Validation of a Toll-like Receptor (TLR)2/TLR1 Activation Assay for Biological Standardization of Arthrospira/Limnospira Immune-Enhancing Potency

Abstract

Arthrospira/Limnospira is a popular botanical dietary supplement throughout the world and has been consumed as a food product for hundreds of years. Ongoing efforts from our research group are focused on evaluating the utility of a Limnospira-derived oral supplement (Immulina) in promoting resilience against influenza viral infection. Like other botanical extracts, Immulina is inherently a complex matrix with variation in the levels of its chemical constituents. Therefore, to ensure therapeutic consistency for future scientific research and clinical studies, we are developing standardization technology using a bioassay and chemical markers. Braun-type lipoproteins, a class of macromolecules that activate the Toll-like receptor (TLR)2/TLR1 signaling pathway, have been identified as a major active component within Immulina. Based on the mechanism of action of the Braun-type lipoproteins, an in vitro bioassay was established using the HEK-Blue hTLR2/TLR1 cell line to quantitate the immune-enhancing potency of Immulina. The objective of the current research was to validate that bioassay for Immulina activity quantification using the U.S. FDA guidance document for botanical drug development and U.S. Pharmacopeia recommendations. System suitability, reference standards and defining potency units were established. Validation of performance parameters included precision, specificity, accuracy, linearity, and range. Validating this bioassay for Immulina activity provides a tool for ensuring product consistency and quantifying the potency of this botanical for use in future research as well as material in the consumer market.

Keywords:

• Arthrospira
• bioassay
• immune
• spirulina
• standardization
• validation

Disclosure statement

NDP and IAK acknowledge financial interest in Immulina. All other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability statement

All data and materials are available upon request by contacting Dr. Mona Haron (email: [email protected]).

Additional information

Funding

Research reported in this publication was supported by the Office of Dietary Supplements and National Center for Complementary and Integrative Health of the National Institutes of Health under Award Number U19AT010838. The content is solely the authors’ responsibility and does not necessarily represent the official views of the National Institutes of Health. Additional funding was also provided by a grant from the USDA, Agricultural Research Service Specific Cooperative Agreement No. 58-6060-6-015.

Notes on contributors

Mona H. Haron

Mona H. Haron, PhD, is a Research Scientist, National Center for Natural Products Research, School of Pharmacy, University of Mississippi. Research focus includes pharmacological properties of medicinal plants and their phytochemical constituents, particularly immune-enhancing and neuro-active agents. A special interest in studying the pharmacokinetic herb-drug interactions and safety of herbal supplements.

Jin Zhang

Jin Zhang, PhD, is a Research Scientist at the National Center for Natural Products Research, School of Pharmacy, University of Mississippi, with extensive research experience in natural products/biological chemistry. Jin Zhang’s interests include immune-enhancing, anticancer, cell signaling, high throughput screening, isolation and structure elucidation of biologically active compounds in natural products.

Amar G. Chittiboyina

Amar G. Chittiboyina, PhD, Assistant Director, National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi. Broad background in organic chemistry, with specific training and expertise in organic and medicinal chemistry, natural products, and analytical chemistry. Holder of several national, international patents, has authored over 120 peer-reviewed research articles in various scientific journals. His research interests include biogenesis of secondary metabolites; development of synthetic methods for commercial viability; safer and greener ways to produce natural products in gram quantities; synthesis and structure-activity relationships (SAR); implementation of orthogonal methods for the quality assessment of phytochemicals in various matrices; development of non-animal alternative methods in dermatotoxicology; application of computational tools to address the adverse effects such as potential herb-herb and herb-drug interactions associated with phytochemicals. Dr. Chittiboyina received his Ph.D. degree from the National Chemical Laboratory, Pune, India, followed by postdoctoral training at the Department of Medicinal Chemistry, University of Mississippi. Dr. Chittiboyina serves as a consultant for several small biotech companies and well versed in grantsmanship and technology transfer aspects.

Ikhlas A. Khan

Ikhlas A. Khan, PhD, is Director and Research Professor of Pharmacognosy at the National Center for Natural Products Research. Main areas of research include phytomedicine, drug discovery, medicinal plants research, analytical fingerprinting for standardization of herbal products, bio-analytical approaches to improvement of product quality and safety, development of analytical methods for phytochemical characterization of biologically active natural products.

Nirmal D. Pugh

Nirmal D. Pugh, PhD, is a Principal Scientist at the National Center for Natural Products Research with a background in pharmacognosy and performs research on botanicals.