IJCRR - 9(11), June, 2017
Pages: 01-05
Date of Publication: 12-Jun-2017
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Biochemical and Nutritional Analysis of the Leaf Extract of Aegle marmelos (L.) Correa.
Author: Samidha M. Pawaskar, K. C. Sasangan
Category: General Sciences
Abstract:Objective: The present study was conducted to investigate the presence of biochemical contents viz., proximate and micronutrient analysis in the leaves of Aegle marmelos (L.) Correa.
Methods: The proximate and micronutrient content was determined by different biochemical methods.
Results: Aegle marmelos (L.) Correa. leaves confirmed the presence of all the essential nutrients, minerals and vitamins in good amounts and possess good nutritive value.
Conclusion: The plant leaf powders can thus be looked forward as the probable sources of food supplementation in future, after further investigation of the anti-nutritive factors present in them and their enzymatic and molecular effect on human health..
Keywords: Aegle marmelos (L.) Correa., Proximate principles, Micronutrients
Full Text:
INTRODUCTION
The tenet “Let food be thy medicine and medicine be thy food,” espoused by Hippocrates nearly 2,500 years ago, is receiving renewed interest. In particular, there has been an explosion of consumer interest in the health enhancing role of specific foods or physiologically-active food components, so-called functional foods1. Clearly, all foods are functional, as they provide taste, aroma, or nutritive value. Within the last decade, however, the term functional as it applies to food has adopted a different connotation -- that of providing an additional physiological benefit beyond that of meeting basic nutritional needs.
The past decade has witnessed intense interest in “nutraceuticals” (or “functional foods”) in which phytochemical constituents can have long-term health promoting or medicinal qualities. Although the distinction between medicinal plants and nutraceuticals can sometimes be vague, a primary characteristic of the latter is that nutraceuticals have a nutritional role in the diet and the benefits to health may arise from long-term use as foods (i.e. chemoprevention)2.
Some of the plants with promising bioactive properties also contain useful minerals and food value for human The tenet “Let food be thy medicine and medicine be thy food,” espoused by Hippocrates nearly 2,500 years ago, is receiving renewed interest. In particular, there has been an explosion of consumer interest in the health enhancing role of specific foods or physiologically-active food components, so-called functional foods1. Clearly, all foods are functional, as they provide taste, aroma, or nutritive value. Within the last decade, however, the term functional as it applies to food has adopted a different connotation -- that of providing an additional physiological benefit beyond that of meeting basic nutritional needs.
Some of the plants with promising bioactive properties also contain useful minerals and food value for human and animal consumption. Each medicinal plant species has its own nutrient composition besides having pharmacologically important phytochemicals. These nutrients are essential for the physiological functions of human body. Such nutrients and biochemicals like carbohydrates, fats and proteins play an important role in satisfying human needs for energy and life processes.
These medicinal plant species are used either as food or food supplements along with their medicinal benefits. Evaluation of the biochemical and nutritional significance of these plants thus can help to understand the worth of these plants species3. As far herbal drug’s standardization is concerned, WHO also emphasizes on the need and importance of determining proximate and micronutrients analysis. Such herbal formulations must pass through standardization processes4.
In the present study, the medicinal plants species viz Aegle marmelos (L.) Correa. was subjected to proximate and micronutrient analysis. The total carbohydrates, reducing sugars, protein and fat were analyzed so also the micronutrient content ie. minerals like Ca, P, Fe and Mg levels were analysed, so also both water soluble and water insoluble vitamins were estimated using biochemical methods.
MATERIAL AND METHODS
1) Analysis of proximate principles of diet
a) Determination of Total Carbohydrates by Anthrone Method5
Carbohydrates are the important components of storage and structural materials in the plants. They exist as free sugars and polysaccharides. The basic units of carbohydrates are the monosaccharides which cannot be split by hydrolysis into more simpler sugars. The carbohydrate content can be measured by hydrolyzing the polysaccharides into simple sugars by acid hydrolysis and estimating the resultant monosaccharides.
b) Determination of Total Reducing Sugars by Folin – Wu Method6, 7, 8
c) Determination of Reducing Sugar (Glucose) by Nelson- Somogyi Method5
Sugars with reducing property (arising out of the presence of of a potential aldehyde or keto group) are called reducing sugars. Some of the reducing sugars are glucose, galactose, lactose and maltose. The Nelson-Somogyi method is one of the classical and widely used methods for the quantative determination of reducing sugar especially glucose.
