LIBRARY OF ABSTRACTS OF PEER-REVIEWED PROFESSIONAL JOURNAL ARTICLES ON STEVIA REBAUDIANA WITH INTRODUCTION AND EDITORIAL COMMENTARY

This site provides a comprehensive and balanced overview of scientific research on Stevia Rebaudiana.  It is extensively referenced for those wishing to pursue further inquiries and study.  It is not intended to provide medical advice or to substitute for the services of a qualified health care professional.  Responsibility is specifically disclaimed for consequences incurred by those using the information reported.

INTRODUCTION
 

Stevia Rebaudiana (Bertoni) is one of a number of medicinal plant species that has been and continues to be voluminously studied.  The plant has many consistently identifiable active compounds.  By and large, the studies focus on one or more such compounds.     

The following bullet points summarize the effects attributable to compounds found in ol.  They are listed in the same sequence as documented by the articles abstracted below. 

•    Internationally (excluding USA) used as a non caloric, safe, and noncariogenic (no

      cavities) sweetener

o       Diabetics needing to limit nutritional sweeteners

o       Phenylketonurics

o       People with obesity limiting caloric intake

•    Non-carcinogenic: cannot cause DNA damage (goes to safety)

•    Many Proven Medical Uses

o       Type 2 diabetes

§         Enhances insulin effect

§         Enhances insulin production

§         Stabilizes glucagon secretion

§         Improves glucose tolerance in both animals and humans and lowers post prandial blood sugars

o       Antihypertensive- works like calcium channel blocking drugs like verapamil

o       Anti-inflammatory/Anti Carcinogenic

o       Antiviral

o       Antibacterial

•    Benefit to oral health

•    Contrasting view on mutagenicity with rebuttal

•    Agronomic information to increase Stevia active ingredient concentration

•    Chemical Composition of Stevia rebaudiana, methods of analysis, and toxicology,

      which further goes to proving safety

Following are the active chemical compounds identified in olive leaf to date      

            Stevioside

            Rebaudioside A

            Seven  glycosidic diterpenes, sterebins B-H

            Six new labdane-type, non-glycosidic diterpenes, sterebins I-N

            The aglycone, Steviol

            alpha-monoglucosylstevioside

            alpha-monoglucosylrebaudioside A   

NARRATIVE
 

Internationally (excluding USA) used as a non caloric, safe, and non-cariogenic (no cavities) sweetener 

Toxicology studies have been performed on Stevia rebaudiana and its standard extracts which yield the diterpinoid compounds stevioside and rebaudioside A.  They have been found to be metabolized in the gut to their aglycon steviol, another derivative compound. These two compounds (stevioside and rebaudioside A) possess up to 250 times the sweetness of ordinary table sugar (sucrose). The conclusion of many studies is that Stevia and stevioside are safe when used as a sweetener. It is suited for diabetics, PKU patients, as well as for obese persons intending to lose weight by avoiding sugar supplements in the diet. No allergic reactions to it seem to exist, and pertinent to oral health the substance is non-cariogenic (meaning it does not promote cavities). 

            Diabetics needing to limit nutritional sweeteners: Certain groups of people would especially benefit from using Stevia as a sweetener. Diabetics need to limit nutritional sweeteners as a major part of their regimen to control glucose blood levels and carbohydrate intolerance.  Stevia not only is a non-caloric sweetener, as will be demonstrated in the library later, it actually contributes to lowering blood sugar in type II diabetes. 

            Phenylketonurics: Phenylketonuria is a disease caused by a genetic inborn error in metabolism that makes it impossible to metabolize the amino acid  phenylalanine.  The partial metabolism of this amino acid leads to neuro toxic byproducts, which cause brain damage in developing brains of infants and children with the disease leading to severe mental retardation.   

            This form of retardation is completely preventable by avoiding the amino acid phenylalanine.  Mandatory testing of infants for this disorder came into being once a rapid test developed by Dr. Robert Guthrie was made available to diagnose the condition at birth.  A number of non-nutritive sweeteners in use today have   the molecule of phenylalanine in the sweetening compound.  Therefore, phenylketonuric patients cannot use those non-nutritive sweeteners.  Stevia   is a completely safe alternative compatible with the special dietary restrictions imposed to prevent the mental retardation. 

            People with obesity limiting caloric intake: Obesity has become an epidemic,          and with it comes increased risk for numerous metabolic and cardiovascular             problems, including hypertension, Type II diabetes mellitus, atherosclerosis, and      so on.  Safe, non-nutritive sweeteners would be of benefit to everyone with a     weight problem, since craving sweet things goes hand in hand with obesity.  For reasons that are not clear, Stevia has not been allowed into the USA as a non-        nutritive sweetener by the FDA.  The multiple benefits of Stevia will be made     clear in the library that follows, including abstracts of articles proving its safety.

Non-carcinogenic: cannot cause DNA damage (goes to safety) 

Genotoxicity is defined as damage to DNA, which in turn leads to errors in genetic reproduction when cells divide.  This process resulting from damaged DNA is a principal cause of cancer.  We include an abstract in this library investigating whether or not Stevia produces DNA damage, which goes to the issue of its safety.  

The genotoxicity of steviol, a metabolite of Stevia extract, was evaluated for its genotoxic potential using the comet assay. In an in vitro study, steviol at 62.5, 125, 250, and 500 micrograms/ml did not damage the nuclear DNA of TK6 and WTK1 cells.  As all studies showed negative responses, Stevia extract and steviol were concluded not to have DNA-damaging activity in cultured cells and mouse organs. 

Proven Medical Uses 

Stevia is not only safe; it has been studied extensively and found to have many medicinal properties worthy of incorporation into healthy regimens. 

            Type 2 Diabetes: Type II Diabetes is the development of a metabolic syndrome of carbohydrate intolerance characterized by rising blood sugar levels,  which then impact upon the integrity of microvascular health over time.  Eventually the diseased micro-vessels lead to kidney damage, nervous system damage, peripheral vascular disease, increased hardening of the arteries, increased risk of heart attack and heart failure, lowered resistance to infection, and even behavioral/cognitive problems in later life.  

             Enhances insulin effect:  In Type 2 Diabetes, at first there is not a deficiency of insulin production as in Type 1 or juvenile onset diabetes.  Rather, there is a loss    of the responsiveness of cell membranes to the  presence of insulin, so that more and more insulin is required to get the desired response to permit glucose to enter the cells from the blood that need it for energy metabolism.  Stevia enhances the insulin effect thereby reducing the demand for ever increasing amounts of insulin, which eventually, if unchecked, leads to the burn out of the beta cells’ ability to produce insulin. 

              Enhances insulin production:  As stated above, at first there is not a deficiency of insulin production as is found in Type I or juvenile onset diabetes. However, because of lost membrane sensitivity to insulin, more  and more is required.  In addition to increasing insulin effect, Stevia also simultaneously stimulates increased insulin production as needed.  Stevia  enhances insulin production from beta cells before burn out occurs. 

              Stabilizes glucagon secretion: Glycogen is a complex molecular structure  resembling a tree with many branches each of which is a molecule of  glucose. Glycogen is stored in the liver.  The regulation of blood glucose levels demands responsive regulation of the release of glucose from these glycogen stores, which is governed by the amount of a substance called glucagon. In Type 2 diabetes there is a loss of the ability to regulate accurately another substance called glucagon that causes glucose to be      released from glycogen stores in the liver.  Stevia stabilizes glucagon secretion. 

                        Improves glucose tolerance in both animals and humans and lowers post prandial blood sugars:  As a result of Stevia’s ability to increase the insulin   effect on cell membranes, increase insulin production, stabilize glucagon secretion, stabilizing blood sugar levels, Stevia has been  demonstrated through all these effects to improve glucose tolerance to ingested carbohydrates, lowering post-prandial blood sugars in both animals and humans.  In other words, Stevia is shown to provide a comprehensive set of mechanisms that counter the mechanics of Type II diabetes and its eventual complications. 

Antihypertensive---works like calcium channel blocking drugs like verapamil “Essential Hypertension” is defined as an increase in blood pressure above certain measured levels.  The definition of high blood pressure begins at a systolic blood pressure of 140mm of Mercury(Hg) and a diastolic blood pressure of 90 mm (Hg).  The more time that a person spends with blood pressures at or in excess of these numbers, the more pathological changes accrue in medium sized and small arteries that cause further increases in blood pressure.  The pathology is a thickening of the walls of these blood vessels so that effectively the diameter of the vessels is diminished.  This causes the heart to work harder to pump enough blood to meet the demands of all the tissues.

Over time the damage to the arteries leads to organ damage as well, most notably heart, kidneys and brain.  With chronic hypertension there is increased risk for heart attack, stroke, and kidney failure.  Brain damage can accrue from “mini-strokes” caused by microvascular clotting in narrowed small arteries deep in the brain, eventually leading to dementia and motor and sensory loss.

