Horsetail’s Mechanism of Action & Proposed Study

PART A – Hypothesis

Horsetail was traditionally used to strengthen bone and is currently recommended to patients suffering from osteoporosis. It is suggested that Equisetum’s silica content increases calcium absorption and usage in the body, although the mechanism of silicon in the role of bone health is unclear. One biochemical model proposes that dietary silicon interacts with osteoblast cells to increasing calcium absorption, thus having a physiological effect of increasing bone density and connective tissue synthesis that prevents osteoporosis (Jugdaohsingh, 2007). The following study explores the biological mechanism of silica extract from horsetail and its interaction with calcium uptake in body.

PART B – Proposed Study 

                     

Introduction

One study shows that horsetail contains 0.78-2.61% of the average daily dietary intake for calcium in one glass of herbal infusion (Raczuk, 2008). This amount of calcium is insignificant to be used as a treatment for osteoporosis since the recommended levels of calcium intake by the National Institutes of Health Consensus Conference on Osteoporosis range from 1000mg/day for men and 1500 mg/day for pregnant and post menopausal women (Truong, 2006). Even with the insufficient amount of calcium, horsetail is still recommended herbal supplement for people suffering from osteoporosis. One proposed role of horsetail in terms of the treatment is that the mineral contents of calcium, magnesium, and copper in horsetail are utilized by prolyl hydroxylase enzyme, a cofactor of gene transcription of type 1 collagen, to increase the synthesis and/or stabilization of collagen (Jugdaohsingh, 2007); again, however, the role of active mineral components found in horsetail on bone health is poorly understood. Instead of focusing on the commonly discussed minerals mentioned beforehand, this study explores the role of silica and silicic acids, which is reported to support bone and collage formation by encouraging calcium absorption and usage in the body, thus increasing bone building and bone density (Williams). As evidence, a recent finding on US and UK cohorts showed a strong positive association between Si intake and bone mineral density (Jugdaohsingh, 2004).

This study hypothesizes that silica content in horsetail encourages bone deposition by stimulating osteoblast proliferation and osteocalcin synthesis (Jugdaohsingh, 2007). Osteocalcin is coded by the BGLAP gene and is known to be secreted by osteoblasts, thus is a good biochemical indicator of bone building (Puchacz, 1989). The study will test the cellular and physiological effect of silica on osteoblast cells in three parts. In the first part, radiolabeled silica will be injected to ovariectomised (OVX) rat (Namkung-Matthai, 2001) and the localization of silica and its affinity to chromatin will be observed. In the second part, oral tablet of silicon will be prepared and osteocalcin gene level will be measured. In he last part, bone density will be determined after the oral administration of the tablet.

Materials

Part 1 - Localizing silicon

Extracted silica from horsetail is ¹⁸O isotope radiolabeled and prepared as a liquid form to be injected in rats.

Part 2 , 3 - Measuring osteocalcin gene expression and determining bone density

Silica is extracted from horsetail and prepared in a tablet form for oral administration. For such therapeutic agent, at least half of valances of organic silicon must be bonded to oxygen and less than quarter of valences are bonded to halogen, such as chlorine and bromine (Wiegand, 1993).  Examples of such compounds are tetraorganoorthosilicates, specifically tetraethylorthosilicate, Si(OC₂H₅)₄ and silicon tetraacetate (Wiegand, 1993). The compound can be prepared by reacting ascorbic acid with silicate ester that has lower alkyl radicals, which are more readily available in metabolism. These compounds are broken to silicic acid in the gastrointestinal mucosa (Wiegand, 1993).

The ideal target subjects will be post-menopausal female with osteoporosis or hypogonadotropic osteoporosis in human males or females (Wiegand, 1993). However, for the safety concern, domesticated warm-blooded animal, OVX rat will be used. 

Method

Extraction of Silicon

All inorganic substances are removed and silica is obtained from stalks of fresh Equisetum averse through acid treatment by submerging stalks in 10% HCl for 2 hours at boiling temperature. Then silica is calcinated to revemove organic components (Sapei, 2007). For the first part of the experiment, the calcinated silica is liquefied and is radioactively labelled with ¹⁸O isotope. For the second and third part, the extracted silica is esterified, and the silicate ester is reacted with ascorbic acid to create an edible form of silicon tetraacetate (Wiegand, 1993). 

