{"id":53777,"date":"2023-12-31T11:46:43","date_gmt":"2023-12-31T11:46:43","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=53777"},"modified":"2024-01-05T05:56:10","modified_gmt":"2024-01-05T05:56:10","slug":"safety-evaluation-of-amaranth-extract-by-acute-sub-chronic-and-chronic-exposure-in-rats","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol16no4\/safety-evaluation-of-amaranth-extract-by-acute-sub-chronic-and-chronic-exposure-in-rats\/","title":{"rendered":"Safety Evaluation of Amaranth Extract by Acute, Sub-Chronic and Chronic Exposure in Rats"},"content":{"rendered":"\n

Introduction<\/strong><\/p>\n\n\n\n

Vegetables\nand fruits in diet are considered essential for long life and human health\nmaintenance. These are considered rich in several vitamins, minerals and other\npotentially metabolically active compounds, e.g. polyphenols1<\/sup>. Apart\nfrom these nutritious compounds, nitrate (NO3<\/sub>\u02c9) present in\nvegetables has unique role in vasodilation2,3<\/sup>. The facultative anaerobic\nbacteria present in the oral cavity, facilitates the conversion of NO3<\/sub>\u02c9\nions into nitrite (NO2<\/sub>\u02c9) ions4<\/sup>. Once NO2<\/sub>\u02c9 is formed,\nit further converts to NO (nitric oxide) via various pathways5<\/sup>. During\nlow oxygen saturation (also called hypoxia) in blood, the conversion of NO2<\/sub>\u02c9\nto NO takes place at faster rate6<\/sup>. Endothelial nitric oxide synthase\n(eNOS or NOS3) is the primary source of NO in the vascular endothelium. Under a\nhypoxia state, the expression of eNOS decreases, resulting in low NO\nproduction. In such state, formation of NO3<\/sub>\u02c9 to NO2<\/sub>\u02c9 and\nthen conversion to NO works as an alternate system for NO production inside the\nbody7<\/sup>. NO is considered as one of the primes signaling molecule with\nmultiple roles in humans. These include maintenance of muscles contraction,\nflow of blood in arteries and veins, homeostasis of certain molecules like calcium\nand glucose etc8,9<\/sup>.<\/p>\n\n\n\n

It\nhas been scientifically proved that NO2<\/sub>\u02c9 level can be improved in\nsignificant manner by ingesting NO3<\/sub>\u02c9 in diet. This helps in lowering\nthe blood pressure by dilating the blood vessels10-12<\/sup>. Consumption\nof NO3<\/sub>\u02c9 in diet also increases endurance to exercise as a response\nto physiological benefits13<\/sup>. In a study by Stokes et al., C-reactive\nprotein level was decreased after NO3<\/sub>\u02c9 and NO2<\/sub>\u02c9 intake in\nhigh cholesterol fed mice. It also decreased the vascular inflammation and\nsignificantly reversed the endothelial dysfunctions14<\/sup>.It\nhas been documented that during old age, the supply of amino acid L-arginine\n(an important NOS-substrate) and tetrahydrobiopterin (one of the cofactors)\nreduces15<\/sup>. This along with poor level of NO2<\/sub>\u02c9, makes the regular\nnitric oxide pathway less efficient during ageing16<\/sup>. Apart from\nthis, overproduction of O2<\/sub>–<\/sup> (free radical superoxide) in\nold age, decreases the bioavailability of NO by formation of peroxy-nitrites17<\/sup>.\nThis reduced availability of NO may increase the chances of endothelial\ndysfunctions during ageing process18<\/sup> and may cause arterial hyper-tension19<\/sup>\nof old age. Thus, increase of NO3<\/sub>\u02c9 consumption in the diet of old\nage peoples may be beneficial for overall vascular health and adequate supply\nof NO in bioavailable form.<\/p>\n\n\n\n

The\nvegetables like Spinach, Cabbage and underground edible parts like beetroot are\nknown to contain high percentage of NO3<\/sub>\u02c9. Amaranth (also known as red\nspinach) is one of the popular vegetables rich in various nutrients along with\nsignificantly higher amount of NO3<\/sub>\u02c9 present in leaves20<\/sup>. It\nis cultivated as gluten free pseudo-cereal primarily in Asia, Mexico, South\nAmerica and all tropical places of the world21<\/sup>. Amaranth is a fast-growing\nplant and easy to maintain and consume as leafy vegetables throughout the year.\nThe leaves as well as seeds of amaranth are considered highly nutritious22,23<\/sup>.\nBoth leaves and seeds are rich sources of various proteins. Quantitatively,\nleaves contain about 15-30% protein whereas seeds contain 15\u201345% of fresh\nmatter. The leaves also contain Vitamin C (one of the widely known antioxidant),\ndietary fibers and traces of essential minerals24,25<\/sup>. The composition\nof amino acid present in amaranth proteins is considered well balanced, highly\nbioavailable and good functional characteristics26<\/sup>. Apart from these\nnutrients, amaranth leaves are also rich in secondary plant metabolites, which may\nprovide potential health benefits27<\/sup>. Recent research has indicated that\nleaves and other aerial parts of amaranth are important sources of phenolic\ncompounds28-30<\/sup>. Among these, hydroxycinnamic acids, benzoic acids, flavonols\nand their glycosides have been reported in amaranth leaves and flowers31<\/sup>.\nOther phytochemicals present in amaranth with antioxidant activity are\nbetalains, especially betacyanins32<\/sup>. The contents of these pigments\nvary among amaranth species and genotypes33<\/sup>.<\/p>\n\n\n\n

