**Figure 18.** Structures of soybean seed and pod. (A) Top view of soybean seed. (B) Side view of soybean seed. (C) Soybean pod with seeds inside. (D) Soybean seed.

As shown in **Table 1**, young pods contained a high concentration of ureides both in the upper and the lower pods. The high accumulation of ureides in the pods may be due to that ureides are tentatively stored in the pods before transporting to the seeds. Seeds contain a high concentration of amino acids especially Asn and GABA (**Table 2**).

**Figure 19** shows the changes in ureide-N and amino acid-N in seeds and pods of nodulated and nonnodulated soybean [34]. The concentrations of ureides in the pods of nodulated soybean were high at 1st September and decreased after 15th September. The ureide-N concentration kept low in the pods of nonnodulated soybean plants. Amino acid-N concentrations were similar between nodulated and nonnodulated soybeans and decreased linearly from 1st September to 10th October at maturing stage. On the other hand, the ureide-N concentrations in the seeds of nodulated and nonnodulated plants were constantly low. The amino acid-N concentrations were similar between nodulated and nonnodulated soybeans, decreased from 1st September to 22nd September, and then constant until maturity at 10th October.

**Figure 19.** Changes in udeide-N and amino acid-N in pods and seeds of nodulated and nonnodulated soybean plants.

Most parts of N stored in matured seeds are storage proteins in the cotyledons (**Figure 20A**) [35]. Soybean seed storage protein consists of glycinin and β-conglycinin (**Figure 20B**). The glycinin is the hexamer, which has acidic and basic subunits. The β-conglycinin is the trimer, which has α′, α, and β subunits.

**Figure 20.** Microscopic picture of soybean cotyledon and soybean seed storage proteins. (A) Microscopic picture of thin slice of soybean cotyledon. Pb: protein body. (B) Soybean seed storage protein separated by SDS-PAGE and stained with CBB.

**Figure 21** summarizes the metabolism and transport of N from pod into seeds. The ureides, allantoin, and allantoic acid, transported from nodules, are tentatively accumulated in the young pods, and these are metabolized into amino acids such as Gln, Asn, and then, these amino acids are secreted into the cavity of seed coat. Then, cotyledons absorb amino acids and synthesize storage protein and sort to the protein body. Asn and amino acids from roots and leaves are also transported to the pods and secreted into seed coat, and then, cotyledons absorb them for storage protein synthesis.

**Figure 21.** Model of the flow of ureides and amino acids in soybean pod to a seed.

## **8. Recycling of nitrogen from shoot to roots**

Most parts of N stored in matured seeds are storage proteins in the cotyledons (**Figure 20A**) [35]. Soybean seed storage protein consists of glycinin and β-conglycinin (**Figure 20B**). The glycinin is the hexamer, which has acidic and basic subunits. The β-conglycinin is the trimer,

A B

Cotyledon cells filled with protein

bodies. (pb: protein body)

**Figure 19.** Changes in udeide-N and amino acid-N in pods and seeds of nodulated and nonnodulated soybean plants.

Nodulated

SDS-PAGE profile of soybean

Seed protein.

**Figure 20.** Microscopic picture of soybean cotyledon and soybean seed storage proteins. (A) Microscopic picture of thin slice of soybean cotyledon. Pb: protein body. (B) Soybean seed storage protein separated by SDS-PAGE and stained with CBB.

glycinin

b-

conglycinin

Non-nodulated

which has α′, α, and β subunits.

190 Amino Acid - New Insights and Roles in Plant and Animal

0.05mm

Recycling of nitrogen from shoot to roots via phloem supports the initial growth of roots and nodules that need external nitrogen nutrients until nitrogen absorption and nitrogen fixation start to meet the N demand. The quantitative measurement of recycling of N in soybean cultivar Williams and hypernodulation mutant lines, NOD1-3, NOD2-4, and NOD3-7, was carried out by split root experiment in which a half root system was in the pot with 15N-labeled solution and the other was in nonlabeled solution (**Figure 22**) [36]. The roots of soybean plants cultivated in culture solution were separated into two pots at 33 days after planting. At the next day, 15N-labeled nitrate (10 mgN L−1) was added in the one side of pot and non-labeled nitrate (10 mgN L−1) in the other side of pot. After 2 days of treatment, plants were harvested and percentage of N from 15N-labeled source (15N%) was determined in each part. The 15N was highest in the roots in 15N-labeled pot (14.0%), followed by stems (6.0%) and leaves (3.9%). A small portion of recycling 15N was detected both in roots (0.7%) and nodules (0.3%) in the nonlabeled pot after 2 days of split root treatment in Williams. The distribution of 15N in sum of nodules and roots was the same in NOD1-3, NOD2-4, and NOD3-7 hypernodulation lines.

**Figure 22.** Recycling of N derived from a half root of soybean plants with 15N-labeled nitrate. Percent in parenthesis is percentage of labeled N in total N in each part.

## **9. Overall nitrogen transport from fixed N and absorbed N**

**Figure 23** shows the model of initial flow of N in soybean plants originated from N2 fixation (A) in nodules and NO3 − absorption from roots (B).

The ureides produced by nitrogen fixation in nodules are transported to leaves and used for the leaf growth and metabolism. Mature leaves do not retransport ureides from phloem, but these are transported as amino acids, especially Asn. Some ureides are directly transported to the pods, and young pods accumulate a large amount of ureides, then it is used for seed growth via seed coat after decomposition into amino acids, such as Gln and Asn.

The absorbed nitrate in the roots is transported as NO3 − or amino acids especially Asn after assimilated in the roots. The absorbed NO3 − is transported to the leaves, then reduced by leaf nitrate reductase and nitrite reductase, then assimilated into amino acids. The amino acids are transported to the pods, and then seeds from the leaves.

