CHEMICAL CONSTITUENTS OF SOME SPECIES OF HOLOTHURIANS FOUND IN SRI LANKA

t The sodium, potassium, calcium, iron, magnesium and phosphorus contents of the six /Holothurian species Holothuria scabra, Holothuria atra, Holothuria nobilis, Holothurin leucospilota, Bohadschia marmorata and Sticopus chloronotus were determined. H. scabra has the highest sodium (4.29%), potassium (1.05%), calcium (7.91%) and magnesium (5.21%) contents. B. marmorata has the lowest amounts of sodium (0.30%) and calcium (2.05%). H. atra has the lowest amount of magnesium (0.73%) and S. chloronotus has the lowest amount of potassium (0.10%). H. scabra has the highest amount of the trace elements copper (9 ppm) and nickel (36 ppm), while B. marmorata contains the highest amount of manganese (7 ppm) and zinc (340 ppm). Of the two commercial species, H. atra (61 65%) has a higher protein content than of H. scabra.(36 38%). The effect of processing on the mineral and protein contents of H. atra and H. scabra is also reported. Processed H. atra and H. scabra from the Northern coast of Sr i Lanka have a higher protein content (73 76%) than the processed H. scabra (59%) from the South Pacific Islands. A possible method of differentiating H. scabra and H. atra on the basis of the mineral content is also suggested.


Introduction
The Holothurians are soft, cylindrical-bodied, usually dull dark-coloured and often warty species much like cucumbers (therefore called sea cucumbers) belonging to the Invertebrate class Holothuroidea (or Holothurioides) of the phylum ~chinodermata.~Holothurians 1 are marine species of economic importance and their export provides valuable foreign exchange to Sri Lanka.Processed Holothurian, Beche-de-mer is considered a delicacy by the Chinese and as a result is exported to the countries where the Chinese population is relatively high.3 The prices of the exported animals depend on qualities such as size, appearance, odour, colour, moisture content and resistance to spoilage.Thus knowledge of the amounts of moisture, protein, carbohydrates, fat, aminoacids and minerals present in different species of Holothurians found in Sri Lanka would be useful in evaluating their food value and the market potential.Holothurians are also used in making poultry feed.3 The water extract of H. scabra, obtained during processing is used to relieve ain and to cure paralysis by some villagers in the Northern part of Sri Lanka.P 117-

Determination of the Amounts o f Sodium and Potassium
Sodium and potassium contents of test solutions were determined Flame ~h o t o m e t r i c a l l ~~ using a Corning 400 Flame Photometer.The Flame intensities for sodium were corrected for interference by calcium by the standard addition method.'

Determination'o f the Amounts o f Calcium and.Magnesium
Calcium and magnesium a contents of test solutions were determined titrimetricallyl l using EDTA with potassium cyanide as the masking agent.Patton Reeders indicator was used for calcium determination.
Magnesium content of test solutions was estimated as follows: The total amount of magnesium and calcium present was determined1' by titrating a known volume of test solution with standard EDTA solution with Eriochrome Black T as indicator and using potassium cyanide as masking agent.''Magnesium content was obtained by subtracting the amount of calcium present from this value.

Determination of Iron
Iron content of test solution was determined colorimetrically.6 1,lO-Orthophenanthroline was used as the complexing agent and the colour intensity was measured using a Corning Model 252 Colorimeter with a 490 nm.filter.

Estimation of Protein Content
The total nitrogen content of oven dried samples of holothurians was determined by the Kjeldhal method6 and protein content was calculated by multiplying the total nitrogen content by 6.25.

Determination df Trace Elements. -
The trace elements copper, manganous, nickel, zinc, cadmium, cobalt and chromium were determined6 using the test solution (prepared as described above) on a Varian Model 1257 Atomic Absorption Spectrophotometer.

Determination of Lead
Oven-dried samples were subjected to wet oxidation with concentrated nitric acid and concentrated sulphuric acid as reported previously.6 The resulting solution was used to prepare the test solution.The amount of lead present was determined using a Coming Model 252 Colorirneter after complexing with dithizone.6

Determination of Phosphorus
Accurately weighed oven-dried samples (1-2g) were ashed with magnesium nitrate6 and the ash was dissolved in 1M H SO (10 ml) and the solution 2 4 made up to 50 ml.Phosphorus content of thls so ution was determined6 by measuring the intensity of the colour produced on complexing with vanadomolybdate reagent using a Corning Model 252 Calorimeter with 430, 470 and 490 nm filters.

Determination of Iodine
Iodine content of accurately weighed oven dried samples was estimated by the alcoholic potash method.'

Identification of aminoacids and carbohydrates
Methanol extracts of fresh samples of H. scabra and H. atra were used for the identification of carbohydrates and aminoacids.Carbohydrates and aminoacids were separated from the methanol extract by using the cation exchange resin, Zeocarb 225.The aminoacids and carbohydrates present were tentatively identified12 by comparing with authentic samples using paper chromatography.

Separation of spicules and their analysis
Spicules were separated3 from the flesh of the specimen by adding saturated solution of sodium hypochlorite.The mixture was left overnight and decanted (The spicules will float in the liquidthe flesh will be at the bottom).The spicules were separated from the decanted solution by centrifuging and decanting the supernatant liquid.The spicules were washed with distilled water until no more chloride ions are detected in the washings.The spicules were then weighed, ashed and dissolved in 0.6M HC1 and the estimation of the minerals were carried out as before.