To\nget clinical benefits, heavy intake of vegetables rich in NO3<\/sub>\u02c9 is practically very difficult in routine\nday-to-day life. Along with nitrates, a large amount of oxalic acid (an\nanti-nutrient) also gets inside the body and may result in kidney damage on prolonged\nuse. Moreover, it has been reported that food rich in NO3<\/sub>\u02c9 didn\u2019t increase the nitrate levels in blood\nwhereas consumption of NO3<\/sub>\u02c9 in the form of dietary supplement increased the same in old age peoples34<\/sup>.\nIn another published study, the absorption of NO3<\/sub>\u02c9 from extract of amaranth leaves (2 g\ndose) was studied where single dose of extract, significantly increased (p<0.001)\nthe plasma NO3<\/sub>\u02c9 levels in healthy adults as compared to the subject\u2019s\nconsumed placebo35<\/sup>. The authors concluded that one dose of extract from\nred spinach leaves can enhance the NO3<\/sub>\u02c9 levels in plasma for more\nthan eight hours. The higher levels of NO3<\/sub>\u02c9 may be beneficial in\nvarious sports activities and routine exercises.<\/p>\n\n\n\n

Apart\nfrom multiple benefits of dietary NO3<\/sub>\u02c9, a few studies have reported some\nadverse effects\/toxicity of synthetic potassium and sodium nitrate in animals36<\/sup>.\nThese were mainly due to development of methemoglobinemia. At the same time, toxic\neffects of potassium nitrate on some biochemical parameters of rats were completely\nameliorated by simultaneous feeding of ascorbic acid37<\/sup>. The rationale\nof current study is to establish safety of extract from amaranth leaves\nwith high dietary nitrates and to show its high safety profile compared to toxicity\nassociated with synthetic NO3<\/sub>\u02c9 and NO2<\/sub>\u02c9. <\/p>\n\n\n\n

Materials and Methods<\/strong><\/p>\n\n\n\n

Test sample and animals <\/strong><\/p>\n\n\n\n

Commercial\nbatch of Oxystorm\u00ae<\/sup> from Arjuna Natural Private Ltd., Kochi, Kerala,\nIndia was used as test sample of Amaranth extract. It is standardized to\ncontain approximately 9% dietary nitrate. Male and female Wistar albino rats\nweighing 150-170 g were kept at animal house conditions (Temperature 24\u00b12\u00b0C;\nRelative humidity 55-70%; 12\/12 h light\/dark cycle). Filtered potable water and\nextruded rodent diet supplied by M\/s. Amruth labs, Bangalore, India was used ad libitum<\/em>. Acute and sub-chronic\ntoxicity studies were approved by IAEC of JSS College of Pharmacy, Ooty, Tamilnadu, India (approval no.\nJSSCP\/IAEC\/CADRAT\/2014-15). SD rats of 160-180 gram were used for the long-term\nchronic toxicity and study was approved by IAEC of Arjuna Natural Private Ltd.,\nKochi, Kerala, India (approval no. ANEL\/ IAEC\/ 2016-I\/ 1607015). SD rats were\nchosen for long term study due to longer life span of SD rats as compared to\nWistar rats. The animal house conditions for long term study were same as\nmentioned for acute and sub-chronic toxicity study. <\/p>\n\n\n\n

Acute toxicity study<\/strong><\/p>\n\n\n\n

OECD\n423 guidelines were followed to conduct this study in stepwise manner38<\/sup>.\nThree rats were used in the each step to determine adequate classification of the\ntest material (amaranth extract) by acute toxicity test. Six Wistar albino rats\n(female only) weighing 150-170 g (Age, 8-10 Weeks) were used in this study and acclimatized\nfor 7 days before each step’s commencement. The amaranth extract was dissolved\nin distilled water and administered to rats by oral route at 2000 mg\/kg body\nweight. A metal canula fitted to a syringe was used for this purpose. Based on\nthe results, the next set of animals were administered with a 2000 mg\/kg dose\nof the test item. The rats were carefully observed by an experienced\nveterinarian for 14 days. Special attention was paid during initial 4 hours on\nday 1 of the study. Body weight of all the rats was noted at baseline, day 7\nand day 14. On the last day of study, necropsy was conducted by a veterinarian\nto see any gross lesions or hemorrhage in major organs. <\/p>\n\n\n\n

