Systems Pharmacology-based strategy to screen new adjuvant for hepatitis B vaccine from Traditional Chinese Medicine Ophiocordyceps sinensis Jingbo Wang+, Rui Liu, Baoxiu Liu, Yan Yang, Jun Xie*, Naishuo Zhu* I Laboratory of Molecular Immunology State Key Laboratory of Genetic Engineering Institute of Biomedical Science, School of Life Sciences, Fudan University, Shanghai, 200438. China *Correspondence and requests for materials should be addresses to NZ.(Email chu@fudan.edu.cn)andJX.(Email:xiejun@fudan.edu.cn) t These authors contributed equally to this work Adjuvants are common component for many vaccines but there are still few licensed for human use due to low efficiency or side effects. The present work adopted Systems Pharmacology analysis as a new strategy to screen adjuvants from traditional Chinese medicine Ophiocordyceps sinensis has been used for many years in China and other Asian countries with many biological properties, but the pharmacological mechanism has not been fully elucidated. First in this study, 190 putative targets for 17 active compounds in Ophiocordyceps sinensis were retrieved and a systems pharmacology-based approach was applied to provide new insights into the pharmacological actions of the drug. Pathway enrichment analysis found that the targets participated in several immunological processes. Based on this, we selected cordycepin as a target compound to serve as an adjuvant of the hepatitis B vaccine because the existing vaccine often fails to induce an effective immune response in
1 Systems Pharmacology-based strategy to screen new adjuvant for hepatitis B vaccine from Traditional Chinese Medicine Ophiocordyceps sinensis Jingbo Wang1+, Rui Liu1+, Baoxiu Liu1 , Yan Yang1 , Jun Xie1 *, Naishuo Zhu1 * 1 Laboratory of Molecular Immunology, State Key Laboratory of Genetic Engineering, Institute of Biomedical Science, School of Life Sciences, Fudan University, Shanghai, 200438, China. *Correspondence and requests for materials should be addresses to N.Z. (Email: nzhu@fudan.edu.cn) and J.X. (Email:xiejun@fudan.edu.cn) + These authors contributed equally to this work. Adjuvants are common component for many vaccines but there are still few licensed for human use due to low efficiency or side effects. The present work adopted Systems Pharmacology analysis as a new strategy to screen adjuvants from traditional Chinese medicine. Ophiocordyceps sinensis has been used for many years in China and other Asian countries with many biological properties, but the pharmacological mechanism has not been fully elucidated. First in this study, 190 putative targets for 17 active compounds in Ophiocordyceps sinensis were retrieved and a systems pharmacology-based approach was applied to provide new insights into the pharmacological actions of the drug. Pathway enrichment analysis found that the targets participated in several immunological processes. Based on this, we selected cordycepin as a target compound to serve as an adjuvant of the hepatitis B vaccine because the existing vaccine often fails to induce an effective immune response in
many subjects. Animal and cellular experiments finally validated that the new vaccine simultaneously improves the humoral and cellular immunity of BALB/c mice without side effects. All this results demonstrate that cordycepin could work as adjuvant to hepatitis b vaccine and systems-pharmacology analysis could be used as a new method to select adjuvants Introduction Adjuvants could elicit immune responses through different signaling pathways thus improve vaccine formulations for better protection, however, there are still very few adjuvants have been licensed for human because of the side effects or other problems. Even the widely used alum adjuvant could cause local reaction and IgE responses during vaccination. Therefore, there is a requirement to develop novel adjuvants for vaccines. Traditional Chinese Medicine (TCM) involves the use of natural products that have been utilized by humans for centuries for good health TCMs contain many bioactive ingredients with beneficial effects, and thus have attracted much attention in recent years. Although herbal medicines are comprised of several ingredients that target multiple organs, it is difficult to pin-point the bioactive compounds by traditional pharmacological methods. In addition, TCMs are not widely used outside of China and other Asian countries because of the lack of scientific data on their mechanisms of drug action. Thus, additional studies are needed to identify the underlying mechanisms Systems pharmacology, an emerging area of pharmacology, combines drug-like
