Emergent quality issues in the supply of Chinese medicinal plants: A mixed methods investigation of their contemporary occurrence and historical persistence

Quality issues that emerged centuries ago in Chinese medicinal plants (CMP) were investigated to explore why they still persist in an era of advanced analytical testing and extensive legislation so that a solution to improve CMP quality could be proposed. This is important for 85% of the world’s population who rely on medicinal plants (MP) for primary healthcare considering the adverse events, including fatalities that arise from such quality issues. CMP are the most prevalent medicinal plants globally. This investigation used mixed-methods, including 15 interviews with CMP expert key informants (KI), together with thematic analysis that identified the main CMP quality issues, why they persisted, and informed solutions. An unexplained case example, Eleutherococcus nodiflorus (EN), was analysed by collection of 106 samples of EN, its known toxic adulterant Periploca sepium (PS), and a related substitute, Eleutherococcus senticosus (ES), across mainland China, Taiwan and the UK. Authenticity of the samples was determined using High-performance thinlayer chromatography. Misidentification, adulteration, substitution and toxicity were the main CMP quality issues identified. Adulteration was found widespread globally with 57.4% EN found authentic, and 24.6% adulterated with cardiotoxic PS, mostly at markets and traditional pharmacies. The EN study further highlighted that the reason CMP quality issues persisted was due to the laboratory-bound nature of analytical methods and testing currently used that leave gaps in detection throughout much of the supply chain. CMP quality could be more effectively tested with patented analytical technology (PAT) and simpler field-based testing including indicator strip tests. Education highlighting the long-term economic value and communal benefit of delivering better quality CMP to consumers was recommended in favour of the financial motivation for actions that lead to the persistence of well-known and recurrent CMP quality issues

In vitro expanded human CD4+CD25+ regulatory T cells suppress effector T cell proliferation.

Regulatory T cells (Tregs) have been shown to be critical in the balance between autoimmunity and tolerance and have been implicated in several human autoimmune diseases. However, the small number of Tregs in peripheral blood limits their therapeutic potential. Therefore, we developed a protocol that would allow for the expansion of Tregs while retaining their suppressive activity. We isolated CD4+CD25 hi cells from human peripheral blood and expanded them in vitro in the presence of anti-CD3 and anti-CD28 magnetic Xcyte Dynabeads and high concentrations of exogenous Interleukin (IL)-2. Tregs were effectively expanded up to 200-fold while maintaining surface expression of CD25 and other markers of Tregs: CD62L, HLA-DR, CCR6, and FOXP3. The expanded Tregs suppressed proliferation and cytokine secretion of responder PBMCs in co-cultures stimulated with anti-CD3 or alloantigen. Treg expansion is a critical first step before consideration of Tregs as a therapeutic intervention in patients with autoimmune or graft-versus-host disease

Evaluation of the efficacy, safety and tolerability of herbal medicine for management of the behavioural and psychological symptoms of dementia