Sub-chronic (28\ndays repeated dose) toxicity study<\/strong><\/p>\n\n\n\n

OECD\n407 guidelines were followed to conduct sub-chronic toxicity study in rats39<\/sup>.\nForty rats (M\/F: 1\/1; 150-170 g; 8-9 weeks old) were included in this study and\nrandomly distributed into four groups. Ten rats comprising of 5 males and 5\nfemales were kept in each group. Acclimatization period was one week in\nstandard animal house conditions. Amaranth extract was dissolved in water and\nadministered at 100, 500 and 1000 mg\/kg to the rats of group 1, 2 and 3 as low,\nmedium and high dose, respectively. Required quantity of extract was daily weighed\nand freshly dissolved in water before administration to the rats. Extract\nfeeding was continued for 28 days using metal cannula attached with syringe. The\nfourth group of ten rats (M\/F: 1\/1) was fed with distill water alone for the\nsame duration (28 days) and was considered as the control group.<\/p>\n\n\n\n

Daily\ncage side observations were done by a qualified veterinarian for any abnormal\nbehaviour or symptom. At the end of the study, sensory reactivity towards\ndifferent stimuli (e.g., various reflexes, visual, auditory and proprioceptive stimuli),\nmeasurement of grip strength and motor coordination assessment were performed as\nper the standards published procedures40-43<\/sup>. In brief, flexion\nreflex (tests spinal cord) was assessed by pinching the toes of rat with forceps,\nthe response was to move the foot away. Grasping reflex (tests cerebral cortex)\nwas assessed by picking up the rat and palm was touched with a wire; the\nresponse was to grip the wire. Righting reflex was tested by putting the rat on\nits back and it turns over immediately. Auditory startle was assessed by\nputting the rat on a level surface in quiet environment and then a loud hand\nclap was given. The rat flexed forelimbs, extended hind-limbs and arched the\nbody. For assessment of grip strength, the animal was kept on the top of the\nwire-bottomed cage. The tail was clenched at the base and the animal was pulled\nalong the surface to measure its capacity to hold on to the wired surface. The\nmotor coordination was evaluated by Rotarod apparatus. At the end of the\nexperiment, animals were fasted for 16 h and blood samples were collected from\nretro orbital plexus of all the animals. The blood samples were used as such (with\nK3EDTA as anti-coagulant) for hematology whereas serum was separated by centrifugation\nat 3000 rpm for 15 min and used for the biochemical estimations. Routine urine analysis\nwas also conducted as per the standard procedures. Body weight of rats was\nrecorded weekly and on the last day of study, all the rats were sacrificed and\nmajor tissues were preserved in 10% buffered formalin for histopathology using\nrotary microtome.<\/p>\n\n\n\n

Chronic toxicity\nstudy<\/strong><\/p>\n\n\n\n

OECD 452 guidelines were followed to\nconduct chronic toxicity study44<\/sup>. Two-hundred rats (100 male\/100 female)\nwere used to conduct this study. The rats were divided into four main groups of\n40 rats in each group. Ratio of male\/female rats was kept 1:1 for all the\ngroups. An acclimatization period of seven days was followed before start of\ndosing of extract. First three group of rats were fed at 45, 90 and 180 mg\/kg\nof amaranth extract as low medium and high dose groups. Distill water was fed\nto the fourth group of rats and designated as control group. Dosing was continued\nonce daily for one year duration.<\/p>\n\n\n\n

Apart\nfrom four main study groups, two groups of 20 animals in each (10M\/10F) were\nfed with amaranth extract 180 mg\/kg and distill water, respectively and\ndesignated as \u2018Recovery groups\u2019. These groups were also fed for the duration of\none year but after the end of one year period, these recovery group animals\nwere observed for one more month (without feeding of extract) for any\nreversible effect or delayed toxicity. <\/p>\n\n\n\n

Daily observations on any abnormal\nbehaviour or toxic symptom were conducted by a qualified veterinarian. Blood samples\nwere collected at the end of study period and serum was separated by\ncentrifugation to conduct biochemistry. Hematology was performed with as such\nwhole blood (EDTA was used as an anti-coagulant). Terminally, the rats were sacrificed\nand all the major organs were collected, preserved in formalin and studied for\nhistopathological changes.<\/p>\n\n\n\n

Statistical analysis<\/strong><\/p>\n\n\n\n

Bartlett’s test was conducted to analyze\nthe homogeneous nature of data by GraphPad Prism Software. The data was further\nanalyzed by ANOVA and if ‘F’ was found significant, an individual comparison of\nmeans of control and treated groups was done using Dunnett’s test. P value\n<0.05 was considered significant.<\/p>\n\n\n\n

