Panax ginseng Pharmacopuncture: Current Status of the Research and Future Challenges

Despite the increasing use of ginseng pharmacopuncture in clinical practice, evidence of its physiological effects, safety, and clinical outcomes is insufficient. The purpose of this review is to summarize previous studies and suggest future challenges for the clinical use of ginseng pharmacopuncture. We systematically searched clinical and animal studies that applied ginseng pharmacopuncture and reviewed the manufacturing processes of ginseng pharmacopuncture solution, safety, physiological responses, and clinical effects. Intravenous or point injection of the ginseng pharmacopuncture solution made by distillation extraction has been commonly used in studies. Ginseng pharmacopuncture does not show any toxicity in animals and humans, while it influenced the heart rate variability, pulse wave velocity, and protein synthesis in human subjects. In 25 case reports, patients with cancer, amyotrophic lateral sclerosis, skin wrinkles, and allergic rhinitis showed significant improvement of clinical outcomes. We found that more evidence is necessary to conclude that ginseng pharmacopuncture is safe and effective. First, the pharmacopuncture manufacturing process should be standardized on the basis of the safety and efficacy tests. Moreover, studies on the quantitative quality of the components of the solution and on the clinical comparison of various injection methods are required to improve clinical outcomes in the future.


Saponin
Traditionally, P. ginseng is used in the decoction form and is decocted with boiling water for a certain number of hours and prepared as a drink [5]. More recently, ginseng concentrates, extracted using water or alcohol, are also used [6]. In addition, ginseng has been transformed into red ginseng or fermented black ginseng through steaming, repeated heating and drying, or fermentation [6,7]. In addition to oral intake of ginseng, whose clinical effects and safety have been demonstrated [8,9], ginseng has been also administered via injection (intravenous or intramuscular on either acupoints or non-acupoints), and this technique is called pharmacopuncture.
Traditionally, P. ginseng is used in the decoction form and is decocted with boiling water for a certain number of hours and prepared as a drink [5]. More recently, ginseng concentrates, extracted using water or alcohol, are also used [6]. In addition, ginseng has been transformed into red ginseng or fermented black ginseng through steaming, repeated heating and drying, or fermentation [6,7]. In addition to oral intake of ginseng, whose clinical effects and safety have been demonstrated [8,9], ginseng has been also administered via injection (intravenous or intramuscular on either acupoints or non-acupoints), and this technique is called pharmacopuncture.
Pharmacopuncture is a relatively new acupuncture therapy in Traditional Korean Medicine (TKM) that combines acupuncture with herbal medicine [10]. Pharmacopuncture involves injection of filtered and sterilized herbal medicine extracts, which are extracted using different techniques (e.g., alcohol immersion, distillation, or pressing) depending on the herbs [11]. Thus, it simultaneously induces mechanical stimulation of acupoints and a pharmacological effect. It was originally developed under the name of 'Aqua-acupuncture' in the 1950s in China [10]. Aqua-acupuncture uses both herbal and non-herbal medicines and has been regarded as a combined therapy with traditional and Western medicine. However, pharmacopuncture in South Korea is exclusively associated with TKM, as it uses herbs that have been used in the form of other formulations (decoction, granule, etc.) and involves injection of the herbal extracts intravenously or into acupuncture points (acupoint), trigger points, or response points. The herbs and acupoints (in the case of herbal extracts injected into acupoints) are selected according to the meridian, Qi, and flavor theories, and the syndrome differentiation diagnosis protocol from TKM [2,11].
Ginseng pharmacopuncture is a typical single-herb pharmacopuncture, which contains diverse substances such as ginsenoside Rg1, ginsenoside Rb2, and phenolic compounds, and the amount of substances vary depending on the extraction methods (e.g., distilled versus ethanol extract [12]). A review of animal studies suggested that ginseng pharmacopuncture is useful in the prevention of diseases and strengthening immune response, especially in Yang insufficiency animal models induced by hydrocortisone acetate injection [13]. Furthermore, several studies have investigated the toxicity of ginseng pharmacopuncture, and its safety has been studied in animals [14,15]. On the basis of these lines of evidence, single-herb ginseng pharmacopuncture has been widely used in clinics as one of the standard TKM therapies in South Korea, while pharmacopuncture with several herbs combined (combination of various herbal extracts; e.g., Shenmai or Shenfu injections) has been more extensively used in China. Despite its wide usage in clinics, evidence is insufficient to prove whether ginseng pharmacopuncture therapy significantly improves the clinical outcomes and risk of adverse events (AEs) in patients.
In this study, we restrictively defined ginseng as the root of Panax ginseng C. A. Meyer, and our aim was to provide a comprehensive review of the clinical application of P. ginseng pharmacopuncture, which we referred to as 'ginseng pharmacopuncture', including its safety, physiological, and clinical responses. To achieve this goal, we (1) provided an overview of the physiological responses to and side effects of ginseng pharmacopuncture in animals or humans (patients and healthy participants), and (2) systematically reviewed previous clinical trials using ginseng pharmacopuncture in patients with various diseases. Lastly, we emphasized that more basic and clinical studies are needed to confirm the effects and safety of ginseng pharmacopuncture, and suggested future directions for developing ginseng pharmacopuncture as a safe and effective treatment for patients seeking TKM therapies.

