Animal Venom for Medical Usage in Pharmacopuncture in Korean Medicine: Current Status and Clinical Implication

Animal venoms, widespread throughout the world, are complex mixtures, the composition of which depends on the venom-producing species. The objective of this study was to contribute to the development of animal venom-based medicines by investigating the use of animal venom pharmacopuncture in Korean medicine (KM) institutions. We surveyed 256 public health centers from 1 through 31 October 2019 as guided by the Ministry of Health and Welfare (MoHW). A questionnaire developed by an expert group was distributed and collected for statistical analysis. The survey identified three types of animal venom-based pharmacopuncture: bee, snake, and toad venoms. The medications are based on a single animal venom ingredient and produced in 11 external herbal dispensaries (EHDs). Each animal venom is processed, refined, and freeze-dried in a cleanroom to produce a powder formulation that is later measured, diluted, filtered, filled, sealed, sterilized, and packaged as pharmacopuncture injections used in KM institutions. Bee venom therapy is effective in treating musculoskeletal pain, snake venom therapy is effective in controlling bleeding during surgery, and toad venom therapy is effective in cancer treatment. The study suggests that bee, snake, and toad venoms could be used in medical institutions and have the potential for drug development.


Introduction
Animal venoms, widespread worldwide, are composed of a variety of proteins and peptides that were developed through millions of years of evolution [1][2][3]. They are complex mixtures that vary depending on the venom-producing species [2]. The most known and studied venoms originate from bees, snakes, scorpions, and spiders [1,2].
Venomous animals are a valuable resource for the development of therapeutics [4]. Bee venom therapy is used to treat musculoskeletal diseases (e.g., low back pain, knee osteoarthritis), Parkinson's disease, adhesive capsulitis, and polycystic ovary syndrome [5]; snake venom-based drugs are used to treat hypertension, heart attack, acute coronary syndrome, stroke, pulmonary embolism, and other diseases [6]. While animal venoms are toxic, they can have therapeutic effects [7].
Korean medicine (KM) institutions have been using animal venoms in their pharmacopuncture therapies: animal venoms are injected into acupunctural points using syringes [8]. Pharmacopuncture, also known as herbal acupuncture, is a new form of acupuncture treatment that injects herbal medicine into acupuncture points [9]. In South

Demographic Characteristics of Pharmacopuncture-EHD (P-EHD)
A total of 11 Pharmacopuncture-EHD (P-EHD) questions were included in this study. The characteristics of the respondents in this survey are shown in Additional file 1 in Supplementary Materials. The average number of pharmacopuncture medicines produced in the 11 P-EHDs was average 18.4.

Preparation Status of Pharmacopuncture
A total of 1,851,896 vials of 46 different pharmacopuncture medicines were produced in 11 P-EHDs in 2018 alone. The status of the top 20 frequently prepared pharmacopuncture medicines produced in P-EHDs and their ingredients are shown in Table 1. The following quantities of animal venom preparations were made: bee venom (99.06%, n = 779,931), snake venom (0.67%, n = 5291), and toad venom (0.27%, n = 2114). All of these were composed of a single venom ( Figure 1). The following quantities of animal venom preparations were made: bee venom (99.06%, n = 779,931), snake venom (0.67%, n = 5291), and toad venom (0.27%, n = 2114). All of these were composed of a single venom ( Figure 1).

The Preparation Process of Animal Venom Pharmacopuncture
Bee venom is extracted from the venom pouch of worker bees (Apis mellifera, ligustica) [15]. Apply a weak current of a low voltage, 3-6V, to a living bee. Then, take the venom coming out of the stinger [15]. Snake venom is extracted from the venom gland of Agkistrodon halys Pallas [15]. Hold a snake by its head and neck. Then, place a vessel under its fangs and press the head where the venom gland is located gently to press the venom out of it. This process is called "snake milking" [15,16]. Toad (Bufo bufo gargarizans Canto) venom is extracted from the skin and parotid glands [15]. The production process of the injectable pharmacopuncture is the same for all three venoms ( Figure 2). Venoms are processed,

