LCMS  spectral analysis of Betacyanin Pigments in Amaranthus L.

 

Pinkie Cherian, D. Sheela

Depatment of Botany, St.Teresa’s College, Ernakulam, Kerala, India, 682011, Ph.no:9288453940

 

 

 

ABSTRACT:

Plant pigments has got wider application in textile and pharmaceutical industry. Betacyanins are classes of purple plant pigments that are useful in plant identification and health monitoring. Pure samples of betacyanin were obtained by extracting and isolating  the pigments found in Amaranthus L. plants. The resulting samples were then used to identify the absorption spectra of the pigment. The sample were analysed for measuring the absorbance at the range of 476nm and 538nm respectively. Betanin, Neobetanin and Amaranthine were found in the red pigments isolated from the plants which are of pharmaceutically important and got medicinal application. These pigments play important role as antioxidant and a natural dye for different formulations. The findings from the  present spectral analysis showed 382 m/z ratio found to be derivative of betanidin component. LCMS analysis of the red pigment were carried out showing positive peak with 389 m/z ratio for betanidin compound among Amaranthus caudatus L. and Amaranthus dubius, Mart along with other peak values. The antioxidant capacity that is to scavenge the free radicals of these two Amaranthus L. species can be taken into account as it is a good dietary supplement in our daily life.

 

 

 

 

INTRODUCTION:

Today Amaranthus L. species are  grown in many tropical, subtropical, and temperate countries. Amaranthaceae family composed of many medicinal plants that are used for various herbal formulations in the present scenario. The genera Amaranthus L. (the amaranths) contain many ornamental and medicinal species, as well as species whose seeds are used as pseudocereals and leaves as leaf vegetables. Green leafy vegetables have been reported as sources of nutrients and folklore remedy for the treatment of infections diseases in different parts of the country.

 

Vegetables are considered as the cheapest and most available sources of important proteins, carbohydrates, vitamins, minerals and essential amino acids¹. Previous studies have shown that the consumption of vegetables is closely related with the decrease risks of diseases that resulted from oxidative stress, including cancer, diabetes and various infectious diseases^2,3 .

 

Various substance  classes have been reported in root, leaf and stem tissues in members of the genera Amaranthus L., i.e. alkaloids, amino acids, minerals, carbohydrates, lipids, saponins, carotenoids, tannins, and other phenolic substances⁴.These metabolites present in the plants make it pharmaceutically important component in traditional system of medicine.

 

While previous reports suggested amaranthine and its epimer as major betalains in A. spinosus seeds⁵, the betanins have so far not been investigated. Furthermore, to the best of our knowledge, information on the phenolic constituents of stem bark extracts of the species is still lacking. The medicinal plants taken for the study include Amaranthus caudatus L. and Amaranthus dubius Mart. Therefore, the present investigation aimed at quantitative estimation of betanins from stem bark extracts and  analysis of betacyanin compound applies spectrometry and LCMS,  that are used traditionally for medicinal and food colouring purposes.

 

MATERIALS AND METHODS:

Plant material

The stem of Amaranthus  L. (Amaranthaceae) collected and  the barks is  removed from the stems with a knife, dried in the laboratory at 30 ^oC for 36 h, and pulverized in a laboratory mortar. The so obtained ground materials are then  stored out of light and dampness.

 

Extraction and purification of extracts

To exactly 5.00 g of ground plant material that is the stem potion of the plant in sea sand before homogenization in a mortar to ease extraction with 25 ml purified water. The betanins and phenolics obtained are separated from the solid material by passing the slurry through a funnel with a filter paper. The pH of the resulting solutions is 5.4 for Amaranthus L., respectively. From an aliquote of these crude extracts, slightly concentrated in vacuo at room temperature, betanin quantifications and colour determinations  is done.

 

Betacyanin determination

Betacyanin content was determined by using a UV- vis  spectrophotometer (Thermoevolution 220) at a wavelength of 538 nm. Phosphate buffer was prepared and it was used to replace with distilled water for betacyanin determination ⁶. The quantification of betacyanin was described⁷. The betacyanin content (mg/100 g of fresh weight) was calculated using A₅₃₈(MW)xVx(DF)/εLWx100.

 

Quantification of betanins

Quantification of betacyanins without prior removal of phenolic compounds is carried out using a UV-Vis spectrophotometer. Samples are then diluted in a 0.05 m phosphate buffer (pH 6.5) as previously described ⁸ using the extinction coefficients of betanin (ε = 60000 l/ mol · cm; λ = 538 nm; molecular weight = 550;and neobetanin (ε = 18200 l/mol · cm; λ = 476 nm; molecular weight = 548;  amaranthine (ε = 56600 l/mol · cm; λ = 538 nm; molecular weight = 726; respectively ^9,10.