d) Estimation of Cellulose Content5
Cellulose, a major structural polysaccharide in plants, is the most abundant organic compound in nature, and is composed of glucose units joined together (β (1→4) glycosidic linkage) in the form of repeating units of the disaccharides cellobiose with numerous cross linkages.
e) Estimation of Crude Fibre Content5
Crude fibre consist largely of cellulose and lignin (97%) plus some mineral matter. It represents only 60% to 80% of the cellulose and 4% to 6% of the lignin. The crude fibre content is commonly used as a measure of the nutritive value of livestock feeds and also in the analysis of various foods and food
products to detect adulteration, quality and quantity.
f) Estimation of Total Protein content by Folin Lowry’s Method5, 9
Proteins can be estimated by different methods as described by Lowry et al.(1951)10 and also by estimating the total nitrogen content (micro-kjeldahl method). No method is 100% sensitive. Hydrolysing the protein and estimating amino acids alone will give the exact quantification. The method developed by Lowry et al. is sensitive enough to give a moderately constant value and hence largely followed10.
g) Estimation of Total Free Amino acids Content5
The amino acids are colourless ionic compounds that form the basic building blocks of proteins. Apart from being bound as proteins, amino acids also exist in the free form in many tissues and are known as free amino acids. They are mostly water soluble in nature. Very often in plants during disease condition the free amino acid composition exhibits a change and hence, the measurement of the total free amino acids gives the physiological and health status of the plants.
h) Extraction of Total Lipid Content9, 11
Lipids are soluble in some organic solvents. This property of specific solubility in nonpolar solvents is utilized for extracting lipids from tissues. In biological materials, the lipids are generally bound to proteins and they are, therefore, extracted either with a mixture of ethanol and diethyl ether or a mixture of chloroform and methanol. Inclusion of methanol or ethanol in the extraction medium helps in breaking the bonds between the lipids and proteins.
i) Estimation of Free Fatty acids Content5
The free fatty acids in an oil is estimated by titrating it against KOH in the presence of phenolphthalein indicator. The acid number is defined as the mg KOH required to neutralize the free fatty acids present in 1g of sample. However, the free fatty acid content is expressed as oleic acid equivalents.
2) Micro-nutrient analysis
a) Mineral Estimation
Preparation of sample for mineral analysis
Biological samples must be appropriately processed before they can be subjected to mineral analysis. There are three methods generally employed for processing the sample prior to mineral analysis as follows
1. Ashing
2. Wet Digestion
3. Direct solution
The ashing method was employed for the analysis. According to this method the entire organic matter (if the tissue is destroyed) and the non-combustible material is recovered as ash. The minerals are then obtained / collected from the ash with an acid (usually dilute HCL), filterted and diluted to a known volume with deionized water and estimated quantitatively. In this method the organic compounds in the sample are decomposed by incineration at high temperatures (5000C - 6000C) for 4-12 hr using muffle furnace.
i) Estimation of Calcium by EDTA Method12
For the estimation of calcium ions the presence of Mg2+ ions is required. The dye Erichrome black-T preferably combines these Mg2+ ions to form a pink coloured Mg – dye complex. During the titration, EDTA first combines with free Ca2+ ions in the solution, and finally EDTA extracts at endpoint Mg2+
from complex . This results in the formation of free uncomplexed dye which in alkaline medium gives blue colour at the endpoint.
ii) Estimation of Phosphorus by Fiske-Subbarow (ANSA) method9, 13
Ammonium acid molybdate reacts with inorganic phosphorous to form phosphomolybdic acid. The Mo6+ of phospho molybdic acid is then reduced to Mo4+ by means of reducing agent like 1- amino – naphthol – 4- sulfonic acid (ANSA), to give deep blue coloured compound which is estimated
colorimetrically. The reducing agent ANSA has only a negligible effect on the Mo6+ ions present in the unreacted acid molybdate reagent.
iii) Estimation of Iron by Wong’s (KCNS) method14, 15
The ferrous ions present in the sample are oxidized to ferric ions by K2S2O8 solution. The ferric ions give a red coloured ‘ferro-sulphocyanide complex’ with KCNS. The intensity of the coloured complex so formed is then estimated colorimetrically at 425 nm.