One of the properties demonstrated in a number of studies included in this library demonstrate that Stevia acts at the cell membrane level much in the same way as a type of medication known as a calcium channel blocking agent. These medicines are routinely prescribed to help control high blood pressure by relaxing the muscular walls of the arteries causing the elevation in blood pressure. The studies included demonstrate clearly that Stevia acts to relax arteries and lower blood pressure. We include one study that actually demonstrated that in an animal model in which both hypertension and Type II diabetes are prominent, Stevia led to improvement in both conditions. 

Anti-inflammatory/anticarcinogenic.  Included is one study on an animal model in which the constituents of Stevia were demonstrated to block inflammation and tumor growth.  This property of Stevia clearly needs further study.

Antiviral Properties.  Rotavirus is the most important cause of severe dehydra-ting diarrhea in infants and young children worldwide. Regardless of the social and economic status, nearly all children will be infected with rotavirus before 3 years of age. Over 500,000 children, primarily from developing countries, die every year from rotavirus infection, and many more have severe diarrhea that requires hospitalization. Given the severity and scope of rotavirus infection, there is an urgent need for a safe and effective vaccine or other suitable and easily administered and affordable treatment.

Included is a study that examined four of the component compounds in Stevia for their ability to inhibit rotavirus infection.  The study determined that infection was blocked by preventing the virus from binding to the target cells it needs for reproducing itself.

Animals like horses, pigs and cattle also are affected by rotavirus.  Included is a study that shows the genetic similarities between the rotavirus strains that attacks pigs and horses.  Based upon the common mechanism for the reproduction of the virus that demands that first the virus has to attach its capsid to the target cell and then inject its genetic material, which commandeers the cells’ genes to make more virus, the blocking action of Stevia should prove to be of veterinary benefit in treating and preventing spread of rotavirus infection in the affected species.

Another family of viruses may well be inhibited by Stevia.  We include a study that demonstrated the microbial digestion of Stevia produces a series of compounds called diterpinoids.  These compounds demonstrated a strong capacity to inhibit the reproduction of the Epstein-Barr Virus which is a DNA virus of the Herpes family of viruses. 

All Herpes viruses reproduce in the same way and attack lymphocytes, neuroglia, and nerve cells of the immune and nervous systems, respectively.  The Epstein Barr virus is the virus most commonly associated with Infectious Mononucleosis, but is also associated with intermittent reactivation and long term   produces the increased risk of developing Hodgkin’s Disease and Non-Hodgkin’s   lymphomas.  Further study is required, but the study enclosed in this library is strongly suggestive that the microflora of the human gut, may well produce the same diterpinoids from Stevia that were produced in the experimental situation.  If the preliminary results hold up in further animal and human studies, Stevia   would be a valuable addition to our medicinal plant arsenal of anti viral and cancer preventive agents.

Antibacterial Properties.   Although we only found one study evaluating the effect of Stevia on bacteria, it turns out to be extremely important and valuable to report. “A fermented aqueous extract from Stevia rebaudiana Bertoni showed strong bactericidal activity towards a wide range of food-borne pathogenic bacteria including enterohemorrhagic Escherichia coli O157:H7.” 

This particular strain of E. coli has been showing up in greater frequency in fast food chains and groceries and creating havoc when it does.  The consequences of E coli 0157 infection from contaminated meat can be lethal.  The discovery that Stevia diterpinoids actually have bacteriocidal impact on these various food borne pathogens demands further study and implementation of preventative as well as therapeutic use.  The thought here is to prevent the beef cattle from bearing the pathogen.  Stevia could be added to their feed to knock out these infections before the beef cattle are slaughtered for their meat.

Benefit to oral health

This topic is very basic and simple. Nutritive sweeteners cause cavities and encourage the growth of harmful bacteria in the mouth, which then contribute to plaque formation and gingivitis.  Stevia, as a non nutritive sweetener with bacteriostatic and bacteriocidal properties could become a benefit to oral health by eliminating the cause of dental decay and gingivitis.

Contrasting view on mutagenicity with rebuttal

Mutagenicity means the propensity of a substance to induce genetic damage that is passed on in subsequent generations of cell divisions.  The result is what is called a mutation.  We present a contrasting view of the properties of Stevia presented that imply that there is a potential for Stevia to induce mutations.  We also include a rebuttal of this position.

The remaining two bullet points are self explanatory, and we refer the reader to the appropriate position where these topics are presented by representative studies.

THE LIBRARY
 

Stevia is safe to use as a sweetener for diabetics, phenylketonuric patients, and people desirous of avoiding sugar to lose weight:

 

Phytochemistry. 2003 Nov;64(5):913-21.

Related Articles, Links

 
Stevioside.

Geuns JM.

Laboratory of Plant Physiology, Catholic University of Leuven, Kasteelpark Arenberg 31, B 3001 Leuven, Belgium. jan.geuns@bio.kuleuven.ac.be

Stevioside is a natural sweetener extracted from leaves of Stevia rebaudiana (Bertoni). The literature about Stevia, the occurrence of its sweeteners, their biosynthetic pathway and toxicological aspects are discussed. Injection experiments or perfusion experiments of organs are considered as not relevant for the use of Stevia or stevioside as food, and therefore these studies are not included in this review. The metabolism of stevioside is discussed in relation with the possible formation of steviol. Different mutagenicity studies as well as studies on carcinogenicity are discussed. Acute and subacute toxicity studies revealed a very low toxicity of Stevia and stevioside. Fertility and teratogenicity studies are discussed as well as the effects on the bio-availability of other nutrients in the diet. The conclusion is that Stevia and stevioside are safe when used as a sweetener. It is suited for both diabetics, and PKU patients, as well as for obese persons intending to lose weight by avoiding sugar supplements in the diet. No allergic reactions to it seem to exist.

 
Metabolism of stevioside and rebaudioside A from Stevia rebaudiana extracts by human microflora.

Gardana C, Simonetti P, Canzi E, Zanchi R, Pietta P.

Department of Food Science and Microbiology, Division of Human Nutrition, University of Milan, Via Celoria 2, 20133 Milan, Italy.

Stevia rebaudiana standardized extracts (SSEs) are used as natural sweeteners or dietary supplements in different countries for their content of stevioside or rebaudioside A. These compounds possess up to 250 times the sweetness intensity of sucrose, and they are noncaloric and noncariogenic sweeteners. The aim of this study was to investigate the in vitro transformation of stevioside and rebaudioside A after incubation with human microflora, the influence of these sweeteners on human microbial fecal community and which specific groups metabolize preferentially stevioside and rebaudioside A. The experiments were carried out under strict anaerobic conditions in batch cultures inoculated with mixed fecal bacteria from volunteers. The hydrolysis was monitored by HPLC coupled to photodiode array and mass spectrometric detectors. Isolated bacterial strains from fecal materials incubated in selective broths were added to stevioside and rebaudioside A. These sweeteners were completely hydrolyzed to their aglycon steviol in 10 and 24 h, respectively. Interestingly, the human intestinal microflora was not able to degrade steviol. Furthermore, stevioside and rebaudioside A did not significantly influence the composition of fecal cultures; among the selected intestinal groups, bacteroides were the most efficient in hydrolyzing Stevia sweeteners to steviol.

Attempts to create DNA damage with Stevia fail to do so, further proof of its safety:

[Genotoxicity studies of Stevia extract and steviol by the comet assay]

[Article in Japanese]

Sekihashi K, Saitoh H, Sasaki Y.

Safety Research Institute for Chemical Compounds Co., Ltd., 363-24 Shin-ei, Kiyota-ku, Sapporo 004-0839, Japan.

The genotoxicity of steviol, a metabolite of stevia extract, was evaluated for its genotoxic potential using the comet assay. In an in vitro study, steviol at 62.5, 125, 250, and 500 micrograms/ml did not damage the nuclear DNA of TK6 and WTK1 cells in the presence and absence of S9 mix. In vivo studies of steviol were conducted by two independent organizations. Mice were sacrificed 3 and 24 hr after one oral administration of steviol at 250, 500, 1000, and 2000 mg/kg. DNA damage in multiple mouse organs was measured by the comet assay as modified by us. After oral treatment, stomach, colon, liver, kidney and testis DNA were not damaged. The in vivo genotoxicity of stevia extract was also evaluated for its genotoxic potential using the comet assay. Mice were sacrificed 3 and 24 hr after oral administration of stevia extract at 250, 500, 1000, and 2000 mg/kg. Stomach, colon and liver DNA were not damaged. As all studies showed negative responses, stevia extract and steviol are concluded to not have DNA-damaging activity in cultured cells and mouse organs.

Medicinal uses for Stevia in addition to its role as a sweetener: 

This next article demonstrates the glucose dependent capacity of rebaudioside A, one of the two most dominant ingredients in Stevia leaves to stimulate insulin secretion in an experimental situation using the islet cells of mouse pancreas.  With a number of critical accompanying conditions defined, rebaudioside A is seen to potentiate the natural response of causing increased insulin secretion when these insulin producing cells are presented with higher concentrations of glucose.  That is what is meant by the term glucose dependent stimulation.  Likewise, extracellular Calcium concentrations at physiologic levels were a necessity to evoke this response; hence the term calcium-dependency.