2 many subjects. Animal and cellular experiments finally validated that the new vaccine simultaneously improves the humoral and cellular immunity of BALB/c mice without side effects. All this results demonstrate that cordycepin could work as adjuvant to hepatitis b vaccine and systems-pharmacology analysis could be used as a new method to select adjuvants. Introduction Adjuvants could elicit immune responses through different signaling pathways thus improve vaccine formulations for better protection1 , however, there are still very few adjuvants have been licensed for human because of the side effects or other problems2-4. Even the widely used alum adjuvant could cause local reaction and IgE responses during vaccination. Therefore, there is a requirement to develop novel adjuvants for vaccines. Traditional Chinese Medicine (TCM) involves the use of natural products that have been utilized by humans for centuries for good health5 . TCMs contain many bioactive ingredients with beneficial effects, and thus have attracted much attention in recent years6 . Although herbal medicines are comprised of several ingredients that target multiple organs, it is difficult to pin-point the bioactive compounds by traditional pharmacological methods. In addition, TCMs are not widely used outside of China and other Asian countries because of the lack of scientific data on their mechanisms of drug action7 . Thus, additional studies are needed to identify the underlying mechanisms. Systems pharmacology, an emerging area of pharmacology, combines drug-like
prediction, absorption, distribution, multiple drug target prediction and network analysis to analyze drugs, drug targets, pathways and drug effects". This approach, which can be used in the discovery of single bioactive ingredients, can help to identify he mechanisms of drug action 0, I Hepatitis B(HB)is an infectious disease caused by the hepatitis B virus(HBv) There are approximately 350 million individuals worldwide that carry the HBv, and China has a high incidence, with approximately 120 million carriers. Chronic HBV infection can lead to hepatitis, cirrhosis and hepatocellular carcinoma(HCC), and effective treatments are lacking. Because of the high morbidity and mortality, hepatitis b vaccination is essential for reducing the carrier rate and preventing viral infection The currently used genetically engineered hepatitis b vaccine with aluminum hydroxide as the conventional adjuvant has low efficacy, a long waiting period to immune response and large individual differences in immune effects. Approximately 5-10% of individuals with a normal immune system cannot achieve effective immunity against hepatitis b virus infection Therefore, developing a quick and effective adjuvant to enhance hepatitis b vaccine immunogenicity is of great practical Ophiocordyceps sinensis(syn. Cordyceps sinensis), placed systematically as Ophiocordycipitaceae, Hypocreales, Hypocreomycetidae, Sordariomycetes, Ascomycota, is a TCM agent that has been widely used as a folk tonic for nearly one thousand years. It has an overwhelming list of pharmacological properties 8-.As one of the traditional and medical fungi, O. sinensis is currently available in China
3 prediction, absorption, distribution, multiple drug target prediction and network analysis to analyze drugs, drug targets, pathways and drug effects8,9. This approach, which can be used in the discovery of single bioactive ingredients, can help to identify the mechanisms of drug action10,11. Hepatitis B (HB) is an infectious disease caused by the hepatitis B virus (HBV). There are approximately 350 million individuals worldwide that carry the HBV, and China has a high incidence, with approximately 120 million carriers12. Chronic HBV infection can lead to hepatitis, cirrhosis and hepatocellular carcinoma (HCC), and effective treatments are lacking. Because of the high morbidity and mortality, hepatitis b vaccination is essential for reducing the carrier rate and preventing viral infection13. The currently used genetically engineered hepatitis b vaccine with aluminum hydroxide as the conventional adjuvant has low efficacy, a long waiting period to immune response and large individual differences in immune effects. Approximately 5-10% of individuals with a normal immune system cannot achieve effective immunity against hepatitis b virus infection14-16. Therefore, developing a quick and effective adjuvant to enhance hepatitis b vaccine immunogenicity is of great practical value. Ophiocordyceps sinensis (syn. Cordyceps sinensis), placed systematically as Ophiocordycipitaceae, Hypocreales, Hypocreomycetidae, Sordariomycetes, Ascomycota, is a TCM agent that has been widely used as a folk tonic for nearly one thousand years17. It has an overwhelming list of pharmacological properties18-21. As one of the traditional and medical fungi, O. sinensis is currently available in China