Analysis

Part 1 – localization of silicon

Radioactive liquid form of silica is injected to OVX rats and the affinity of silica is observed under Positron emission tomography (PET) scan (Phelps, 2006). A small section of tissue where silica is located can be cut out to examine the molecular interaction between silica and chromatin using chip-on-chip to identify the binding site (Buck, 2004). It has been reported that silica may have a role in gene transcription since silicon increased the mRNA of certain proteins (Jugdaohsingh, 2007). It is hypothesized that silica will localize around connective tissues and cartilage of bones, as well around matrix to activate transcription factors.

Part 2 – Osteocalcin expression level

Silica tablet is prepared as mentioned under the extraction section and is orally administered to OVX rats. A global gene regulation expression of transcription factors and proteins on bone formation can be determined using DNA microarray. The appropriate genetic information of proteins involved in osteoblast activities can also be obtained from cDNA library. A special attention can be paid to BGLAP gene that codes osteocalcin, and further information on this gene expression can be obtained by complementing DNA microarray with RT-PCR or with northern blotting (Tardif, 2004). It is expected to observe a significant up-regulation of BGLAP gene expression. The translation of up-regulated gene expression to protein synthesis can be determined using ELISA kit to measure osteocalcin and osteoblast protein level (Kim, 2009).

Part 3 – Bone Mineral Densities (BMD)

After orally administrating the prepared silica tablet, BMD of OXV rats are determined by dual-energy X-ray absorptiometry for rats (Kim, 2009). It is expected to see an increase in BMD.

This design is to propose an experiment that will determine the biochemical and physiological role of horsetail’s silica. The implication of the study is that if silicon extract from horsetail is proven to have an essential biological role in bone health, horsetail can be a potential therapeutic herb used to preventive measure and a treatment for osteoporosis.

Reference

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Jugdaohsingh, R.(2007). Silicon and Bone Health. J Nutr Health Aging. 11(2): 99-110.

Jugdaohsingh R, Tucker KL, Qiao N, Cupples LA, Kiel DP, Powell JJ. (2004). Dietary silicon intake is positively associated with bone mineral density in men and premenopausal women of the Framingham Offspring cohort J Bone Miner Res. 19(2):297-307.

Kim, MH. Bae, YJ.  Choi, MK. Chung, YS. (2009) Silicon Supplementation Improves the Bone Mineral Density of Calcium-Deficient Ovariectomized Rats by Reducing Bone Resorption.  Biol Trace Elem Res (2009) 128:239–247

Namkung-Matthai H, Appleyard R, Jansen J, Hao Lin J, Maastricht S, Swain M, Mason RS, Murrell GA, Diwan AD, Diamond T (2001). Osteoporosis influences the early period of fracture healing in a rat osteoporotic model. Bone 28 (1): 80–6.

Phelps, Michael E. (2006). PET: physics, instrumentation, and scanners. Springer. pp. 8–10.

Puchacz E, Lian JB, Stein GS, Wozney J, Huebner K, Croce C (1989). Chromosomal localization of the human osteocalcin gene. Endocrinology 124 (5): 2648–50.

Raczuk J, Biardzka E, Daruk J. (2008). The content of Ca, Mg, Fe and Cu in selected species of herbs and herb infusions. [Pubmed abstract] Rocz Panstw Zakl Hig. 59(1):33-40. Available from Epub.

Sapei, L. Gierlinger, N. Hartmann, J. Nöske, R Strauch, P. Paris, O. (2007). Structural and analytical studies of silica accumulations in Equisetum hyemale. Anal Bioanal Chem.  389:1249–1257

Tardif, G. Hum, D. Pelletier, JP. Boileau, C. Ranger, P. Martel-Pelletier, J. (2004).

Differential Gene Expression and Regulation of the Bone Morphogenetic Protein Antagonists Follistatin and Gremlin in Normal and Osteoarthritic Human Chondrocytes and Synovial Fibroblasts. ARTHRITIS & RHEUMATISM. 50(8):2521-2530

Truong, Michael (Interviewee).  (2006, Dec 11). Osteoporosis Prevention & Treatment [Interview transcript]. Retrieved from MedicineNet.Com Web site: http://www.medicinenet.com/script/main/art.asp?articlekey=6850&page=2

Wiegand, Karl E. (Baton Rouge, LA), Hu, Patrick C. (Baton Rouge, LA).  (1993) Silicon compounds in bone treatment         United States  Ethyl Corporation (Richmond, VA)   5183061 http://www.freepatentsonline.com/5183061.html

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