 
Rebaudioside A potently stimulates insulin secretion from isolated mouse islets: studies on the dose-, glucose-, and calcium-dependency.

Abudula R, Jeppesen PB, Rolfsen SE, Xiao J, Hermansen K.

Department of Endocrinology and Metabolism, Aarhus University Hospital, Denmark.

Extracts of leaves of the plant Stevia rebaudiana Bertoni (SrB), have been used for many years in traditional treatment of diabetes in South America. Stevia leaves contain diterpene glycosides, stevioside and rebaudioside A being the most abundant. Recently, it was demonstrated that stevioside stimulates the insulin secretion both in vitro and in vivo. Subsequently, we wanted to elucidate the influence of rebaudioside A on the insulin release from mouse islets using static incubations, as well as perifusion experiments. Rebaudioside A (10(-16) to 10(-6) mol/L) dose-dependently stimulated the insulin secretion in the presence of 16.7 mmol/L glucose (P < .05). The stimulation of insulin release occurs at a concentration of 10(-14) mol/L rebaudioside A, and maximal insulin response was obtained at 10(-10) mol/L (P < .01). Rebaudioside A stimulates insulin secretion in a glucose-dependent manner (3.3 to 16.7 mmol/L) and only potentiated insulin secretion at glucose > 6.6 mmol/L. The effect of rebaudioside A is critically dependent on the presence of extracellular Ca2+, ie, rebaudioside A-induced insulin stimulation at high glucose disappears in the absence of extracellular Ca2+. In conclusion, rebaudioside A possesses insulinotropic effects and may serve a potential role as treatment in type 2 diabetes mellitus.

How does Stevia work to improve the ability of skeletal muscle to utilize insulin to allow glucose transport into the cell for energy metabolism?  The following study from “Metabolism” helps demonstrate how it works:

 
Effects of stevioside on glucose transport activity in insulin-sensitive and insulin-resistant rat skeletal muscle.

Lailerd N, Saengsirisuwan V, Sloniger JA, Toskulkao C, Henriksen EJ.

Muscle Metabolism Laboratory, Department of Physiology, University of Arizona College of Medicine, Tuscon, USA.

Stevioside (SVS), a natural sweetener extracted from Stevia rebaudiana, has been used as an antihyperglycemic agent. However, little is known regarding its potential action on skeletal muscle, the major site of glucose disposal. Therefore, the purpose of the present study was to determine the effect of SVS treatment on skeletal muscle glucose transport activity in both insulin-sensitive lean (Fa/-) and insulin-resistant obese (fa/fa) Zucker rats. SVS was administered (500 mg/kg body weight by gavage) 2 hours before an oral glucose tolerance test (OGTT). Whereas the glucose incremental area under the curve (IAUC(glucose)) was not affected by SVS in lean Zucker rats, the insulin incremental area under the curve (IAUC(insulin)) and the glucose-insulin index (product of glucose and insulin IAUCs and inversely related to whole-body insulin sensitivity) were decreased (P<.05) by 42% and 45%, respectively. Interestingly, in the obese Zucker rat, SVS also reduced the IAUC(insulin) by 44%, and significantly decreased the IAUC(glucose) (30%) and the glucose-insulin index (57%). Muscle glucose transport was assessed following in vitro SVS treatment. In lean Zucker rats, basal glucose transport in type I soleus and type IIb epitrochlearis muscles was not altered by 0.01 to 0.1 mmol/L SVS. In contrast, 0.1 mmol/L SVS enhanced insulin-stimulated (2 mU/mL) glucose transport in both epitrochlearis (15%) and soleus (48%). At 0.5 mmol/L or higher, the SVS effect was reversed. Similarly, basal glucose transport in soleus and epitrochlearis muscles in obese Zucker rats was not changed by lower doses of SVS (0.01 to 0.1 mmol/L). However, these lower doses of SVS significantly increased insulin-stimulated glucose transport in both obese epitrochlearis and soleus (15% to 20%). In conclusion, acute oral SVS increased whole-body insulin sensitivity, and low concentrations of SVS (0.01 to 0.1 mmol/L) modestly improved in vitro insulin action on skeletal muscle glucose transport in both lean and obese Zucker rats. These results indicate that one potential site of action of SVS is the skeletal muscle glucose transport system.

Human studies confirm animal study results as to therapeutic benefits of Stevia in treating Type 2 diabetes mellitus by lowering post prandial (after eating) blood sugar levels:

 
Antihyperglycemic effects of stevioside in type 2 diabetic subjects.

Gregersen S, Jeppesen PB, Holst JJ, Hermansen K.

Department of Endocrinology and Metabolism C, Aarhus University Hospital, Denmark.

Stevioside is present in the plant Stevia rebaudiana Bertoni (SrB). Extracts of SrB have been used for the treatment of diabetes in, for example, Brazil, although a positive effect on glucose metabolism has not been unequivocally demonstrated. We studied the acute effects of stevioside in type 2 diabetic patients. We hypothesize that supplementation with stevioside to a test meal causes a reduction in postprandial blood glucose. Twelve type 2 diabetic patients were included in an acute, paired cross-over study. A standard test meal was supplemented with either 1 g of stevioside or 1 g of maize starch (control). Blood samples were drawn at 30 minutes before and for 240 minutes after ingestion of the test meal. Compared to control, stevioside reduced the incremental area under the glucose response curve by 18% (P =.013). The insulinogenic index (AUC(i,insulin)/AUC(i,glucose)) was increased by approximately 40% by stevioside compared to control (P <.001). Stevioside tended to decrease glucagon levels, while it did not significantly alter the area under the insulin, glucagon-like peptide 1, and glucose-dependent insulinotropic polypeptide curves. In conclusion, stevioside reduces postprandial blood glucose levels in type 2 diabetic patients, indicating beneficial effects on the glucose metabolism. Stevioside may be advantageous in the treatment of type 2 diabetes.

More evidence that Stevia and/or derivatives may be useful as therapy in type 2 diabetes mellitus because of combined effects of lowering blood sugar and enhancing insulin production, and maintaining glucagon levels. Glucagon is a substance that is secreted for the purpose of mobilizing glucose for release from glycogen stores in the liver.  In type II diabetes, there is decreased sensitivity to detect the need for glucagon control, the ability to diminish glucagon release once started, and the insulin sensitivity by the cells that require insulin to absorb and use the glucose that is available for energy.  

 
Stevioside induces antihyperglycaemic, insulinotropic and glucagonostatic effects in vivo: studies in the diabetic Goto-Kakizaki (GK) rats.

Jeppesen PB, Gregersen S, Alstrup KK, Hermansen K.

Department of Endocrinology and Metabolism C, Aarhus University Hospital, Denmark. pbj@mail-telia.dk

Extracts of leaves from the plant Stevia rebaudiana Bertoni have been used in the traditional treatment of diabetes in Paraguay and Brazil. Recently, we demonstrated a direct insulinotropic effect in isolated mouse islets and the clonal beta cell line INS-1 of the glycoside stevioside that is present in large quantity in these leaves. Type 2 diabetes is a chronic metabolic disorder that results from defects in both insulin and glucagon secretion as well as insulin action. In the present study we wanted to unravel if stevioside in vivo exerts an antihyperglycaemic effect in a nonobese animal model of type 2 diabetes. An IV glucose tolerance test (IVGT) was carried out with and without stevioside in the type 2 diabetic Goto-Kakizaki (GK) rat, as well as in the normal Wistar rat. Stevioside (0.2 g/kg BW) and D-glucose (2.0 g/kg BW) were administered as IV bolus injections in anaesthetized rats. Stevioside significantly suppressed the glucose response to the IVGT in GK rats (incremental area under the curve (IAUC): 648 +/- 50 (stevioside) vs 958 +/- 85 mM x 120 min (control); P < 0.05) and concomitantly increased the insulin response (IAUC: 51116 +/- 10967 (stevioside) vs 21548 +/- 3101 microU x 120 min (control); P < 0.05). Interestingly, the glucagon level was suppressed by stevioside during the IVGT, (total area under the curve (TAUC): 5720 +/- 922 (stevioside) vs 8713 +/- 901 pg/ml x 120 min (control); P < 0.05). In the normal Wistar rat stevioside enhanced insulin levels above basal during the IVGT (IAUC: 79913 +/- 3107 (stevioside) vs 17347 +/- 2882 microU x 120 min (control); P < 0.001), however, without altering the blood glucose response (IAUC: 416 +/- 43 (stevioside) vs 417 +/- 47 mM x 120 min (control)) or the glucagon levels (TAUC: 5493 +/- 527 (stevioside) vs 5033 +/- 264 pg/ml x 120 min (control)). In conclusion, stevioside exerts antihyperglycaemic, insulinotropic, and glucagonostatic actions in the type 2 diabetic GK rat, and may have the potential of becoming a new antidiabetic drug for use in type 2 diabetes.