and South East Asia22230 sinensis contains several bioactive components, and many reports have carefully analyzed its make-up of polysaccharides, sterols, nucleosides and protein 2224. Many of these components have been found to possess immunological, anti-tumorigenic, anti-oxidative, anti-inflammatory, anti-fatigue anti-fungal and anti-hypertensive properties, as well as to protect the kidney, liver and lung2526. However, there is no systematic analysis of the drug-target network of O sinensis. Therefore, the aim of the current study was to explain how the immunoregulation function was implemented and find a component that can act as an adjuvant of the hepatitis b vaccine through systems pharmacology analysis. Briefly, as seen in Figure 1, we first use a network pharmacology approach to determine the active ingredients of O. sinensis. A molecular-target network was then developed followed by enrichment analysis and functional classification. After screening,a single component was selected and validated by in vivo and in vitro experiments Results Candidate component identification. It has been difficult to identify the mechanisms of action of different TCM agents due to their complex biochemical make-up, and specific methods to identify the bioactive compounds have not been available until now. In this study, we used oral bioavailability(OB) screening and drug-likeness(DL) property evaluation or drug half-life(HL) prediction to identify the bioactive compounds".All the ingredients of O. sinensis were obtained from the Traditional Chinese Medicines Systems Pharmacology Database and Analysis
4 and South East Asia22,23. O. sinensis contains several bioactive components, and many reports have carefully analyzed its make-up of polysaccharides, sterols, nucleosides and protein21,22,24. Many of these components have been found to possess immunological, anti-tumorigenic, anti-oxidative, anti-inflammatory, anti-fatigue, anti-fungal and anti-hypertensive properties, as well as to protect the kidney, liver and lung25,26. However, there is no systematic analysis of the drug-target network of O. sinensis. Therefore, the aim of the current study was to explain how the immunoregulation function was implemented and find a component that can act as an adjuvant of the hepatitis b vaccine through systems pharmacology analysis. Briefly, as seen in Figure 1, we first use a network pharmacology approach to determine the active ingredients of O. sinensis. A molecular-target network was then developed, followed by enrichment analysis and functional classification. After screening, a single component was selected and validated by in vivo and in vitro experiments. Results Candidate component identification. It has been difficult to identify the mechanisms of action of different TCM agents due to their complex biochemical make-up, and specific methods to identify the bioactive compounds have not been available until now27. In this study, we used oral bioavailability (OB) screening and drug-likeness (DL) property evaluation or drug half-life (HL) prediction to identify the bioactive compounds7,9. All the ingredients of O. sinensis were obtained from the Traditional Chinese Medicines Systems Pharmacology Database and Analysis
Platform2 and the CancerHSP Database2 Twenty potential compounds with OB 30%, DL 20.18 or HL 4 were selected. Additionally, three compounds in the CancerhSP database with potential anti-cancer activities and ob 30% were also selected as candidate compounds for further analysis. Duplicate compounds in the two databases were eliminated, yielding 23 readily-absorbed compounds from a total of 40 compounds. The satisfied compounds are presented in Supplementary Table SI Target prediction and functional analysis. Generally, TCMs could prevent diseases through synergistic effects of different compounds and targets. Therefore, the potential therapeutic targets of the multiple compounds were important for its synergistic effects. After eliminating six additional compounds without targets, 190 targets in the database were assembled and ranked for 17 components contained in O sinensis. Detailed enrichment analysis and functional pathway classification showed that the bioactive components were involved in several cellular events, including signal transduction and cell differentiation( Figure 2a and Supplementary Table $2) Network construction. O. sinensis exerts extensive biological and pharmacological effects through multiple compound and target interactions. To understand these effects at the systemic level, a compound-target network was constructed based on the candidate components and targets. Figure 2b shows the results of the compound-target-function network, which consisted of 17 compounds, 190 candidate targets and seven functional annotations. The results displayed an average degree of 9.4 per compound and 3. 8 per target protein, respective relationships between compounds and targets, as shown in Figure 2c, the
