Review Reports

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1. The introduction provides limited information on amyrin itself.

I strongly recommend including a background on its chemical structure, discovery history, and classification within terpenoids.

Additionally, the reason for focusing on amyrin, among many terpenoids, remains unclear. It would be beneficial to explain what makes α- and β-amyrins unique or promising compared to other structurally related compounds. A brief comparison of their chemical characteristics and pharmacological potential would help justify the focus of this review. Furthermore, the structural differences and biological implications between α- and β-amyrins should be briefly mentioned.

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Triterpenoids are compounds found in numerous plants, belonging to the isoprenoid group. These compounds are often present in plants as glycosides and saponins. Due to their diverse bioactivities and abundant availability, triterpenoids are considered a promising source of raw materials for the pharmaceutical industry

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Figure 1. Molecular structure of α-amyrin and β-amyrin (Viet et al., 2021)

 α-Amyrin and β-amyrin are two representative compounds within the triterpenoid group. Two compounds are similar in chemical formula and difirent about molecular structure of α. While α-amyrin has (CH3-29) attached at carbon position C₁₉ facing forward and (CH3-30) group is attached facing backward at carbon position C₂₀. β-Amyrin has (CH3-29) and (CH3-30) group groups are attached at carbon position C₂₀ and facing forward (Figure 1) [20]. α-Amyrin (with an ursane skeleton) and β-amyrin (with an oleanane skeleton) are two typical compounds with similar chemical structures, both classified within the triterpene acid group. Among these, ursane-type triterpenes, derived from α-amyrin, are the most common triterpenes, while oleanane-type triterpenes are produced from β-amyrin . Amyrins occur in the surface wax of tomato fruit, dandelion coffee. Parterculary, these two compounds have been extensively researched and successfully extracted by the author in significant quantities (10.75 g/kg) from the leaves of Celastrus hindsii. α-Amyrin and β-amyrin are easy to find in plants, and authors have been successfully extracted from Celustrus hindsii in previous research. However, the pharmaceutical values of these compounds have not been commonly known, and the isolation of these compounds is difficult. Therefore, this study clarified the values ​​and activities of these two compounds. It serveed as a comprehensive summary of the value of these compounds, aiming to maximize the inherent potential of this mixture, as a basis for the construction and development of compounds into pharmaceutical products.

Thank you very much again for your kind suggestion.

2, For each subsection in the Results section (e.g., 2.1, 2.2, 2.3, etc.), I suggest adding a summary figure. Visual representation of key findings would greatly enhance reader understanding and provide an overview of the data. Moreover, an integrative diagram illustrating the biological systems or pathways modulated by amyrin may help identify shared or unique mechanisms of function across various conditions. I believe the addition of such figures will significantly improve the impact of the manuscript.

Figure 2. The summary diagram of α- and β-amyrins's pharmaceutical potential

Figure 2 showed the synthesis of α- and β-amyrin activities that have been studied with the aim to clarify the values of these compounds can contribute to the pharmaceutical industry.

The pharmacological activities of the α- and β-amyrin mixture have been extensively studied in vitro and in vivo to support future medicinal applications.

Research has demonstrated that α- and β-amyrin possess a wide range of therapeutic properties, including analgesic, anti-inflammatory,anticonvulsant,antidepressant, gastroprotective, hepatoprotective,anti-pancreatitis,antihyperglycemic,and hypolipidemic effects.

α- and β-Amyrin have also been shown to exhibit anti-cancer and anti-inflammatory properties, cardiovascular disease, diabetes and arthritis .

Notably, these compounds have been shown to be non-toxic to normal cells. Therefore, α- and β-amyrin represented a promising source of raw materials for the development of future medicinal products.

Thank you very much again for your kind suggestion.

3, The Discussion section currently includes merely general information about diseases such as cancer and inflammation. As the Discussion should focus on interpreting the current findings rather than restating general knowledge, I recommend removing or relocating unrelated content. Any necessary background should be briefly addressed in the Introduction or in the relevant Results subsection. This will help maintain focus on amyrin and provide deeper scientific discussion.

We have removed a lot of the basic general information and replaced it with more specific and sharp discussion content. Please check from line 761 to line 1036

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4, Please discuss the mechanisms of function of α- and β-amyrins. Are there any shared or distinct molecular pathways through which these compounds exert their effects? If yes, what might explain their ability to effect on a diverse range of diseases through common or unique mechanisms? This discussion would enhance the depth and originality of the Discussion.