iv) Estimation of Magnesium by Titan Yellow method16, 17
Titan yellow reacts with magnesium in alkaline medium and gives an orange red colored complex. The intensity of the coloured complex so formed is then estimated colorimetrically at 540nm. The intensity of the colour produced is proportional to concentration of magnesium. The procedure was
developed by Neil and Neely (1956) 16.
b) Estimation of Vitamins
i) Estimation of Thiamine by Thiochrome method5
Alkaline potassium ferricyanide oxidizes thiamine to thiochrome which is a fluorescent compound. The thiochrome is extracted in isobutyl alcohol and measured in a Fluorimeter.
ii) Estimation of Riboflavin5
Riboflavin is used in veterinary and medical practices for supplementation of animal feeds and as natural coloring agent in food products. It is estimated in urine and with an average diet, the daily losses amount to 12% of the intake. A fall in the level of Riboflavin excretion occurs before deficiency symptoms of Vitamin B2 are noticed. Under protein deficiency the urinary output of Vitamin B2 increases. Riboflavin fluoresces at wavelength 440 nm to 500 nm. The intensity of fluorescence is proportional to the concentration of Riboflavin in the solution. The Riboflavin is measured these Mg2+ ions to form a pink coloured Mg – dye complex. During the titration, EDTA first combines with free Ca2+ ions in the solution, and finally EDTA extracts at endpoint Mg2+ from complex . This results in the formation of free uncomplexed dye which in alkaline medium gives blue colour at the endpoint.
ii) Estimation of Phosphorus by Fiske-Subbarow (ANSA) method9, 13
Ammonium acid molybdate reacts with inorganic phosphorous to form phosphomolybdic acid. The Mo6+ of phospho molybdic acid is then reduced to Mo4+ by means of reducing agent like 1- amino – naphthol – 4- sulfonic acid (ANSA), to give deep blue coloured compound which is estimated
colorimetrically. The reducing agent ANSA has only a negligible effect on the Mo6+ ions present in the unreacted acid molybdate reagent.
iii) Estimation of Iron by Wong’s (KCNS) method14, 15
The ferrous ions present in the sample are oxidized to ferric ions by K2S2O8 solution. The ferric ions give a red coloured ‘ferro-sulphocyanide complex’ with KCNS. The intensity of the coloured complex so formed is then estimated colorimetrically at 425 nm.
iv) Estimation of Magnesium by Titan Yellow method16, 17
Titan yellow reacts with magnesium in alkaline medium and gives an orange red colored complex. The intensity of the coloured complex so formed is then estimated colorimetrically at 540nm. The intensity of the colour produced is proportional to concentration of magnesium. The procedure was
developed by Neil and Neely (1956) 16.
b) Estimation of Vitamins
i) Estimation of Thiamine by Thiochrome method5
Alkaline potassium ferricyanide oxidizes thiamine to thiochrome which is a fluorescent compound. The thiochrome is extracted in isobutyl alcohol and measured in a Fluorimeter.
ii) Estimation of Riboflavin5
Riboflavin is used in veterinary and medical practices for supplementation of animal feeds and as natural coloring agent in food products. It is estimated in urine and with an average diet, the daily losses amount to 12% of the intake. A fall in the level of Riboflavin excretion occurs before deficiency
symptoms of Vitamin B2 are noticed. Under protein deficiency the urinary output of Vitamin B2 increases. Riboflavin fluoresces at wavelength 440 nm to 500 nm. The intensity of fluorescence is proportional to the concentration of Riboflavin in the solution. The Riboflavin is measured these Mg2+ ions to form a pink coloured Mg – dye complex. During the titration, EDTA first combines with free Ca2+ ions in the solution, and finally EDTA extracts at endpoint Mg2+
from complex . This results in the formation of free uncomplexed dye which in alkaline medium gives blue colour at the endpoint.
iv) Estimation of Magnesium by Titan Yellow method16, 17
Titan yellow reacts with magnesium in alkaline medium and
gives an orange red colored complex. The intensity of the
coloured complex so formed is then estimated colorimetrically
at 540nm. The intensity of the colour produced is proportional
to concentration of magnesium. The procedure was
developed by Neil and Neely (1956) 16.
b) Estimation of Vitamins
i) Estimation of Thiamine by Thiochrome method5
Alkaline potassium ferricyanide oxidizes thiamine to thiochrome
which is a fluorescent compound. The thiochrome
is extracted in isobutyl alcohol and measured in a Fluorimeter.
ii) Estimation of Riboflavin5
Riboflavin is used in veterinary and medical practices for
supplementation of animal feeds and as natural coloring
agent in food products. It is estimated in urine and with an
average diet, the daily losses amount to 12% of the intake.
