Carbohydrate Analysis Research Articles

MEDI: Macronutrient Extraction and Determination from invertebrates, a rapid, cheap and streamlined protocol.

Macronutrients, comprising carbohydrates, proteins and lipids, underpin many ecological processes, but their quantification in ecological studies is often inaccurate and laborious, requiring large investments of time and bulk samples, which make individual‐level studies impossible. This study presents Macronutrient Extraction and Determination from Invertebrates (MEDI), a protocol for the direct, rapid and relatively low‐cost determination of macronutrient content from single small macroinvertebrates.Macronutrients were extracted by a sequential process of soaking in 1:12 chloroform:methanol solution to remove lipid and then solubilising tissue in 0.1 M NaOH. Proteins, carbohydrates and lipids were determined by colorimetric assays from the same individual specimens.The limits of detection of MEDI with the equipment and conditions used were 0.067, 0.065 and 0.006 mg/ml for proteins, carbohydrates and lipids respectively. Adjusting the volume of reagents used for extraction and determination can broaden the range of concentrations that can be detected. MEDI successfully identified taxonomic differences in macronutrient content between five insect species.Macronutrient Extraction and Determination from Invertebrates can directly and rapidly determine macronutrient content in tiny (dry mass ~3 mg) and much larger individual invertebrates. Using MEDI, the total macronutrient content of over 50 macroinvertebrates can be determined within around 3 days of collection at a cost of ~$1.35 per sample.

Tandem Breastfeeding: A Descriptive Analysis of the Nutritional Value of Milk When Feeding a Younger and Older Child.

Breastfeeding is a gold standard of feeding of newborns and infants. Tandem breastfeeding (TBF) is feeding two children of different ages at the same time. The knowledge about the composition of human milk in prolonged lactation is still scarce. Milk from tandem breastfeeding women and after weaning was examined. Milk samples were collected from 13 TBF mothers. A 24-h milk collection was done. Analyses of fat, protein, carbohydrate and energy content were performed using MIRIS. Sociodemographic characteristics of TBF mothers was done. Higher fat content, energy value and total protein concentration was found in TBFM milk during tandem breastfeeding, than in milk after weaning the older child. The carbohydrate content remained stable. The composition of breastmilk, in terms of macronutrients, changes after weaning, taking into account the nutritional requirements of the younger child. The milk of nursing mothers in tandem did not show diurnal variability in individual components. These findings suggest an adaptive role of human milk to nutrient requirements of newborn and older children. The results may support the promotion of long breastfeeding, including tandem breastfeeding.

In situ synthesis of fluorescent silicon nanodots for determination of total carbohydrates in a paper microfluidic device combined with laser prepared graphene heater

We report a simple, rapid, low-resource and one-step method for formation of fluorescent silicon nanodots (SNDs) in a microfluidic paper-based analytical device (μPAD) incorporated in a reusable, portable and flexible heater for determination of total carbohydrates. The synthesis of SNDs is based on the redox reaction between (3-aminopropyl) triethoxysilane (APTS) reagent and carbohydrates, which act as a reducer. The graphene-based heater was fabricated by laser ablation of Kapton polyimide. Thereby, the developed system heat the μPAD during the synthesis of SNDs at 80 °C for 30 min. The blue emitting SNDs formed have an emission peak wavelength at 475 nm. We used a digital camera and smartphone for the quantitative analysis of total carbohydrates expressed such as index of glucose or fructose with grey scale value as the analytical parameter. Under the optimal conditions, the method has a low detection limit (0.80 μM for glucose and 0.51 μM for fructose, respectively), and a linear response (10–200 μM for glucose and 10–100 μM for fructose). The method has been applied to the determination of glucose in biological fluids (serum and urine samples). In addition, determination of total carbohydrates in commercial juices and teas have been carried out.

Could the Content of Soluble Carbohydrates in the Young Shoots of Selected Willow Cultivars Be a Determinant of the Plants’ Attractiveness to Cervids (Cervidae, Mammalia)?