Search Strategy
We used a systematic search strategy following the preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines for systematic reviews. Electronic searches of articles were conducted from their inception to January 2018, without language restrictions in PubMed, the Research Information Sharing Service (RISS), the Korean Studies Information Service System (KISS), KoreaMed, and the China National Knowledge Infrastructure (CNKI). The search terms used were "ginseng" including Panax ginseng Radix, Panax ginseng, ginseng radix, or red ginseng; and "pharmacopuncture", including aqua acupuncture, herbal/herb acupuncture, point-injection, or yakchim. "Red ginseng", which is the most common processed type of P. ginseng, was added as a search term. Furthermore, a secondary search was performed by screening the reference lists of articles that met the inclusion criteria.

Study Selection, Data Extraction, and Data Analysis
Three categories of studies were included in the search strategy if they evaluated pharmacopuncture using Panax ginseng C. A. Meyer roots as follows: (1) preclinical trial in animals, (2) study of physiological responses in human subjects, and (3) clinical trial in patients to identify the clinical effects of ginseng pharmacopuncture.
We followed the process of systematic literature review. First, searched articles were screened on the basis of the title and abstract before the full text was assessed. Second, articles identified as duplicates or non-original studies such as reviews, opinions, or protocols were removed. Third, publications that did not meet our definition of ginseng pharmacopuncture (single-herb ginseng administered via intravenous or intramuscular injection), studies that used pharmacopuncture consisting multiple herbs, and articles not written in English, Chinese, or Korean were excluded. The included studies were classified as animal studies, studies on the physiological responses of healthy participants, or clinical trials in patients. After the selection of the articles to be analyzed, we extracted data from the included clinical trials by using a predefined form that contained the following items presented in a separate table: manufacturing process of pharmacopuncture solution and characteristics of participants (e.g., age, sex, disorder or symptoms), intervention details (injection site, volume of injection, number and duration of treatment and co-intervention), and clinical outcomes. Results of animal studies and physiological responses are summarized narratively in the manuscript.

The Manufacturing Process of the Ginseng Pharmacopuncture Solution
Distillation extraction, alcohol immersion, the combined method of distillation and alcohol immersion, compression, and dilution methods are used for the manufacture of pharmacopuncture solution [2,11]. Among these methods, the ginseng pharmacopuncture solution is generally produced using the distillation method. In the included studies, ginseng plant was rinsed with clean water (in some cases, the rhizome part of the ginseng is removed) followed by distillation and/or decoction, filtering, and sterilization of the solution. Rarely, in some cases, extracts of certain active or target substances such as polysaccharides from ginseng were used to make the solution [16] (Figure 1). Recently, the Accreditation System of External Herbal Dispensaries (EHDs) was announced. The quality of pharmacopuncture should be managed according to the EHDs and clinicians and patients can verify whether the pharmacopuncture solution is produced in herbal dispensaries with an EHD certification mark [17].
Results of animal studies and physiological responses are summarized narratively in the manuscript.