The mechanism of action is further elucidated below in the next and older study in which it is proven that there is a direct action on the beta cells of the pancreas to directly cause increased insulin production:

Stevioside acts directly on pancreatic beta cells to secrete insulin: actions independent of cyclic adenosine monophosphate and adenosine triphosphate-sensitive K+-channel activity.

Jeppesen PB, Gregersen S, Poulsen CR, Hermansen K.

Department of Endocrinology and Metabolism, Aarhus University Hospital, Denmark.

The natural sweetener stevioside, which is found in the plant Stevia rebaudiana Bertoni, has been used for many years in the treatment of diabetes among Indians in Paraguay and Brazil. However, the mechanism for the blood glucose-lowering effect remains unknown. To elucidate the impact of stevioside and its aglucon steviol on insulin release from normal mouse islets and the beta-cell line INS-1 were used. Both stevioside and steviol (1 nmol/L to 1 mmol/L) dose-dependently enhanced insulin secretion from incubated mouse islets in the presence of 16.7 mmol/L glucose (P < .05). The insulinotropic effects of stevioside and steviol were critically dependent on the prevailing glucose concentration, i.e., stevioside (1 mmol/L) and steviol (1 micromol/L) only potentiated insulin secretion at or above 8.3 mmol/L glucose (P < .05). Interestingly, the insulinotropic effects of both stevioside and steviol were preserved in the absence of extracellular Ca2+. During perfusion of islets, stevioside (1 mmol/L) and steviol (1 micromol/L) had a long-lasting and apparently reversible insulinotropic effect in the presence of 16.7 mmol/L glucose (P < .05). To determine if stevioside and steviol act directly on beta cells, the effects on INS-1 cells were also investigated. Stevioside and steviol both potentiated insulin secretion from INS-1 cells (P < .05). Neither stevioside (1 to 100 micromol/L) nor steviol (10 nmol/L to 10 micromol/L) influenced the plasma membrane K+ adenosine triphosphate ((K+)ATP)-sensitive channel activity, nor did they alter cyclic adenosine monophosphate (cAMP) levels in islets. In conclusion, stevioside and steviol stimulate insulin secretion via a direct action on beta cells. The results indicate that the compounds may have a potential role as antihyperglycemic agents in the treatment of type 2 diabetes mellitus.
 

Antihypertensive properties:

Additional benefits of Stevia may accrue because of the similarities in its physiological impact on vascular smooth muscle to antihypertensive medication called calcium channel blockers:

 
Inhibitory effect of stevioside on calcium influx to produce antihypertension.

Lee CN, Wong KL, Liu JC, Chen YJ, Cheng JT, Chan P.

Department of Medicine, Taipei Medical University-Wan Fang Hospital, Wen Shan, Taipei, Taiwan.

Stevioside is a sweet-tasting glycoside occurring abundantly in the leaves of Stevia rebaudiana (Compositae). It has been used popularly in Japan and Brazil as a sugar substitute for decades. Previous study has shown that it lowered blood pressure in spontaneously hypertensive rats (SHRs) when administered intravenously. This study shows that intraperitoneal injection of stevioside 25 mg/kg also has antihypertensive effect in SHRs. In isolated aortic rings from normal rats, stevioside could dose-dependently relax the vasopressin-induced vasoconstriction in both the presence and absence of endothelium. However, stevioside had no effect on phenylephrine- and KCl-induced phasic vasoconstriction. In addition, stevioside lost its influence on vasopressin-induced vasoconstriction in Ca(2+)-free medium. The results indicate that stevioside caused vasorelaxation via an inhibition of Ca(2+) influx into the blood vessel. This phenomenon was further confirmed in cultured aortic smooth muscle cells (A7r5). Using 10(-5) M methylene blue for 15 min, stevioside could still relax 10(-8) M vasopressin-induced vasoconstriction in isolated rat aortic rings, showing that this vasorelaxation effect was not related to nitric oxide. The present data show that the vasorelexation effect of stevioside was mediated mainly through Ca(2+) influx inhibition.

Antihypertensive study confirms Stevia as antihypertensive agent in spontaneously hypertensive rats:

Antihypertensive effect of stevioside in different strains of hypertensive rats.

Hsu YH, Liu JC, Kao PF, Lee CN, Chen YJ, Hsieh MH, Chan P.

Department of Medicine, Taipei Medical University-Wan Fang Hospital, Taiwan, ROC.

BACKGROUND: Stevioside is a natural sweet-tasting glycoside isolated from the herb Stevia rebaudiana, composed of stevia, a diterpenic carboxylic alcohol with three glucose molecules, mainly used commercially as sugar substitute. Previous study has shown that it can lower blood pressure in anesthetized spontaneously hypertensive rats (SHR). This study was undertaken to evaluate the antihypertensive effect of stevioside in different strains of hypertensive rats and to observe whether there is difference in blood pressure lowering effect. METHODS: Noninvasive tail-cuff method was employed to measure blood pressure. Stevioside at the concentrations of 50, 100 and 200 mg/kg were administered intraperitoneally (IP) to normotensive Wistar-Kyoto rats (NTR), SHR, deoxycorticosterone acetate-salt (DOCA-NaCl) sensitive hypertensive rats (DHR) and renal hypertensive rats (RHR). RESULTS: Significant hypotensive effect of stevioside administered IP as noted in different strains of rats at the dose of 50 mg/kg. When stevioside was increased to the concentrations of 100 and 200 mg/kg, IP it also caused slow and persistent lowering of blood pressure in SHR and NTR. Data also showed that stevioside given at the concentrations of 100, 200 and 400 mg/kg ip resulted in lowering of blood pressure in SHR dose-dependently. Blood pressure returned to previous levels after the drug was discontinued for 2-3 days. Drinking of 0.1% stevioside solution in mature SHR could have antihypertensive effect and also prevented hypertension in immature SHR. CONCLUSIONS: This study reconfirmed stevioside has hypotensive effect and the effect is more prominent in hypertensive rats.

A more sophisticated set of studies documents and further elucidates the antihypertensive effectiveness of Stevia and the mechanism by which it occurs:

Editor’s note:  This study clearly demonstrates an intricate relationship between two active transport mechanisms and demonstrates the priority ranking of importance.  In membrane physiology there is a requirement for the normal functioning of all cells, but especially electrophysiologically active cells such as muscle and nerve cells, which depend upon the maintenance of resting membrane electrical potential difference (voltage) between the inside and the outside of the cell.  The tone of vascular smooth muscle is dependent upon this delicate balance between charged ion concentrations inside and outside the cell. The pairs of charged ions that are most critical to this electrical function are sodium/potassium balance and calcium/magnesium balance.  This next abstract is of a study that demonstrates an additional definition of function regarding the antihypertensive mechanism of Stevia in which the energy dependent function of  potassium channel (ATP-sensitive) was inseparable from the calcium channel modulation, which when inhibited induced relaxation of the vascular smooth muscle, thereby, reducing blood pressure.   The key finding in this study is a decrease of [Ca (2+)]i in vascular smooth muscle cells by isosteviol is mainly mediated by the selective opening of K (ATP) channel or/and SK (Ca) channel. Alteration in the Kv channel also plays a critical role in the inhibitory action of isosteviol.

 
Isosteviol as a potassium channel opener to lower intracellular calcium concentrations in cultured aortic smooth muscle cells.

Wong KL, Yang HY, Chan P, Cheng TH, Liu JC, Hsu FL, Liu IM, Cheng YW, Cheng JT.

Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan, R.O.C.

Isosteviol is a derivative of stevioside, a constituent of Stevia rebaudiana, and is commonly used as a non-caloric sugar substitute in Japan and Brazil. The present study attempted to elucidate the role of potassium (K (+)) channels in the action of isosteviol on intracellular calcium concentrations ([Ca (2+)]i) in cultured vascular smooth muscle (A7r5) cells using the Ca (2+)-sensitive dye Fura-2 as an indicator. The increase of [Ca (2+)]i in A7r5 cells produced by vasopressin (1 micromol/L) or phenylephrine (1 micromol/L) was attenuated by isosteviol from 0.01 micromol/L to 10 micromol/L. The attenuation by isosteviol of the vasopressin- and phenylephrine-induced increase in [Ca (2+)]i was inhibited by glibenclamide, apamin and 4-aminopyridine but not by charybdotoxin. Furthermore, the inhibitory action of isosteviol on [Ca (2+)]i was blocked when A7r5 cells co-treated with glibenclamide and apamin in conjunction with 4-aminopyridine were present. Therefore, not only did the ATP-sensitive potassium (K (ATP)) channel affect the action of isosteviol on [Ca (2+)]i modulation in A7r5 cells, but also those on the small conductance calcium-activated potassium (SK (Ca)) channels and voltage-gated (Kv) channels. However, the blockers of large-conductance Ca (2+)-activated potassium channels failed to modify the inhibitory action of isosteviol on [Ca (2+)]i. The obtained results indicated that a decrease of [Ca (2+)]i in A7r5 cells by isosteviol is mainly mediated by the selective opening of K (ATP) channel or/and SK (Ca) channel. Alteration in the Kv channel also plays a critical role in the inhibitory action of isosteviol.