5 Platform28 and the CancerHSP Database29. Twenty potential compounds with OB ≥ 30%, DL ≥ 0.18 or HL ≥ 4 were selected. Additionally, three compounds in the CancerHSP database with potential anti-cancer activities and OB ≥ 30% were also selected as candidate compounds for further analysis. Duplicate compounds in the two databases were eliminated, yielding 23 readily-absorbed compounds from a total of 40 compounds. The satisfied compounds are presented in Supplementary Table S1. Target prediction and functional analysis. Generally, TCMs could prevent diseases through synergistic effects of different compounds and targets. Therefore, the potential therapeutic targets of the multiple compounds were important for its synergistic effects. After eliminating six additional compounds without targets, 190 targets in the database were assembled and ranked for 17 components contained in O. sinensis. Detailed enrichment analysis and functional pathway classification showed that the bioactive components were involved in several cellular events, including signal transduction and cell differentiation (Figure 2a and Supplementary Table S2). Network construction. O. sinensis exerts extensive biological and pharmacological effects through multiple compound and target interactions. To understand these effects at the systemic level, a compound-target network was constructed based on the candidate components and targets30. Figure 2b shows the results of the compound-target-function network, which consisted of 17 compounds, 190 candidate targets and seven functional annotations. The results displayed an average degree of 9.4 per compound and 3.8 per target protein, respectively. With regard to the relationships between compounds and targets, as shown in Figure 2c, the
compound-target network embodied 207 nodes(17 potential compounds and 190 potential targets). The mean degree value(the number of associated targets)of candidate components was 18.1, and 1l components possessed a mean degree value greater than 12, suggesting that most components associated with multiple targets to exert different biological and pharmacological effects. The major components and its target numbers are shown in Table S3. Specifically, components such as caffeine, beta-sitosterol(Sito) and arachidonic acid(AA), which acted on 54, 38 and 38 targets respectively, were the crucial active components for O. sinensis in this network. The second major components are oleic acid, cordycepin and nicotinic acid mmune regulation mode. Previous data showed that the targets of O. sinensis were involved in immune function that included positive regulation of the immune system to bacterial infection, and T and B cell function. Cytokines also play an important role in the activation and regulation of immune responses. As can be seen from Figure 3, marked target genes make a contribution to the production of interferon which are detected to connect with anti-virus or bacterial responses. On the other hand, components in O. sinensis also possess anti-inflammatory effects during both our prediction and some other studies. For instance, cordycepin was observed to suppress LPS-stimulated release of pro-inflammatory cytokines TNFa and IL-1B through NF kB pathway, Sito was observed to induce cancer cell line apoptosis through decrease the expression of apoptosis regulator Bcl-2 which makes it a candidate for cancer chemotherapy. Besides, cordycepin was predicted to interact with ROCK which related to leukocyte migration and this may contribute to the formation of germinal
6 compound-target network embodied 207 nodes (17 potential compounds and 190 potential targets). The mean degree value (the number of associated targets) of candidate components was 18.1, and 11 components possessed a mean degree value greater than 12, suggesting that most components associated with multiple targets to exert different biological and pharmacological effects. The major components and its target numbers are shown in Table S3. Specifically, components such as caffeine, beta-sitosterol (Sito) and arachidonic acid (AA), which acted on 54, 38 and 38 targets, respectively, were the crucial active components for O. sinensis in this network. The second major components are oleic acid, cordycepin and nicotinic acid. Immune regulation mode. Previous data showed that the targets of O. sinensis were involved in immune function that included positive regulation of the immune system to bacterial infection, and T and B cell function. Cytokines also play an important role in the activation and regulation of immune responses. As can be seen from Figure 3, marked target genes make a contribution to the production of interferon which are detected to connect with anti-virus or bacterial responses. On the other hand, components in O. sinensis also possess anti-inflammatory effects during both our prediction and some other studies. For instance, cordycepin was observed to suppress LPS-stimulated release of pro-inflammatory cytokines TNFα and IL-1β through NF- κB pathway31; Sito was observed to induce cancer cell line apoptosis through decrease the expression of apoptosis regulator Bcl-2 which makes it a candidate for cancer chemotherapy32. Besides, cordycepin was predicted to interact with ROCK which related to leukocyte migration and this may contribute to the formation of germinal