The Manufacturing Process of the Ginseng Pharmacopuncture Solution
Distillation extraction, alcohol immersion, the combined method of distillation and alcohol immersion, compression, and dilution methods are used for the manufacture of pharmacopuncture solution [2,11]. Among these methods, the ginseng pharmacopuncture solution is generally produced using the distillation method. In the included studies, ginseng plant was rinsed with clean water (in some cases, the rhizome part of the ginseng is removed) followed by distillation and/or decoction, filtering, and sterilization of the solution. Rarely, in some cases, extracts of certain active or target substances such as polysaccharides from ginseng were used to make the solution [16] (Figure 1). Recently, the Accreditation System of External Herbal Dispensaries (EHDs) was announced. The quality of pharmacopuncture should be managed according to the EHDs and clinicians and patients can verify whether the pharmacopuncture solution is produced in herbal dispensaries with an EHD certification mark [17].

Physiological Response to Ginseng Pharmacopuncture in Human Subjects
In this review, 13 studies measured various physiological responses before and after receiving ginseng pharmacopuncture. In the healthy subjects, intravenous injection of wild ginseng pharmacopuncture did not change the blood pressure, pulse, temperature, respiration, and blood test indexes [18,19]. Wild ginseng pharmacopuncture injected at acupoints (ST36 and ST37) did not significantly change the acupuncture sensations as compared with other types of complex-herb pharmacopuncture (e.g., BUM pharmacopuncture made of Calculus Bovis, Fel Ursi, Moschus) and saline injection [20]. Another study showed that only the volume of injected solution, but not the stimulation methods (perpendicular versus transverse injection) at acupoints, affected the subjective intensity reporting of pharmacopuncture stimulation [21].
By contrast, randomized controlled trials (RCTs) showed that the sympathetic nervous system in healthy participants was activated significantly by wild ginseng pharmacopuncture injected at acupoints as compared with saline injection [22][23][24][25]. Intravenous wild ginseng pharmacopuncture decreased the mean heart rate variability (HRV) and pulse wave velocity (PWV) and increased the mean and standard deviation of normal R-R intervals and very low frequency oscillation power in patients with breast cancer [26], while it decreased the mean HRV and PWV and increased the mean normal R-R intervals in healthy volunteers [27]. Intravenous wild ginseng pharmacopuncture injection also affected pulse-related factors (e.g., increased the stability of the pulse wave) [28]. It also increased the level of proteins CR2-C3d, Ral-A, proapolipoprotein, apolipoprotein, transferrin, human hemoglobin, and vitamin D binding protein and reduced the transthyretin and antitrypsin levels in blood samples [29,30].
Current evidence suggests that ginseng pharmacopuncture may activate the sympathetic nervous system and change the protein synthesis mechanisms, while the sensory stimulation aspect (e.g., De-Qi sensation induced by needle injection at acupuncture points) may not specifically be involved in the effect of ginseng pharmacopuncture. However, the underlying mechanisms and physiological actions of ginseng pharmacopuncture have not yet been fully explained owing to insufficient evidence.

Safety Tests of Ginseng Pharmacopuncture
Several studies have tested the safety of ginseng pharmacopuncture in animals using different dosages and treatment frequencies. For example, both single administration (20 or 10 mL/kg) and repeated administration (10, 5 or 2.5 mL/kg for 4 weeks, once a day) of ginseng pharmacopuncture evoked no significant toxic responses in Sprague-Dawley rats (e.g., changes in mortality, histological observations, body weight, clinical signs, and food consumption behavior) [14,15]. In addition, pharmacopuncture using intravenous or intramuscular injection of radix ginseng (dried root of P. ginseng) at a single dose (0.1 0.5, or 1.0 mL/animal) did not change body weight, general condition, and hematological or biochemistry test results in rats [31,32].
In a case report, drug-induced liver injury (increased alkaline phosphatase level, white blood cells, and platelets; all indexes were outside the normal range) was suspected after intravenous wild ginseng pharmacopuncture treatment in a patient with shoulder pain, however, the causal influence of the wild ginseng pharmacopuncture treatment on the liver injury was not proven in the study [33]. In the RCT that compared the combination therapy of ginseng polysaccharides pharmacopuncture, Bupleurum pharmacopuncture, and paroxetine (pharmacopuncture group) with paroxetine monotherapy (control group), seven cases of AEs such as headache, dry mouth, or tremor were reported in the pharmacopuncture group. However, 21 cases of AEs such as diarrhea, constipation, weakness, or anorexia were reported in the control group, including nine cases of the same type of AEs as the treatment group [16].
In summary, the safety of ginseng pharmacopuncture injection has been mainly studied in animals, but more empirical investigations are necessary to prove its safety in humans. Figure 2 summarizes the results of the literature search process for analysis of clinical studies. Our research strategy retrieved a total of 595 articles, 245 of which were duplicates. An additional 324 studies were discarded after screening the full text or abstract including 95 not related to pharmacopuncture, 47 not related to P. ginseng, 53 that used pharmacopuncture with combined herbs. Finally, 25 case reports (24 studies written in English [14, and one study in Chinese [56]) and one RCT (written in Chinese) [16] met the inclusion criteria and were incorporated in the systematic review ( Figure 2 and Table 4).