The Preparation Process of Animal Venom Pharmacopuncture
Bee venom is extracted from the venom pouch of worker bees (Apis mellifera, ligustica) [15]. Apply a weak current of a low voltage, 3-6V, to a living bee. Then, take the venom coming out of the stinger [15]. Snake venom is extracted from the venom gland of Agkistrodon halys Pallas [15]. Hold a snake by its head and neck. Then, place a vessel under its fangs and press the head where the venom gland is located gently to press the venom out of it. This process is called "snake milking" [15,16]. Toad (Bufo bufo gargarizans Canto) venom is extracted from the skin and parotid glands [15]. The production process of the injectable pharmacopuncture is the same for all three venoms ( Figure 2). Venoms are processed, refined, and freeze-dried in a clean room into a powder formulation [15]. The venom powder goes through purification and dilution using distilled water in a specific concentration [15]. The venom is then filled, sealed, sterilized, and packaged as pharmacopuncture injections for clinical use ( Figure 2). refined, and freeze-dried in a clean room into a powder formulation [15]. The venom powder goes through purification and dilution using distilled water in a specific concentration [15]. The venom is then filled, sealed, sterilized, and packaged as pharmacopuncture injections for clinical use ( Figure 2).

Discussion
To our knowledge, this study is the first to report on the three animal venom therapies used in KM institutions in South Korea. It covers the current status of the production of pharmacopuncture medicines based on animal venoms, their production processes, and indications. The government played a leading role in this study, which aimed to understand the current status of the production of animal venom pharmacopuncture medicines in EHDs. All of the pharmacopuncture medicines used in KM institutions are being produced in EHDs; this study covered 11 of the 16 EHDs that produced pharmacopuncture medicines (68.8%). Since EHDs prepared these medicines following the prescriptions issued by KM institutions, the production records at EHDs can be considered to represent the current status of the pharmacopuncture medicines used in KM institutions. Therefore, this study provides an overall status of the use of animal venoms for therapeutic applications by KM institutions.
Bee venom products represent the second-largest quantity of pharmacopuncture medicines in South Korea, accounting for 99.06% of the total animal venom-based pharmacopuncture. While bee venom causes neurotoxic symptoms including local paresthesias, headache, dizziness, nausea and vomiting, muscle aches, and rarely, cerebrovascular infarcts [62][63][64][65], recent non-clinical studies demonstrated that bee venom has antiinflammatory [66], anti-nociceptive [67], and anti-cancer activities [68]. The toxic reactions of bee venom therapy in clinical studies ranged from fatigue [69], erythematous plaques [70], pallor face [71], nausea [71], vomiting [71], and other minor side effects to more serious ones such as limb paralysis [72], dyspnea [72], unconsciousness [73], and death [73]. There have been many clinical and non-clinical studies reporting the side effects and benefits of bee venom therapy. However, due to the possibility of variation in the species of bees and the nutrients they take, the composition of bee venom can differ depending on geography. Therefore, it is necessary to standardize the material, evaluate its toxicity, and examine its benefits.
Toad venom is the least frequently used animal venom in KM institutions (0.27%). While toad venom has toxic effects and causes gastrointestinal, mental, cardiac conduction, and arrhythmic disturbances [80], non-clinical studies have reported anti-inflammatory [81], antiplasmodial activity [82], and antiproliferative effects on cancer cells [83]. The treatment toxicity is the most important factor limiting its use [84]. Further studies should be carried out to evaluate the benefits and side effects of its use to treat inflammatory diseases.
As is the case with bee stings, animal venom was used as it was, in some cases. However, in most cases, it was used as diluted to an appropriate concentration for clinical purposes. Animal venoms may cause idiosyncratic reactions depending on the individual in addition to their toxic effect [85]. For this reason, it is recommended to have the patient take a certain amount before clinical applications, in order to determine whether the use or administration of such a substance causes adverse effects or not. Also, according to the analysis on the 1,192,667 cases of accidents caused by animal venom over 12 years in Brazil by Chippaux [86], snake bites were linked to the highest fatality rate of 0.43%, followed by bee stings, of which the fatality rate was 0.33%. Since animal venom has toxic effects, it is necessary to refine, dilute, or otherwise process it before clinical use.
This study has several limitations. First, this study was a literature review of the indications and benefits of pharmacopuncture medicines based on animal venoms, without any reviews on the clinical indications in the real world. Further surveys of KM doctors are needed to cover the clinical indications and side effects. Second, a literature review of indications and side effects used only articles published in South Korea, and only articles dealing with injectables were reviewed. International databases will need to be reviewed to evaluate animal venoms factors such as the species, extraction process, and administration routes. Third, this survey was conducted by the government, which measured its accuracy and trustworthiness. However, the validity of the questionnaire was not confirmed.
Despite these limitations, this is the first study reviewing the production volume and current status of animal venom-based pharmacopuncture medicines used in clinical practice in South Korea. Collecting further data is necessary for the standardization of pharmacopuncture medicine and safety of dosages and clinical applications.

