G.Athinrynn,A.J.A.Rnjitsingh,A.Ush Rj Nnthini,C.Pmlth

a Department of Zoology,Sri Paramakalyani College,Alwarkurichi,MS University,Tamil Nadu,India

b JP College of Arts and Science,Tenkasi,MS University,Tamil Nadu,India

c Department of Biotechnology,Mother Teresa University,Kodaikanal,Tamil Nadu,India

d UGC,New Delhi,India

Abstract Natural dyes taken from the barks of the tree Caesalpinia sappan has been used in many consumer products.Hence it is imperative to test the toxicity of this dye.In the present study an investigation was conducted to fin out the toxic effect of aqueous extract of the dye C.sappan in test animal Wister albino rats.Acute oral toxicity showed no clinical signs of toxicity and no mortality even at a dose level of 100–2000 mg/kg in 14 days observation period.When a dose level above 2500 mg/kg was given for 28 days,no death was noticed up to the dose level 5000 mg/kg body weight.The weight of the tested rats was not significantl reduced,when compared with the control group.The organ-body weight ratio of kidney,liver and abdomen did not change when compared to the control group in the observation period.The less toxicity of the dyes of C.sappan.? 2017 Beijing Academy of Food Sciences.Production and hosting by Elsevier B.V.This is an open access article under the CC BY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Keywords:C.sappan;Natural dye;Dye toxicity;Food grade dye;Consumer products

1.Introduction

Natural dyes are reported to be biodegradable,less toxic to human health as compared to synthetic dyes and cause no skin dermatitisandallergy[1].Toxicityandmutagenicityofsynthetic dyes have increasingly become a major occupational hazard and challenge in regard to their use and safety in the textile industry[2].However,studies on toxicity of natural dyes are very limited.The vast majority of natural dye constituents are non-toxic,and have a long history of use for treatment of different diseases and complaints.Several natural dyes are also used as colouring matters in food,for which a low toxicity is obviously crucial.Even so,annatto,cohineal(carmine),saffron and turmeric appear on lists of permitted food additives in the EU and USA[3,4].

Caesalpinia sappanis a traditional medicinal plant cultivated in India,Mayanmar,Vietnam,Sri Lanka,and the Malay Peninsula,and is distributed domestically in China,Fujia and Taiwan.Many pharmacological activities ofC.sappanheart wood are usedaspH-sensitiveacid-baseindicatorandamedicinallyuseful dye[5,6].It has also been used to treat diabetic complications[7]and to improve blood circulation[8].Extracts ofC.sappanhave been shown to exert various pharmacological effects,including anti-hypercholesterolemia,sedation,and depression of central nervous system[9].

According to Ayurveda,the heart wood is useful in vitiated conditions of Pitta,burning sensation,wounds,ulcers,leprosy,skin diseases,diarrhoea,dysentery,diabetes etc[10].The decoction of the wood is a powerful emmenagogue being used in India,Brazil,Chinaandseveralothercountries.InChinaandMalaysia,it is used for disturbances of menstrual function.The heart wood is reputed to have blood vitalizing activity.In Malaysia it is used as a antimalarial and in Philippines as anti-anaemic agent.The small core of heart wood produces a dark red solution in water and is being used as herbal drinking water in Kerala[11].

However,studies on toxicity of natural dyes are very limited and not only are the vast majority of natural dye constituents non-toxic but have a long history of use on the African continent for treatment of different diseases and complaints[12].In this study,non-toxic and eco-friendly dye of this plant was extracted and were examined for toxicity using healthy white albino rats.The aim of the present study was to demonstrate whether this natural dye is toxic or non-toxic with an evidence of histological investigations.

2.Materials and methods

2.1.Plant collection

TheC.sappanbark was obtained from a siddha medicinal shop at Nagercoil(Tamilnadu).The plant identity was confirme by Department of Botany,Sri Paramakalyani College.

2.2.Preparation of aqueous extract

The preparation of the aqueous plant extract was carried out as described by Yakubu et al.[13]and Mariselvam et al.[14–16].TheC.sappanbark were sundried and macerated into uniform powder.Approximately 300 g of the powder was extracted with 500 ml distilled water using soxhlet apparatus and concentrated by rotator evaporator at 50?C.This was transferred into a suitable container and lyophilized (freeze dried).The yield of the crude aqueous plant extract was stored in desiccators until required for use.The extract was dissolved in appropriate volume of distilled water to the desired concentration.

2.3.Experimental procedure

This study was carried out in healthy male Wister rats,weighing 240–260 g.The animals were housed under standard laboratory conditions oflight,temperature(21±2?C)and relative humidity (55±5%).The animals were given standard rat pellets and tap water ad libitum (For the use of rats,prosper permission was obtained from the Ethical committee of Sri Paramakalyani College,Alwarkurichi).

2.4.Acute toxicity study

The acute toxicity study was conducted in accordance with Lorke’s method [17].The study was conducted in two phase using a total of 21 rats.In the firs phase,nine rats were divided into 3 groups of 3 rats each.Groups 1,2 and 3 animals were given 100,1000 and 2000 mg/kg body weight(b.w)of the extract respectively to possibly establish the range of doses producing any acute toxic effect.Each rat was given a single dose after at least 5 days of adaptation.In addition,a fourth group of three rats was setup as control group and animals in the group were not given the extract.