Systematic Review of Clinical Studies
3.4.1. Search Results Figure 2 summarizes the results of the literature search process for analysis of clinical studies. Our research strategy retrieved a total of 595 articles, 245 of which were duplicates. An additional 324 studies were discarded after screening the full text or abstract including 95 not related to pharmacopuncture, 47 not related to P. ginseng, 53 that used pharmacopuncture with combined herbs. Finally, 25 case reports (24 studies written in English [14, and one study in Chinese [56]) and one RCT (written in Chinese) [16] met the inclusion criteria and were incorporated in the systematic review ( Figure 2 and Table 4).        Intervention group was significantly improved symptoms more than control group at 2, 4, 6 weeks after treatment (The Hamilton Depression Scale (HAMD), p < 0.05). Intervention group showed significantly higher total effective rate (98%) than control group (82%). Intervention group reported significantly lower adverse events (7 cases) than control group (21cases).

Outcome Measures
Clinical outcomes were classified into three categories as follows: objective measurement (e.g., tumor size in patients with cancer measured using computed tomography, blood test results, and intake of analgesics), subjective reporting of symptom changes, and reports of side effects or death. Ginseng pharmacopuncture improved the objective clinical outcomes in patients with skin wrinkles

Outcome Measures
Clinical outcomes were classified into three categories as follows: objective measurement (e.g., tumor size in patients with cancer measured using computed tomography, blood test results, and intake of analgesics), subjective reporting of symptom changes, and reports of side effects or death. Ginseng pharmacopuncture improved the objective clinical outcomes in patients with skin wrinkles (width and depth), cervical dysplasia, cancer (in 13 of 18 studies), and amyotrophic lateral sclerosis. The stabilized tumor size in the patients with cancer was reported in two studies. Subjective improvements of symptoms were reported in patients with allergic rhinitis, Behçet's disease, drug-induced hepatitis, acute demyelinating encephalomyelitis, depression (one RCT), and cancer (11 of 18 studies). Four studies reported the aggravation of diseases such as increase in tumor mass size, metastasis to other organs, and death (one patient) after the treatment session.