Please check from line 1012 to line 1022 with Table 14

 α- and β-amyrin are triterpenoids, a class of compounds that are composed of three isoprene units, have physical properties such as large molecular weights, typically between 400 and 500 daltons which enable them to interact with other large molecules in the environment [202]. The strong anti-inflammatory mechanism of α- and β-amyrin is a valid explanation for the many pharmaceutical potentials of these compounds [203]. Since most diseases originate from inflammation [204], under the effective anti-inflammatory effects of α- and β-amyrin, these compounds affect the functioning of many organs in the body including the liver, kidney, heart, brain and reducing the risk of cancer. [205]. α- and β-amyrin also can strengthen the body's immune system to prevent the growth of cancer cells [206]. α- and β-amyrin also have antioxidant effects, helping to protect cells from damage caused by free radicals. Therefore, α- and β-amyrin can help reduce the risk of cardiovascular disease, diabetes and neuroprotective effects 's disease [207]. α- and β-amyrin could inhibit the production of certain enzymes that play an important role in the aging process of the body. Therefore, α- and β-amyrin can help slow down the aging process and maintain the health of the body [208]. α- and β-amyrin have strong reducing properties, helping to purify the body by removing harmful substances in the body. This helps reduce the risk of many diseases such as cancer, arthritis and cardiovascular disease [209]. α- and β-amyrin have antioxidant properties, thus slowing down the aging process and preventing diseases related to oxidation such as Parkinson’s disease [210]. α- and β-Amyrin are acidic organic compounds in the triterpenoid group, so they have high antioxidant properties, good at eliminating free radicals, destroying and eliminating bacteria.

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5, I recommend that the authors address the key challenges associated with the pharmaceutical development of amyrin. For example, is large-scale production of α- or β-amyrin feasible? Are there any reported human clinical trials involving these compounds? Including a discussion on these points would provide a more comprehensive and practical perspective and help position this review as a useful reference for guiding future research in the field.

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Table 15 summarizes studies investigating the pharmaceutical potential of α- and β-amyrins. The majority of this research has been carried out at the in vitro and in vivo levels. Furthermore, significant attention has been given to non-cytotoxicity assays to evaluate the safety profile of α- and β-amyrins. However, clinical trials specifically assessing these compounds have yet to be conducted or reported. Consequently, the absence of clinical studies represents both a challenge and a promising opportunity for future research on α- and β-amyrins. Notably, a specialized extraction method detailed in Table 11 has yielded the highest reported global quantities of α- and β-amyrins. These findings collectively highlight and reinforce the significant value of these compounds for pharmaceutical development.

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Comments and Suggestions

Responses

The manuscript would benefit from thorough English language editing. There are frequent grammatical and syntactic issues that hinder readability and reduce the professional tone expected in a scientific review.

Example: Phrases like “This document served as a vital reference...” (Abstract) should be replaced with more academic and objective language such as “This review highlights the therapeutic relevance...”.

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Thank you very much for your comments.

The manuscript has been thoroughly edited in English, with grammatical and syntactical issues professionally corrected, ensuring the quality of a scientific review. The editing was carried out by professors and research experts with excellent English proficiency.

Example: Thank you very much. Please check the edit from line 30 to line 32.

This review highlighted the therapeutic of α- and β-amyrin compounds in the prevention and treatment of various serious diseases worldwide, potentially opening new opportunities and directions for the pharmaceutical industry.

The Introduction section contains excessive repetition about the global importance of pharmaceuticals, often stated in different ways without adding new insight. Consider condensing this section to make it more concise and focused.

Thank you very much. I have edited and condensed the information to be as simple and concise as possible but still convey the content Please check from line 38 to line 45

Pharmaceuticals are commodities of high social significance [1], are an eternal need of the world [2]. The role of pharmaceuticals in treating, preventing, improving health, prolonging and enhancing the quality of life of billions of people worldwide is indisputable [3, 4]. The global demand for pharmaceuticals is growing rapidly [5]. The global demand for pharmaceuticals is increasing rapidly, necessitating the expansion and advancement of production capabilities [6]. The global pharmaceutical market was valued at approximately USD 230.03 billion in 2021 and is expected to reach USD 430.05 billion by 2028, reflecting a compound annual growth rate (CAGR) of 11.32% [7].