2.5.Repeated dose 28-day oral toxicity study

In the second phase,further specifidoses(2500,3500 and 5000 mg/kg) body weight of the extract were administered to nine rats(3 rat per dose).The extract was dissolved in distilled water and given via orally.All animals were observed frequently on the day of treatment and surviving animals were monitored daily for signs of oral toxicity.At the end of 28 days,all surviving rats were sacrifice and then autopsied in the department of Zoology,Sri Paramakalyani College,Alwarkurichi.The internal organswereexaminedmacroscopicallyforpathologicalchanges compared to the control group.The weights of these organs were also taken and the mean organ-body weight ratios werecalculated and compared with those of the control group.

2.6.Statistical analysis

The statistical analyses were carried out using statistical package for social sciences (SPSS—computer package).Percentage organ-body weight ratios and rat’s body weights were expressed as mean±SD.Values in all groups were compared using the analysis of variance (ANOVA).For all analyses the level of statistical significanc was f xed at p<0.05[18].

2.7.Histology

The kidney and liver tissue were excised from the animals,washed with normal saline to remove blood,fied in 10%buffered neutral formalin for 1 h and processed for 1 h and processed for paraffiembedding.Section of 5 μ thickness was cut using rotary microtome.The sections were processed and through graded alcohol series,stained with haematoxylin and eosin[19],cleaned in xylene and cover slipped in DPX.Histological examination was done under 10× magnificatio using Trinocular Research zeiss Microscope(Gottingen,Germany).

3.Results

No abnormalities were observed during the experimental period.Death of the animals is used to determine an LD50 value using the ADT test.As there was no death (Table1) resulting from the oral treatment of the test samples even up to the highest dose of 5000 mg/kg of body weight.No changes occurred in urine,faecal matter,body and organ body weight(Table2–4)at all dose levels in all the animals used in the test.

4.Discussion

The toxicity evaluation of the dye taken fromC.sappanshowed its safety nature and it can be recommended to use in consumer products.The organ indices,activity and haematological parameters of the tested rat did not show any change when compared with control group(Fig.1a–1d and 2a–2b)

The safety of the dye is further confirme by histological observation of the tissues of rat treated with the dye.Fig.1a–1d shows the histological architecture of the kidney of control rats and rats treated with dye.When the internal organisms of thekidney of rats exposed to dye is compared with that of control rat no marked changes were noticed.The kidney of both treated and control group shows normal histological features.

Table1 Effect of Lethal concentration of aqueous extract of the wood of C.sappan administered orally to Wister albino rats.

Table2 Impact of the extract of C.sappan on the body weights of rat during acute toxicity experiment.

Table3 Effect of oral treatment of aqueouswood extract of C.sappan on percent organ-body weight ratio of rats.

Table4 Effect of aqueous heartwood extract of C.sappan on haematological parameters in repeated 28 days oral toxicity study.

Like kidney,the section through the liver of rats treated with the dye did not show any histopathological change.In all the tested animals the histology of the liver is normal showing its normal functioning(Fig.1,2a–2d).

The histological study further strengthens the finding that the dose of the dye up to the level of 5000 mg/kg body weight in Wister rat did not give any harm,this was further supported by the chemical with LD50 value greater than 5000 mg/kg b.w is thought to be safe as suggested by Garg[20].So the dye can be incorporated to the food shift as turmeric is used in Indian kitchens.

The toxicity of synthetic dyes currently being used in the textile industry has been widely reported[21]and many of them have been known to be toxic and dangerous to human and the environment as compared to many natural dyes which have been reported to be largely eco-friendly and less harmful[22].

5.Conclusion

At the end of each experiment,there were no deaths recorded as a result of the oral treatment to the test plant sample materials at dose levels of up to 5000 mg/kg body weight.None of the test dye samples exhibited any signs of toxicity/adverse reactions in the animal models used in the study.Assessment of toxicity of natural dyes for textile use is therefore of utmost importance and also additive to the food items.The test samples were not illness to the animals and were not likely to cause allergic reactions.This plant extract will be used in future to give colourful food like kaesari,lottu,idly,idiyappam,and lemon food (yellow colour food) etc.In south India,“kesari”is one of the famous sweet at festival time.It is made up of different colours,such as synthetic food dyes.This dyes cause many ill effects like diarrhoea,fever,cancer etc.,to human body especially children’s.

Fig.1.Section through the kidney and liver of Wister rats treated with different doses of C.sappan bark aqueous dye for 28 days.

Instead of this synthetic dye colourant powder,will useC.sappanbark intense red colour natural powder in future to kaesari and also otherwise.TheC.sappanplant dye is alternative for synthetic red dyes used in food colorant and it is good for human health.

Acknowledgement

The authors are grateful to Department of Zoology,Sri Paramakalyani College,Alwarkurichi,Manonmaniam Sundaranar University for providing Research specialities.