**Figure 23.** Model of the fate of N from N2 fixation and NO3 − absorption in soybean plants. (A) Fate of N from N2 . (B) Fate of N from NO3 − .

## **Author details**

**9. Overall nitrogen transport from fixed N and absorbed N**

absorption from roots (B).

(A) in nodules and NO3

−

percentage of labeled N in total N in each part.

192 Amino Acid - New Insights and Roles in Plant and Animal

The absorbed nitrate in the roots is transported as NO3

transported to the pods, and then seeds from the leaves.

assimilated in the roots. The absorbed NO3

**Figure 23** shows the model of initial flow of N in soybean plants originated from N2

growth via seed coat after decomposition into amino acids, such as Gln and Asn.

−

nitrate reductase and nitrite reductase, then assimilated into amino acids. The amino acids are

The ureides produced by nitrogen fixation in nodules are transported to leaves and used for the leaf growth and metabolism. Mature leaves do not retransport ureides from phloem, but these are transported as amino acids, especially Asn. Some ureides are directly transported to the pods, and young pods accumulate a large amount of ureides, then it is used for seed

**Figure 22.** Recycling of N derived from a half root of soybean plants with 15N-labeled nitrate. Percent in parenthesis is

−

or amino acids especially Asn after

is transported to the leaves, then reduced by leaf

Takuji Ohyama1 \*, Norikuni Ohtake1 , Kuni Sueyoshi1 , Yuki Ono1 , Kotaro Tsutsumi1 , Manabu Ueno1 , Sayuri Tanabata2 , Takashi Sato3 and Yoshihiko Takahashi1

\*Address all correspondence to<EMAIL_ADDRESS>
1 Faculty of Agriculture, Niigata University, Niigata, Japan

2 Agricultural Research Institute, Ibaraki University, Ibaraki, Japan

3 Faculty of Bioresource Sciences, Akita Prefectural University, Akita, Japan

## **References**

fixation

[1] Sato T, Ohtake N, Ohyama T, Ishioka NS, Watanabe S, Osa A, Sekine T, Uchida H, Tsuji A, Matsuhashi S, Ito T, Kume T. Analysis of nitrate absorption and transport in non-nodulated and nodulated soybean plants with 13NO3 − and 15NO3 − . Radioisotopes. 1999;**48:**450–458

[16] Ohyama T, Kumazawa K. Incorporation of 15N into various nitrogenous compounds in intact soybean nodules after exposure to 15N2 gas. Soil Science and Plant Nutrition. 1978;**24**:525–533

[2] Fujikake H, Yamazaki A, Ohtake N, Sueyoshi K, Matsuhashi S, Ito T, Mizuniwa C, Kume T, Hashimoto S, Ishioka NS, Watanabe S, Osa A, Sekine T, Uchida H, Tsuji A, Ohyama T. Quick and reversible inhibition of soybean root nodule growth by nitrate involves a decrease in sucrose supply to nodules. Journal of Experimental Botany. 2003;**54**:1379–1388

[3] Tempest DW, Meers JL, Brown CM. Synthesis of glutamate in *Aerobacter aerogenes* by

[4] Kumazawa K, Yoneyama T, Arima Y, Muhammad SS. Assimilation of nitrogen by rice plant as revealed with 15N. Proceedings of the Japan Academy Series B. 1987;**63**:219–222

[5] Yoneyama T, Kumazawa K. A kinetic study of the assimilation of 15N-labelled ammo-

[6] Yoneyama T, Kumazawa K. A kinetic study of the assimilation of 15N-labelled nitrate in

[7] Arima Y, Kumazawa K. Evidence of ammonium assimilation via glutamine synthetase-glutamate synthase system in rice seedling roots. Plant & Cell Physiology.

[8] Muhammad S, Kumazawa K. Assimilation and transport of nitrogen in rice. I. 15N-labelled

[9] Rosentahal GA. Plant Nonprotein Amino and Imino Acids: Biological, Biochemical, and

[10] Done J, Fowden L. A new amino-acid amide in the groundnut plant (*Arachis hypogea*): Evidence of the occurrence of ɤ-methylene glutamine and ɤ-methylene glutamic acid.

[11] Zacharius RM, Polland JK, Steward FC. ɤ-methylene glutamine and ɤ-methylene glutamic acid in the tulip (*Tulipa gesneriana*). Journal of the American Chemical Society.

[12] Ohyama T, Kera T, Ikarashi T. Occurrence of 4-methyleneglutamine and 2-oxo-4 methyl-3-pentene-1,5-dioic acid in leaves and stem of tulip plants. Soil Science and Plant

[13] Ohyama T. Comparative studies on the distribution of nitrogen in soybean plants sup-

[14] Lea PJ, Miflin BJ. Transport and metabolism of asparagine and other nitrogen compounds within the plants. In: The Biochemistry of Plants. Vol. 5. Academic Press; San