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The review summarizes numerous studies without offering sufficient critical analysis. For example, differences in methodologies, limitations of in vivo vs. in vitro studies, and the translational gap to clinical application should be discussed.

A comparative table of bioavailability, toxicity, and delivery methods for α- and β-amyrins would significantly improve the translational value of the review.

Thank you very much. I have edited and condensed the information to be as simple and concise as possible but still convey the content Please check from line 1039 to line 1047

Table 15. α- and β-Amyrins 's Pharmaceutical Potential Research Level Accreditation

(✔: Researched); (-: Not Researched)

Table 14 presents studies on the pharmaceutical potential of α- and β-amyrins. Most of the research has been conducted at the in vitro and in vivo levels. Additionally, non-cytotoxicity experiments have received significant attention regarding the safety of α- and β-amyrins. However, clinical trials have not yet been specifically conducted or evaluated. Therefore, the lack of clinical studies presents both a challenge and an opportunity for future research on α- and β-amyrins.

Figures like the PRISMA diagram are present but inadequately described or referenced in the text. Ensure that all figures and tables are numbered, titled, and discussed meaningfully in the main text.

Thank you very much. I have edited and condensed the information to be as simple and concise as possible but still convey the content Please check from line 104 to line 112

Figure 1. PRISMA flow diagram of the literature included in the review.

This study was carried out based on a comprehensive review of 550 scientific articles concerning α- and β-amyrins. Among these, 350 articles were screened and identified as relevant to the objectives of this research. From these, 300 articles with content most closely aligned with the research objectives were selected. Subsequently, 89 articles were excluded due to limitations in transparency, inadequate language clarity, or lack of indexing within the Science Citation Index (SCI) systemUltimately, 211 articles containing the most comprehensive and clearly presented content and data were selected to serve as the foundation for this research project (Figure 1).

The conclusion should go beyond summarizing the findings. Include limitations, future directions, and potential applications or challenges in pharmaceutical development of α- and β-amyrins.

Thank you very much. I have edited and condensed the information to be as simple and concise as possible but still convey the content Please check from line 1054 to line 1068

The increasing global demand for pharmaceuticals underscores the importance of compounds like α- and β-amyrins, which have demonstrated a wide range of therapeutic benefits. These compounds have shown promise in treating various conditions, exhibiting anti-inflammatory, antidiabetic, anti-atherosclerotic, antinociceptive, anti-gout, and neuroprotective effects.

They also exhibited potential in addressing serious health concerns such as Parkinson's disease, cancer, bacterial infections, and HIV, without inducing cytotoxicity. Although these studies were conducted at the in vivo and in vitro levels, they open up opportunities for future clinical trials, paving the way for the development of products to support the treatment of various diseases. This study reaffirmed the value of previous research that led to the development of a highly efficient extraction method for α- and β-amyrins, yielding an impressive 10.75 g/kg from raw material. The compounds were accurately identified and verified using advanced techniques such as NMR, GC-MS, and ESI-MS, ensuring the precision of the findings. This study served as a valuable reference for further research into utilizing α- and β-amyrins as raw materials for pharmaceutical production, providing a foundation for developing effective treatments for various diseases.

Several tables and in-text citations are inconsistently formatted.

Check uniformity across the manuscript, particularly with table numbering and units (e.g., µg/mL vs. μg/mL).

We sincerely appreciate your valuable comment.

-                    According to your suggestion, I have revised the relevant units to ensure consistency. For example, values previously expressed in g/mL have been converted to µg/mL where appropriate

-                    Nonetheless, it is important to acknowledge that variations in unit conversion may occur depending on the level of research and the nature of the experiments, as authors typically choose units that are most appropriate and relevant to their study objectives

-                    Hence, applying a uniform unit conversion across all studies is difficult and may misrepresent the original findings.

For example:

-At the in vivo level, the unit mg/kg reflects the amount of drug (in milligrams) administered per kilogram of the subject’s body weight.

-At the in vitro level, µg/mL indicates the concentration of the substance (in micrograms) dissolved in 1 mL of solvent used in the experiment.

-µM (micromolar) is a standard unit of concentration in chemistry and biology, representing the number of moles of solute per liter of solution.

Clarify the use of abbreviations such as “TNBS”, “DSS”, “VEGF” upon first use and use consistently throughout the manuscript.