Ten willow cultivars grown in experimental plots were evaluated for performance, attractiveness to foragers, and the content and composition of soluble carbohydrates. The survival of willow cuttings in a thicket and in browse plots differed subject to cultivar, soil quality, and soil moisture content. The number of stump sprouts varied considerably, from 1.1 shoots in the weakest soils in Słonin, Poland, to 3.43 in the plot in Czempin, Poland. Browse plots were established in 2017. They were cut, and fencing was removed in early spring of 2019. Young shoots (10 cm shoot tip with buds, preferably eaten by animals) were sampled for analyses of soluble carbohydrates as potential attractors for foraging cervids. All willow cultivars contained the same soluble carbohydrates: glucose, fructose, galactose, sucrose, myo-inositol, galactinol, and raffinose. Total carbohydrate content ranged from 21.31 (S. amygdalina 1045) to 69.37 mg/g−1 DM (dry matter) (S. purpurea). Glucose was the predominant soluble sugar in the shoots of all willow cultivars, excluding S. viminalis. The fructose content of the shoots was approximately twice lower than their glucose content in all willow cultivars. Smaller differences were observed in the content of myo-inositol, which ranged from 4.61 (S. amygdalina 1045) to 8.26 mg/g−1 DM (S. fragilis cv. Kamon/Resko). The phloem of all willow species contained small quantities of galactinol and trace amounts of raffinose. Weak negative correlations were noted between total carbohydrate content, the content of glucose, fructose, and galactose vs. the attractiveness of willow shoots to foraging cervids. The remaining carbohydrates that occurred in smaller quantities in willow shoots were not correlated with their attractiveness to cervids.

Structural Changes in Milled Wood Lignin (MWL) of Chinese Quince (Chaenomeles sinensis) Fruit Subjected to Subcritical Water Treatment.

Subcritical water treatment has received considerable attention due to its cost effectiveness and environmentally friendly properties. In this investigation, Chinese quince fruits were submitted to subcritical water treatment (130, 150, and 170 °C), and the influence of treatments on the structure of milled wood lignin (MWL) was evaluated. Structural properties of these lignin samples (UL, L130, L150, and L170) were investigated by high-performance anion exchange chromatography (HPAEC), FT-IR, gel permeation chromatography (GPC), TGA, pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS), 2D-Heteronculear Single Quantum Coherence (HSQC) -NMR, and 31P-NMR. The carbohydrate analysis showed that xylose in the samples increased significantly with higher temperature, and according to molecular weight and thermal analysis, the MWLs of the pretreated residues have higher thermal stability with increased molecular weight. The spectra of 2D-NMR and 31P-NMR demonstrated that the chemical linkages in the MWLs were mainly β-O-4′ ether bonds, β-5′ and β-β′, and the units were principally G- S- H- type with small amounts of ferulic acids; these results are consistent with the results of Py-GC/MS analysis. It is believed that understanding the structural changes in MWL caused by subcritical water treatment will contribute to understanding the mechanism of subcritical water extraction, which in turn will provide a theoretical basis for developing the technology of subcritical water extraction.

Analysis of Non-Structural Carbohydrate in Relation with Shoot Elongation of Rice under Complete Submergence

Regulation of non-structural carbohydrates (NSCs) are important for plants in response to submergence. In this study, the difference in non-structural carbohydrates in relation with shoot elongation between Sub1A and non-Sub1A rice genotypes was investigated. Two rice genotypes, namely Inpari30 (Sub1A genotype) and IR72442 (non-Sub1A genotype), were submerged completely for 6 days and re-aerated by lowering water level up to stem base for 6 days of post submergence. In addition, non-submerged plants (control) was treated with water level up to stem base during the experiment. Photosynthesis rate decreased in both submerged Inpari30 and IR72442 genotypes 71% and 96% lower than their control, respectively. Submerged IR72442 declined Fv/Fm 15.6% lowest than its control and both control and submerged Inpari30. Investigation of the distribution of starch and soluble sugar content in plant organs suggested that shoot elongation of non-Sub1A genotype led to starch and sugar consumption that distributed faster to the new developed organ during submergence. In contrast, Sub1A genotype of Inpari30, which did not exhibit shoot elongation and showed slower NSCs distribution during submergence, performed better on post submergence by maintaining NSCs and distributing to the new developed organ faster than IR72442. These results suggest that Sub1A genotype managed elongation and NSCs during submergence more efficiently than non-Sub1A genotype.

Determination of Bifidobacterial Carbohydrate Utilization Abilities and Associated Metabolic End Products.