center or other cellar immune responses There have been many studies in the 3 key components in O. sinensis, for example caffeine was used to defense depression and AA could promote secretion of pro-Inflammatory leukotrienes 33, 34 After screening the major components of o sinensis, the nucleoside antibiotic cordycepin was selected for further experimental alidation based on our ad has d much attention, mostly because of its anti-tumor and antiviral activities in the inhibition of viral DNA/RNA synthesis 5-37. For example, cordycepin induces apoptosis of tumorigenic Leydig cells in the mouse via several signaling pathways. It also plays roles in platelet aggregation and inflammation, 142, however, there are few reports on the use of cordycepin as an adjuvant to enhance the immune response. We here in combined cordycepin with HBsAg to generate a new vaccine and tested its immunologic activity in mice. Cordycepin improves HBsAg-specific antibody production without side effects Serum HBV antibodies were measured by elisa to determine the effects of the adjuvant on the humoral immune response. As shown in Figure 4, cordycepin(0. 2, 1 and 2 mg/kg)adjuvanted with HBsAg led to an increase in the serum antibody level in a dose-dependent manner. The IgM level remained high on day 21 in the 2 mg/kg cordycepin group but not in the other groups(Figure 4a). On day 14, the IgG titers nduced by 2 mg/kg cordycepin adjuvanted with HBsAg were similar to the vaccine group on day 21 after the last injection(Figure 4b). However, there was no difference in the IgG level in this group on days 14 and 21. Furthermore, there was no significant
7 center or other cellar immune responses. There have been many studies in the 3 key components in O. sinensis, for example, caffeine was used to defense depression and AA could promote secretion of pro-inflammatory leukotrienes33,34. After screening the major components of O. sinensis, the nucleoside antibiotic cordycepin was selected for further experimental validation based on our adjuvant screen purpose. Cordycepin has received much attention, mostly because of its anti-tumor and antiviral activities in the inhibition of viral DNA/RNA synthesis35-37. For example, cordycepin induces apoptosis of tumorigenic Leydig cells in the mouse via several signaling pathways38-40. It also plays roles in platelet aggregation and inflammation36,41,42; however, there are few reports on the use of cordycepin as an adjuvant to enhance the immune response. We here in combined cordycepin with HBsAg to generate a new vaccine and tested its immunologic activity in mice. Cordycepin improves HBsAg-specific antibody production without side effects. Serum HBV antibodies were measured by ELISA to determine the effects of the adjuvant on the humoral immune response. As shown in Figure 4, cordycepin (0.2, 1 and 2 mg/kg) adjuvanted with HBsAg led to an increase in the serum antibody level in a dose-dependent manner. The IgM level remained high on day 21 in the 2 mg/kg cordycepin group but not in the other groups (Figure 4a). On day 14, the IgG titers induced by 2 mg/kg cordycepin adjuvanted with HBsAg were similar to the vaccine group on day 21 after the last injection (Figure 4b). However, there was no difference in the IgG level in this group on days 14 and 21. Furthermore, there was no significant
different in body weight in mice from all groups(Figure Sla). There was also no apparent histological change in the liver and spleen of mice from all groups on day 28 by haematoxylin-eosin staining and routine light microscopy(Figure SIb) Cordycepin adjuvanted vaccine promotes lymphocyte proliferation and cytokine production in vivo and in vitro. The enhancement of cellular immunity is also important barrier for HBV infection. HBSAg adjuvanted with I or 2 mg/kg cordycepin resulted in significantly higher proliferative activity than that in the other group. The cell activity of the 2 mg/kg cordycepin group was similar to that of the positive control (Figure 4c), indicating that it elicits an effective cellular immune response. The increased