Results <\/strong><\/p>\n\n\n\n

Acute toxicity study<\/strong><\/p>\n\n\n\n

The\nbody weight gain of all the rats was similar and in normal range over the study\nduration of 2 weeks (Table 1). All the animals were healthy and no animal died\nafter feeding of amaranth extract at 2000 mg\/kg dose. The behavior of all the\nrats was normal as observed by veterinarian. The animals didn\u2019t show any\nsymptom of toxicity or abnormality throughout the study period. The cavities\nand orifices were normal when observed on the day of sacrifice. There was no\nchange in skin or eye color and mucous membrane was also normal. The gross\nnecropsy revealed that animals were healthy and all the internal organs were\nnormal. The LD50<\/sub> of amaranth extract was calculated as >2000 mg\/kg\nin rats. As per the OECD 423 guidelines, the extract falls in the category 5 of\nGlobally Harmonized System.<\/p>\n\n\n\n

Table 1: Body weight (g) and mortality data in acute toxicity study of amaranth extract<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Dose in mg\/kg<\/strong><\/p>\n<\/td>\n

\n

Rat <\/strong><\/p>\n<\/td>\n

\n

M\/F<\/strong><\/p>\n<\/td>\n

\n

Body weight (g)<\/strong><\/p>\n<\/td>\n

\n

No. dead \/ No. tested<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Initial<\/strong><\/p>\n<\/td>\n

\n

Day 8<\/strong><\/p>\n<\/td>\n

\n

Weight change (day 8 \u2013 Initial)<\/strong><\/p>\n<\/td>\n

\n

Day 15<\/strong><\/p>\n<\/td>\n

\n

Weight change (day 15 \u2013 Initial)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

2000<\/p>\n<\/td>\n

\n

1<\/p>\n<\/td>\n

\n

Female<\/p>\n<\/td>\n

\n

158<\/p>\n<\/td>\n

\n

165<\/p>\n<\/td>\n

\n

7<\/p>\n<\/td>\n

\n

169<\/p>\n<\/td>\n

\n

11<\/p>\n<\/td>\n

\n

0\/6<\/p>\n

 <\/p>\n<\/td>\n<\/tr>\n

\n

2<\/p>\n<\/td>\n

\n

Female<\/p>\n<\/td>\n

\n

161<\/p>\n<\/td>\n

\n

170<\/p>\n<\/td>\n

\n

9<\/p>\n<\/td>\n

\n

176<\/p>\n<\/td>\n

\n

15<\/p>\n<\/td>\n<\/tr>\n

\n

3<\/p>\n<\/td>\n

\n

Female<\/p>\n<\/td>\n

\n

157<\/p>\n<\/td>\n

\n

163<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

174<\/p>\n<\/td>\n

\n

17<\/p>\n<\/td>\n<\/tr>\n

\n

4<\/p>\n<\/td>\n

\n

Female<\/p>\n<\/td>\n

\n

149<\/p>\n<\/td>\n

\n

152<\/p>\n<\/td>\n

\n

3<\/p>\n<\/td>\n

\n

163<\/p>\n<\/td>\n

\n

14<\/p>\n<\/td>\n<\/tr>\n

\n

5<\/p>\n<\/td>\n

\n

Female<\/p>\n<\/td>\n

\n

150<\/p>\n<\/td>\n

\n

155<\/p>\n<\/td>\n

\n

5<\/p>\n<\/td>\n

\n

172<\/p>\n<\/td>\n

\n

22<\/p>\n<\/td>\n<\/tr>\n

\n

6<\/p>\n<\/td>\n

\n

Female<\/p>\n<\/td>\n

\n

149<\/p>\n<\/td>\n

\n

154<\/p>\n<\/td>\n

\n

5<\/p>\n<\/td>\n

\n

171<\/p>\n<\/td>\n

\n

22<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n

Sub-chronic\ntoxicity study<\/strong><\/p>\n\n\n\n

There was no mortality in extract treated or\ncontrol rats. All the rats were clinically fit and their behavior was normal\nthroughout the study period. Gain in body weight was almost similar in all the\ngroups. Similarly, there was no significant difference in the food intake\npattern of all the groups. In this study, no significant changes in the haematology\n(Table 2-3) and bio-chemistry profile of control and treated rats were observed\nafter 28 days. These parameters for treated groups were comparable to respective\ncontrol group of rats (Table 4-5).
<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Table 2: Hematology profile for male rats in 28 days sub-chronic toxicity study of amaranth extract<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Table 3: Hematology profile for female rats in 28 days sub-chronic toxicity study of amaranth extract<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Table 4: Clinical chemistry data for male rats in 28 days sub-chronic toxicity study of amaranth extract<\/strong>.<\/p>\n\n\n\n\n\n\n\n\n\n
\n

