualitative and quantitative detection of the instrument. The primary goal of the enzymatic hydrolysis process is to dissolve, suspend, or glue the intracellular components into the solvent in order to achieve the goal of extraction. This is done by using one or more enzyme solutions to dissolve the plant cell wall and break macromolecular chains like cellulose and pectin through hydrolysis. The removal of impurity interference and a rise in the concentration of the material under test can be accomplished through purification, which facilitates qualitative and quantitative detection by the equipment. Effective pre-treatment technology may increase target separation, minimize loss, and boost extraction rate[43–45].The methods for preparing and analyzing KR samples to identify iso-flavone components are presented in Table2.

Q3: In paragraph 2.1.1 I would like more details on the use of deep eutectic fluids. It is quite popular today and fast growing sector of green chemistry. Including an additional table with NADES examples would be appreciated.

A3: Thank you for your precious suggestions. We supplemented the application of new green solvents in the extraction process of Pueraria isoflavones and the related information of DES. The paragraph in manuscript appears as follows:

More and more green solvents are emerging to replace conventional volatile harmful solvents as science and technology advance. The application of novel green solvents in flavonoids extraction are presented in Table3. For instance, the extraction of Pueraria isoflavone using ILS, nonionic surfactant micelle solution, and NADES. Deep eutectic solvents （DES） is a class of green solvents commonly associated with ionic liquids because of common properties, such as high thermal stability, low volatility, and low vapor pres-sure[81]. NADES are composed of natural com-pounds produced by cell metabolism, and they have similar characteristics to DES. For the extraction of puerarin isoflavones, Saied A[82] created NADESs with a molar ratio of choline chloride to citric acid of 1: 2. High performance liquid chromatography (HPLC)-diode array detector (DAD) coupled with high resolution (HR) mass spectrometry (MS) was used to detect the extract of KR, and 10 isoflavones were detected. The amount of isoflavones in KR was measured to be 1.09 ± 0.006% overall. Puerarin was extracted from KR by Fan[83] using ultrasonic aided extraction with IL as the extraction solvent using the response surface optimization approach. The quality of KR, the kind and concentration of IL, the strength and duration of ultrasonic extraction, and all of these factors were optimized. Finally, 0.43 g of KR raw materials were added to 10 mL of 1.06 mol/L 1-butyl-3-methylimidazolium bromide aqueous solution. The best extraction effect could be reached by extracting for 27.43 min while using 480 W of ultrasonic power.

Table 3. Application of novel green solvents in flavonoids extraction.

-: Not reported.

Q4: Paragraphs 3.1 and 3.2 should be supplemented with a list of spectral properties such as UV-Vis maxima and m/z and MS/MS characteristic fragments.

A4: Thanks for the suggestion of improvement. We supplemented the relevant information of the target compounds in Table 5 and Table 6.

Table5. The UV and LC information of Pueraria isoflavones.[118]

Table6. The MS information of Pueraria isoflavones.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

author has revised all my major concern

Reviewer 2 Report

The authors responded positively to my comments, which, in my opinion, significantly improved the manuscript. I Recommend accepting the manuscript in its current form.