[15] Ohyama T, Minagawa R, Ishikawa S, Yamamoto M, Hung NVP, Ohtake N, Sueyoshi K, Sato T, Nagumo Y, Takahashi Y. Soybean seed production and nitrogen nutrition. In: El-Shemy HA, editor. Soybean Physiology and Biochemistry. Rieka, Croatia: InTech; 2011

enous constituents. Soil Science and Plant Nutrition. 1984;**30**:219–229

at the pod filling stage. II. Assimilation and transport of nitrog-

itherto unknown route. Biochemical Journal. 1970;**117**:405–407

rice seedling roots. Plant & Cell Physiology. 1975;**16**:21–26

ammonium nitrogen. Plant & Cell Physiology. 1974;**15**:747–758

Toxicological Properties. New York: Academic Press; 1982. pp. 1–273

1977;**18**:1121–1129

194 Amino Acid - New Insights and Roles in Plant and Animal

1954;**76**:1961–1962

plied with N2

Nutrition. 1988;**34**:613–620

Biochemical Journal. 1952;**51:**451–458

and NO3

−

Diego, California, USA. 1980. pp. 569–607

nium in rice seedling roots. Plant & Cell Physiology. 1974;**15**:655–661

## **Amino Acid Changes during Energy Storage Compounds Accumulation of Microalgae under the Nitrogen Depletion**

Cao Xupeng, Xue Song and Fan Xuran

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.68540

#### **Abstract**

[30] Lersten NR, Carlson JB. Vegetative morphology. In: Wilcox JR, editor. Soybeans: Improvement, Production, and Uses. 2nd ed. Madison, Wisconsin, USA: ASA, CSSA,

[31] Carlson JB, Lersten NR. Reproductive morphology. In: Wilcox JR, editor. Soybeans: Improvement, Production, and Uses. 2nd ed. Madison, Wisconsin, USA: ASA, CSSA,

[32] Ohyama T, Kawai S. Nitrogen assimilation and transport in soybean leaves: Investigation by petiole girdling treatment. Soil Science and Plant Nutrition. 1983;**29**:227–231

[33] Pate JS, Sharkey PJ, Lewis OAM. Phloem bleeding from legume fruits-A technique for

[34] Ohtake N, Suzuki M, Takahashi Y, Fujiwara T, Chino M, Ikarashi T, Ohyama T. Differential expression of β-conglycinin genes in nodulated and non-nodulated isolines

[35] Ohtake N, Ikarashi Y, Ikarashi T, Ohyama T. Distribution of mineral elements and cell morphorogy in nodulated and non-nodulated soybean seeds. Bulletin of the Facululty

[36] Ohyama T, Ueno M, Ono Y, Ohtake N, Sueyoshi K, Sato T, Tanabata S. Recycling of nitrogen from shoots to underground parts in hypernodulation mutant lines of soybean by split-root experiment. Bulletin of the Faculty of Agricululture, Niigata University. (in

SSSA publishers; 1987

196 Amino Acid - New Insights and Roles in Plant and Animal

SSSA publishers; 1987

press) 2017.

study of fruit nutrition. Planta. 1974;**120**:229–243

of soybean. Physiology Plant. 1996;**96**:101–110

of Agriculture, Niigata University. 1997;**49**:93–101

The nitrogen depletion stress is widely used to promote energy storage compound (ESC) production of microalgae, such as starch and lipids. Previous reports show that the fast ESC's accumulation happens around the overall nitrogen content lowered to the half of normal cells. It indicates that the cells take an active nitrogen reassembly to rebalance the requirement of nitrogen, in which the amino acid conversion should play an important role. So here, using a marine strain, Isochrysis zhanjiangensis, as the model to give a detail view on metabolic, transcriptomic and proteomic levels during ESC's fast accumulation. The intracellular metabolite fluctuation within 32 h was profiled by GC-MS and LC-MS. These techniques identified and quantified the levels of 14 SMAs, 2 carbohydrates involved in the TCA cycle and glycolysis, and 28 free amino acids (AAs). The pulsed increase of pyruvate indicated a potential to produce more FAs. Although overall AAs showed a decreasing trend, Ala and Phe increased initially. Meanwhile, the transcriptomic and proteomic studies were utilized to show the nitrogen metabolic pathways changes. It is found that gamma-aminobutyric acid (GABA) and other nonprotein AAs play important roles in the regulation of energy metabolism.

**Keywords:** nitrogen reassembly, microalgae, energy storage compound, *Isochrysis*

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **1. Introduction**

As the potential producer of the third-generation biofuels [1], and the origin of plenty of high-value products [2], microalgae have attracted more and more attentions from the last decade. An ideal algal biofuel production process should have high biofuel yield, with economic raw material utilization and proper biomass formulation. To meet these requirements, a two-stage culture method has been widely utilized, with nitrogen deficiency (N-deficiency) stress applied after the growth stage of the culture [3–7]. Nitrogen is the key element among the creatures, and the response to nitrogen deficiency varies among different plants [8]. For example, *Isochrysis zhangjiangensis* (synonym *Isochrysis zhanjiangensis*), a marine microalga with high carbon fixation capacity, will accumulate both polysaccharides and lipids as ESCs under N-deficiency conditions [3, 6, 9], while wild *Chlamydomonas reinharditii* stores starch and *Nannochloropsis oceanica* stores lipid preferentially.

However, the regulatory mechanism beneath the coordination of carbon and nitrogen metabolism is unclear, which is important for the design of high efficient process. For photoautotroph microalgae, carbohydrates are from the complex photosynthesis system. The synthesis of lipids is a more complex process than that of carbohydrates, which needs more ATP together with the reduction power from NADPH [10]. During the accumulation of ESCs, it can be reasonably assumed that plenty of enzymes are involved and a considerable portion of amino acids (AAs) are consumed or turned over to produce proteins (enzymes) for metabolism adjustment. Otherwise, some AAs involve the ESCs production directly. For instance, branched AAs (leu, Ile and Val) take part in the Ac-CoA production [11, 12], which is the raw material for the fatty acid synthesis; Glu is the precursor of chlorophyll synthesis. The clear understanding of carbohydrate and lipid accumulation coordinating process will contribute to oriental enrichment of bio-products according to the interest of industry.

The fast development of various "-omics" analyses provides versatile tools for probing complex biological problems. Different levels of "-omics" are combined to show a multidimensional information and widen our views on the essence of biological processes. Therefore, it gives us the opportunity to investigate the facts involving in this "golden period" of ESC's production, especially here, focused on the AA-related processes.