Group &<\/strong>
Dose<\/strong><\/p>\n<\/td>\n

\n

FBS (mg\/dl)<\/strong><\/p>\n<\/td>\n

\n

ALKP<\/strong><\/p>\n

(U\/L)<\/strong><\/p>\n<\/td>\n

\n

CHO (mg\/dl)<\/strong><\/p>\n<\/td>\n

\n

BUN<\/strong><\/p>\n

(mg\/dl)<\/strong><\/p>\n<\/td>\n

\n

Cre (mg\/dl)<\/strong><\/p>\n<\/td>\n

\n

T.Pro (gm\/dl)<\/strong><\/p>\n<\/td>\n

\n

Alb (gm\/dl)<\/strong><\/p>\n<\/td>\n

\n

SGOT<\/strong><\/p>\n

(U\/L<\/strong><\/p>\n<\/td>\n

\n

SGPT<\/strong><\/p>\n

(U\/L))<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Control (0 mg\/kg)<\/p>\n<\/td>\n

\n

85.2\u00b15.9<\/p>\n<\/td>\n

\n

144.3\u00b18.1<\/p>\n<\/td>\n

\n

118.7\u00b18.5<\/p>\n<\/td>\n

\n

44.8\u00b114.1<\/p>\n<\/td>\n

\n

0.9\u00b10.0<\/p>\n<\/td>\n

\n

11.9\u00b11.9<\/p>\n<\/td>\n

\n

7.3\u00b10.6<\/p>\n<\/td>\n

\n

119\u00b112.9<\/p>\n<\/td>\n

\n

74\u00b111.2<\/p>\n<\/td>\n<\/tr>\n

\n

Low dose (100 mg\/kg)<\/p>\n<\/td>\n

\n

84.8\u00b16.2<\/p>\n<\/td>\n

\n

146.6\u00b112.3<\/p>\n<\/td>\n

\n

112.1\u00b17.6<\/p>\n<\/td>\n

\n

46.9\u00b114.4<\/p>\n<\/td>\n

\n

0.9\u00b10.0<\/p>\n<\/td>\n

\n

12.6\u00b12.0<\/p>\n<\/td>\n

\n

7.7\u00b11.0<\/p>\n<\/td>\n

\n

130.5\u00b112.7<\/p>\n<\/td>\n

\n

69.4\u00b110.5<\/p>\n<\/td>\n<\/tr>\n

\n

Medium dose (500 mg\/kg)<\/p>\n<\/td>\n

\n

87.0\u00b14.6<\/p>\n<\/td>\n

\n

151.5\u00b18.8<\/p>\n<\/td>\n

\n

108.1\u00b16.9<\/p>\n<\/td>\n

\n

51.3\u00b17.1<\/p>\n<\/td>\n

\n

0.9\u00b10.1<\/p>\n<\/td>\n

\n

11.1\u00b11.6<\/p>\n<\/td>\n

\n

7.7\u00b11.0<\/p>\n<\/td>\n

\n

126.4\u00b120.8<\/p>\n<\/td>\n

\n

68.8\u00b17.0<\/p>\n<\/td>\n<\/tr>\n

\n

High dose (1000 mg\/kg)<\/p>\n<\/td>\n

\n

85.5\u00b14.2<\/p>\n<\/td>\n

\n

149.3\u00b110.6<\/p>\n<\/td>\n

\n

113.0\u00b18.1<\/p>\n<\/td>\n

\n

42.8\u00b19.0<\/p>\n<\/td>\n

\n

0.8\u00b10.0<\/p>\n<\/td>\n

\n

12.6\u00b12.4<\/p>\n<\/td>\n

\n

7.5\u00b10.7<\/p>\n<\/td>\n

\n

125.9\u00b15.4<\/p>\n<\/td>\n

\n

74.5\u00b13.6<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Data presented as Mean\u00b1SD; n=5. ANOVA, p>0.05 as compared to control in each case. There was no significant difference between treated and control groups.<\/p>\n

FBS=Fasting Blood Sugar; ALKP=Alkaline phosphatase; CHO=Cholesterol; Cre=Creatinine; BUN=Blood urea nitrogen; SGOT=Serum glutamic oxaloacetic transaminase; SGPT=Serum glutamic pyruvic transaminase; T.Pro=Total Protein; Alb=Albumin.<\/p>\n\n\n

Table 5: Clinical chemistry data for female rats in 28 days sub-chronic toxicity study of amaranth extract<\/strong>.<\/p>\n\n\n\n\n\n\n\n\n\n
\n

