Introduction:

Glucose formed through diet is converted to glycogen by glycogen synthase enzyme. Glycogen is one of the food storage in body. Whenever body requires energy it is hydrolyzed to glucose and energy is created from glucose. If excess of glucose remain in body then it reaches to different parts of body while blood circulation. It affects metabolic reactions. There are many disadvantages of excess sugar in blood stream. It is observed that many factors in daily life are responsible for increasing tension which affect normal secretion of hormones. Which in turn affect biological activities in body.

Insulin is enhancer for glycogen synthesis. Extra insulin is supplied to body for more conversion of glucose to glycogen. There are many other factors are responsible for glycogen synthesis. Ayuvedic medicines, some food material and alopathic medicines are supplied to body for inhancing glycogen synthesis. There are many references where use of plants and vegetables is done for treatment of diabetes [Preethi (2013)¹, Rizvi and Mishra (2013)²]. Yet it is not solved satisfactorily. Insulin like signaling is observed in yeast. Muller et al. (1997, 2000)^3,4 have studied effect of insulin on synthesis of yeast-glycogen. Therefore study on yeast glycogen synthase is applicable to human glycogen synthase. There are so many factors for increasing blood pressure. One of the factors is that increase in angiotensin-II concentration increases blood pressure. Angiotensin-I is hydrolyzed by angiotension-I converting enzyme (ACE) to angiotensin-II. Generally medicines are used to inhibit ACE activity. These medicines have some side effects. Some diet material have ingradients which inhibits ACE activity therefore blood pressure is reduced. Angiotensin II is hydrolyzed to angiotensin III by enzyme called amino peptidase A (angiotensinase A, hypertensinase). Angiotensin III increases blood pressure but lesser than angiotension II. Therefore overall blood pressure in decreased due to formation of angiotensin III. It is known that our food potato, onion, soya bean etc. able to reduce blood pressure. Gollan et al. (1948)⁵ studied hypertensinase (angiotensinase, aminopeptidase) from plant and observed hypertensinase is able to degrade angiotensin II. Plant amino peptidases degrade angiotensin-II to other products which increases blood pressure lesser than angiotensin-II. In this study we have used yeast system for study of glycogen synthesis. Saccharomyces cerevisiae has same insulin receptor as in human body. Glycogen synthesis study carried out using yeast cells. Effect of additives on glycogen synthesis was studied. In this paper we have studied amino peptidase from some plants. These amino peptidase also known to degrade angiotensin-II in human. It is also reported in literature these plant extract reduces blood pressure. Amino peptidase from onion, soya bean, potato etc. were studied in this paper.

MATERIALS AND METHODS:

Chemicals:

Yeast extract, malt extract, peptone and agar were purchased from Himedia Chemicals, Mumbai. Other chemicals were used form S,D-Fine Chemicals, Mumbai. Onion, potato, soyabean etc. were purchased from local market.

Culture:

Saccharomyces cerevisiae NCIM 3215 was brought from NCIM (National Collection of Industrial Microorganism, N.C.L., Pune). It was maintained on MGYP-agar medium (Malt extract 0.3, glucose 2, yeast extract 0.3 and Peptone 0.5 %, pH adjusted to 6.5, 2.0% agar).

Growth:

Saccharomyces cerevisiae NCIM 3215 was grown on MGYP broth. Preinoculum was used. Growth was carried out at 220rpm and 28⁰C for 24h-48h. Cells were separated by centrifugation.

Analysis:

Sugar:

Sugar was determined by DNSA method [Miller (1959)⁶].

Glycogen:

Glycogen was determined by iodine-method described by Quain and Tubb (1983)⁷. Sample was suitably diluted. Freshly prepared iodine reagent (KI-I₂solution) was added and optical density was measured at 480nm. Appropriate blanks were used. Optical density of standard glycogen was taken using iodine-method. The method was verified by using iodine-sodium thiosulphate titration.

Amino acids:

Analysis of amino acid was done by ninhydrin method (Wang)⁸. Sample solution was heated with ninhydrin reagent in boiling water bath and optical density was measured at 570nm.

Glycogen synthesis:

Resting cells were used for glycogen synthesis Additives were used for enhancing glycogen synthesis. Glucose and glycogen was analysed.