## **2. The methods used for "-omics" studies of** *I. zhangjiangensis*

#### **2.1. Strain and cultivation**

*I. zhangjiangensis* FACHB-1750 was maintained by the Freshwater Algae Culture Collection of the Institute of Hydrobiology, Chinese Academy of Sciences. The microalga had been previously cultivated in F/2 medium without silica under 14:10 light/dark cycle [3]. Algal cells were harvested in the exponential phase and re-suspended in nitrate-free medium to a final concentration of 3.0–4.0 × 106 cells/mL. For flask cultures, 200 mL seeds were inoculated in 500-mL glass shaking flasks with hand shake after inoculation and sampling. For bioreactor cultures, 500 mL seeds were inoculated in 600-mL glass bubbling reactors and aired by CO2 enriched air (2%, v/v) at the speed of 100 mL/min. The cultivation was under the control of self-made Algal Station Platform for reproducible growth (http://www.mbpe.dicp.ac.cn/yjcg/ kyjz/kyjzpage.html).

#### **2.2. "-omics" analysis procedures**

**1. Introduction**

198 Amino Acid - New Insights and Roles in Plant and Animal

and *Nannochloropsis oceanica* stores lipid preferentially.

bio-products according to the interest of industry.

**2.1. Strain and cultivation**

concentration of 3.0–4.0 × 106

production, especially here, focused on the AA-related processes.

**2. The methods used for "-omics" studies of** *I. zhangjiangensis*

As the potential producer of the third-generation biofuels [1], and the origin of plenty of high-value products [2], microalgae have attracted more and more attentions from the last decade. An ideal algal biofuel production process should have high biofuel yield, with economic raw material utilization and proper biomass formulation. To meet these requirements, a two-stage culture method has been widely utilized, with nitrogen deficiency (N-deficiency) stress applied after the growth stage of the culture [3–7]. Nitrogen is the key element among the creatures, and the response to nitrogen deficiency varies among different plants [8]. For example, *Isochrysis zhangjiangensis* (synonym *Isochrysis zhanjiangensis*), a marine microalga with high carbon fixation capacity, will accumulate both polysaccharides and lipids as ESCs under N-deficiency conditions [3, 6, 9], while wild *Chlamydomonas reinharditii* stores starch

However, the regulatory mechanism beneath the coordination of carbon and nitrogen metabolism is unclear, which is important for the design of high efficient process. For photoautotroph microalgae, carbohydrates are from the complex photosynthesis system. The synthesis of lipids is a more complex process than that of carbohydrates, which needs more ATP together with the reduction power from NADPH [10]. During the accumulation of ESCs, it can be reasonably assumed that plenty of enzymes are involved and a considerable portion of amino acids (AAs) are consumed or turned over to produce proteins (enzymes) for metabolism adjustment. Otherwise, some AAs involve the ESCs production directly. For instance, branched AAs (leu, Ile and Val) take part in the Ac-CoA production [11, 12], which is the raw material for the fatty acid synthesis; Glu is the precursor of chlorophyll synthesis. The clear understanding of carbohydrate and lipid accumulation coordinating process will contribute to oriental enrichment of

The fast development of various "-omics" analyses provides versatile tools for probing complex biological problems. Different levels of "-omics" are combined to show a multidimensional information and widen our views on the essence of biological processes. Therefore, it gives us the opportunity to investigate the facts involving in this "golden period" of ESC's

*I. zhangjiangensis* FACHB-1750 was maintained by the Freshwater Algae Culture Collection of the Institute of Hydrobiology, Chinese Academy of Sciences. The microalga had been previously cultivated in F/2 medium without silica under 14:10 light/dark cycle [3]. Algal cells were harvested in the exponential phase and re-suspended in nitrate-free medium to a final

500-mL glass shaking flasks with hand shake after inoculation and sampling. For bioreactor

cells/mL. For flask cultures, 200 mL seeds were inoculated in

*I. zhangjiangensis* cells from standard 7 day's cultivation were used as sample pools for the transcriptomic database construction. Sequencing was performed on Illumina HiSeq™ 2000 by BGI Tech (Shenzhen, China). Reads were assembled to unigenes (7511 clusters and 16,712 singleton transcripts) by Trinity [13] and further annotated against NCBI NR database (nonredundant protein database) and using Nt (non-redundant nucleotide database), SwissProt, KEGG and COG database by blastx (e-value <10−5). The calculation of unigene expression uses FPKM method (Fragments Per kb per Million fragments) [14]. The COG cluster enrichment analysis was performed basing on the expression. The RNA-seq data are available with accession No. PRJNA217946 on NCBI.

The proteomics analysis was performed according to previous report [15] against above RNAseq database. Totally 1862 proteins were identified and quantified.

The AAs and other small molecular acids (SMAs) were inspected by SIM LC-MS and GC-MS, respectively. The experimental details can be found from Zhang et al. [16]. Furthermore, by the pulse-isotope label of 15N on nitrogen containing compounds (NCCs) during the "golden period," the turnover of nitrogen between AAs was investigated.

#### **2.3. Growth, photosystem II (PS II) activity and other biochemical composition analyses**

Other biochemical and physiological parameters were detected as previous description [3, 6, 9]. In brief, dry weight was determined gravimetrically after filtration by Whatman GF/C filters (47 mm diameter) and air dried in the air until constant weight achieved. Nitrate analysis was conducted with SEAL Analytical AutoAnalyzer 3 following the manufacturer's instructions. Lipid analysis was performed using Nile Red or GC according to Wang's method [6, 9]. The chlorophyll fluorescence measurements were performed using a chlorophyll fluorometer (Water-PAM, Heinz Walz GmbH).