Group &<\/strong>
Dose<\/strong><\/p>\n<\/td>\n

\n

FBS (mg\/dl)<\/strong><\/p>\n<\/td>\n

\n

ALKP
<\/strong>(U\/L)<\/strong><\/p>\n<\/td>\n

\n

CHO (mg\/dl)<\/strong><\/p>\n<\/td>\n

\n

BUN
<\/strong>(mg\/dl)<\/strong><\/p>\n<\/td>\n

\n

Cre (mg\/dl)<\/strong><\/p>\n<\/td>\n

\n

T.Pro (gm\/dl)<\/strong><\/p>\n<\/td>\n

\n

Alb (gm\/dl)<\/strong><\/p>\n<\/td>\n

\n

SGOT
<\/strong>(U\/L<\/strong><\/p>\n<\/td>\n

\n

SGPT
<\/strong>(U\/L))<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Control (0 mg\/kg)<\/p>\n<\/td>\n

\n

88.7\u00b16.2<\/p>\n<\/td>\n

\n

145.0\u00b13.7<\/p>\n<\/td>\n

\n

105.4\u00b14.2<\/p>\n<\/td>\n

\n

43.6\u00b110.0<\/p>\n<\/td>\n

\n

0.9\u00b10.1<\/p>\n<\/td>\n

\n

13.8\u00b12.1<\/p>\n<\/td>\n

\n

7.3\u00b10.9<\/p>\n<\/td>\n

\n

118.4\u00b114.3<\/p>\n<\/td>\n

\n

68.1\u00b16.3<\/p>\n<\/td>\n<\/tr>\n

\n

Low dose (100 mg\/kg)<\/p>\n<\/td>\n

\n

85.7\u00b15.4<\/p>\n<\/td>\n

\n

137.5\u00b14.2<\/p>\n<\/td>\n

\n

112.0\u00b112.0<\/p>\n<\/td>\n

\n

46.4\u00b113.0<\/p>\n<\/td>\n

\n

0.9\u00b10.1<\/p>\n<\/td>\n

\n

11.6\u00b12.0<\/p>\n<\/td>\n

\n

7.5\u00b10.8<\/p>\n<\/td>\n

\n

124.0\u00b116.0<\/p>\n<\/td>\n

\n

71.9\u00b16.5<\/p>\n<\/td>\n<\/tr>\n

\n

Medium dose (500 mg\/kg)<\/p>\n<\/td>\n

\n

83.2\u00b16.2<\/p>\n<\/td>\n

\n

150.3\u00b113.4<\/p>\n<\/td>\n

\n

111.8\u00b19.6<\/p>\n<\/td>\n

\n

42.7\u00b16.3<\/p>\n<\/td>\n

\n

0.9\u00b10.0<\/p>\n<\/td>\n

\n

13.2\u00b12.5<\/p>\n<\/td>\n

\n

7.4\u00b11.0<\/p>\n<\/td>\n

\n

123.7\u00b121.7<\/p>\n<\/td>\n

\n

69.0\u00b18.1<\/p>\n<\/td>\n<\/tr>\n

\n

High dose (1000 mg\/kg)<\/p>\n<\/td>\n

\n

87.3\u00b12.3<\/p>\n<\/td>\n

\n

141.7\u00b114.0<\/p>\n<\/td>\n

\n

110.2\u00b112.7<\/p>\n<\/td>\n

\n

45.9\u00b111.5<\/p>\n<\/td>\n

\n

0.9\u00b10.1<\/p>\n<\/td>\n

\n

11.6\u00b11.8<\/p>\n<\/td>\n

\n

7.5\u00b10.4<\/p>\n<\/td>\n

\n

125.4\u00b113.9<\/p>\n<\/td>\n

\n

71.6\u00b15.5<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Data presented as Mean\u00b1SD; n=5. ANOVA, p>0.05 as compared to control in each case. There was no significant difference between treated and control groups.<\/p>\n

FBS=Fasting Blood Sugar; ALKP=Alkaline phosphatase; CHO=Cholesterol; Cre=Creatinine; BUN=Blood urea nitrogen; SGOT=Serum glutamic oxaloacetic transaminase; SGPT=Serum glutamic pyruvic transaminase; T.Pro=Total Protein; Alb=Albumin.<\/p>\n\n\n

The histological observations of all the vital organs in control and treated groups were same. There was no treatment related abnormality (Figure 1-2). Some haemorrhage and alveolar oedema was observed in the lungs of extract treated (high dose) as well as control rats. In few rats hydronephrosis and cysts in kidney was also noted but it was similar for control and high dose extract treated rats. Liver of a few rats in both the groups have shown vacuolation and cellular swelling at some places. These findings were considered normal\/incidental by the histopathologist and concluded as non-toxic nature of the test extract.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 1: Microscopic sections of major organs in 28 days sub-chronic toxicity (X100). <\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Figure 2: Microscopic sections of major organs in 28 days sub-chronic toxicity (X100).<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