A fall in the level of Riboflavin excretion occurs before deficiency
symptoms of Vitamin B2 are noticed. Under protein
deficiency the urinary output of Vitamin B2 increases. Riboflavin
fluoresces at wavelength 440 nm to 500 nm. The
intensity of fluorescence is proportional to the concentration
of Riboflavin in the solution. The Riboflavin is measured in terms of the difference in fluorescence before and after
chemical reduction.
iii) Estimation of Niacin by Cyanogen bromide method5
Niacin reacts with cyanogen bromide to give a pyridinium
compound which undergoes rearrangement yielding derivatives.
These derivatives couple with aromatic amines to
give yellow colored pigment. Under proper conditions the
intensity of the yellow color produced is proportional to the
amount of Niacin present.
iv) Estimation of Ascorbic acid (Vitamin C) by DNPH
method5
Ascorbic acid is first dehydrated by bromination. The dehydroascorbic
acid is then reacted with 2, 4-Dinitriphenyl
hydrazine to form osazones and dissolve in sulphuric acid to
give an orange red color solution which is measured colorimetrically
at 540nm.
v) Estimation of Retinol (Vitamin A) by Carr-Price method12,
18, 19
The retinol and carotenes are extracted into light petroleum
by soxhlet method. It is than evaporated to dryness to obtain
a residue which is reconstituted in n-Heptane. On addition
of chloroform and Carr-price reagent, different intensities of
blue color are obtained which is read at 465nm.
vi) Estimation of Tocopherol (Vitamin E)12, 20
Vitamin E activity is shown by four naturally occurring tocopherols
of which oe -Tocopherol is the most potent tocopherol
give Emmeric-Engel reaction which is based on on a
reduction by tocopherols of ferric to ferrous ions which then
form a red complex with oe, oe’-Dipyridyl. Tocopherols and
carotenes are first extracted into xylene and the extinction
read at 460nm to measure the carotenes. A correction is made
for these after adding ferric chloride and reading at 520nm
RESULTS
The results of the biochemical analysis of various nutritional
parameters viz., proximate principles and micronutrients are
represented in table 1 and table 2, respectively.
DISCUSSION
Since many of the herbal products are used orally, knowledge
of proximate and nutrient analysis of these products and raw
materials used there in plays a crucial role in assessing their
nutritional significance and health effects3, 21, 22.
The biochemical analysis of the leaf extracts of Aegle marmelos
(L.) Correa. (AML) showed considerably high levels of most of the estimated nutritional elements.
The micronutrients analysis of the leaf powders of the Aegle
marmelos (L.) Correa. (AML) showed significant variation
among different micronutrients. Magnesium content was
found to be the highest followed by calcium, as compared
to the rest of the tested minerals. However, phosphorus and
iron contents were found to be comparatively less. Phosphorus
content was found to be the least among all the tested
minerals. Most of the vitamins were also found to be in good
quantity.
No proper previous study reports were found on the nutritional/
biochemical analysis of the leaf powders of Aegle
marmelos (L.) Correa. indicating their essential nutrients,
minerals and vitamins content. Hence our study may be considered
as the first to report the same.
CONCLUSION
From the results of the study, it can be concluded that the
leaf powders of Aegle marmelos (L.) Correa. was found to
be having all the essential nutrients, minerals and vitamins
in good amounts and possess good nutritive value. These
plant leaf powders can thus be looked forward as the probable
sources of food supplementation in future, after further
investigation of the anti-nutritive factors present in them and
their enzymatic and molecular effect on human health.
ACKNOWLEDGEMENT
Authors acknowledge the immense help received from the
scholars whose articles are cited and included in references
of this manuscript. The authors are also grateful to authors /
editors / publishers of all those articles, journals and books
from where the literature for this article has been reviewed
and discussed.
Financial support and sponsorship: Nil
Conflicts of interest: The author declares no competing interests.
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