cell supernatant cytokine levels(Figure 4d) suggest that both Thl and Th2 cell responses were activated by cordycepin Lymphocyte differentiation in the spleen after immunization. To assess t and b cell differentiation after immunization, spleen cells were collected and analyzed by flow cytometry( Figure 5). The percentage of CD3 CD4 Th cells and CD3 CD8 CTL cells were all increased by the supplementary of cordycepin. The CD80 and CD86 expression on CD19+ B cells were also enhanced in the adjuvant groups, indicating hat b cells were activated more effectively. The increase in Th and B cell number indicates that the increase in antibody production and CTL cell number enhanced the cytotoxic effect, which might associate with IFN-Y Discussion Complex composition of TCMs makes it hard to understand its therapeutic mode from a molecular level. In recent years, several herbal components with different
8 different in body weight in mice from all groups (Figure S1a). There was also no apparent histological change in the liver and spleen of mice from all groups on day 28 by haematoxylin-eosin staining and routine light microscopy (Figure S1b). Cordycepin adjuvanted vaccine promotes lymphocyte proliferation and cytokine production in vivo and in vitro. The enhancement of cellular immunity is also an important barrier for HBV infection. HBsAg adjuvanted with 1 or 2 mg/kg cordycepin resulted in significantly higher proliferative activity than that in the other group. The cell activity of the 2 mg/kg cordycepin group was similar to that of the positive control (Figure 4c), indicating that it elicits an effective cellular immune response. The increased cell supernatant cytokine levels (Figure 4d) suggest that both Th1 and Th2 cell responses were activated by cordycepin. Lymphocyte differentiation in the spleen after immunization. To assess T and B cell differentiation after immunization, spleen cells were collected and analyzed by flow cytometry (Figure 5). The percentage of CD3+ CD4+ Th cells and CD3+ CD8+ CTL cells were all increased by the supplementary of cordycepin. The CD80 and CD86 expression on CD19+ B cells were also enhanced in the adjuvant groups, indicating that B cells were activated more effectively. The increase in Th and B cell number indicates that the increase in antibody production and CTL cell number enhanced the cytotoxic effect, which might associate with IFN-γ. Discussion Complex composition of TCMs makes it hard to understand its therapeutic mode from a molecular level. In recent years, several herbal components with different
pharmacological activities have been identified as the development of biological analysis, these components constitute a substantial percentage of todays new drugs#4. One of the most commonly used method to investigate new drugs is to start from a disease and search from natural products which could treat it, however, the limitation of technical difficulties and suitable animal models makes it difficult to understand their mode of action 5,46. Recently, systems pharmacology offers a new approach to study bioactive components of TCM agents, as well as their drug-target interactions. Herein, we try to selective adjuvant from O. sinensis for its remarkable immunoregulation effects. Although there have been many studies about effective components in O. sinensis, the exact action mode was not fully elicited at a system level In the present study, with the help of OB, DL and HL screening, 17 active compounds with 190 potential targets were identified in O. sinensis. The target enrichment analysis shows that these targets take part in many biological pathways such as cytokine secretion, drug binding, blood circulation and so on. Several targets were lso involved in different types of cancer, supporting an earlier study which showed O sinensis extracts to exhibit anti-cancer effects"-49. It was not a surprise that several ts have been studied clearly in ches. Caffeine is well-known phosphodiesterase 3(PDE3)inhibitor and antagonist of adenosine receptors. Caffeine can inhibit the production of tumor necrosis factor alpha(tNF-a in lipopolysaccharide-stimulated human whole blood and modulate specific biological parameters associated with depression, thereby preventing the disease 2. 53