Amino peptidase enzyme extraction:

Amino peptidase enzyme was extracted from sources reported in literature. Onion (red, white), potato, soya bean etc. were used. Tulsi and betel were also used for experiment. Extraction was done in 0.2M K-phosphate buffer pH 7.8 at 30⁰C and 220 rpm for 1h. Extraction was done with different source concentration.

Aspartame hydrolysis:

5-20% enzyme concentration and 0.005-0.02% substrate (aspartame) concentration was used for reaction. Reaction was carried out at pH 7.4. It was kept at 37⁰C for 2h. Sample was analyzed for amino acid by ninhydrin reagent.

Experimental:

Glycogen synthesis using resting cells:

Cells of Saccharomyces cerevisiae NCIM 3215 for study. Cells were added to reaction mixture containing 0.1% glucose. Cell concentration was 4% wet weight basis. 6 mL reaction volume was used. Reaction was carried out at pH 7.4 and 30⁰C without shaking for 3h. List of additives is given in table 1. Some additives are added directly. Aqueous extract of some additive prepared and used. Comparison is shown in figure 1.

Table 1: Additives used for enhancing glycogen synthesis

Additives	Concentration (%)
Ibuprofen, crocin, action-500, amla, jamun seed powder	0.33
karela, okra	0.33, 1.66, 3.33, 5
neem	0.833, 1.66
garlic, onion, ginger	1.66
coriader, cinnamon	0.33, 1.66,3.33
jeera	0.33, 1.66
Insulin	3.33, 16.66, 33.33, 50 I.U./100mL

Figure 1: Effect of karela, insulin and neem on glycogen synthesis by cell.

Amino peptidase:

We have used different plant material and amino peptidase from it extracted. Aspartame was 0.005-0.02%. Enzyme was extracted from sources. Enzyme is used in reaction mixture. Amount of source required for the enzyme used in reaction mixture was calculated. From reaction volume, source amount is converted into %. It is termed as enzyme concentration. Effective enzyme concentration was 5-20% in the experiment. Results are shown in figure 2-4.

Figure 2: Hydrolysis of aspartame by R-Onion (red onion) and W-Onion (white onion)

Figure 3:Comparison of different enzyme sources: white onion, potato and soya bean.

Figure 4: Effect of enzyme/substrate ratio on hydrolysis. Enzyme sources: white onion and, potato; Substrate=Aspartame (ASTM). Enzyme /substrate ratio= g of enzyme source/mg of aspartame.

RESULTS AND DISCUSSION:

Glycogen synthase:

Results obtained using Saccharomyces cerevisiae NCIM 3215 are applicable to human glycogen synthesis. Every experiment was done with control. Amla, jamun seed powder, karela, okra, neem, garlic, onion, ginger, coriader, cinnamon, jeera, insulin, iboprufen, crocin and action-500 were used as additives (table 1). Resting cells were used for glycogen synthesis at 30⁰C. Insulin, neem extract and karela have shown better results (figure 1). Abdel and Omnia (2014)⁹ has reported neem as antidiabetes. Muller et al. (1997)³ have reported that insulin has increased glycogen by 40-60% in yeast. Raza et al. (2004)¹⁰ have reported karela is useful in diabetes. There is little increase in glycogen synthesis by cinnamon, jeera, crocin, action-500 and coriander. Ibuprofen, amla and ginger have low activity. Jamun seed powder, onion and garlic had no activity.

Amino peptidase:

Amino peptidase specificity:

Enzyme converting angiotensin II (Asp-Arg-Val-Try-lle-His-Pro-Phe) to angiotensin III (Arg-Val-Try-lle-His-Pro-Phe) called angiotensinase A (Herzig et al. 1992)¹¹

Angiotensinase A

Asp-Arg-Val-Try-lle-His-Pro-Phe

Arg-Val-Try-lle-His-Pro-Phe + Aspartic acid

Where, Asp=aspartic acid; Arg= arginine; Val= valine; Try= tryptophan; lle=isoleucine; His= histidine; Pro=proline; Phe= phenylalanine.

