Bibliometric Analysis of Literature in Snake Venom-Related Research Worldwide (1933–2022)

Simple Summary Around the world, snake envenomation poses a serious health risk. Proteins with pharmacological effects are present in snake venom. Recent studies elaborate snake venom and its potential application, including as a cancer drug and antibacterial substances. Our study aimed to analyze the global profile of the literature in snake venom research from documents indexed in the Scopus database between 1933 and 2022. In total, 2999 documents were published with Brazil showing the highest productivity. Antivenom, proteomics, and transcriptomics are emerging as hot topics on a global scale. The present study offers a distinctive overview of snake venom research conducted worldwide. Abstract Snake envenomation is a severe economic and health concern affecting countries worldwide. Snake venom carries a wide variety of small peptides and proteins with various immunological and pharmacological properties. A few key research areas related to snake venom, including its applications in treating cancer and eradicating antibiotic-resistant bacteria, have been gaining significant attention in recent years. The goal of the current study was to analyze the global profile of literature in snake venom research. This study presents a bibliometric review of snake venom-related research documents indexed in the Scopus database between 1933 and 2022. The overall number of documents published on a global scale was 2999, with an average annual production of 34 documents. Brazil produced the highest number of documents (n = 729), followed by the United States (n = 548), Australia (n = 240), and Costa Rica (n = 235). Since 1963, the number of publications has been steadily increasing globally. At a worldwide level, antivenom, proteomics, and transcriptomics are growing hot issues for research in this field. The current research provides a unique overview of snake venom research at global level from 1933 through 2022, and it may be beneficial in guiding future research.


Introduction
Venom glands are considered a unique morphological and physiological adaptation developed by animals during evolution to increase the efficacy of capturing prey and as part of a defense system against predators [1][2][3][4][5]. A growing body of research attempting to dissect the composition and possible application of animal venoms has been accumulating for decades [6][7][8][9]. In particular, snake venoms, consisting of various types of proteins and small peptide cocktails, have been gaining significant attention as novel sources of drug discovery in recent years [10][11][12]. The main reason for understanding the compositions of snake venom is that snake bites are considered serious health and economic problems Between 1933-2022, 138 countries contributed to the literature on snake venom. The top 10 most productive countries listed a publication share ranging from 24.3% for Brazil to 3.33% for Germany. Table 1 illustrates the top ten countries in terms of their proportionate contribution to the total number of documents on a worldwide scale. Brazil produced the most documents with 729 (24.3%) documents, followed by the United States (n = 548; 18.2%), Australia (n = 240; 8%), Costa Rica (n = 235; 7.83%), and the United Kingdom (n = 208; 6.93%). The United States (n = 24) listed the highest number of international collaborations, followed by Australia, Germany, and the United Kingdom (n = 21) (Table 1; Figure 2).  