Changes in Clinical Outcomes: Examples from Case Reports
To investigate changes in clinical outcomes by treatment session, we summarized two case reports that reported symptoms and clinical indexes over time. Ryu et al. [53] reported three clinical cases of ALS ( Figure 4A). In Case 1, combined therapy of three different pharmacopunctures showed the improved general condition after 2-3 months of treatment; however, symptoms and muscular activity were aggravated after 6-7 months of treatment. In Case 2, muscular strength increased after 6-9 weeks of treatment, although the circumference of the limbs decreased. The subject also reported subjective improvement of general condition. In Case 3, pain fluctuated over time, and some symptoms were relieved such as low back pain and discomfort, while muscular strength was decreased. Kim et al. [50] reported a single case of hepatocellular carcinoma, and subjective symptoms (abdominal pain, sleep disorder, and loss of appetite) and ascites were reduced by the combined therapy of wild ginseng pharmacopuncture, acupuncture, and herbal medicine. The alkaline phosphatase (ALP) level returned to normal, and the subject stopped receiving other treatments except medications for hepatitis B ( Figure 4B) [50]. In summary, ginseng pharmacopuncture induced improvement of symptoms might vary in each individual and also change over time. To investigate changes in clinical outcomes by treatment session, we summarized two case reports that reported symptoms and clinical indexes over time. Ryu et al. [53] reported three clinical cases of ALS ( Figure 4A). In Case 1, combined therapy of three different pharmacopunctures showed the improved general condition after 2-3 months of treatment; however, symptoms and muscular activity were aggravated after 6-7 months of treatment. In Case 2, muscular strength increased after 6-9 weeks of treatment, although the circumference of the limbs decreased. The subject also reported subjective improvement of general condition. In Case 3, pain fluctuated over time, and some symptoms were relieved such as low back pain and discomfort, while muscular strength was decreased. Kim et al. [50] reported a single case of hepatocellular carcinoma, and subjective symptoms (abdominal pain, sleep disorder, and loss of appetite) and ascites were reduced by the combined therapy of wild ginseng pharmacopuncture, acupuncture, and herbal medicine. The alkaline phosphatase (ALP) level returned to normal, and the subject stopped receiving other treatments except medications for hepatitis B ( Figure 4B) [50]. In summary, ginseng pharmacopuncture induced improvement of symptoms might vary in each individual and also change over time.

Discussion
This study provides an overview of the current knowledge of the clinical usage and safety of the use of ginseng pharmacopuncture in place of the traditional oral intake of ginseng. To the best of our knowledge, this is the first review article that covers the clinical usage of ginseng pharmacopuncture by summarizing results of previous clinical studies as well as the toxicity test results in animals and physiological responses in humans. Based on the safety test results in animals, physiological responses to ginseng pharmacopuncture have been conducted, and ginseng pharmacopuncture has been demonstrated to significantly increase the sympathetic nervous system activities and influence protein synthesis in humans. According to the 26 clinical studies, ginseng pharmacopuncture is widely used in clinics, primarily in patients with cancer (lung cancer, hepatocellular carcinoma, prostate cancer, colorectal cancer, pancreatic cancer, cervical cancer, thymus cancer, breast cancer, etc.). In addition to cancer, ginseng pharmacopuncture has been used for the treatment of rhinitis,