And from the same group more on mechanism of vasodilatation (relaxation of smooth muscle in hypertension):

 
Isosteviol acts on potassium channels to relax isolated aortic strips of Wistar rat.

Wong KL, Chan P, Yang HY, Hsu FL, Liu IM, Cheng YW, Cheng JT.

Graduate Institute of Medical Science, Taipei Medical University, Taipei 106, Taiwan.

Isosteviol is a derivative of stevioside, a constituent of Stevia rebaudiana, which is commonly used as a noncaloric sugar substitute in Japan and Brazil. In the present study, the role of potassium channels in the vasodilator effect of isosteviol was investigated using potassium channel blockers on isosteviol-induced relaxation of isolated aortic rings prepared from Wistar rats. Isosteviol dose-dependently relaxed the vasopressin (10(-8) M)-induced vasoconstriction in isolated aortic rings with or without endothelium. However, in the presence of potassium chloride (3x10(-2) M), the vasodilator effect of isosteviol on arterial strips disappeared. Only the inhibitors specific for the ATP-sensitive potassium (K(ATP)) channel or small conductance calcium-activated potassium (SK(Ca)) channel inhibited the vasodilator effect of isosteviol in isolated aortic rings contracted with 10(-8) M vasopressin. Also; since the isosteviol-induced relaxation was unchanged by methylene blue, a role of nitric oxide and/or endothelium in the vasodilatation produced by isosteviol could be ruled out. The obtained results indicated that vasodilatation induced by isosteviol is related to the opening of SK(Ca) and K(ATP) channels.

And further more recently:

More evidence of the antihypertensive effect in dogs:

 
Mechanism of the antihypertensive effect of stevioside in anesthetized dogs.

Liu JC, Kao PK, Chan P, Hsu YH, Hou CC, Lien GS, Hsieh MH, Chen YJ, Cheng JT.

Department of Medicine, Taipei Medical University--Wan Fang Hospital, Taipei, Taiwan.

Stevioside is a sweet-tasting glycoside isolated from the leaves of Stevia rebaudiana. It has been used as a noncaloric sugar substitute in Japan and Brazil for decades. Previous studies have shown that it lowered blood pressure in spontaneously hypertensive rats by intravenous injection. This study was designed to evaluate the hypotensive effect of stevioside in dogs and to define the underlying mechanism. After nasogastric administration of stevioside powder (200 mg/kg), the blood pressure of healthy mongrel dogs began to significantly decrease at 60 min and returned to baseline level at 180 min. The reduction of blood pressure was more rapid (at 5-10 min) and effective after intravenous injection. However, no significant change of blood pressure was noted after injection through left vertebral artery, implicating that the hypotensive effect is not related to the central nervous system. Stevioside also showed significant hypotensive effects in renal hypertensive dogs, in a dose-dependent manner. In cultured rat aortic smooth muscle cells (A7r5 cell line), stevioside can dose-dependently inhibit the stimulatory effects of vasopressin and phenylephrine on intracellular Ca(2+) in a calcium-containing medium. However, no intracellular Ca(2+) inhibitory effect was observed in calcium-free medium, implicating that stevioside may inhibit the Ca(2+) influx from extracellular fluid. Our present data show that stevioside did not influence the calcium ionophore (A23187) induced Ca(2+) influx, indicating that the antagonistic effect was through Ca(2+) channels. This study confirmed that stevioside is an effective antihypertensive natural product, and its hypotensive mechanism may be probably due to inhibition of the Ca(2+) influx. Copyright 2003 S. Karger AG, Basel

The following  is an animal study in which Stevia is shown to be effective in the same model for effectively treating both Hypertension and Diabetes:

 
Antihyperglycemic and blood pressure-reducing effects of stevioside in the diabetic Goto-Kakizaki rat.

Jeppesen PB, Gregersen S, Rolfsen SE, Jepsen M, Colombo M, Agger A, Xiao J, Kruhoffer M, Orntoft T, Hermansen K.

Department of Endocrinology and Metabolism, Molecular Diagnostic Laboratory, Aarhus Amtssygehus, Aarhus University Hospital, Aarhus, Denmark.

Stevioside, a glycoside present in the leaves of the plant, Stevia rebaudiana Bertoni (SrB), has acute insulinotropic effects in vitro. Its potential antihyperglycemic and blood pressure-lowering effects were examined in a long-term study in the type 2 diabetic Goto-Kakizaki (GK) rat. Rats were fed 0.025 g x kg(-1) x d(-1) of stevioside (purity > 99.6%) for 6 weeks. An intra-arterial catheter was inserted into the rats after 5 weeks, and conscious rats were subjected to arterial glucose tolerance test (2.0 g x kg(-1)) during week 6. Stevioside had an antihyperglycemic effect (incremental area under the glucose response curve [IAUC]): 985 +/- 20 (stevioside) versus 1,575 +/- 21 (control) mmol/L x 180 minutes, (P <.05), it enhanced the first-phase insulin response (IAUC: 343 +/- 33 [stevioside] v 136 +/- 24 [control] microU/mL insulin x 30 minutes, P <.05) and concomitantly suppressed the glucagon levels (total AUC: 2,026 +/- 234 [stevioside] v 3,535 +/- 282 [control] pg/mL x 180 minutes, P <.05). In addition, stevioside caused a pronounced suppression of both the systolic (135 +/- 2 v 153 +/- 5 mm Hg; P <.001) and the diastolic blood pressure (74 +/- 1 v 83 +/- 1 mm Hg; P <.001). Bolus injections of stevioside (0.025 g x kg(-1)) did not induce hypoglycemia. Stevioside augmented the insulin content in the beta-cell line, INS-1. Stevioside may increase the insulin secretion, in part, by induction of genes involved in glycolysis. It may also improve the nutrient-sensing mechanisms, increase cytosolic long-chain fatty acyl-coenzyme A (CoA), and downregulate phosphodiesterase 1 (PDE1) estimated by the microarray gene chip technology. In conclusion, stevioside enjoys a dual positive effect by acting as an antihyperglycemic and a blood pressure-lowering substance; effects that may have therapeutic potential in the treatment of type 2 diabetes and the metabolic syndrome. Copyright 2003, Elsevier Science (USA). All rights reserved.

And Stevia has been confirmed to lower blood pressure in humans with mild essential hypertension:

 
A double-blind placebo-controlled study of the effectiveness and tolerability of oral stevioside in human hypertension.

Chan P, Tomlinson B, Chen YJ, Liu JC, Hsieh MH, Cheng JT.

Division of Cardiovascular Medicine, Taipei Medical College and affiliated Taipei Wan Fang Hospital, Taiwan.

AIMS: Stevioside is a natural plant glycoside isolated from the plant Stevia rebaudiana which has been commercialized as a sweetener in Japan for more than 20 years. Previous animal studies have shown that stevioside has an antihypertensive effect. This study was to designed to evaluate the effect of stevioside in human hypertension. METHODS: A multicentre, randomized, double-blind, placebo-controlled study was undertaken. This study group consisted of 106 Chinese hypertensive subjects with diastolic blood pressure between 95 and 110 mmHg and ages ranging from 28 to 75 years with 60 subjects (men 34, women 26; mean +/- s.d., 54.1+/-3.8 years) allocated to active treatment and 46 (men 19, women 27; mean +/- s.d., 53.7+/-4.1 years) to placebo treatment. Each subject was given capsules containing stevioside (250 mg) or placebo thrice daily and followed-up at monthly intervals for 1 year. RESULTS: After 3 months, the systolic and diastolic blood pressure of the stevioside group decreased significantly (systolic: 166.0+/-9.4-152.6+/-6.8 mmHg; diastolic: 104.7 +/- 5.2-90.3+/-3.6 mmHg, P<0.05), and the effect persisted during the whole year. Blood biochemistry parameters including lipid and glucose showed no significant changes. No significant adverse effect was observed and quality of life assessment showed no deterioration. CONCLUSIONS: This study shows that oral stevioside is a well tolerated and effective modality that may be considered as an alternative or supplementary therapy for patients with hypertension.

Confirmation of the above results and conclusions in a number of other human double blind placebo controlled studies:

 
Efficacy and tolerability of oral stevioside in patients with mild essential hypertension: a two-year, randomized, placebo-controlled study.

Hsieh MH, Chan P, Sue YM, Liu JC, Liang TH, Huang TY, Tomlinson B, Chow MS, Kao PF, Chen YJ.

Department of Medicine, Taipei Medical University--Wan Fang Hospital, Taipei City, Taiwan.