Bifidobacteria are able to utilize a diverse range of host-derived and dietary carbohydrates, the latter of which include many plant-derived oligo- and polysaccharides. Different bifidobacterial strains may possess different carbohydrate utilization abilities. These metabolic abilities can be studied using classical bacterial growth assessment methods, such as measurement of changes in optical density or acidity of the culture in the presence of the particular carbohydrate to generate growth and acidification curves, respectively. Scientists may also be interested in the growth rate during the exponential growth phase, and the maximum OD that is reached on a particular sugar, or the length of the lag phase. Furthermore, high-performance liquid chromatography (HPLC) and high-performance anion exchange chromatography coupled to pulsed amperometric detection (HPAEC-PAD) are extensively used in carbohydrate and metabolic end-product analysis due to their versatility and separation capabilities.

The Determination of Carbohydrates by High-Performance Anion-Exchange Chromatography Coupled with Pulsed Amperometric Detection (HPAEC-PAD).

Chromatography techniques are widely used to separate, identify, and quantify molecules depending on their physicochemical properties. Standard methods range from simple size exclusion to separation based on affinity or ion exchange. Here, we present a method for the direct analysis of carbohydrates in Magnaporthe oryzae using high-performance anion-exchange chromatography (HPAEC) coupled with pulsed amperometric detection (PAD). The combination of HPAEC with PAD provides the highest selectivity and sensitivity with minimal sample preparation and cleanup time. Utilizing our HPAEC-PAD approach, we obtain reliable and highly reproducible determination of carbohydrates produced as osmotic stress response by M. oryzae. Thus, the method described provides a fast, precise, and comprehensive analysis of stress-dependent metabolic adjustments of carbohydrates not only relevant for M. oryzae but also applicable in other systems.

Linkage Analysis of Oligosaccharides and Polysaccharides: A Tutorial.

Polysaccharides and oligosaccharides are a diverse group of natural polymers with important biological functions. The diversity of carbohydrate polymers is vast, ranging from small oligosaccharides of defined composition decorating proteins, to large, complex heteropolymers comprising integral cell wall components of plants, fungi and bacteria. An important step in the elucidation of unknown carbohydrate structures in a sample is the assessment of the various linkages present. This is accomplished by performing linkage analysis of the sample. The analysis proceeds as a successive series of chemical steps in which unlinked carbohydrate hydroxyls are marked with methyl groups, the sample is hydrolyzed into monosaccharides and reduced to alditols, and finally free hydroxyls are acetylated. Gas chromatography-mass spectrometry (GC-MS) analysis is employed to analyze the resultant partially methylated alditol acetates (PMAAs). The following paper reviews the major literature pertaining to the specific protocol for linkage analysis of carbohydrates outlined herein. The review details additional steps necessary for the completion of uronic acid linkage analysis, as well as analysis of chitin containing polymers. It also gives chromatographic examples of common erroneous results which the first time practitioner will want to be aware of. Our hope is that this protocol will serve as a definitive guide, allowing novice researchers to perform linkage analysis of carbohydrates in their own lab.

Capillary Electrophoresis-Based N-Glycosylation Analysis in the Biomedical and Biopharmaceutical Fields.

Glycomics has a growing interest in the biopharmaceutical industry and biomedical research requiring new high-performance and high-sensitivity bioanalytical tools. Analysis of N-glycosylation is very important during the development of protein therapeutics and it also plays a key role in biomarker discovery. The most frequently used glycoanalytical methods are capillary electrophoresis, liquid chromatography, and mass spectrometry. In this chapter, the capillary electrophoresis-based N-linked carbohydrate analysis methods are conferred with emphasis on its use in the biopharmaceutical and biomedical fields.

Determination of carbohydrates in the herbal antidiabetic mixtures by GC-MC.

