Sample-4

5 Oct

Sample-4

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Natural compound (containing a mixture of bio-active molecules) that has anecdotally been claimed to have health-promoting benefits

1.0 Introduction:

Fenugreek (Trigonella foenum graecum) is a plant from the Leguminaceae family. The spice has been used in food as well as for the medicinal uses. The fenugreek seeds hold an important place in the Ayurvedic medicinal system.  The drug fenugreek is used as a common part of the diet in the Asian countries as it is a coomonly used spice. The plant is used and consumed in many forms like the leaves, plant as a whole, the seeds or the extract. Therefore a list of chemical constituent ought to be huge as the fenugreek as a characteristic smell odour and color which is imparted because of the different chemical constituents resent in the plant. This paper focuses on the related researches conducted on the plant Fenugreek and a list of all the therapeutic properties has also been reviwed in the paper. This paper focuses on the major use of modern day technologies like HPLC, TLC, Enzyme Linked Immunosorbent Assay (ELISA), qRT-PCR (Quantitaive Real Time Polymerase Chain reaction) and Cell viability methods using hemocytometer and variable dyes.

The fenugreek seeds are used as a spice worldwide and are known for its great medicinal properties worldwide. This spice is used immensely in food preparations and are an integral part of the traditional medicine system (Syeda et al, 2008)

The research has proven that the seeds are helpful in treating great neutraceutical value (Srinivasan 2006).  Similarly a lot of health benefits have been reported which suggests that Fenugreek seeds have a bio-active molecules responsible for proving the health benefits.

In this paper we have reviewed the reported health benefits of fenugreek and we have researched about the bio-active molecules by isolation and detection techniques like ELISA, Flow cytometry, qRT-PCR techniques, HPLC, TLC and Free radical scavenging methods.

1.1 Seed:

The fenugreek seeds are bitter in taste and they are mainly available in both the seed form as well as the ground from. The Fenugreek seed has a core yellow colored hard embryo which is surrounded by semi transparent endosperm area (Betty 2008)

1.2 Phytochemistry:

The chemical components of fenugreek seeds are the steroid sapogenins. Diosgenin has been found as an essential component in the embryo of the Fenugreek seeds. The alkaloids present in fenugreek are trigocoumarin, tri methyl coumarin, trigonelline and nicotinic acid. The mucilage present in the fenugreek seeds is the major constituent of the seed. (Khare 2004)

Table 1: Chemical constituents of fenugreek. (Chatterjee et al., 2010) Yadav et al. (2011), Sowmya and Rajyalakshmi (1999).

S. NoChemical Constituents
AlkaloidsTrimethylamine, Neurin, Trigonelline, Choline, Gentianine, Carpaine and Betain
Lipids Triacylglycerols, diacylglycerols, monoacylglycerols, phosphatidylcholine phosphatidylethanolamine, phosphatidylinositol
Amino acidsIsoleucine, 4-Hydroxyisoleucine, Histidine, Leucine, lysine, L-tryptophan, Arginine
SaponinsGraecunins, fenugrin B, fenugreekine, trigofoenosides A–G
Steroidal sapinogensYamogenin, diosgenin, smilagenin, sarsasapogenin, tigogenin, neotigogenin, gitogenin, neogitogenin, yuccagenin, saponaretin
FlavonoidsQuercetin, rutin, vitexin, isovitexin
Other Coumarin, lipids, vitamins, minerals. 28% mucilage; 22% proteins; 5% of a strongerswelling, bitter fixed oil.

1.3 Leaves:

The leaves of fenugreek contain seven types of saponins known as graecunins. These graecunins are glycosides of diosgenin. The leaves contain about 4.4% protein, 0.9% fat, 86.1% moisture, 1.5% minerals, 1.1% fiber and 6% carbohydrates. The leaves of fenugreek have minerals and vitamins present in it which are calcium, zinc iron, phosphorous, thiamine, niacin and vitamin C, ribo- flavin, carotene. (Rao, 2003)

In the experiment conducted by Yadav and Sehgal (1997) they found that the fresh leaves of fenugreek have ascorbic acid of 220.97 mg per 100 g of leaves, whereas the b-carotene was estimated to be about 19 mg per 00 gm of leaves. The fresh leaves are used as an intergral part of vegetables in the diet. It is suggested that the retention of nutrition in the fenugreek leaves is great.  Therefore the leaves of fenugreek can easily be boiled or stored or refrigerated as used as a nutrient in the diet.