BACKGROUND: Stevioside, a natural glycoside isolated from the plant Stevia rebaudiana Bertoni, has been used as a commercial sweetening agent in Japan and Brazil for >20 years. Previous animal and human studies have indicated that stevioside has an antihypertensive effect. OBJECTIVES: This study was undertaken to investigate the long-term (2-year) efficacy and tolerability of stevioside in patients with mild essential hypertension. Secondary objectives were to determine the effects of stevioside on left ventricular mass index (LVMI) and quality of life (QOL). METHODS: This was a multicenter, randomized, double-blind, placebo-controlled trial in Chinese men and women aged between 20 and 75 years with mild essential hypertension (systolic blood pressure [SBP] 140-159 mm Hg and diastolic blood pressure [DBP] 90-99 mm Hg). Patients took capsules containing 500 mg stevioside powder or placebo 3 times daily for 2 years. Blood pressure was measured at monthly clinic visits; patients were also encouraged to monitor blood pressure at home using an automated device. LVMI was determined by 2-dimensional echocardiography at baseline and after 1 and 2 years of treatment. QOL was assessed using the Medical Outcomes Study 36-Item Short-Form Health Survey. Electrocardiographic, laboratory, and QOL parameters were assessed at the beginning of treatment, and at 6 months, 1 year, and 2 years. RESULTS: One hundred seventy-four patients (87 men, 87 women) were enrolled in the study, and 168 completed it: 82 (42 men, 40 women; mean [SD] age, 52 [7] years) in the stevioside group and 86 (44 women, 42 men; mean age, 53 [7] years) in the placebo group. After 2 years, the stevioside group had significant decreases in mean (SD) SBP and DBP compared with baseline (SBP, from 150 [7.3] to 140 [6.8] mm Hg; DBP, from 95 [4.2] to 89 [3.2] mm Hg; P < 0.05) and compared with placebo (P < 0.05). Based on patients' records of self-monitored blood pressure, these effects were noted beginning approximately 1 week after the start of treatment and persisted throughout the study. There were no significant changes in body mass index or blood biochemistry, and the results of laboratory tests were similar in the 2 groups throughout the study. No significant difference in the incidence of adverse effects was noted between groups, and QOL scores were significantly improved overall with stevioside compared with placebo (P < 0.001). Neither group had a significant change in mean LVMI. However, after 2 years, 6 of 52 patients (11.5%) in the stevioside group had left ventricular hypertrophy (LVH), compared with 17 of 50 patients (34.0%) in the placebo group (P < 0.001). Of those who did not have LVH at baseline, 3 of 46 patients (6.5%) in the stevioside group had developed LVH after 2 years, compared with 9 of 37 patients (24.3%) in the placebo group (P < 0.001). CONCLUSIONS: In this 2-year study in Chinese patients with mild hypertension, oral stevioside significantly decreased SBP and DBP compared with placebo. QOL was improved, and no significant adverse effects were noted.

Anti-inflammatory/anticarcinogenic

The following study suggests Stevia may be anti-inflammatory as well as anticarcinogenic, as in protective against carcinogens:

 
Inhibitory effect of stevioside on tumor promotion by 12-O-tetradecanoylphorbol-13-acetate in two-stage carcinogenesis in mouse skin.

Yasukawa K, Kitanaka S, Seo S.

College of Pharmacy, Nihon University, Chiba, Japan. yasukawa@pha.nihon-u.ac.jp

Four steviol (ent-kaurene-type diterpenoid) glycosides, stevioside, rebaudiosides A and C, and dulcoside A, have been isolated from Stevia rebaudiana BERTONI. These compounds showed strong inhibitory activity against 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation in mice. The 50% inhibitory dose of these compounds for TPA-induced inflammation was 54.1-291.6 micro g/ear. Furthermore, at 1.0 and 0.1 mg/mouse of stevioside mixture, the mixture of these compounds markedly inhibited the promoting effect of TPA (1 micro g/mouse) on skin tumor formation initiated with 7,12-dimethylbenz[a]anthracene (50 micro g/mouse).
 

Antiviral Properties:

Editor’s note: Rotaviruses are the leading cause of severe diarrheal disease in young children.  Intestinal mucosal IgA responses play a critical role in protective immunity against rotavirus reinfection. Rotaviruses consist of three concentric capsid layers surrounding a genome of 11 segments of double-stranded RNA. The outer layer capsid proteins, VP4 and VP7, which are responsible for viral attachment and entry, are targets for protective neutralizing antibodies. However, IgA monoclonal antibodies directed against the intermediate capsid protein VP6, which do not neutralize the virus, have also been shown to protect mice from rotavirus infection and clear chronic infection in SCID mice.

Rotavirus is the most important cause of severe dehydrating diarrhea in infants and young children worldwide. Regardless of the social and economic status, nearly all children will be infected with rotavirus before 3 years of age. Over 500,000 children, primarily from developing countries, die every year from rotavirus infection, and many more have severe diarrhea that requires hospitalization. Given the severity and scope of rotavirus infection, there is an urgent need for a safe and effective vaccine or other suitable and easily administered and affordable treatment.  The abstract following the very next one discusses results  demonstrating that Stevia inhibits rotavirus replication.

            During the rotavirus replication cycle, virions attach to host cells as triple- layered particles (TLPs)and subsequently enter the cytoplasm by either plasma    membrane or endosomal membrane penetration. As a result of cell entry, the outer layer of VP4/VP7 is lost, and the resulting double-layered particles (DLPs)  become transcriptionally active, releasing mRNA transcripts through a system of   channels that penetrate the VP6 and inner VP2 capsid layers at each of the icosahedral vertices. ¹

^{Note that the reference for a more detailed discussion of rotavirus is available to download as a free full text article footnoted below:}

 
Inhibition of rotavirus replication by a non-neutralizing, rotavirus VP6-specific IgA mAb.

Feng N, Lawton JA, Gilbert J, Kuklin N, Vo P, Prasad BV, Greenberg HB.

Department of Gastroenterology, Stanford University School of Medicine, Stanford, California 94304, USA.

The next study demonstrated that Stevia is inhibitory to Rotavirus reproduction and may well be more generally examined for its anti viral properties:

 
Analysis of anti-rotavirus activity of extract from Stevia rebaudiana.

Takahashi K, Matsuda M, Ohashi K, Taniguchi K, Nakagomi O, Abe Y, Mori S, Sato N, Okutani K, Shigeta S.

Department of Microbiology, School of Medicine, Fukushima Medical University, 1 Hikarigaoka, Fukushima-shi 960-1295, Japan. k-tak@fmu.ac.jp
(HRV= Human Rotavirus)
Anti-human rotavirus (HRV) activity of hot water extracts from Stevia rebaudiana (SE) was examined. SE inhibited the replication of all four serotypes of HRV in vitro. This inhibitory effect of SE was not reduced on the prior exposure of SE to HCl for 30 min at pH 2. Binding assay with radiolabeled purified viruses indicated that the inhibitory mechanism of SE is the blockade of virus binding. The SE inhibited the binding of anti-VP7 monoclonal antibody to HRV-infected MA104 cells. The inhibitory components of SE were found to be heterogeneous anionic polysaccharides with different ion charges. The component analyses suggested that the purified fraction named as Stevian with the highest inhibitory activity consists of the anionic polysaccharide with molecular weight of 9800, and contains Ser and Ala as amino acids. Analyses of sugar residues suggest uronic acid(s) as sugar components. It did not contain amino and neutral sugars and sulfate residues. These findings suggest that SE may bind to 37 kD VP7 and interfere with the binding of VP7 to the cellular receptors by steric hindrance, which results in the blockade of the virus attachment to cells.

Since animals such as pigs, cattle, and horses are vulnerable to their own strains of diarrhea inducing rotaviruses, one would think based upon the study below that Stevia may well be a natural remedy for use in veterinary medicine. The genetic study below suggests this possibility:

 
Antigenic and molecular analyses reveal that the equine rotavirus strain H-1 is closely related to porcine, but not equine, rotaviruses: interspecies transmission from pigs to horses?

Ciarlet M, I a P, Conner ME, Liprandi F.

Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas 77030, USA. mciarlet@bcm.tmc.edu

We have sequenced the genes encoding the inner capsid protein VP6 and the outer capsid glycoprotein VP7 of the subgroup (SG) I equine rotavirus strain H-1 (P9[7], G5). The VP6 and VP7 proteins of the equine rotavirus strain H-1 shared a high degree of sequence and deduced amino acid identity with SG I porcine strains and serotype G5 porcine strains, respectively. Previous sequence analyses of the genes encoding the outer capsid spike protein VP4 and the nonstructural proteins NSP1 and NSP4 of equine H-1 strain also revealed a high degree of sequence and deduced amino acid homology with the prototype porcine rotavirus strain OSU (P9[7], G5). We have also confirmed and extended the VP4 and VP7 antigenic relatedness of equine rotavirus strain H-1 to porcine strains of P9[7] and G5 serotype specificities isolated in the United States, Venezuela, Argentina, and Australia based on cross-neutralization studies. In addition, the pathogenicity of tissue culture-adapted equine H-1, H-2, FI-14, FI-23, and L338, and porcine OSU rotavirus strains was compared in the neonatal mouse model. The 50% diarrhea dose (DD50) of equine H-1 was similar to that of porcine OSU and equine H-2 and L338 strains, while the DD50 of equine H-2 was > or = 50 or 315-fold lower than those of equine FI-14 or FI-23, respectively. Our sequence comparison of NSP4 of the rotavirus strains tested potentially identified amino acid residue 136, within the variable region spanning amino acids 130 to 141, as playing a role in virulence. Taken together, there is strong support to suggest that the equine rotavirus strain H-1 may represent an example of interspecies transmission from pigs to horses.