Due to the wide range of biologically active substances, the herbal mixtures can influence the development of diabetes mellitus and its complications. Carbohydrates attract particular attention due to their hypoglycemic, hypolipidemic, anticholesterolemic, antioxidant, antiinflammatory and detoxifying activities. The aim of this study was to investigate the content of carbohydrates through their monomeric composition in the herbal mixture samples: a) Urtica dioica leaf, Cichorium intybus roots, Rosa majalis fruits, Elymys repens rhizome, Taraxacum officinale roots, b) Arctium lappa roots, Elymys repens rhizome, Zea mays columns with stigmas, Helichrysum arenarium flowers, Rosa majalis fruits, c) Inula helenium rhizome with roots, Helichrysi arenarium flowers, Zea mays columns with stigmas, Origanum vulgare herb, Rosa majalis fruits, Taraxacum officinale roots, d) Cichorium intybus roots, Elymys repens rhizome, Helichrysum arenarium flowers, Rosa majalis fruits, Zea mays columns with stigmas and e) Urtica dioica leaf, Taraxacum officinale roots, Vaccinium myrtillus leaf, Rosa majalis fruits, Mentha piperita herb, which were used in Ukrainian folk medicine for the prevention and treatment of diabetes mellitus type 2.The carbohydrates were separated by gas chromatography-mass spectrometry after conversion into volatile aldononitrile acetate derivatives. The monomeric composition of polysaccharides was studied after their hydrolysis to form monosaccharides and poly-alcohols.Quantitative analyses of free carbohydrates showed that the predominant sugars were fructose, glucose and disaccharide - sucrose, in all samples. Concerning the determination of polysaccharide monomers after hydrolysis, glucose was the most abundant in all samples. The chromatographic study revealed a number of polyalcohols that are important for the treatment and prevention of progression of diabetes mellitus and its complications, namely, mannitol, pinitol and myo-inositol.

CRISPR/Cas9-Mediated Knockout of Galactinol Synthase-Encoding Genes Reduces Raffinose Family Oligosaccharide Levels in Soybean Seeds.

Raffinose family oligosaccharides (RFOs) are major soluble carbohydrates in soybean seeds that cannot be digested by human and other monogastric animals. Hence, a major goal is to reduce RFO levels to improve the nutritional quality of soybean. In this study, we utilized a dual gRNAs CRISPR/Cas9 system to induce knockouts in two soybean galactinol synthase (GOLS) genes, GmGOLS1A and its homeolog GmGOLS1B. Genotyping of T0 plants showed that the construct design was efficient in inducing various deletions in the target sites or sequences spanning the two target sites of both GmGOLS1A and GmGOLS1B genes. A subset of induced alleles was successfully transferred to progeny and, at the T2 generation, we identified null segregants of single and double mutant genotypes without off-target induced mutations. The seed carbohydrate analysis of double mutant lines showed a reduction in the total RFO content of soybean seed from 64.7 mg/g dry weight to 41.95 mg/g dry weight, a 35.2% decrease. On average, the stachyose content, the most predominant RFO in soybean seeds, decreased by 35.4% in double mutant soybean, while the raffinose content increased by 41.7%. A slight decrease in verbascose content was also observed in mutant lines. Aside from changes in soluble carbohydrate content, some mutant lines also exhibited increased protein and fat contents. Otherwise, no difference in seed weight, seed germination, plant development and morphology was observed in the mutants. Our findings indicate that GmGOLS1A and GmGOLS1B contribute to the soybean oligosaccharide profile through RFO biosynthesis pathways, and are promising targets for future investigation, as well as crop improvement efforts. Our results also demonstrate the potential in using elite soybean cultivars for transformation and targeted genome editing.

Characterization of Buckwheat Beverages Fermented with Lactic Acid Bacterial Cultures and Bifidobacteria.

This study aimed to examine the effect of four different industrial starter cultures containing lactic acid bacteria (LAB) and bifidobacteria on selected characteristics of beverages prepared from buckwheat and stored at 4 °C for 28 days. This study included the determination of pH during fermentation and during refrigerated storage, determination of the number of LAB and bifidobacteria, and chromatographic analysis of carbohydrates. This study showed that the tested starter cultures effectively fermented the buckwheat beverage. There was a sufficient number of viable cells in the starter microflora for the obtained beverages to exhibit potential health-promoting properties. Beverages had stable pH values during refrigerated storage. The stored beverages showed changes in the content of selected carbohydrates, which indicates the constant biochemical activity of the present starter microflora. This study provides useful references on the metabolism of LAB in plant-based beverages.

Effective separation of carbohydrate isomers using metal cation and halogen anion complexes in trapped ion mobility spectrometry

Comprehensive analysis of carbohydrates, one of the critical steps towards the fundamental understanding of biological systems, has been hindered by the identifications of their complex isomeric structures differing in monosaccharide constituents, linkage positions, and configurations. While ion mobility spectrometry-mass spectrometry (IMS-MS) based methods have shown their utilities in performing glycomics measurements, there is still a lack of analytical methods allowing effective isomeric carbohydrate separation. Here, we systematically investigated the effect of alkaline earth cations (Ca2+ and Ba2+) and halogen anions (Cl−, Br− and I−) addition on the separation of 21 pairs of isomeric oligosaccharides using trapped ion mobility spectrometry-mass spectrometry (TIMS-MS). [M + Ca + I]+ complex (M is an oligosaccharide) has shown experimentally to be the optimum complex for the separation of all 21 pairs of isomeric oligosaccharides used in this study with a calculated average resolution (r) ~ 1.82 and resolving power (R) ~ 100–150. The separation capability of this complex was further demonstrated by using mixtures of 15 oligosaccharides. The optimum [M + Ca + I]+ complex can be potentially used as an effective complex for enhancing the IMS separation resolution of isomeric carbohydrates.