Chemical properties and the Therapeutic Importance:

1.4 Nutraceutical Properties:

Fenugreek acts as a cleanser when it is used in flu, cold or bronchitis. Any blockade in the lymphatic system in the body is cleared when you consume the Fenugreek seeds or the active extract which basically helps in clearing away any blockage Example: in lungs, throat during urge to clear the cough with thick mucus. Fenugreek seeds have been reported by researches that they are a great held in medical conditions like hay fever, cold, flu, influenza, asthma, constipation, pneumonia and laryngitis. (Anon 2013)

1.5 Blood Pressure:

The fenugreek seeds also play an important role in controlling the blood pressure by maintaining the cholesterol levels in the body. The seeds and the leaves of fenugreek are responsible for decreasing the blood pressure. So anyone suffering from High blood pressure problems or cardiac diseases with frequent shoot ups in blood pressure can be treated very well using fenugreek. (Sowmya 1999)

1.6 Anti bacterial and Anti Fungal:

The anti bacterial as well as anti fungal action of fenugreek has been reported. In a research conducted by Haouala et al 2008, an aqueous extract of fenugreek plant parts was taken and then it was tested against the bacterial and fungal growth of different strains like Fusarium Graminearum, Rhizoctonia Solani, Botrytis Cinerea and Pythium Aphanidermatum.

1.7 Digestion:

The use of fenugreek plant in aiding digestion has been proved by researchers as they induce the secretion of enzymatic and digestive enzymes. A research experiment conducted by Patel and Srinivasan (2000) proved that fenugreek seeds as well as other herbal plants have markedly reported the release of digestive secretions.

1.9 Immunological Activity:

Any agent which is responsible for increasing or decreasing the immune response inside the bosy is called as Immunomodulator. The researches have been conducted access the immunulomodulator effect of fenugreek seeds. The Swiss albino mice were treated with a dose of aqueous extract of fenugreek seeds as 50 , 100, 200 mg per kg.The immune responses were studied and accessed for ten continuous days. The responses concluded from the research showed and increase in the immunity response in the body of Swiss albino mice. The parameters on which the conclusion was drawn about the immunomodulatory action was judged were, the body weight of animal, Thymus weight, phagocytosis, hemolytic assay,  cellularity of lymphoid organs, lymph proliferation and increase in phagocytic index. (Meghwal and Goswami 2012)

1.10 Lactation Aid:

The Lactation aids are the substances which increase the Breasts and modify the sweat glands. The researches on fenugreek plant have proved that they stimulate the sweat as well as the milk production. The response has been because of the increased hormone precursor in the breasts which ultimately leads to increased milk production and therefore secretion. Some scientist has proven that the fenugreek used as a herbal medicine for the mother has reportedly increased the milk secretion within an administration of 24-72 hours of fenugreek. (Snehlata and Payal, 2012).

The fenugreek lactation stimulation benefits have also been studied on goats where the lactating goats from a dairy were daily fed with fenugreek in the diet for 9 weeks in a lactating dairy goat. There were three groups maintained to prove the research and they were fed with 0%, 25% and 50% fenugreek in the diet respectively. All diet plans were similar for all the groups except for the fenugreek percentages. The milk yield was recorded daily and then a conclusion was drawn which showed that the  group receiving 25% fenugreek showed a significantly higher milk as compared to the group receiving no fenugreek whereas the group receiving 50% fenugreek marked a record increase in the milk yield and fat in the body of goat. (Al-Shaikh et al., 1999).

1.11 Fenugreek in extruded product:

Fenugreek leaf powder as well as the fenugreek seed flour has been used as part of the extruded snack. When in a mixture of 1.78% fenugreek seed powder and 0.66% of the fenugreek powder was mixed with a powder base then the color parameters, physical parameters and the functional parameters were highly increased (Wani and Kumar, 2015, 2016).

Due to the bitter taste of fenugreek when it was added to a mixture chickpea based food a relatively bitter taste was observed beyond a percentage of 2%. (of fenugreek).

The addition of fenugreek to the chickpea paste caused a slight decrease in the radial expansion as well as the longitudinal expansion property increased. The water absorption index was markedly increased while the water solubility was decreased in comparison to the control wher no fenugreek paste was added.