4 Costa Rica 235 (7.83) 18 Between 1933-2022, 138 countries contributed to the literature on snake venom. The top 10 most productive countries listed a publication share ranging from 24.3% for Brazil to 3.33% for Germany. Table 1 illustrates the top ten countries in terms of their proportionate contribution to the total number of documents on a worldwide scale. Brazil produced the most documents with 729 (24.3%) documents, followed by the United States (n = 548; 18.2%), Australia (n = 240; 8%), Costa Rica (n = 235; 7.83%), and the United Kingdom (n = 208; 6.93%). The United States (n = 24) listed the highest number of international collaborations, followed by Australia, Germany, and the United Kingdom (n = 21) (Table 1; Figure 2).   were the most prolific journals on the subject of snake venom. Research articles with the highest number of citations in Table 3 highlight the landmark studies in snake venomrelated research and can be used as references in determining the current trends and future directions.   were the most prolific journals on the subject of snake venom. Research articles with the highest number of citations in Table 3 highlight the landmark studies in snake venomrelated research and can be used as references in determining the current trends and future directions.   Table 4 shows the global performance of the top 10 productive institutions in the field of snake venom from 1933 to 2022, with a total of 1192 (39.74%) documents. The Universidad de Costa Rica in Costa Rica is the most prolific contributor with 240 (8%) snake venomrelated documents. The Instituto Butantan in Brazil (n = 228; 7.60%), the Universidade de São Paulo in Brazil (n = 213; 7.10%), the Universidade Estadual de Campinas in Brazil (n = 95; 3.16%), and the National University of Singapore in Singapore (n = 77; 2.56%) were listed second through fifth.  Figure 3 maps the occurrence of terms retrieved from 2999 documents related to snake venom indexed by Scopus. Among the 15,498 extracted terms, 255 were detected to be present in more than 50 occurrences, resulting in 5 distinguished clusters: red, blue, green, yellow, and purple ( Figure 3a). Cluster 1 (red color) includes terms such as amino acid sequence, metalloproteinase, blood clotting; cluster 2 (green color): envenomation, animal model, mice; cluster 3 (blue color): viperidae, mass spectrometry, proteomics; cluster 4 (yellow color): drug effect, human cell, metabolism; cluster 5 (purple color): crotalid venoms, bothrops. In Figure 3b, VOSviewer categorizes the extracted terms into a color gradient from blue to yellow, representing old to new publication years. The early years of snake venom-related studies elaborated on several key terms such as drug effect, venom, disintegrin, phospholipase A2, cytotoxicity, and amino acid sequence. Meanwhile, the emerging topics in recent years includes antivenom, proteomics, and transcriptome.
Animals 2022, 12, x FOR PEER REVIEW 6 of 21 Figure 3 maps the occurrence of terms retrieved from 2999 documents related to snake venom indexed by Scopus. Among the 15,498 extracted terms, 255 were detected to be present in more than 50 occurrences, resulting in 5 distinguished clusters: red, blue, green, yellow, and purple ( Figure 3a). Cluster 1 (red color) includes terms such as amino acid sequence, metalloproteinase, blood clotting; cluster 2 (green color): envenomation, animal model, mice; cluster 3 (blue color): viperidae, mass spectrometry, proteomics; cluster 4 (yellow color): drug effect, human cell, metabolism; cluster 5 (purple color): crotalid venoms, bothrops. In Figure 3b, VOSviewer categorizes the extracted terms into a color gradient from blue to yellow, representing old to new publication years. The early years of snake venom-related studies elaborated on several key terms such as drug effect, venom, disintegrin, phospholipase A2, cytotoxicity, and amino acid sequence. Meanwhile, the emerging topics in recent years includes antivenom, proteomics, and transcriptome.