In\nthe treated as well as control group of rats, body weight and organ weight on\n28th<\/sup> day was in normal range. The organ weight to body weight ratios\nwere comparable to respective control rats for males as well as females. In the\nmedium dose (500 mg\/kg) males, there was significant reduction in fasting body\nweight and a significant enhancement in weights of gonads and heart. In addition,\nmedium dose females showed increase in absolute gonads weight. However, the\nsame was not found with high dose rats (treated at 1000 mg\/kg) and there was no\ndose correlation was observed, therefore, considered incidental.<\/p>\n\n\n\n

In\nthe 28-days repeated dose study with amaranth extract, the maximum dose of 1000\nmg\/kg in rats didn\u2019t produce any toxic effect and all the findings were comparable\nto respective control counterparts. The NOEL (No Observed Adverse Effect Level)\nin this study was found as 1000 mg\/kg in rats.<\/p>\n\n\n\n

Chronic toxicity\nstudy<\/strong><\/p>\n\n\n\n

The chronic toxicity study was conducted\nat slightly low dosages to observe the accumulated effect over a period of one\nyear. There was no death of rats at any dose level of extract treated or control\ngroup of animals. The body weight and food intake of all the rats was in normal\nrange. When compared to the control rats, there was no abnormality noted with extract\ntreated rats as far as visible clinical symptoms are concerned. <\/p>\n\n\n\n

The\nlevel of RBC, WBC, Hemoglobin etc was in normal range in extract treated as\nwell as control group of rats. Full hematological profile is presented as Table\n6-7 for the rats of both the sexes. Furthermore, the liver functions, kidney\nfunctions and lipid profile of all the rats was normal after one year of extract\ntreatment. The whole biochemistry data of control and amaranth extract fed rats\nwere similar when side by side comparison was conducted (Table 8-9). The gross\npathology and histopathology of all the collected organs was normal in all the\ncontrol and treated rats.
\n<\/strong><\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Table 6: <\/strong>Hematology profile for male rats in chronic toxicity study of amaranth extract<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Table 7: <\/strong>Hematology profile for female rats in chronic toxicity study of amaranth extract<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Table 8: Clinical chemistry data for male rats in chronic toxicity <\/strong>study of amaranth extract.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Table 9: Clinical chemistry data for female rats in chronic toxicity <\/strong>study of amaranth extract.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Discussion<\/strong><\/p>\n\n\n\n

The\ntraditional Japanese and Mediterranean food habits are considered most healthy\nall over the world. These are found to increase the life span and less occurrence\nof heart related disorders45<\/sup>. Both of these diets contain plenty of\nfresh vegetables and fruits. Japanese diet is also rich in fish whereas olive\noil is one of the ingredients in Mediterranean diet. These diets do not contain\nred meat except a few occasional dishes. One of the common things is the\npresence of high dietary nitrate content in both of these diets. In Mediterranean\ndiet, vegetables (leafy) are used as such. These may include red-spinach,\nlettuce and rocket salad etc. Whereas Japanese diet contains ta cai, spinach, garland\nchrisantemum, etc46<\/sup>. Food rich in vegetables is normally considered\nto bring down the blood pressure and less occurrence of fatal coronary heart\ndisease, nonfatal myocardial infarction or acute stroke47<\/sup>.<\/p>\n\n\n\n

The\nADI (Acceptable Daily Intake) for nitrate is 3.7 mg per kg of body weight as set\nby the European Food Safety Authority. This equals to 0.06\u2009mmol\/kg body weight\nin human. For an average built human of roughly 70 kg body weight, it will be\nequal to 260\u2009mg daily48<\/sup>. This ADI was set on the basis of toxicity\nand safety studies conducted with synthetic nitrate. Since the natural nitrate supplements\nlike amaranth or red spinach extract contains a lot of anti-oxidants and other\nphyto-nutrients, these extracts are safer than synthetic nitrates. Moreover,\nthe recommended dose of amaranth extract is 1000 to 2000 mg\/day for an adult.\nThis equates to around 90 to 180 mg of nitrate per day which is well within the\nset ADI limits.<\/p>\n\n\n\n

There\nare a number of side effects are known due to ingestion of synthetic nitrates. These\ninclude toxicity to reproductive organs, methylation of hemoglobin (methemoglobinemia)\nand other endovrine or metabolic disorders. In one study, male Wistar rats were\ntreated with sodium nitrate at 19 mg\/kg, 66 mg\/kg and 150 mg\/kg once daily by\noral route for a period of 10 days49<\/sup>. Methemoglobinemia was observed\nin all the rats and nitrate got accumulated in the liver of high dose rats. The\nfunctions of liver were also got impaired as evidenced by increased levels of transferases,\nlactates, triglycerides, and glucose in medium (66 mg\/kg) as well as high dose\n(150 mg\/kg) groups. In their investigations, histopathology further confirmed\nthe inflammation of liver cells, necrosis, steatosis etc in high dose group. In\ncontrast to the study by Gonz\u00e1lez et al., in present study 28 days repeated feeding\nof amaranth extract at highest dose of 1000 mg\/kg daily (equivalent to 90 mg\nnitrate\/kg per day) to rats did not induce any observable toxic effect or liver\ninjury. The polyphenols and other anti-oxidants present in the amaranth extract\nmight have a protective effect on liver and other organs by avoiding production\nof nitrosamines.<\/p>\n\n\n\n