## **3. Overall of cell growth and turnover of AAs**

## **3.1. The newly synthesized AAs during the fast accumulation of ESCs by pulsing label**

The assimilation of nitrogen starts from the reduction of nitrate and the formation of glutamate. Without nitrate supply from the medium, free AAs were turn over between different NCCs, including proteins, nucleic acids and AAs themselves. By pulsing adding 12.3 mg/L 15N (in form of Na15NO3 ) at day 2, when initial 24.6 mg/L N was nearly used out, the labeled ratio of free AAs was detected within 32 h.

It has been reported, while the external nitrogen supply depleted, the protein, especially ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), was partially degrade as a nitrogen pool to reassemble AAs and other NCCs [17]. So, the final labeled ratio of AAs was expected near 33% (according to total nitrogen supplied). Most of AAs (15/23) consisted with the theoretical value with about 33% labeled ratio. However, some AAs showed interesting changing patterns (**Figure 1**).

The labeled AAs were newly synthesized, and therefore, most of free AAs show a high labeled ratio at the beginning (day 3-1 of **Figure 1**), except Arg, which increased stepwise. The Arg is mostly from the conversion of ornithine. The low level of labeled Arg indicates a slower than average AAs' synthesis rate of it in the early of exponential growth phase.

Among 23 AAs detected, 6 of them (His, taurine, Asn, Gln, Arg, and Trp) show a the final label ratio higher than 35%, indicating more than average newly synthesized of them were left inform of free AAs. Together with the low label level of Arg, the nitrogen assimilation may mostly form Gln branch.

The first undetectable labeled AA was methionine sulfoxide, which disappeared within 24 h. It is the oxidation form of methionine and formed post-translationally. The content of methionine sulfoxide is about one-fifth of that of Met in *I. zhangjiangensis* [16].

The second disappeared labeled AA was ornithine. It may be the result of the increasing labeled Arg and function involving in the urea cycle [11].

**Figure 1.** 15N labeling percentage of AAs during the ESC fast accumulation period. (A) The labeling ratio of AAs changed within 32 h. The theoretical labeled ratio is about 33%. (B) The nitrogen changes during a standard bioreactor cultivation. The dotted line is nitrate concentration in medium of pulsing label experiment. (C) The dry weight, *Fv /Fm* and neutral lipids content changes in above cultivation. The black arrow indicates the start of pulsing label of 15N.

#### **3.2. The change of free AAs together with SMAs**

It has been reported, while the external nitrogen supply depleted, the protein, especially ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), was partially degrade as a nitrogen pool to reassemble AAs and other NCCs [17]. So, the final labeled ratio of AAs was expected near 33% (according to total nitrogen supplied). Most of AAs (15/23) consisted with the theoretical value with about 33% labeled ratio. However, some AAs showed interesting changing

The labeled AAs were newly synthesized, and therefore, most of free AAs show a high labeled ratio at the beginning (day 3-1 of **Figure 1**), except Arg, which increased stepwise. The Arg is mostly from the conversion of ornithine. The low level of labeled Arg indicates a slower than

Among 23 AAs detected, 6 of them (His, taurine, Asn, Gln, Arg, and Trp) show a the final label ratio higher than 35%, indicating more than average newly synthesized of them were left inform of free AAs. Together with the low label level of Arg, the nitrogen assimilation may

The first undetectable labeled AA was methionine sulfoxide, which disappeared within 24 h. It is the oxidation form of methionine and formed post-translationally. The content of methio-

The second disappeared labeled AA was ornithine. It may be the result of the increasing

**Figure 1.** 15N labeling percentage of AAs during the ESC fast accumulation period. (A) The labeling ratio of AAs changed within 32 h. The theoretical labeled ratio is about 33%. (B) The nitrogen changes during a standard bioreactor cultivation.

*/Fm* and neutral

The dotted line is nitrate concentration in medium of pulsing label experiment. (C) The dry weight, *Fv*

lipids content changes in above cultivation. The black arrow indicates the start of pulsing label of 15N.

average AAs' synthesis rate of it in the early of exponential growth phase.

nine sulfoxide is about one-fifth of that of Met in *I. zhangjiangensis* [16].

labeled Arg and function involving in the urea cycle [11].

patterns (**Figure 1**).

200 Amino Acid - New Insights and Roles in Plant and Animal

mostly form Gln branch.

The AAs metabolism relates to other SMAs closely. Zhang et al. have reported a detailed metabolic network change, with 18 protein AAs, 11 non-protein AAs, 14 SMAs and 2 carbohydrates [16]. As shown in **Figure 1B**, the total nitrogen was steady during the fast ESC accumulation stage, so the per cell-based qualification was used to tracing the changes. The cell number doubled after nitrogen depletion, and it is reasonable that the AA content level decreased to about half level. However, Phe had an evident 3-time increase during the early stage of nitrogen depletion. Other AAs maintained a steady state or increased in quantity slightly at the beginning and then decreased below their initial amounts.

The relative constant of protein AAs may contribute to the release of AAs from Rubisco. **Table 1** is the comparison of AA content in Rubisco large subunit and whole cell of *I. zhangjiangensis*. The data for Rubisco are from the putative AA sequence translated based on RNAseq and that for whole cell is measured by MS from nitrogen-rich cultured cells. They share a similar composition. Only Glu shows a great difference.

In non-protein AAs, only Gamma-aminobutyric acid (GABA) shows a profoundly increase after nitrogen depleted, indicating an important role in the response of nitrogen stress.

Together with the changes of AAs, including the metabolites of glycolysis (d-glucose-1p,dihydroxyacetone-p, glyceraldehyde-3p, d-fructose-6p) and the glyoxylate or TCA cycles (citrate, cis-aconitate, isocitrate, α-ketoglutarate) showed a pattern of initial increase in quantity followed by subsequent decrease. Malate, fumarate and succinate, all from the TCA cycle, shared the same tendency of steady decline. Glucose and lactate showed steady increase in quantity [16].