Discussion
The current study thoroughly examined global research output on the topic of snake venom. According to our findings, snake venom has garnered much interest from scientists all around the world in the last 89 years. The gradual increase in snake venomrelated documents since the 1960s could be associated with the funding of the International Society on Toxinology (IST) in 1962 [110,111]. Toxicon, the official journal of IST, was listed as the most prolific journal with the highest number of documents related to snake venom in this study. The earliest document from Toxicon retrieved in this study was published in 1962 titled "Hemolytic action of indirect lytic snake venom in vivo" by De Vries et al. from Israel [112]. In total, Toxicon journal contributed 22.74% (n = 682) of

Discussion
The current study thoroughly examined global research output on the topic of snake venom. According to our findings, snake venom has garnered much interest from scientists all around the world in the last 89 years. The gradual increase in snake venom-related documents since the 1960s could be associated with the funding of the International Society on Toxinology (IST) in 1962 [110,111]. Toxicon, the official journal of IST, was listed as the most prolific journal with the highest number of documents related to snake venom in this study. The earliest document from Toxicon retrieved in this study was published in 1962 titled "Hemolytic action of indirect lytic snake venom in vivo" by De Vries et al. from Israel [112]. In total, Toxicon journal contributed 22.74% (n = 682) of the total documents extracted from the Scopus database, indicating the significant impact of Toxicon in the development of snake venom-related studies. Interestingly, despite the fact that the oldest article related to snake venom in Toxins journal was published in 2009 [113], Toxins, by Multidisciplinary Digital Publishing Institute (MDPI), was recognized as the journal with the second highest number of published documents (n = 115; 3.83%). The designation of snake bites as a priority neglected tropical disease by WHO in 2017 also affects the growth of documents related to snake venom research [20]. In the period 2017-2021, with an average of 119 documents per year, a total of 596 (19.87%) documents were published, indicating a high research productivity in the field of snake venom in recent years.
Our results showing that Brazil is the most prolific country in terms of snake venom research could be explained by the fact that Brazil is the home to a highly diverse species of snakes [114,115]. Additionally, the high prevalence of snake envenomation in Brazil promotes extensive efforts for prevention and management of snake bites, as well as elaborating the potential application of snake venom in medicine in this country [116][117][118][119][120][121]. Consistent with the result showing that Brazil is the most productive country, Brazil is home to 6 of the 10 institutions with the highest number of documents in snake venomrelated research. Other developing countries, such as Costa Rica and India, were among the most productive countries in the field of snake venom research, which could be linked to multiple reports of the snake biting cases in these countries [122][123][124][125][126][127][128]. Our results also demonstrate that developing countries published a relatively high percentage of research articles, indicating that snake venom-related research is not limited to developed countries. Taken together, these findings suggest that the study of snake venom is currently emerging as a global effort.
The number of citations obtained by research articles might be used to determine the central topics in a certain field [129][130][131]. The "Astacins, serralysins, snake venom and matrix metalloproteinases exhibit identical zinc-binding environments (HEXXHXXGXXH and Met-turn) and topologies and should be grouped into a common family, the 'metzincins'" article by Bode et al., from Germany, published in FEBS Letters, was the most frequently cited article [101]. Importantly, our bibliometric analysis also revealed that snake venom-related articles and reviews were published in reputable journals such as Nature and Science [105,106,[132][133][134].
Up to 2010, researchers reported various studies related to disintegrin, venom, amino acid sequence, phospholipase A2, cytotoxicity, and drug effect. Recent focus on snake venom-related research has been gradually shifting to antivenom, proteomics, and transcriptome, providing hints to the emerging subjects in snake venom-related research in the future. In the last few years, there has been an increase in the publication of snake venom proteomes, especially from the families of Elapidae and Viperidae (Table S3)  . To estimate the protein diversity and abundance, characterization of snake venom proteomes involves two main steps: identification of the proteins and peptides followed by quantification [214]. In general, to improve the efficiency of the protein identification step, de-complexing procedures were highly recommended before performing mass spectrometry [215][216][217]. The established protocols generally involve the following workflows: Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC), 1D SDS-PAGE, and in-gel trypsin digestion followed by mass spectrometry (MS) [218][219][220]. The coverage of proteome identification might be improved by incorporating various approaches, including the utilization of venom gland transcriptome libraries and a top-down/bottom-up combination of mass spectrometry [216,221].
In the field of transcriptomics, cloning technology served as the foundation for the early studies of venom gland transcriptome [170,222]. High-throughput RNA sequencing obtained from venom glands is now possible due to the development of next generation sequencing (NGS) technologies [223]. A growing number of venom gland transcriptomes of numerous species of snakes have been constructed (Table S4) [5,136,143,147,148,170,182,189,193,222,[224][225][226][227][228]. The possibilities of examining various genes are one of the most powerful applications of transcriptomics from the snake venom gland. Through comprehensive profiling, identification of novel protein or peptides in snake venom and interspecies comparison is possible [143,236,237,250,253,254]. Additional transcriptomics studies can also be employed to analyze genetic varieties within snake families [234,247]. Notably, a comprehensive analysis of venom gland transcriptome libraries might help in accelerating the discovery of novel antivenoms.
Lastly, the limitation of the current study, similar to previous bibliometric analysis [278,279], is that it excluded documents published in journals not indexed by Scopus.

Conclusions
The current study presents a comprehensive review of snake venom-related research, spanning nearly eight decades of global literature output. According to our findings, Brazil produced the highest number of documents, followed by the United States, Australia, Costa Rica, and the United Kingdom. Studies in the areas of antivenom, proteomics, and transcriptome are expected to gather a considerable amount of interest in the near future. To summarize, the data offered in this study paints a clear picture of the progress made in the field of snake venom research from 1933 to 2022, and it may be helpful in providing insights for future research.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ani12162058/s1. Table S1. Snake venom components as novel drug candidates to eliminate drug resistant bacteria. Table S2. Anticancer properties of snake venom and its components. Table S3. Elapidae and Viperidae families as representatives of the wellcharacterized proteomics of snake venoms. Table S4. List of snake species for which transcriptomics libraries are available. Table S5. List of promising antivenom molecules.