Acute\ntoxicity in rats and mice is considered first step in toxicity evaluation. In\nthe present study, non-toxic nature of amaranth extract at 2000 mg\/kg in rats\nconfirmed the safety of the test material as LD50 was computed as >2000\nmg\/kg in rats. The 28 days study by daily feeding to rats at maximum dose of\n1000 mg\/kg in rats is another evidence of safety of amaranth extract. Therefore,\nthe NOEL of amaranth extract in rats was found as 1000 mg\/kg. This corresponds\nto 11.2 g for a 70 Kg human50<\/sup>.<\/p>\n\n\n\n

The\nsub-chronic toxicity had certain limitations. A few histopathological findings in\nlungs, kidney and liver of control as well as amaranth extract treated group\nwere noted. These were considered to be safe as seen in such studies, and were\nconsidered as incidental. Similarly, the male rats of 500mg\/kg dose had slight\ndecrease in fasting weight and an enhancement in weights of gonads and heart. The\nfemales of 500 mg\/kg group showed a remarkable enhancement in the weight of gonads.\nHowever, same was not found with 1000 mg\/kg daily dose group and there was no\ncorrelation with dose was observed, therefore, considered incidental.<\/p>\n\n\n\n

Since\nfood supplements are supposed to be taken for long term, the chronic toxicity\nstudy was also conducted by feeding amaranth extract to rats for one year\nduration. There was no toxic sign or symptoms observed in chronic toxicity\nstudy at low, medium or high dose of amaranth extract. The biochemical,\nhematological and histopathological observations further confirmed the safety\nof amaranth extract in rats. In this study, the three dosages 45, 90 and 180\nmg\/kg in rats were chosen to represent the human equivalent dose of 500, 1000 and\n2000 mg. In this study, the gross observation of behavior, appearance and toxicological\nfindings like changes in pupil size, color of skin and unusual respiratory\npattern was normal, thus detailed ophthalmic examination using ophthalmoscope\nwas not done. In sub-chronic toxicity study, the neurological and functional\nexaminations (proprioceptive stimuli)\nwas normal in all rats examined, thus detail examination on these parameters\nwas not conducted in the chronic toxicity study. These parameters are optional\nas stated in the OECD 452 guidelines.<\/p>\n\n\n\n

Several\nhuman clinical studies have also been reported with amaranth extract (red spinach\nextract). In one such study, acute effect of amaranth extract extract (1000 mg\ndose) was determined on vascular reactivity in peripheral conduit and resistance\narteries51<\/sup>. In another study on healthy human subjects, amaranth\nextract (1000 mg dose which equals to 90 mg nitrate) was orally given as a\nsingle dose to study the exercise performance and endurance. The authors found\nthat amaranth extract delayed the ventilatory threshold and response was ergogenic52<\/sup>.\nInterestingly, there were no adverse effects reported in the above human studies\nwhich further confirm the safety of amaranth extract in human. In chronic toxicity\nstudy 180mg\/kg was the maximum dose in rats. While converting to human dose, it\nwill be approximately 2 g per day for a 70 Kg human. Overall, the results of these\nstudies confirm the safety of amaranth extract in rats at the tested dosages.<\/p>\n\n\n\n

Conclusion<\/strong><\/p>\n\n\n\n

The\namaranth extract tested in the present study has shown non-toxicity of the\nproduct at 2000 mg\/kg in acute toxicity test. There were no adverse effects at\n1000 mg\/kg daily dosing for 28-days in sub-chronic toxicity study and this dose\nwas considered as NOEL. In the chronic toxicity study also, the extract was non-toxic\nat 180 mg\/kg daily in rats. Thus, it can be concluded that amaranth extract is\nsafe at the doses tested in this study as per the guidelines laid by OECD.<\/p>\n\n\n\n

Acknowledgement<\/strong><\/p>\n\n\n\n

The authors from JSS College of Pharmacy, Ooty, Tamilnadu acknowledge Arjuna Natural Private Ltd., Aluva, Kerala for providing Amaranth extract (Oxystorm\u00ae<\/sup>) as gift sample.<\/p>\n\n\n\n

Conflict of Interest<\/strong><\/p>\n\n\n\n

The authors declare that there is no conflict of interest.<\/p>\n\n\n\n

Funding\nsource<\/strong><\/p>\n\n\n\n

This research received no specific grant from\nany funding agency in the public, commercial, private, or not-for-profit sectors.<\/p>\n\n\n\n

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