Table 2: General Uses of the drug Fenugreek:

S.NoPart of FenugreekProductResearches who have reported
1.Seeds The fenugreek is consumed in Bread(Isikli and Karababa, 2005; Thomas et al., 2011; Raju et al., 2001)
2.Fenugreek seeds, leavesThe fenugreek is consumed in form of Biscuits (Hussein et al., 2011)
3.Seeds The fenugreek is consumed as Extruded product (Shirani and Ganesharanee, 2009)
4.Seed
The fenugreek is used as Food stabilizer, adhesive and emulsifying agent (Jani et al., 2009; Sowmya and Rajyalakshmi, 1999)
5.Seed, leaves The fenugreek is used in Culinary use (color, flavor, aroma) (Ramesh et al., 2001)
6.Seed The fenugreek is consumed as a Dietary fiber (Blank, 1996)

Techniques used to isolate the Bio-active compound and prove the Therapeutic importance if the drug as whole or leaf extract, seed extract, ethanolic extractor as a whole in part of the diet or administration through variable methods is listed in the following:

2.0 Silica Gel Plate Method:

The Silica Gel 60254 plates were used to serve the stationary phase which was freshly prepared using the solvent mixture of hexane: ethyl acetate (85:15) which served as the mobile phase. Now the TLC chamber was used to develop a chromatogram from where we can easily calculate the Rf values and therefore identify various bio-active compounds.

2.1 HPLC Analysis:

The extraction of Octa decanoic acid can be done from fenugreek plant using C-18 column. Octa decanoic acid is extracted from the roots of the fenugreek plant. In this HPLC method we prepared the column using the acetonitrile/water (65:35, v/v) which was maintained at a pH of 2.5. The sample was identified by using the melting point method (Toshniwal Apparatus) and the mass spectroscopy. (Yamauchi LY TN 2002)

2.2 Isolation and Structural Elucidation of the Active Compound:

When 24 gm extract was chromatographed using the normal phased silica gel the ethanolic extract of fenugreek seeds was found to be enhancing the GLP-1R signaling. The fenugreek fraction was divided in 30 fractions and further grouped into four major groups depending on their ability to potentiate the GLP-1 induced endocytosis.( Klim King, Nai-Pin Lin 2015)

2.3 qRT-PCR:

Single copies of specific sequences can be detected and amplified using the quantitative Polymerase chain reactions (qRT-PCR). The Polymerase chain reactions can easily generate copies of DNA exponentially. It further results in developing a quantitative relationship between the initial targeted sequence and the PCR product which has been accumulated at every cycle end. The duplication reactions create multiple copies of the product.

The first use of real time polymerase chain reactions has been of immense help in multiplying the copies therefore there has been an increase in number of usage as well as diverse list of applications including the numbers of DNA copies produced, mRNA expression studies in the viral or genomic DNA.

Since the first documentation of real-time PCR (Higuchi R 1993), it has been used for an increasing and diverse number of applications including mRNA expression studies, DNA copy number measurements in genomic or viral DNAs (Kariyazono H,2001; Ingham DJ 2005)

The qRT-PCR technique is used in expressing the levels of genes which are involved in the metabolism of glucose molecules in the liver. The Streptozotocin induced diabetic rats were used. When Trigonella was administered orally after an intra-gastric incubation for two months the diabetic induced rats were experimented to access insulin like growth factor-1 (IGF- 1), hepatic glucokinase (GCK) glucose transporters (GLUT-2) genes expressions using the RTR-PCR assay method.

All such experiments using the qRT-PCR technology showed the insulin mimetic activity of fenugreek seeds. (Alaa-Eldin Salah-Eldin, 2015)

2.4 ELISA (Enzyme linked immune sorbent assay):

ELISA stands for the Enzyme linked immune sorbent assay. It is useful and powerful tool used in estimating the cultures and samples form ng/ml to pg/ml. (Savige, 1998). The ELISA technique has been widely used by researches since a long time in isolation and detection of components from any biological sample (Hongbao Ma, 2004).

The basic principle of the Enzyme linked Immunosorbent assay technology is use some enzyme to detect the proper binding of Antigen and antibody. The enzyme used in the ELISA technique converts the colorless substrates called as chromogens into the colored products. This therefore indicates the presence of Antigen: Antibody binding. Therefore an ELISA test can be used to detect the presence of Antigens or Antibodies in the sample depending upon the test which is designed for the process.

The hypoglycemic actions of the fenugreek seeds have been very well studied and investigated using the ELISA technique. Here the researchers have used the Alloxan administered rabbits, diabetic rabbits and human subjects. The animals were treated with a dose of 100m/kg for the duration of fifteen days in the rabbits and there was a major improvement in the GHbA1c and lipid profile (Sharma RD 1990).