Conclusions
This study reports that bee, snake, and toad animal venoms are being used in medical institutions and have the potential for drug development. Based on the clinical evidence for animal venom summarized in this study, it is expected that the development of therapeutic agents for target diseases will be conducted.

Overview of the Study
A survey of community health centers across the country was conducted with the cooperation of the Ministry of Health and Welfare to understand the nationwide status of the production and use of animal venom medicinal products in the country.
For this purpose, a systematically developed questionnaire was distributed to participating organizations. The process of producing animal venom-based pharmacopuncture medicines was analyzed using textbooks, research papers, and data from the EHDs currently producing such medicines.

Study Sample
The study sample consisted of 256 community health centers across the country that were responsible for establishing and supervising EHDs in South Korea.

Questionnaire Development and Distribution
To develop the questionnaire, a group of five experts was formed (including two KM specialists with an average of 10+ years of clinical experience of pharmacopuncture (J.K.P., S.B.C.), one PhD with 10+ years of experience in the KM field (G.L.), one expert in the GMP, and one expert in hazard analysis critical control point (HACCP)). The draft of the questionnaire was developed based on previous studies [87][88][89]. The final version of the questionnaire was agreed upon through a review and revision process (Additional file 2 in Supplementary Materials).
To ensure nationwide representation, the questionnaire was sent together with official letters to 256 community health centers in South Korea through the Ministry of Health and Welfare over the period from 1 through 31 October 2019. A community health center has the authority to approve the establishment and supervision of an EHD. The centers were asked to complete the questionnaire based on the status in 2018; the community health centers were also instructed to complete the questionnaire only when their EHD produced pharmacopuncture medicines. To enhance the accuracy of the survey, it was requested that the questionnaire be completed by the KM pharmacist or the head of the KM institution that established the EHD.

Questionnaire Items
To obtain accurate information on the use of animal venoms in pharmacopuncture, the survey items consisted of two items concerning the basic status of EHDs (i.e., the type of the medical institution that set up the EHD and the location) and three items relating the production of pharmacopuncture medicines (the type of pharmacopuncture, herbal ingredients, and production volume). The questionnaire was designed so that the five items could be completed in a Microsoft Office Excel file (verson 16.0, 2013, Microsoft ® , Redmond, WA, USA).

Data Collection
Completed questionnaires that were sent back to us from the Ministry of Health and Welfare were reviewed for the integrity of the completed data in Excel files and sorted out by the relevant items. For any missing or incomplete data, the EHD was called or contacted via email to request additional or missing data and correct the incorrect data as much as possible.

Data Analysis
The data gathered using the questionnaire were processed using SPSS version 21.0 (IBM, Armonk, NY, USA). An explorative data analysis (EDA) was conducted. A summary of the data was prepared including the average, standard deviations, and frequency (n, %). Any incomplete data were treated as missing.

Literature Search of the Production Process and Clinical Effectiveness of Animal Venom Medications
The survey identified 46 types of pharmacopuncture medicines; three animal venoms (bee, snake, and toad venoms) were selected by the reviewing experts. The external experts were a professor of KM university who is a pharmaceutical board member at the national hospital, a KM professor who majored in herbal medicines, a general manager of an EHD with 8+ years of experience in preparing pharmacopuncture, and a quality control (QC) team leader from a pharmaceutical company that manufactures herbal drugs.
The researcher also reviewed textbooks used in South Korean medical schools for pharmacopuncture and related academic papers to review the production process and indications of the three selected animal venoms. The following electronic databases using keywords "bee venom", "snake venom", and "toad venom" were searched to identify relevant studies for inclusion in the review from inception to September 2020: Pubmed, EMBASE, Medline, Cochrane library, and six Korean databases (National Digital Science Library (NDSL), the Korean Traditional Knowledge Portal, KoreaMed, the Oriental Medicine Advanced Searching Integrated System (OASIS), the Research Information Sharing Service (RISS), and The National Library of Korea). All identified clinical studies and systematic reviews were examined to understand the clinical indications and effects of animal venom-based therapy. Two researchers (S.S.H. and J.E.H.) independently reviewed and screened the titles and abstracts of the retrieved studies based on predefined eligibility criteria. Two independent reviewers (J.W.K. and B.C.S.) extracted the treated diseases and the effectiveness of animal venom-based therapy. The systematic review reflected the results of the meta-analysis.  Institutional Review Board Statement: Ethical review and approval were waived for this study, because this study did not include personal information and was for evaluating public services.

Informed Consent Statement: Not applicable.
Data Availability Statement: The data will be made available upon reasonable request.