In general, nitrogen has positive effects on the growth of microalgae, while having a negative impact on the accumulation of lipids, for aims of biofuel production. When carbon is not a limiting factor, intracellular energy substrates will accumulate under nitrogen starvation. Evidence shows that glutamate dehydrogenase as a metabolism shunt plays an important role in ensuring that the nitrogen metabolism does not affect the function of mitochondria and nitrogen recycling [18]. As a consequence, Gln, Glu, Asp, and Asn form the basis of several other organic nitrogen compounds, especially AAs [18].

#### **3.3. AA metabolism–related transcriptomics and proteomics change**

The overall transcriptomics and proteomics annotation results by KEGG pathways show opposite changes, up-regulated in transcriptomics while down-regulated in proteomics (**Figure 2**). The transcription and translation are controlled complex mechanisms. Their trends difference is a result of different level responses to the nitrogen deficiency.

To have a more global view of cell response, basing on the Clusters of Orthologous Groups (COGs) enrichment analysis of differentially expressed genes, amino acid transport and metabolism cluster (E) is the most significantly down-regulated cluster (**Figure 3**).

**Table 1.** The AA profiling in Rubisco large subunit and whole cell\*.

#### **3.4. Glu vs. α-ketoglutarate**

Glu is the key AA for photosynthesis cell as the precursor of chlorophyll. Also, it is the second AA products after the series reduction of nitrate to ammonia. It is the most abundant AA (in weight ratio) in *I. zhangjiangensis* (**Table 1**). α-Ketoglutarate plays two vital roles in the biochemical pathways of microalgae. One role is its participation in the TCA cycle, and the other is its involvement in the synthesis of glutamate. α-Ketoglutarate accumulates in the early stage of nitrogen deficiency in *Phaeodactylum tricornutum* and *I. zhangjiangensis* [10, 16]. α-Ketoglutarate accumulated incipiently instead of participating in the synthesis of glutamate, as indicated by the smooth decline in glutamine levels during the same time period. Then, both the level of glutamate and glutamine decreased significantly and the high level of α-ketoglutarate then resulted in feedback inhibition for its precursors, leading to their accumulation and apparent Amino Acid Changes during Energy Storage Compounds Accumulation of Microalgae under the Nitrogen Depletion http://dx.doi.org/10.5772/intechopen.68540 203

**Figure 2.** The mapping of significant changed AA metabolism related genes and proteins on KEGG metabolic pathways (map01100). The thin line indicates the significantly down-regulated elements, while the thick line indicates the significantly up-regulated elements.

increase (isocitrate, cis-aconitate, citrate) consequently [10, 16]. Nitrogen supply status showed directly influence on nitrogen assimilation, and by way of α-ketoglutarate intrigued the rebalance the carbon metabolism on a certain degree. AKG-Gln-Glu is the linker between nitrogen assimilation and central carbon metabolism in *I. zhangjiangensis*' response to nitrogen starvation.

**3.4. Glu vs. α-ketoglutarate**

\*The bold italic underlined AAs are first 7 abundant AA in each group.

**Table 1.** The AA profiling in Rubisco large subunit and whole cell\*.

**AA Mole % in Mass % in**

202 Amino Acid - New Insights and Roles in Plant and Animal

**Rubisco large subunit Whole cell Rubisco large subunit Whole cell**

His 1.5% 0.3% 1.8% 0.4% Cys 1.8% 0.4% 1.7% 0.4% Trp 1.8% 0.0% 2.9% 0.0% Glu 3.4% *9.0%* 3.8% *10.9%* Met 3.7% 2.1% 4.2% 2.6% Phe 3.8% 4.8% 4.9% 6.5% Asn 4.0% / 4.1% / Ile 4.1% 5.3% 4.2% 5.7% Tyr 4.3% 2.1% *6.0%* 3.1% Gln 4.4% 0.7% 5.0% 0.8% Pro 4.7% 4.5% 4.2% 4.2% Lys 4.9% *6.2%* 5.5% *7.4%* Ser 5.0% *7.9%* 4.1% *6.9%* Arg *6.0%* 4.8% *8.1% 6.9%* Thr *6.4%* 4.8% *5.9%* 4.7% Asp *6.6%* 4.5% *6.8%* 4.9% Gly *7.3%* 13.0% 4.3% *8.0% Val 7.6% 7.4% 6.9% 7.2% Leu 8.0% 9.0% 8.1% 9.7% Ala 10.6% 13.2% 7.3% 9.7%*