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The anti-inflammatory properties of α- and β-amyrin were assessed in a TNBS-induced murine model of colitis, with the compounds administered in a 1:1 ratio. Following induction via rectal administration of trinitrobenzene sulphonic acid, the animals were monitored for 72 hours to evaluate clinical and biochemical parameters. Systemic treatment with α- and β-amyrin (3 mg/kg, intraperitoneally) was compared to dexamethasone and vehicle-treated control groups. Disease progression was evaluated through both macroscopic and microscopic assessments of colonic lesions, as well as measurements of myeloperoxidase (MPO) activity and cytokine levels. Immunohistochemical analysis was conducted to examine the expression of cyclooxygenase-2 (COX-2), vascular endothelial growth factor (VEGF), phosphorylated NF-κB (phospho-p65), and phosphorylated cAMP response element-binding protein (phospho-CREB). TNBS-induced colitis was characterized by severe tissue damage, marked neutrophil infiltration, and elevated levels of pro-inflammatory mediators. Treatment with α- and β-amyrin resulted in notable improvements in colonic tissue morphology, a significant reduction in polymorphonuclear cell infiltration, decreased levels of interleukin-1β (IL-1β), and restoration of the anti-inflammatory cytokine interleukin-10 (IL-10) in the colon. Furthermore, α- and β-amyrin markedly suppressed the expression of vascular endothelial growth factor (VEGF), COX-2, phosphorylated NF-κB (phospho-p65), and phosphorylated CREB. These results highlight their potent anti-inflammatory activity in TNBS-induced colitis and underscore their promise as therapeutic candidates for the treatment of inflammatory bowel disease (IBD) [26].

α- and β-Amyrin exhibit potent anti-inflammatory activity, notably via modulation of the endocannabinoid system. In a murine model of colitis induced by dextran sulfate sodium (DSS), administration of these compounds significantly attenuated the severity of colonic lesions. This therapeutic effect was associated with decreased activity of inflammatory enzymes, including myeloperoxidase (MPO) and N-acetylglucosaminidase. Administration of the triterpenes also significantly reduced levels of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), and various chemokines, while enhancing the expression of the anti-inflammatory cytokine interleukin-4 (IL-4). Moreover, α- and β-amyrin downregulated the mRNA expression of several adhesion molecules involved in leukocyte recruitment and inflammation, including intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), platelet endothelial cell adhesion molecule-1 (PECAM-1), β2-integrin, CD68, and P-selectin. Importantly, these compounds also inhibited cannabinoid receptor type 1 (CB1), suggesting their role in modulating cannabinoid-mediated signaling. Additionally, they suppressed the expression of endocannabinoid-degrading enzymes such as monoglyceride lipase (MGL) and fatty acid amide hydrolase (FAAH), thereby enhancing endocannabinoid tone. Collectively, these findings support the therapeutic potential of α- and β-amyrin as novel agents for the treatment of inflammatory diseases, particularly those involving dysregulation of the cannabinoid system [27].

α- and β-Amyrin have demonstrated considerable therapeutic potential in the treatment of acute pancreatitis. In a model of pancreatitis induced by L-arginine, treatment with these triterpenes significantly reduced the elevated wet weight to body weight ratio of the pancreas, an established marker of inflammation and edema. Furthermore, administration of α- and β-amyrin led to substantial decreases in serum amylase and lipase levels, which are key biochemical indicators of pancreatic injury. The compounds also markedly lowered concentrations of pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6). In pancreatic tissues, treatment with α- and β-amyrin reduced the activity of inflammation and oxidative stress markers, including myeloperoxidase (MPO), thiobarbituric acid reactive substances (TBARS), and nitrate/nitrite levels. Immunohistochemical analysis further confirmed decreased expression of tumor necrosis factor-alpha (TNF-α) and inducible nitric oxide synthase (iNOS), reinforcing the observed anti-inflammatory effects. Collectively, these findings indicate that α- and β-amyrin exert both antioxidant and anti-inflammatory actions, highlighting their potential as therapeutic agents for the management of acute pancreatitis [28]

Author affiliations and contact details are overly detailed and may need to follow journal formatting guidelines more strictly.

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We sincerely appreciate your valuable comment. We have revised the author contact information to ensure full compliance with the journal’s formatting requirements.

Comments on the Quality of English Language

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The English and scientific style of the manuscript have been carefully revised with the assistance of field experts proficient in academic writing.