Dementia involves a gradual loss of memory and cognitive skills. Over 50% of people with dementia also suffer from the behavioural and psychological symptoms of dementia (BPSD). BPSD refer to disturbed perception, thought content and mood associated with dementia, and include psychosis, agitation, aggression, irritability, depression, anxiety and abnormal motor activity. BPSD can have a negative impact on the progression of Alzheimer’s disease and related disorders and are associated with a greater level of caregiver distress. Currently recommended pharmacological treatments for dementia, including acetylcholinesterase inhibitors and memantine, focus on relieving cognitive symptoms, while BPSD are managed according to the presenting symptoms. Guidelines recommend non-pharmacological approaches for BPSD but selective serotonin reuptake inhibitors, analgesics and second generation antipsychotics may be used when other approaches fail. Pharmacological treatments for BPSD have limited benefit and safe prescribing is difficult as some may produce severe adverse effects and may worsen cognitive symptoms. Consequently, there is a pressing need for new approaches to BPSD management. Multiple clinical studies have reported that herbal medicines (HMs), such as Ginkgo biloba leaf extract, can alleviate BPSD as well as improve cognition in dementia. The present project aimed to determine the current state of evidence for HMs and propose future directions for the development of plant-based therapeutics for managing BPSD. Both historical use and contemporary clinical trials provide evidence for the use of herbs for management of memory impairment, cognitive symptoms of dementia and BPSD. Based on these findings, there is potential for identifying effective herbal interventions that could be fast-tracked into clinical use for this unmet need. Identification of useful compounds and their possible mechanisms of action may contribute to development of new therapeutic approaches and/or drug discovery. Notably, the acetylcholinesterase inhibitors galantamine and rivastigmine were discovered from plant-derived compounds. The objectives of this project were to assess the current state of evidence and its limitations regarding the efficacy, safety and tolerability of HMs for BPSD by systematically reviewing and analysing the results of clinical trials and the classical Chinese medicine literature on BPSD; identify any HMs that show potential benefit for BPSD; based on the best available evidence, select a herbal intervention suitable for further clinical investigation; and design a rigorous randomised controlled trial (RCT) to test the intervention that addresses the limitations of previous clinical studies. The comprehensive systematic review and meta-analysis included 31 controlled clinical trials testing 19 different HMs. Meta-analysis of well-designed, placebo-controlled studies indicated that the G. biloba leaf extract EGb 761® was safe and well-tolerated. Significant and clinically meaningful improvements in BPSD and cognition were detected at 24 weeks. However, independently funded studies of G. biloba leaf extract are needed to confirm these findings. Meta-analysis of randomised, comparative effectiveness studies of the Japanese multi-herb formula Yokukansan (Chinese: Yi gan san) showed no significant differences on BPSD outcomes when compared to standard pharmacotherapies used in Japan for BPSD management and superiority to no treatment. However, the only placebo-controlled study of Yokukansan for BPSD did not find any difference in BPSD outcomes at the end of four weeks treatment. Important limitations were identified in this trial which could have resulted in a false negative. These included its short duration, relatively small sample size, large improvement in the placebo group, and the use of a simplified outcome measure which could be less likely to detect changes in symptoms. Overall, the clinical evidence for Yokukansan suggested improvements in BPSD, notably in the clinically important symptoms of irritability/lability and agitation/aggression. An issue identified with Yokukansan was the increased risk of liquorice-induced hypokalaemia, which requires monitoring. Lack of replication and methodological issues in the studies testing other HMs precluded any conclusions for these other interventions. The classical Chinese medical literature was evaluated using the Zhong Hua Yi Dian database, using similar methods to a previous study which evaluated the herbs used for treatment of the cognitive symptoms of dementia. No specific term in the literature corresponded with BPSD, although terms for specific symptoms such as anxiety, depression and agitation were frequently mentioned in conjunction with terms for memory impairment. Some of the HMs described in the classical Chinese medical literature for treatment of memory impairments with mood and behavioural symptoms had also been tested in the clinical trial literature, including Glycyrrhiza uralensis, Poria cocos and Angelica species, which are ingredients of Yokukansan, but the most frequently cited herbs in the classical literature were generally not the same as the frequently tested HMs in clinical trials. The in vitro and in vivo literature showed evidence of relevance to treatment of BPSD for G. biloba, Yokukansan and their constituent compounds. For both these HMs, animal studies have reported anti-aggression-like, antidepressant-like, anxiolytic-like effects as well as benefits on abnormal motor activities and reduction in cognitive impairments and mental stress. Important activities of G. biloba leaf and Yokukansan of relevance to BPSD include modulation of neurotransmission, neuroendocrine regulation and antioxidant effects. The issue of wide variation in placebo effect sizes in BPSD studies was explored through meta-analysis of placebo data. Results showed that placebo effect sizes for BPSD have increased over time. Proposed new studies may therefore require larger sample sizes in order to be adequately powered. Based on the available evidence, it appears that EGb 761® provides small improvements in cognitive symptoms and reduces BPSD while Yokukansan can improve BPSD but not cognition, although it does not appear to have any negative effect on cognition. Both HMs are available commercially, are well characterised and are in widespread use but their combination has not been tested in a clinical trial. Notably, most studies of EGb 761® were conducted in Europe or Russia while Yokukansan was tested in Japan. Therefore, it was proposed that the combination of EGb 761® and Yokukansan at conservative dosages should be tested through an adequately powered, randomised, placebo-controlled clinical trial. A clinical trial protocol was designed which utilised validated diagnostic criteria and assessments relevant to an older population with BPSD, including assessments of caregiver distress associated with BPSD. Issues relating to informed consent from participants and their caregivers were addressed. Safety was an important consideration and was addressed through inclusion and exclusion criteria, careful monitoring of adverse events and strategies to reduce the risk of liquorice-induced hypokalaemia. The results of the RCT would provide useful data on the safety, tolerability and efficacy of this combined intervention in an Australian population with BPSD. The results would assist with clinical decision-making in the management of BPSD

Cultivation and quality studies of Danshen (Salvia miltiorrhiza) in Australia

Danshen (Salvia miltiorrhiza Bunge) is a commonly used Chinese medicinal herb for treating cardiovascular and cerebrovascular diseases. This thesis aims to determine which Danshen varieties are most suitable for local cultivation in Australia, including the optimum harvesting times, growth densities and fertiliser conditions. A two-year field trial was conducted. Among the eight varieties (V1-V8), significant variations were found in root yields and contents of the bioactive marker compounds. The contents of tanshinone IIA and salvianolic acid B in V1, V2, V3 and V6 reached the standards as set in the Chinese Pharmacopoeia. In particular, V6 was the best variety for tanshinones, and V4 and V5 were best varieties for salvianolic acid B. V4 showed a great variation in the growth of roots and aerial parts， and the quantities of the four marker compounds and other main chemical components. The optimum harvesting time for V4 was at 250 days for the roots with the best root yield and the highest salvianolic acid B content, and 290 days for the roots with the best root yield and the highest tanshinone IIA content. Moreover, V4 roots showed a higher content of tanshinones in the first year cultivation, but a higher content of salvianolic acid B in the second year cultivation. V4, grown at 30 × 30 cm, produced the highest root yield per square meter and the highest content of the three tanshinones (e.g. 0.210% tanshinone IIA, w/w); in contrast, when grown at 45 × 30 cm, it produced a high salvianolic acid B content (>3%) and the highest root yield per square meter in three densities (30 × 40 cm, 45 × 30 cm and 45 × 40 cm). Different fertiliser treatments significantly increased the root yield of V1. In particular, a high level phosphorus and nitrogen showed a synergistic effect on root yield. However, an optimum fertiliser treatment for maximum root yield and content of tanshinones was not determined. Both qualitative and quantitative analysis, using marker compounds and chromatographic fingerprint, by TLC and/or HPLC, were developed for assessing the quality of Australian grown Danshen. The findings showed that the three tanshinone markers or their corresponding peaks correlated linearly with each other as well as with other main peaks in chromatographic fingerprints of methanol extracts. Similarly, salvianolic acid B or its corresponding peak correlated linearly with the other main peaks in chromatographic fingerprints of aqueous extracts. In conclusion, the present findings indicate that under proper conditions of harvesting time, fertilisation and plant density, high quality Danshen varieties can be cultivated in Victoria, Australia