Further evidence of antiviral/anticarcinogenesis properties of Stevia:

Editor’s note: It is important for the reader to recognize that although these microbes used to transform isosteviol into subsequent derivatives were used out of the body, the same or similar organisms and/or other organisms normally present in human gut have been shown to create the same or similar compounds in vivo. These and other diterpenes present as a result of microbial transformation in the human gut, implies the same or similar inhibitory action on Epstein-Barr virus and probably other Herpes family viruses as well.  This study is also significant because of the known relationship between E-B virus activity and the tumor promotion.  Also there was direct inhibition of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate by these diterpenes.

 
Microbial transformation of isosteviol and inhibitory effects on Epstein-Barr virus activation of the transformation products.

Akihisa T, Hamasaki Y, Tokuda H, Ukiya M, Kimura Y, Nishino H.

College of Science and Technology, Nihon University, 1-8 Kanda Surugdai, Chiyoda-ku, Tokyo 101-8308, Japan. akihisa@chem.cst.nihon-u.ac.jp

Microbial transformation of isosteviol (2), a beyerane-type diterpenoid obtained from stevioside (1) by acid hydrolysis, yielded 7beta-hydroxyisosteviol (3), 11beta-hydroxyisosteviol (5), and 12beta-hydroxyisosteviol (6) by the fungus Aspergillus niger, 17-hydroxyisosteviol (7) by the fungus Glomerella cingulata, and 3 and 7-oxoisosteviol (4) by the fungus Mortierella elongate. The five metabolites, 3-7, along with 1 and 2 were evaluated for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells as a primary screening test for inhibitors of tumor promoters. All the diterpenes tested showed potent inhibitory effects, with the five metabolites 3-7 exhibiting more potent effects.

Editor’s note: There is an assumption that these metabolites noted above are similar to those encountered in the intestinal metabolism of Stevioside and Steviol recorded elsewhere in the library.  It is reasonable given additional evidence to suggest that there is similar inhibitory capacity of Stevia as relates to Herpes family virus activation, of which the Epstein Barr Virus is only one of eight known family members all of which reproduce similarly and have similar characteristics when it comes to antiviral susceptibilities.

Antibacterial Properties:

The next study demonstrates yet another property of Stevia: It is clearly bacteriocidal against E. coli 0157 without detrimental effects against normal intestinal flora:

Bactericidal activity of a fermented hot-water extract from Stevia rebaudiana Bertoni towards enterohemorrhagic Escherichia coli O157:H7 and other food-borne pathogenic bacteria.

Tomita T, Sato N, Arai T, Shiraishi H, Sato M, Takeuchi M, Kamio Y.

Faculty of Agriculture, Tohoku University, Sendai, Miyagi, Japan. tomita@biochem.tohoku.ac.jp

A fermented aqueous extract from Stevia rebaudiana Bertoni showed strong bactericidal activity towards a wide range of food-borne pathogenic bacteria including enterohemorrhagic Escherichia coli O157:H7. The colony-forming ability of the food-borne pathogenic bacteria tested so far was reduced to < 10(-7) when exposed to > or = 40% (v/v) solutions of the fermented extract at 37 C for 2 hr. Secretion of verocytotoxin 1 and 2 by enterohemorrhagic E. coli was also diminished by fermented extract at a concentration of > or = 10% (v/v). In contrast, the fermented extract did not significantly kill Bifidobacteria or Lactobacilli. The active principle(s) of the fermented Stevia extract were bactericidal under acidic conditions.

There is a benefit to oral health by using non-cariogenic sweeteners (Stevia):

Update on low-calorie sweeteners to benefit dental health.

Grenby TH.

Department of Oral Medicine & Pathology, United Medical School, Guy's Hospital, London, UK.

Ten different types of high-intensity, low-calorie sweeteners that have potential dental applications as sugar substitutes are appraised, taking into consideration their general properties, safety and toxicological assessment, metabolic fate in the body, regulatory status and any dental research that has been carried out on them. Attention focuses on the continuing expansion in the wide diversity of types of sweeteners becoming available, the multiple sweetener concept, the growing interest in materials of natural origin, and the distinction between non-cariogenic and actively anti-cariogenic properties of the individual sweeteners.

A contrasting view of mutagenicity of one of the metabolites of Stevia; Later studies prove it is not mutagenic; e.g. J Toxicol Sci. 2002 Dec;27 Suppl 1:1-8. (see above).

Metabolically activated steviol, the aglycone of stevioside, is mutagenic.

Pezzuto JM, Compadre CM, Swanson SM, Nanayakkara D, Kinghorn AD.

Stevioside, a constituent of Stevia rebaudiana, is commonly used as a noncaloric sugar substitute in Japan. Consistent with reports in the literature, we have found that stevioside is not mutagenic as judged by utilization of Salmonella typhimurium strain TM677, either in the presence or in the absence of a metabolic activating system. Similar negative results were obtained with several structurally related sweet-tasting glycosides. However, steviol, the aglycone of stevioside, was found to be highly mutagenic when evaluated in the presence of a 9000 X g supernatant fraction derived from the livers of Aroclor 1254-pretreated rats.*** Expression of mutagenic activity was dependent on both pretreatment of the rats with Aroclor 1254 and addition of NADPH; unmetabolized steviol was not active. The structurally related species, isosteviol, was not active regardless of metabolic activation. Similarly, chemical reduction of the unsaturated bond linking the carbon-16 and -17 positions of steviol resulted in the generation of two isomeric products, dihydrosteviol A and B that were not mutagenic. In addition, ent-kaurenoic acid was found to be inactive. It is therefore clear that a metabolite of an integral component of stevioside is mutagenic; structural features of requisite importance for the expression of mutagenic activity include a hydroxy group at position 13 and an unsaturated bond joining the carbon atoms at positions 16 and 17. A potential metabolite of steviol, steviol-16 alpha,17-epoxide, was synthesized chemically and found to be ineffective as a direct-acting mutagen. Thus, although stevioside itself appears innocuous, it would seem prudent to expeditiously and unequivocally establish the human metabolic disposition of this substance.

*** A HIGHLY UNNATURAL MANIPULATION

Agronomic Information to enhance Stevia Production:

 
Features of development of Stevia rebaudiana shoots cultivated in the roller bioreactor and their production of steviol glycosides.

Bondarev N, Reshetnyak O, Nosov A.

Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Moscow, Russia.

Growth and development of Stevia rebaudiana shoots cultivated in the roller bioreactor and their production of steviol glycosides (SGs) were investigated. It was found that, owing to the highly favorable conditions of shoot cultivation created in such an apparatus, the intensity of shoot growth and SG production appeared to be 1.5 - 2.0 times higher than those of the shoots grown in tubes. These results indicate the existence of a positive correlation between these two processes. The data obtained suggest that the enhanced SG production is due to the differentiation of chlorenchyma cells and formation of specific subcellular structures for the glycoside to be accumulated.

A functional genomics approach proposes how to create a model for industrial synthesis of Stevia:

Functional genomics uncovers three glucosyltransferases involved in the synthesis of the major sweet glucosides of Stevia rebaudiana.

Richman A, Swanson A, Humphrey T, Chapman R, McGarvey B, Pocs R, Brandle J.

Agriculture and Agri-Food Canada, Southern Crop Protection and Food Research Centre, 1391 Sandford St, London, Ontario, Canada N5V 4T3.

Summary Stevia rebaudiana leaves accumulate a mixture of at least eight different steviol glycosides. The pattern of glycosylation heavily influences the taste perception of these intensely sweet compounds. The majority of the glycosides are formed by four glucosylation reactions that start with steviol and end with rebaudioside A. The steps involve the addition of glucose to the C-13 hydroxyl of steviol, the transfer of glucose to the C-2' and C-3' of the 13-O-glucose and the addition of glucose to the hydroxyl of the C-4 carboxyl group. We used our collection of ESTs, an UDP-glucosyltransferase (UGT)-specific electronic probe and key word searches to identify candidate genes resident in our collection. Fifty-four expressed sequence tags (ESTs) belonging to 17 clusters were found using this procedure. We isolated full length cDNAs for 12 of the UGTs, cloned them into an expression vector, and produced recombinant enzymes in Escherichia coli. An in vitro glucosyltransferase activity enzyme assay was conducted using quercetin, kaempferol, steviol, steviolmonoside, steviolbioside, and stevioside as sugar acceptors, and (14)C-UDP-glucose as the donor. Thin layer chromatography was used to separate the products and three of the recombinant enzymes produced labeled products that co-migrated with known standards. HPLC and LC-ES/MS were then used to further define those reaction products. We determined that steviol UGTs behave in a regioselective manner and propose a modified pathway for the synthesis of rebaudioside A from steviol.