Angiotensin III has less effect on blood pressure than angiotensin II. Therefore blood pressure is reduced. Angiotensinase A [EC 3.4.11.7) is also named by various other names e.g. amino peptidase A, glutamyl amino peptidase. Gollan et al. (1948)⁵ have reported that corn, peas, potatoes, wheat bran, onion etc. contain hypertensinase enzyme. There are some other amino peptidases which degrade angiotensin II. There reaction products are different than angiotensin III. Prolylendopeptidase (EC 3.4.21.26), angiotensin converting enzyme-2 (EC 3.4.17.23) and leucine amino peptidase (EC 3.4.11.1) [google site-2¹², Lambert et al. (2008)¹³, Watanabe et al. (2003)¹⁴]. Various sources were used for extraction of amino peptidase enzyme. Enzyme is extracted from onion (red, white), potato, soya bean, tulsi etc. Enzyme was used for hydrolysis of aspartame (ASTM). Aspartic acid is linked with phenylalanine-methylester in aspartame. Some amino peptidases degrade aspartame. Angiotensin II is peptide. It is degraded by some amino peptidase mentioned above. Leucine amino peptidase (EC 3.4.11.1) present in some microbes degrades aspartame. Leucine amino peptidase (EC 3.4.11.1) present in human also degrades angiotensin II. Therefore leucine amino peptidase (EC 3.4.11.1) able to degrade aspartame and angiotensin II. Substrate specificity is different. Amino peptidase A (EC 3.4.11.7) present in microbe and human able to degrade aspartame. Amino peptidase A (EC 3.4.11.7) present in human degrades angiotensin II in human. Substrate specificity is different.

Onion:

Leucine amino peptidase (EC 3.4.11.1) is also called leucyl amino peptidase. It cleaves N- terminal peptide bond. Leucine or proline is cleaved preferably. Arima et al. ((2012)¹⁵ has shown it cleaves aspartame also. Watanabe et al. (2003)¹⁴have shown cleavage of angiotensin II in human by leucine amino peptidase. Onion contain leucine amino peptidase and some other enzymes [Shigyo et al. (1995)¹⁶ ]. This leucine amino peptidase is resposible for breaking of angiotensin II in human resulting in reduction in blood pressure. We observed 31.63% hydrolysis of aspartame by amino peptidase from onion (white). There are some other reports which predict that onion contain some ingradients which inhibit angiotensin I to angiotensin II conversion followed by reduction in blood pressure. Larson et al. (2010)¹⁷ have discussed effect of quercetin from onion on angiotensin I converting enzyme. Therefore less angiotensin II formed and blood pressure is reduced. It is reported in literature that onion reduces blood pressure.

Potato:

Potato contain leucine amino peptidase [Vujcic et al. (2010)¹⁸]. Leucine amino peptidase able to hydrolyzes angiotensin II, hence in blood pressure is reduced by potato. Enzymes from potato was used for hydrolysis of ASTM. 26.36% hydrolysis was observed by potato. It is reported in literature that potato reduces blood pressure.

Soya bean:

Amino peptidase A (EC 3.4.11.7) in microbe and human cleaves aspartame [Hooper et al. (1994)¹⁹ ]. Soya bean contain glutamyl amino peptidase (amino peptidase A) (EC 3.4.11.7) (Asano et al. (2010)²⁰ . Aminopeptidase in soya bean degrade angiotensin II hence reduction in blood pressure. It is reported in literature that soya bean reduces blood pressure. Results are shown in figure 2-4. From figure 2 it is clear that white onion is better than red onion. As per figure 3 white onion is has shown more results than potato and soya bean. Ratio of enzyme and substrate is plotted in figure 4. As ratio is increased hydrolysis of aspatame is increased. Red onion has shown 5.94% hydrolysis of ASTM. We did not found hydrolysis of aspartame by peas, corn, betel and tulsi. Potato and white onion has shown 26.63 and 31.63% hydrolysis respectively. White onion has shown highest results.

CONCLUSION:

Resting yeat cells at 30⁰C used for glycogen synthesis. Neem extract, insulin and karela were used in glycogen synthesis. All have shown encouraing results. Neem has shown maximum 17.05% synthesis. Amino peptidase from white onion, soya bean, potato, etc. were used hydrolysis of ASTM (aspartame). Onion (red) has shown 5.94% hydrolysis. We have compared hydrolysis of ASTM by soya bean, potato and white onion. Hydrolysis obtained was 17.08, 17.52 and 19.6% respectively. White onion is better among the all sources used. White onion had maximum 31.63% hydrolysis.

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Received on 22.01.2016 Modified on 02.02.2016

Research J. Science and Tech. 8(1): Jan.– Mar. 2016; Page 51-55

DOI: 10.5958/2349-2988.2016.00007.3