 

LC-MS analysis                                

Betacyanin content were analysed using Waters 3100 Single Quadrupole LCMS System. The gradient used were methanol and water at a wavelength ranges from 300 to 700nm  having  ion trap mass spectrometer fitted with an ESI source and  were characterized in the positive and negative ionization mode.

 

RESULT:

The present work evaluated the betacyanin pigment among the two species of Amaranthus L. showed good betanin content that has potent antioxidant capacity. The spectral analysis data can determine the bio-molecules present in the selected species in the genus Amaranthus L. Based on the observation using UV-VIS spectrophotometer, all  samples showed absorbance at  536 indicates the presence of betacyanin compound. The highest content of betanin were reported in A. dubius (57.38µg/gm) and A. caudatus showed 29.79µg/gm. Analysis using LCMS  predict the compound based on the molecule weight and fragmentation pattern. The analysis on species A. caudatus found that it contain compounds with molecular mass 305, 349, 365, 393, 409, 437, 453, 481, 497, 607. Similarly A. dubius showed molecular mass 312, 367, 607 and 608. The compounds are found to be similar like amaranthine, 637 (phylocactin) and 389 (betanidin) from the similar studies ¹¹. A. dubius showed 382.87,367.22 m/z ratio while A.caudatus showed 377, 365m/z ratio on both the ionization mode. These components analysed will be useful for the chemical profiling of the Amaranthus L. sps. Thus this study  isolate the compounds that may partly contribute to traditional applications of these plants as a drug for modern medicines. Thus, it seems likely that these compounds may partly contribute to the pharmacological effects of their traditional applications. Finally, the obtained data present a valuable contribution for the scientific evaluation of pharmacologically active principles in Amaranthus L. species.

 

DISCUSSION:

Based on the observation using UV-VIS spectrophotometer, all  samples showed absorbance at  536 indicates the presence of betacyanin compound. The highest content of betanin were reported in A. dubius (57.38µg/gm) and A. caudatus showed 29.79µg/gm. Betanins are reported to exhibit preventive anticarcinogenic properties ^12,13. They constitute a prospective group of compounds, not only for food, but also pharmaceutical or cosmetic industries ¹⁴. The Amaranthine content ranges from 41.68 µg/gm to 80.20 µg/gm whereas in the case of Neobetanin the value ranged from 74.67 µg/gm to 120 µg/gm among the Amaranthus L. species selected in the present study(Fig. 1).

 

Fig. 1: Comparison of pigment content among A. caudatus and A. dubius

 

Analysis using LCMS  predict the compound based on the molecule weight and fragmentation pattern. The analysis on species A. caudatus found that it contain compounds with molecular mass 305, 349, 365, 393, 409, 437, 453, 481, 497, 607. Similarly A. dubius showed molecular mass 312,367,607 and 608.According to the reports, these compounds are amaranthine, 637 (phylocactin) and 389 (betanidin)¹⁵. A. dubius showed 382.87,367.22 m/z ratio while A.caudatus showed 377, 365m/z ratio on both the ionization mode(Fig.2a, 2b and Fig.3a, 3b). Thus from the above mass ratios it can be concluded that betanidin compound with similar mass/charge ratio were reported in the literature reviewed. The present spectral analysis showed 382 m/z ratio found to be derivative of betanidin component, which can be further more validated by conducting IR and LCMS/MS studies. 

 

Fig 2a: LCMS analysis of Amaranthus caudatus L.(Positive mode)

 

 

Fig 2b: LCMS analysis of Amaranthus caudatus L.(Negative mode)

 

Fig 3a: LCMS analysis of  Amaranthus dubius Mart.(Positive mode)

 

 

Fig 3b: LCMS analysis of  Amaranthus dubius Mart.(Negative mode)

 

The red pigments of Amaranthus L. sp. are well characterized, there are only three reports on the occurrence of betanins¹⁶. Another work showed that betacyanins and phenolic compounds from Amaranthus spinosus L. and Boerhavia erecta L.  has therapeutic property¹⁷. The antioxidant, antimicrobial and anti-inflammatory properties have only very recently been suggested for the betalains^18,19 .Thus the present study throw a light on the application of betacyanins synthesised from Amaranthus L. species, that  can be useful for the commercial industry to explore this natural  pigment producing plants for the future.

 

ACKNOWLEDGEMENT:

The authors show their gratittude to Department of applied chemistry, CUSAT, Cochin for undergoing LCMS analysis.

 

CONFLICT OF INTEREST:                                              

Authors shall declared that there is no conflict of interest in the work done for the  submission of manuscript for publication.

 

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Received on 29.04.2017       Modified on 02.06.2017

Accepted on 18.08.2017       ©A&V Publications All right reserved