Chemical composition of Stevia rebaudiana:

 
New non-glycosidic diterpenes from the leaves of Stevia rebaudiana.

McGarvey BD, Attygalle AB, Starratt AN, Xiang B, Schroeder FC, Brandle JE, Meinwald J.

Agriculture and Agri-Food Canada, Southern Crop Protection and Food Research Centre, 1391 Sandford Street, London, Ontario, Canada N5V 4T3. mcgarveyb@agr.gc.ca

Six new labdane-type, non-glycosidic diterpenes, sterebins I-N (1-6), were isolated from the leaves of Stevia rebaudiana. Their structures, analogous to those of the previously described sterebins A-H, were elucidated on the basis of spectroscopic and chemical studies.

 
Rebaudioside F, a diterpene glycoside from Stevia rebaudiana.

Starratt AN, Kirby CW, Pocs R, Brandle JE.

Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, ON, N5V 4T3, London, Canada. starratta@em.agr.ca

The sweet diterpenoid glycoside, rebaudioside F, was isolated from leaves of a high rebaudioside C producing line of Stevia rebaudiana, and its structure was established by chemical and spectral studies.

 
Analysis of sweet diterpene glycosides from Stevia rebaudiana: improved HPLC method.

Kolb N, Herrera JL, Ferreyra DJ, Uliana RF.

Facultad de Ciencias Exactas, Quimicas y Naturales, Universidad Nacional de Misiones, Felix de Azara 1552, 3300 Posadas (Misiones), Argentina. nkolb@fceqyn.unam.edu.ar

An improved analytical method was developed which may be applied to quality control of stevioside and rebaudioside A contents in dried leaves of Stevia rebaudiana before processing; in a selective sampling program searching for plants of higher yield in diterpene glycosides content; or when a large number of samples are sent to the laboratory for analysis. The procedure developed involves two steps: solvent extraction followed by an isocratic HPLC analysis. The sample, 1 g of dried leaves of S. rebaudiana, is ground and solvent-extracted with EtOH 70% (w/w) in Erlenmeyer flasks by shaking for 30 min in a 70 degrees C water bath. After the extract was cooled, it was filtered and analyzed by HPLC using an NH(2) column (250 x 4.6 mm) and a mixture of acetonitrile/water (80:20, v/v) as mobile phase, pH 5 adjusted with acetic acid. The detection was in the UV range at 210 nm (0.04 AUFS). Quantitation was performed by means of an external standard calibration curve for each analyte which had been obtained from standard solutions of pure stevioside and rebaudioside A. Working under these conditions there were no observed interference effects. The method saves time in sample preparation, and reduces sample handling and chromatographic analysis time, while having little loss of precision [coefficient of variation (CV%) between 1.8% and 3.0%] and recovery [between 98.5% and 100.5%]. The method was applied to 30 samples of S. rebaudiana from Misiones (Northeastern Argentina), and the stevioside content found ranged between 3.78 and 9.75% (weight) whereas Rebaudioside A content ranged between 1.62 and 7.27% (weight).

How Stevia is metabolized in the liver of humans and rats is so similar as to be insignificantly different.  This study, in addition to the value for toxicology of Stevia further amplifies the importance of other studies demonstrating the various medicinal applications of Stevia in rat models in that those results become more significant affirmation of the transference of principle to humans.  For those interested in the toxicology of Stevia we include the following:

 
Absorption and metabolism of glycosidic sweeteners of stevia mixture and their aglycone, steviol, in rats and humans.

Koyama E, Sakai N, Ohori Y, Kitazawa K, Izawa O, Kakegawa K, Fujino A, Ui M.

Kashima Laboratory, Mitsubishi Chemical Safety Institute Ltd., 14 Sunayama, Hasaki-machi, Ibaraki, 314-0255, Kashima-gun, Japan. e-koyama@ankaken.co.jp

Stevia mixture, sweeteners extracted from the leaves of Stevia rebaudiana Bertoni, consists mainly of the glycosides of the diterpene derivative steviol. The aims of this study were to investigate the absorption (in rats) and the hepatic metabolism (in rats and humans) of both Stevia mixture and steviol. Absorption was investigated both in vivo and ex vivo. In ex vivo experiments using the rat everted sac method, no absorption of Stevia mixture was observed, but significant absorption of steviol was noted (equivalent to approximately 70% of the absorption reference- salicylic acid- value). In the in vivo experiment, rats received a single oral administration of either steviol or Stevia mixture; a peak steviol concentration in plasma was observed 15 min after its oral administration, demonstrating rapid absorption. However, after oral administration of Stevia mixture, the steviol concentration in plasma increased steadily over 8 h, suggesting that Stevia mixture components are first degraded and then absorbed as steviol in the rat intestine. Steviol metabolism in humans and rats was examined by incubating steviol with liver microsomes from the two species. Oxidative (monohydroxy and dihydroxy) metabolites of steviol were observed by LC-ESI/MS after incubation with both human and rat liver microsomes. The intrinsic clearance of steviol in human liver microsomes was 4-times lower than that found in rat liver microsomes. In conclusion, this study suggests that there are no major species differences in steviol hepatic metabolism between rats and humans. Absorption from the human intestine can be predicted to occur in an analogous manner to that from the rat intestine.

And the following study confirms the above findings:

 
In vitro metabolism of the glycosidic sweeteners, Stevia mixture and enzymatically modified Stevia in human intestinal microflora.

Koyama E, Kitazawa K, Ohori Y, Izawa O, Kakegawa K, Fujino A, Ui M.

Department of Biology, Kashima Laboratory, Mitsubishi Chemical Safety Institute Ltd, 14 Sunayama, Hasaki-machi, Kashima-gun, Ibaraki, 314-0255, Japan.

Stevia mixture, sweeteners extracted from the leaves of Stevia rebaudiana Bertoni, consists mainly of stevioside and rebaudioside A (glycosides of the diterpene derivative steviol). The aim of this study was to investigate human intestinal metabolism of Stevia mixture and its alpha-glucose derivative (known in Japan as enzymatically modified Stevia) by LC/MS/ESI analysis. Degradation was examined by incubating Stevia mixture, enzymatically modified Stevia, stevioside, rebaudioside A, alpha-monoglucosylstevioside, alpha-monoglucosylrebaudioside A and the aglycone, steviol with pooled human faecal homogenates (obtained from five healthy volunteers) for 0, 8 and 24 h under anaerobic conditions. Stevia mixture, enzymatically modified Stevia, stevioside and rebaudioside A (0.2 mg/ml) were completely eliminated within 24 h, whereas no degradation of steviol (0.08 and 0.2 mg/ml) appeared to be found during the incubation period. Stevia mixture, stevioside and rebaudioside A appeared to be hydrolyzed to steviol by human intestinal microflora: this observation is consistent with previous rat metabolism studies. Similarly, enzymatically modified Stevia appeared to be metabolized via Stevia components and, finally, to steviol. This study suggests that there are apparently no species differences in intestinal metabolism of Stevia mixture between rats and humans.

This next study implies that there is only a remote possibility that there can be an overdose of Steviol, especially in the light of the previous studies showing no significant interspecies difference in the metabolism of Stevia mixture and or its component diterpenoid derivatives.  However, at 15g/kg LD 50s causing renal damage as the cause of death, the ingested dose to compare for a 150 kg man would be 2.25 kg ingested or almost 5 pounds;  That is an awful lot of Steviol, suggesting as in previous studies a very substantial margin of safety: Again safe means safe!

Acute toxicity of stevioside, a natural sweetener, and its metabolite, steviol, in several animal species.

Toskulkao C, Chaturat L, Temcharoen P, Glinsukon T.

Department of Physiology, Faculty of Science, Mahidol University, Bangkok, Thailand.

The acute toxicity of stevioside and steviol (a product of enzymatic hydrolysis of stevioside) was investigated in three animal species including rat, mouse and hamster. The susceptibility to stevioside and steviol acute toxicity in both sexes of these animal species was compared. The animals were treated intragastrically with stevioside or steviol and general signs and symptoms were observed. The numbers of dead animals were recorded within a period of 14 days after administration for estimation of LD50. Stevioside at a dose as high as 15 g/kg BW was not lethal to either mice, rats or hamsters. Hamsters were found to be more susceptible to steviol than rats or mice. LD50 values of steviol in hamsters were 5.20 and 6.10 g/kg BW for males and females, respectively. In rats and mice, LD50 values of steviol were higher than 15 g/kg BW in both sexes.(Editor’s note: Refer back to above studies demonstrating no difference between human and rat metabolism of Stevia, implying that LD 50s would be roughly equivalent as well, meaning that the above figure quoted above strongly suggests the article is accurate for humans) Histopathological examination in the kidney of hamsters induced by steviol revealed severe degeneration of the proximal tubular cells. These structural alterations were correlated with the increases in serum blood urea nitrogen (BUN) and creatinine. Therefore, the possible cause of death induced by steviol might be due to acute renal failure.