Unravelling Chemical Composition of Agave Spines: News from Agave fourcroydes Lem.

Spines are key plant modifications developed to deal against herbivores; however, its physical structure and chemical composition have been little explored in plant species. Here, we took advantage of high-throughput chromatography to characterize chemical composition of Agave fourcroydes Lem. spines, a species traditionally used for fiber extraction. Analyses of structural carbohydrate showed that spines have lower cellulose content than leaf fibers (52 and 72%, respectively) but contain more than 2-fold the hemicellulose and 1.5-fold pectin. Xylose and galacturonic acid were enriched in spines compared to fibers. The total lignin content in spines was 1.5-fold higher than those found in fibers, with elevated levels of syringyl (S) and guaiacyl (G) subunits but similar S/G ratios within tissues. Metabolomic profiling based on accurate mass spectrometry revealed the presence of phenolic compounds including quercetin, kaempferol, (+)-catechin, and (−)-epicatechin in A. fourcroydes spines, which were also detected in situ in spines tissues and could be implicated in the color of these plants’ structures. Abundance of (+)-catechins could also explain proanthocyanidins found in spines. Agave spines may become a plant model to obtain more insights about cellulose and lignin interactions and condensed tannin deposition, which is valuable knowledge for the bioenergy industry and development of naturally dyed fibers, respectively.

Determinant morphological features of flax plant products and their contribution in injection moulded composite reinforcement

The use of biomass in injection moulded or extruded thermoplastic composites is an important issue, especially when trying to add value to low-cost co-products. The objective of this work was to conduct a complete study on the morphological characterisation and carbohydrate analysis of a range of co-products obtained during the processing of flax straw. Thus, the morphology of (i) cut flax fibres, (ii) fragmented shives, and (iii) scutching and carding dusts is characterised using a dynamic image analyser with a sieving approach. These different fractions are then used to produce injection moulded composite materials. Their mechanical performances are discussed in relation to the morphology of the reinforcements, as well as their carbohydrate compositions and fine particle contents. Co-products, based on their reinforcement properties, can be classified into three categories. In all cases, a reinforcing effect is demonstrated for the tensile Young's modulus with an increase from +24 to +137% depending of the material. A linear relationship was observed between the cellulose content of reinforcing material and the tensile strength at break of the injection moulded composites. The results are promising for adding value to all flax co-products in plastics processing, targeting industrial applications in line with their intrinsic performances.

Improvements in the Extractive and Carbohydrate Analysis of Sugarcane Bagasse

Generic procedures for the determination of extractives and carbohydrates (glucose, xylose, galactose, arabinose, mannose) were improved specifically for sugarcane bagasse. The extraction time was optimized using a specific design of experiments. The optimal condition for sugarcane biomass with up to 20% extractives was 11.5 h of extraction in water, followed by 9 h in ethanol. For the carbohydrate analysis, a separation method using liquid chromatography was improved and optimized to reduce the retention time of furanic compounds. The reduction obtained was approximately 30%. The optimal condition was the isocratic elution of the mobile phase with an aqueous solution containing 15% acetonitrile. The percentages of glycans, xylans, and arabinans in the extractive-free biomass was 33%, 22%, and 6.5%, respectively. The galactans and mannans were not detected. Acetyl groups and uronic acids were not determined.