Discussion
This study provides an overview of the current knowledge of the clinical usage and safety of the use of ginseng pharmacopuncture in place of the traditional oral intake of ginseng. To the best of our knowledge, this is the first review article that covers the clinical usage of ginseng pharmacopuncture by summarizing results of previous clinical studies as well as the toxicity test results in animals and physiological responses in humans. Based on the safety test results in animals, physiological responses to ginseng pharmacopuncture have been conducted, and ginseng pharmacopuncture has been demonstrated to significantly increase the sympathetic nervous system activities and influence protein synthesis in humans. According to the 26 clinical studies, ginseng pharmacopuncture is widely used in clinics, primarily in patients with cancer (lung cancer, hepatocellular carcinoma, prostate cancer, colorectal cancer, pancreatic cancer, cervical cancer, thymus cancer, breast cancer, etc.). In addition to cancer, ginseng pharmacopuncture has been used for the treatment of rhinitis, skin wrinkles, ALS, hepatitis, hepatocirrhosis, and depression, and it has shown significant clinical improvements in patients. Almost all studies applied ginseng pharmacopuncture intravenously, and only a few studies injected it at acupoints.
Previous studies showed the inhibitory effect of ginseng pharmacopuncture on the growth of human non-small cell lung cancer cells (NCI-H460)-induced solid tumor [57] and on the inflammation-related cytokine levels in hepatic metastatic mice model using colon carcinoma cells [58]. The results suggest that ginseng pharmacopuncture could be used in patients with cancer in combination with conventional therapies for cancer (e.g., chemotherapy) by improving the quality of life and general conditions and reducing the AEs of conventional therapies. However, the underlying mechanisms, safety, and clinical efficacy of ginseng pharmacopuncture remain controversial, which necessitates obtaining empirical data from large-scale and well-designed clinical studies.
Although the present review showed that clinical and basic research on ginseng pharmacopuncture has been carried out on many diseases, but it is still far from sufficient. As with other therapies, ginseng pharmacopuncture needs more evidence of its underlying mechanisms and safety at a fundamental level, and requires better evidence of its clinical efficacy and effectiveness from large-scale clinical studies. For example, whether the marker substances, ginsenosides (saponins), of ginseng pharmacopuncture depend on the manufacturing process is controversial. A standard method for extracting the active ingredients of ginseng for pharmacopuncture solution has not been developed, and the extracted substances vary depending on the extraction process (e.g., no index compounds were extracted in the distilled extract of ginseng while ethanol extraction successfully extracted the ginsenosides Rg1 and Rb1 [12]). Baek et al. [59] tested the marker substances of a combined ginseng pharmacopuncture solution comprising distilled extract and alcohol-extracted liquid using high-performance liquid chromatography. The combination method extracted all marker substances (ginsenosides Rg1, Rb1, and Rg3) and did not show significant toxicological changes in rats [59]. However, the clinical effects of ginseng pharmacopuncture manufactured using the combined extraction method (mixture of water-distilled solution and alcohol-extracted compound) have not been studied, while the water-distilled extracts have been mainly tested in our included clinical trials.
As only one RCT using ginseng polysaccharide extract as a pharmacopuncture solution has been reported [16], the clinical effect and safety of ginseng pharmacopuncture described in this review can be interpreted as the complex effects of multiple components in the whole-plant extracts, instead of a single compound. Studies on the pharmacokinetics of active components such as ginsenosides, polysaccharides, fatty acids, essential oils, and phenolic compounds and interactions between multiple components (e.g., a synergistic or antagonistic effect) of ginseng solutions for pharmacopuncture are necessary to demonstrate the pharmacological mechanisms of ginseng pharmacopuncture and develop the most effective solution in the future.
Moreover, among the 26 clinical trials, 15 used intravenous injections and 10 used the acupoint injection method. However, the pharmacokinetics of ginseng pharmacopuncture injected at different injection sites remain unclear. Future studies on various extraction and injection methods in terms of safety, mechanisms, and clinical effects will allow us to improve the efficacy and safety of ginseng pharmacopuncture therapy.
Although not included in this review, the ginseng-related pharmacopuncture research conducted in China often used ginseng extract in combination with various herbs. However, the different types of ginseng pharmacopuncture solution have never been compared to their clinical efficacy and safety. Therefore, clinical studies are necessary to test the effects and toxicity of the many different ginseng extraction methods and injection procedures (intravenous, intramuscular, and acupoints injection), combination of extracts from various herbs as compared with single ginseng extraction, and substance extracted solution as compared with whole plant extracted solution. Future studies should begin with testing the pharmacokinetics and pharmacological interactions of each substance in the ginseng pharmacopuncture solution and compare the substances and effects of ginseng solutions made by various extraction processes. In addition, various toxicity tests in animals and humans should be conducted. These approaches will allow us to find the best manufacturing method to produce the safest and most effective ginseng pharmacopuncture solution. The final and key challenges in the future application of the ginseng pharmacopuncture technique are proving its safety by various toxicity tests such as gene toxicity, carcinogenicity, developmental toxicity, toxicity of single or repeated administration of various doses, and efficacy in patients through a large-scale RCT with an appropriate placebo control. Although ginseng pharmacopuncture has been widely used in TKM clinics, we strongly argue that more concrete evidence is necessary for a safer and more prevalent use of the therapeutic modality in the future. Moreover, quality checks, management of the manufacturing facility, and relevant regulations are required, as pharmacopuncture techniques basically use direct intravenous or intramuscular injection of solutions in the human body.

Conclusions
In conclusion, although clinical trials and animal experiments showed that ginseng pharmacopuncture treatment is safe and effective for various diseases such as cancer, a higher level of evidence is still needed to confirm its safety and effects before using it widely in the clinical setting. Considering that injection of pharmacopuncture solution could be an alternative to the oral intake of herbal decoction in patients in a coma state or patients with liver damage, we believe that further studies on ginseng pharmacopuncture will bring benefits.