Glu is the key AA for photosynthesis cell as the precursor of chlorophyll. Also, it is the second AA products after the series reduction of nitrate to ammonia. It is the most abundant AA (in weight ratio) in *I. zhangjiangensis* (**Table 1**). α-Ketoglutarate plays two vital roles in the biochemical pathways of microalgae. One role is its participation in the TCA cycle, and the other is its involvement in the synthesis of glutamate. α-Ketoglutarate accumulates in the early stage of nitrogen deficiency in *Phaeodactylum tricornutum* and *I. zhangjiangensis* [10, 16]. α-Ketoglutarate accumulated incipiently instead of participating in the synthesis of glutamate, as indicated by the smooth decline in glutamine levels during the same time period. Then, both the level of glutamate and glutamine decreased significantly and the high level of α-ketoglutarate then resulted in feedback inhibition for its precursors, leading to their accumulation and apparent **Figure 3.** Enrichment of COG clusters analysis. Bars indicated the number of identified genes in different COG clusters and up or down regulated gene number in each cluster. The significant enrichment classes were mark with asterisk (\*) on the top of the bar. The meaning of letters (A–Z) is listed below and significantly up or down regulated clusters were mark with up\* or down\* after their names correspondingly and the number after \* was the *Q*-value. RNA processing and modification (A); chromatin structure and dynamics (B); energy production and conversion (up\*, 2.95E−02) (down\*, 1.00E−05) (C); cell cycle control, cell division, chromosome partitioning (up\*, 1.05E−02) (D); amino acid transport and metabolism (down\*, 0.0) (E); nucleotide transport and metabolism (F); carbohydrate transport and metabolism (G); coenzyme transport and metabolism (up\*, 2.95E−02) (H); lipid transport and metabolism (down\*, 1.88E−03) (I); translation, ribosomal structure and biogenesis (J); transcription (K); replication, recombination and repair (up\*, 1.93E−03) (L); cell wall/membrane/envelope biogenesis (up\*, 2.42E−11) (M); cell motility (N); posttranslational modification, protein turnover, chaperones (O); inorganic ion transport and metabolism (P); secondary metabolites biosynthesis, transport and catabolism (up\*, 6.57E−05) (down\*, 1.54E−07) (Q); general function prediction only (R); function unknown (up\*, 3.60E−07) (S); signal transduction mechanisms (T); intracellular trafficking, secretion, and vesicular transport (U); defense mechanisms (V); extracellular structures (W); nuclear structure (Y); Cytoskeleton (up\*, 2.54E−02) (Z).

It's reported that Gln and Glu subject influence on the intermediates of the TCA cycle. The transcription repressor of aconitase, CcpC, can be suppressed by glutamate, when nitrate is assimilated in phototrophic eukaryotes, glutamate or its precursors can arrest the glutamate synthase operon, which is closely affected by both carbon and nitrogen sources, and induced in the presence of ammonium and glycolytic carbon sources to form a feedback inhibition cycle [19]. The nitrogen source for the increased glutamine was considered as the recycling of internal nitrogen, indicating by the higher level of a key intermediate in the urea cycle, citrulline. Proteolysis and AA metabolism consume plenty of energy and release CO2 , resulting in the low level of intermediates in central carbon metabolism in the early stage of nitrogen deficiency [16].

Furthermore, in *I. zhangjiangensis*, the photosynthesis brought enough glucose to cell after the growth was inhibited by nitrogen depletion, and further stored in form of β-1,3 glucan [9, 16]. The wholly increased intermediates in glycolysis process inhibit glyoxylate cycle from the very beginning. By sharing the intermediates with TCA, the influence also causes the transitory increase of α-ketoglutarate, and further influence Gln and Glu metabolism.

#### **3.5. Succinate and Asp, pyruvate and Ala: two secondary intersections of carbon and nitrogen metabolism**

In most microalgae species, aspartate and glutamate usually constitute a large proportion of the total amino acid content, while GABA, Trp, Met, His, ornithine and others only represent a small proportion [20]. Asp makes up 6.9% or more of the total AA content and approximately 1.5–1.8% of total detected free AAs in *I. zhangjiangensis*. Asp serves as the source of nitrogen for transaminations and as the early product of the carbon fixation pathway for carbon storage in blue-green algae [21]. Succinate and Asp are intermediates between the glyoxylate cycle and the TCA cycle in cells. The glyoxylate cycle is involved in the formation of glucose and further AAs and nucleotides, whereas the TCA cycle targets the generation of ATP as an energy carrier. For *I. zhangjiangensis*, when nitrogen was limited, some cell physiological activities related to growth receded or stopped, and the product of carbon fixation, glucose, accumulated, while the surplus energy was stored in the form of polysaccharose or lipids [3, 6, 9]. The increase of intermediates of glycolysis and the TCA cycle, as well as AMP and ADP [10], blocked the glyoxylate and TCA cycles. Most of metabolites of the TCA cycle in the inhibitor-treated sample showed a stable concentration at the beginning, except succinate and fumarate. Subsequently, with a further decrease in photosynthetic activity, the TCA cycle activity increased to produce more energy, and the levels of all TCA cycle metabolites decreased. While lipid accumulation increased lately, malate, α-ketoglutarate and other metabolites in TCA decreased. More energy consumed in lipid synthesis than that of polysaccharose may be the reason for this.

Due to the share of intermediates of above cycles, citrate, cis-aconitate and isocitrate accumulated rapidly. In one glyoxylate cycle, two molecules of Ac-CoA are consumed, while one molecular Ac-CoA is consumed during one TCA cycle. Otherwise, from the stable level of pyruvate, it is postulated the initiative synthesis of Ac-CoA was intact. Therefore, the block of above two cycles induced a transitory net accumulation of Ac-CoA, which redirected to FAs, which also consists with our previous work [6, 9].

Pyruvate and Ala are another pair of metabolites at the carbon-nitrogen metabolism intersection. Pyruvate is the key linker between different sub cell component (**Figure 4**), and the precursor of acetyl-CoA, the precursor for FAs synthesis and TCA cycle. As the most abundant AA in Rubisco, which contributes about 3% dry weight of normal *I. zhangjiangensis* cells, Ala was considered to release ammonium after nitrogen depleted by consuming α-ketoglutarate and producing Glu and pyruvate. The free Ala kept decrease in this period.

**Figure 4.** Postulated carbon partition of *I. zhangjiangensis* under during the "golden period." While exogenous nitrogen depleted, the nitrogen assimilation stopped and α-ketoglutarate as the reactant of Gln-Glu cycle had a transitory increase. The synthesis of Gln was reduced, and proteolysis was enhanced, while GABA acted as a transient N buffer as well as a signal to indicating the nitrogen level in cells to stimulate subsequent response. The slowdown of growth without exogenous nitrogen also caused the accumulation of carbohydrates, such as glucose or glucan, which and whose derivants in glycolysis together with α-ketoglutarate inhibited the activities in TCA and glyoxylate cycle. Partial of carbon flowed to pyruvate for further FAs or lipids. Words with doubled underline: increased, others: no significant change. Drafted based on Zhang et al.