The standard curves were generated by using the ELISA plates where the plotting of each concentration was done for the known protein samples and hence the absorbance values of the proteins can be well accessed using a molecular device apparatus which will estimate the concentrations. The ELISA values when plotted against the log values it was found out that there were no detectable proteins. Therefore, providing the fact that the type 2 diabetes can be well monitored and treated using the fenugreek seeds.

All the researches on variable animal and human subjects using the ELISA technique have pointed out the hypoglycemic action. The results have proven that the induced serum insulin levels as well as the glucose tolerance in GTT have increased.

In another research conducted on forty two patients three groups were formed and for group 1 patients a powdered fenugreek drug was given on each day of the research. Then Group 2 was administered 20 gm of fenugreek powder per day and subsequently for Group 3 no fenugreek powder was administered. All these groups received the same diet and at the end of six weeks it was clearly observed using the ELISA method of detection that the Group 1 which was given a lesser dose of the fenugreek powder showed no decrease in the fasting blood sugar whereas the Group 2 which received a large dose of the Fenugreek powder has reflected a significantly large drop in the fasting blood sugar levels. (Raghuram TC 1992)

Direct Lectin ELISA: In this study they have conducted the primary screening of the urine samples who were administered fenugreek and then the collected urine samples from each group Group 1: control group, Group 2: IFG, Group 3: IGT and Group 4: NDM . The Direct Lectin ELISA has been performed by the researches to determine then changes which were occurring in specific glycosylation patterns of the urine proteome. In this research the urine samples were plated evenly on the ELISA plates and the major changes in the saccharide glycotopes were identified using probes of bio inylated plant lectins. With the conclusions of the research it was found out that there are weak signals for LTL (a-1,2 fucose), GSL-2(GlcNAc-GlcNAc), VVL (O-linked GalNac) and HHL (polymannose). (Paturi V Rao 1989)

2.5 Cell Viability:

The cell numbers and cell viability method is used as a parameter to measure the activity of fenugreek. The cell viability can be easily accessed by taking the help of a hemocytometer which calculates the cell percentage which are able to exclude trypan blue.

This technique is practiced by treating a small portion of the cells from the complete sample in the wells for a specific duration and then the 0.4% of trypan blue was added to it. Then the solution is allowed to stay for two to three minutes where the viable cells are counted with the help of hemocytometer. The percentage of viable cells is calculated on the basis of a conclusion drawn from the viable cells in untreated well which is considered to be 100%. The cell numbers of viable cells with standard error was finally calculated as a mean of the three separate or independent experiments conducted. (Nasser M Al-Daghri, 2012)

The Jurkat cells were incubated in a research conducted by Nasser M Al-Daghr et al and they took concentrations of 30 to 1500 μg/mL with a 50%  ethanolic extract of the dry fenugreek seeds which were accessed for the changes in viability using the trypan blue assay. The morphological examination was also performed as well as the RT-PCR studies were conducted using the autophagic marker LC3 transcript. The results have suggested that the Fenugreek extract acted as an inducer of cellular death autophagy in human the T lymphoma Jurkat cells. (Nasser M Al-Daghri, 2012)

3.0 Conclusion:

The fenugreek plant has proved its medicinal importance as they have found to be healthy for many disease conditions. The herb has proved healthy and reported to cure and treat diseases like diabetes due to its anti- diabetic properties, Anti –fertility actions, anti parasitic functions and lactation stimulant actions. The techniques used for studying the active constituent are HPLC, TLC, Enzyme Linked Immunosorbent Assay (ELISA), qRT-PCR (Quantitaive Real Time Polymerase Chain reaction) and Cell viability methods using hemocytometer and dyes as a marker to judge the activity of the drug.

The herb has listed properties as anti-bacterial and an anti- fungal agent in the viable bacterial and microbial infection as well therefore it is claimed as a potential anti – bacterial as well as anti-fungal agent.

The drug fenugreek has found to be biologically active and it has been reported by many researchers that the immune stimulant action, hypocholesterolemic effects were marked by the extracts of the drug when they were administered to patients or animals for a set span of time. The previous researches have also made it clear that the drug has been used as a stabilizer, has food adhesive properties, emulsification properties as well. The findings have also made it very clear that the chemical constituents isolated from the drug have individual bio-active actions which make the herbal drug are useful in all diseases and disorders. The article has also pointed out the three important techniques cell viability method, qRT-PCR( quantitative real time polymerase chain reaction) and ELISA( Enzyme Linked Immunosorbent Assay) as a model of platform to prove the researches conducted using them. The therapeutic actions have been well researched on human subjects, rats, rabbits as well as diseased individuals.

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