9 pharmacological activities have been identified as the development of biological analysis; these components constitute a substantial percentage of today’s new drugs43,44. One of the most commonly used method to investigate new drugs is to start from a disease and search from natural products which could treat it, however, the limitation of technical difficulties and suitable animal models makes it difficult to understand their mode of action45,46. Recently, systems pharmacology offers a new approach to study bioactive components of TCM agents, as well as their drug-target interactions. Herein, we try to selective adjuvant from O. sinensis for its remarkable immunoregulation effects. Although there have been many studies about effective components in O. sinensis, the exact action mode was not fully elicited at a system level. In the present study, with the help of OB, DL and HL screening, 17 active compounds with 190 potential targets were identified in O. sinensis. The target enrichment analysis shows that these targets take part in many biological pathways such as cytokine secretion, drug binding, blood circulation and so on. Several targets were also involved in different types of cancer, supporting an earlier study which showed O. sinensis extracts to exhibit anti-cancer effects47-49. It was not a surprise that several major components have been studied clearly in previous researches. Caffeine is a well-known phosphodiesterase 3 (PDE3) inhibitor and antagonist of adenosine receptors50. Caffeine can inhibit the production of tumor necrosis factor alpha (TNF-α) in lipopolysaccharide-stimulated human whole blood51 and modulate specific biological parameters associated with depression, thereby preventing the disease52,53
Interestingly, caffeine can also protect against the production of free radicals 4. Sito is a functional phytosterol with a chemical structure similar to that of cholesterol. Sito alone or combined with other plant sterols is known to reduce blood cholesterol levels by blocking cholesterol absorption 6,7 Sito has also been reported to possess antimicrobial activity". AA, an unsaturated fatty acid that modulates the activities of various ion channels, functions as a second messenger. As the adjuvant selection purpose, we choose all immune related function to get a component-target -function network. Results show that all components participate in these processes. Then,an immune-related pathway was constructed to further dissect the potential immunoregulation activities of components in O. sinensis. Cordycepin was finally selected because of its significant functions to suppress inflammation and regulate leukocyte migration as well as cell proliferation in the networks. Although it was not the only key component in O. sinensis, other components may interference with nutrient absorption or inhibit cytokine secretion, may have an adverse effect on healthy people or just suppress immune responses There have been many studies on cordycepin these days, mainly concentrate on its anti-tumor and immunoregulation effects, but few reports pay attention to its adjuvanticity to vaccines In the experimental validation part, cordycepin triggered IgM and Igg antibody responses in mice by 7 days in a dose dependent manner after combination with a hepatitis b vaccine. The high cordycepin group showed no difference in IgG titers between days 14 and 21 suggesting that the third injection might be unnecessary. Certainly, this would reduce the overall cost and the immune
10 Interestingly, caffeine can also protect against the production of free radicals54. Sito is a functional phytosterol with a chemical structure similar to that of cholesterol55. Sito alone or combined with other plant sterols is known to reduce blood cholesterol levels by blocking cholesterol absorption56,57. Sito has also been reported to possess antimicrobial activity58. AA, an unsaturated fatty acid that modulates the activities of various ion channels, functions as a second messenger59. As the adjuvant selection purpose, we choose all immune related function to get a component-target-function network. Results show that all components participate in these processes. Then, an immune-related pathway was constructed to further dissect the potential immunoregulation activities of components in O. sinensis. Cordycepin was finally selected because of its significant functions to suppress inflammation and regulate leukocyte migration as well as cell proliferation in the networks. Although it was not the only key component in O. sinensis, other components may interference with nutrient absorption or inhibit cytokine secretion, may have an adverse effect on healthy people or just suppress immune responses. There have been many studies on cordycepin these days, mainly concentrate on its anti-tumor and immunoregulation effects60-62, but few reports pay attention to its adjuvanticity to vaccines. In the experimental validation part, cordycepin triggered IgM and IgG antibody responses in mice by 7 days in a dose dependent manner after combination with a hepatitis b vaccine. The high cordycepin group showed no difference in IgG titers between days 14 and 21 suggesting that the third injection might be unnecessary. Certainly, this would reduce the overall cost and the immune