Biogas potential and methanogenic community shift in in-situ anaerobic sewage sludge digestion with food waste leachate additions

The objective of this study was to determine methane yields (MY) of organic wastes in biogasification facilities according to the mixing ratio of food waste/food waste leachate and sewage sludge. One biogasification facility that treated sewage sludge only was compared with three biogasification facilities treating sewage sludge and food waste. The theoretical MY was derived based on analyses of carbohydrate, fat, and protein to examine the efficiency of the biogasification facility. The average actual MY was 0.424 Sm3CH4/kg volatile solids, which corresponded to 83.7% of theoretical MY. In the case of combined anaerobic digestion (CD) mixing with food waste/food waste leachate, inhibitory factors (volatile fatty acids [VFAs], total nitrogen [TN], and organic matter contents) showed the tendency to have relatively higher values in CD facilities than in the biogasification facility treating sewage sludge only. Mean concentrations of VFAs and TN in the anaerobic digester effluent, and the organic loading rate were 406 mg/L, 3,721 mg/L, and 1.62 kg volatile solids/m3 day, respectively. The influence of anaerobic digester effluent was in charge of 10% within the influent environmental loading rate from the sewage treatment plants associated with the biogasification facilities. Analyses of the microbial community showed that a remarkable change in the structure of methanogens was directly related to different MY in each plant. In particular, Methanoculleus and Methanosaeta increased with an increasing ratio of food waste/food waste leachate to sludge, while Methanococcus and Methanosarcina decreased. In conclusion, CD showed steady operational conditions and high efficiency of MY by injecting food waste/food waste leachate into the anaerobic digester. It met the current criteria for integrated treatment of organic waste in biogasification facilities in South Korea.

Short Communication: Lugol’s iodine test on Rafflesia patma–Tetrastigma leucostaphylum intersection tissue for preliminary starch visualization

Abstract. Wicaksono A, Mursidawati S. 2020. Short Communication: Lugol’s iodine test on Rafflesia patma-Tetrastigma leucostaphylum intersection tissue for preliminary starch visualization. Nusantara Bioscience 12: 91-96. As holoparasitic plant, Rafflesia has no recognizable plastid genome, but it has plastid-like organelle. Despite the fact that it obtains nutrients from host plant, it is unknown if Rafflesia stores primary metabolites, such as carbohydrates, from its host. A study was performed to visualize the starch in Rafflesia patma Blume proximal tissue which was intersected to its host root, Tetrastigma leucostaphylum (Dennst.) Alston, using modified Sachs’ test with Lugol’s iodine. The result revealed the absence of blackening in the R. patma tissue caused by starch reaction with the iodine, but occurred in the root cortical tissue of T. leucostaphylum. The absence of starch in R. patma tissue indicated that possibly the plastid-like organ has no similar function to amyloplast, and starch is not used for storage in the flower. It is likely that R. patma relies completely on the host’s photosynthate to maintain the flower metabolism during anthesis period. However, detailed histochemical analysis for starch or carbohydrate is needed for confirmation whether the starch is existing even in small quantity, and molecular genetic observation on sucrose intake and flowering (anthesis) regulatory genes will also be required to confirm if Rafflesia takes the photosynthate directly from its host and possibility if Rafflesia coordinates the formation of flower bud and anthesis between the endophytic tissues to prevent overexploitation of nutrients from its host.

Comprehensive nutritional profiling and activity directed identification of lead antioxidant, antilithiatic agent from Macrotyloma uniflorum (Lam.) Verdc

Macrotyloma uniflorum (Lam.) Verdc. (family Fabaceae) is traditionally important pulse used for kidney stone degradation, cold,cough, asthma and in bronchial issues. It is well reported for the antilithiatic activity and nutritional profiling but no attempt has been made to find out the mechanism and nutrient responsible for the activity in its seed decoction. Therefore, lead high altitude variety responsible for the highest nutrient content and antilithiatic activity was selected for the study. Boiled spray dried extract (M1), lead fractionation (Spray dried residual water fraction: M2), and crude powder extract (M3) were nutritionally profiled using proximate composition analysis, Atomic absorption spectroscopy (minerals and trace elements), Ion exchange chromatography (carbohydrate analysis) and NMR spectroscopy (polar and non polar metabolite analysis). These extracts were also screened for the antilithiatic activity and found significantly important. M1 showed more activity than M3. Further, boiled extract was fractionated with different polarity of solvents (hexane, chloroform, n-butanol, residual water extract). All fractions and reference standard (cystone) were screened for the antilithiatic activity. M2 showed highest activity followed by ethyl acteate while hexane and chloroform did not show any activity in comparison to standards. Thus, lead fraction and market reference standard (cystone) were validated for the presence of nutritional components. Moreover, the selected nutritional components were individually and in combinations screened for the activity which revealed that kidney stone degradation was due to the synergetic action of components than the individual. The results clearly pave a way that Macrotyloma uniflorum is best for combating with malnutrition and kidney stone related problems.