Results and Discussion
The habitats of the six holothurian species studied are given in Table 1 and their localities are given in Table 2. Of the six species studied, H. atra is the smallest (usually 8 -15 cm in length) and H. leucospilota is the largest (30 -80 cm).Our results on the estimation of the amounts of sodium, potassium, calcium and magnesium present in six species of Holothurians are given in Table 3.The amounts of iron, phosphorus and iodine are given in It is interesting to note that seaweeds from the Mandaitivu coast also have high iodine ~o n t e n t .~ H. scabra from the South Pacific lslands10 has higher amounts of sodium and iron than the Holothurian species from Sri Lankan coast but its calcium and phosphorus contents are much lower.Tables 3 and 4 also show the amounts of the same minerals present in different species of HoIothurians after processing.Processing removes 87 -90% of sodium and potassium from both H. scabra and H. atra.
RelativeIy smaller amounts of calcium, magnesium, iron and phosphorus are lost during processing and iodine is lost completely.However there is a large difference in the amount of calcium, magnesium and phosphorus lost by H. atra and H. scabra.Table 5 gives the percentage of the original amounts of the minerals remaining in the animals after processing to the original value.

Sticopus cbloronotus
36.47  4.  One of the reasons for the greater loss of alkali metals during processing could be the higher solubility of their salts.Another possibility is that these salts are present in relatively large amounts in the outer skin and spicules which are removed during the processing.The difference in the loss of calcium, magnesium and phosphorus between H. scabra and H. atra could be attributed t o the difference in the distribution of these elements in the animal body wall, the skin and spicules of H. atra containing relatively larger amounts of the minerals than H. scabra.Table 6 gives the amount of minerals left in the animal after each stage in processing for the two species H. scabra and H. atra.

Rajeswary Mageswarun and Vaseetha Balakrishnan
The trace elements present in different species of holothurians were also estimated using an Atomic absorption spectrophotometer.The elements chromium and cobalt could not be detected in any of the species.The Table 7 shows the amounts of copper, manganous, zinc, lead, nickel and cadmium present in four species.The elements copper, manganous and zinc are essential and nutritive, whereas lead and cadmium are toxic.Elements such as copper and zinc although essential for life in trace amounts, have an toxit action when ingested in higher amounts.The recommended levels of copper and zinc are 20 and 50 ppm respectively.The amounts of copper and zinc present in the commercial species H. scabra and H. atra are well below the recommended level.The non commercial species B. marmorata and H. leucospilota contain zinc in quantities well above the recommended level.On processing the percentage of the nutritive elements [Cu, Mn and Zn] increases slightly but remain well within the recommended level.The percentage of toxic elements [Pb and Cd] decreases [by about 30 -46%] on processing.The mineral contents of the spicules of five species of holothurians are given in Table 8.The spicules of the commercial species H. scabra and H. atra have a relatively high calcium content.The spicules of B. marmorata has relatively high magnesium content while that of S. chloronotus is low in calcium but contain the highest amount of potassium and iron.These findings may be of chemotaxonomic significance.I t is relevant to note that shape of spicules among other properties is used3 in identification of different species of holothurians.The protein contents of fresh and processed H:scabra and H. atra species are given in Table 9.I t is of interest to note that although unprocessed H. atra has a very much higher percentage of protein than unprocessed H. scabra, the processed species have nearly the same percentage of protein.This may be due to a greater loss of non-protein material from H. scabra than from H. atra during ptocessing.Also the local H. scabra and H. atra appear to have higher percentage of protein than the H. scabra from South Pacific Islands. 10   The total free aminoacid and carbohydrate contents of H. atra and H. scabra have also been estimated by the usual method.The values of the total amount of substances extractable into methanol.,andthe percentages of free aminoacids and carbohydrates present are given inTable 10.The carbohydrates present in both the species are in the polysaccharide form, as simple sugars were not detected by chromatographic analysis.Comparative two dimensional paper chromatographic analysis has shown that H. atra contains seventeen free aminoacids.Fifteen of them have been identified as aspartic acid, glutamic acid, serine, glycine, threonine, alanine, tyrosine, valine, methionine, histidine, lysine, phenylalanine, arginine, leucine and isoleucine.H. scabra contains fifteen aminoacids.Fourteen of these have been identified as aspartic acid, glutamic acid, serine, glycine, alanine, tyrosine, threonine, vdine, arginine, methinine, proline, phenylalanine, leucine and isoleucine.I t is interesting to note that H. atra contains nine of the ten essential aminoacids whereas H. scabra contains only seven of them.

Conclusion
Processed H. scabra and H. atra from the coastal area of Northern Sri Lanka have a higher protein content [73-76%] than the processed H. scabra from the South Pacific Islands [59.12%] .Generally H. scabra is richer in alkali, alkaline earth and trace metals than H. atra whereas the reverse is true in the case of the elements iron and phosphorus.Both H. scabra and H. atra have similar Na/Ca ratio.But the K/Ca and Mg/Ca ratios of E-I, scabra are very much higher [> 60%] than those of H. atra.Thus it is possible to differentiate H. scabra and H. atra on the basis of the mineral constituents.
Processing increases the protein content' and the amounts of copper, manganese and zinc and decreases the alkali, alkaline earth and toxic elements.

* 2 samples
Samples from Miss P. Elanganayagam; Department of Zoology.Sun dried mixture of several animals was used (moisture content of these sun dried

Table 4
. Of the six species investigated, H. scabra contains the highest amounts of sodium, calcium and magnesium.But H. atra, and the non-commercial species H. leucospilota and S. chloronotus contain relatively high amounts of iron and phosphorus.H. scabra from the Mandaitivu coast and S. chloronotus from Trincomalee have appreciable amounts of iodine while H. atra, H. leucospilota and B. marmorata do not have detectable amounts of iodine.

Table 1 .
The habitats of the holothurian species studied.

Table 10 . Percentage of free amino acids and carbohydrates
56 (Mandaitivu)