## **4. Conclusion**

It's reported that Gln and Glu subject influence on the intermediates of the TCA cycle. The transcription repressor of aconitase, CcpC, can be suppressed by glutamate, when nitrate is assimilated in phototrophic eukaryotes, glutamate or its precursors can arrest the glutamate synthase operon, which is closely affected by both carbon and nitrogen sources, and induced in the presence of ammonium and glycolytic carbon sources to form a feedback inhibition cycle [19]. The nitrogen source for the increased glutamine was considered as the recycling of internal nitrogen, indicating by the higher level of a key intermediate in the urea cycle, citrul-

in the low level of intermediates in central carbon metabolism in the early stage of nitrogen

Furthermore, in *I. zhangjiangensis*, the photosynthesis brought enough glucose to cell after the growth was inhibited by nitrogen depletion, and further stored in form of β-1,3 glucan [9, 16]. The wholly increased intermediates in glycolysis process inhibit glyoxylate cycle from the very beginning. By sharing the intermediates with TCA, the influence also causes the transi-

, resulting

line. Proteolysis and AA metabolism consume plenty of energy and release CO2

tory increase of α-ketoglutarate, and further influence Gln and Glu metabolism.

**3.5.  Asp makes up 6.9% or more of the total AA content and approximately 1.5–1.8% of total detected free AAs in *I. zhangjiangensis*. Asp serves as the source of nitrogen for transaminations and as the early product of the carbon fixation pathway for carbon storage in blue-green algae [21]. Succinate and Asp are intermediates between the glyoxylate cycle and the TCA cycle in cells. The glyoxylate cycle is involved in the formation of glucose and further AAs and nucleotides, whereas the TCA cycle targets the generation of ATP as an energy carrier. For *I. zhangjiangensis*, when nitrogen was limited, some cell physiological activities related to growth receded or stopped, and the product of carbon fixation, glucose, accumulated, while the surplus energy was stored in the form of polysaccharose or lipids [3, 6, 9]. The increase of intermediates of glycolysis and the TCA cycle, as well as AMP and ADP [10], blocked the glyoxylate and TCA cycles. Most of metabolites of the TCA cycle in the inhibitor-treated sample showed a stable concentration at the beginning, except succinate and fumarate. Subsequently, with a further decrease in photosynthetic activity, the TCA cycle activity increased to produce more energy, and the levels of all TCA cycle metabolites decreased. While lipid accumulation increased lately, malate, α-ketoglutarate and other metabolites in TCA decreased. More energy consumed in lipid synthesis than that of polysac-

Due to the share of intermediates of above cycles, citrate, cis-aconitate and isocitrate accumulated rapidly. In one glyoxylate cycle, two molecules of Ac-CoA are consumed, while one molecular Ac-CoA is consumed during one TCA cycle. Otherwise, from the stable level of pyruvate, it is postulated the initiative synthesis of Ac-CoA was intact. Therefore, the block of above two cycles induced a transitory net accumulation of Ac-CoA, which redirected to FAs,

deficiency [16].

204 Amino Acid - New Insights and Roles in Plant and Animal

**nitrogen metabolism**

charose may be the reason for this.

which also consists with our previous work [6, 9].

The AA metabolism changes during the fast ESCs accumulation process of *I. zhangjiangensis* was detailed illustrated by "-omics" analysis. An overall changing model is raised from above data, which will help us to understand the function of different AAs and promote the new regulation methodology for the bioenergy development. A draft global change of AA-related metabolism is shown in **Figure 5**.

**Figure 5.** The possible response of AA metabolism of *I. zhangjiangensis* based on omics analysis in the fast ESCs accumulation period.

## **Author details**

Cao Xupeng\*, Xue Song and Fan Xuran

\*Address all correspondence to<EMAIL_ADDRESS>
Marine Bioengineering Group, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China

## **References**

**Author details**

period.

Sciences, Dalian, China

**References**

Cao Xupeng\*, Xue Song and Fan Xuran

206 Amino Acid - New Insights and Roles in Plant and Animal

\*Address all correspondence to<EMAIL_ADDRESS>
Marine Bioengineering Group, Dalian Institute of Chemical Physics, Chinese Academy of

**Figure 5.** The possible response of AA metabolism of *I. zhangjiangensis* based on omics analysis in the fast ESCs accumulation

[1] Gonzalez-Fernandez C, Ballesteros M. Linking microalgae and cyanobacteria culture conditions and key-enzymes for carbohydrate accumulation. Biotechnology Advances.

[2] Vidoudez C, Pohnert G. Comparative metabolomics of the diatom *Skeletonema marinoi* in different growth phases. Metabolomics. 2011;**8**(4):654-669. DOI: 10.1007/s11306-011-0356-6

[3] Feng D, Chen Z, Xue S, Zhang W. Increased lipid production of the marine oleaginous microalgae *Isochrysis zhangjiangensis* (Chrysophyta) by nitrogen supplement. Bioresource

[4] Chisti Y. Constraints to commercialization of algal fuels. Journal of Biotechnology.

[5] Hu Q, Sommerfeld M, Jarvis E, Ghirardi M, Posewitz M, Seibert M, et al. Microalgal triacylglycerols as feedstocks for biofuel production: Perspectives and advances. The Plant

Technology. 2011;**102**(12):6710-6716. DOI: 10.1016/j.biortech.2011.04.006

Journal. 2008;**54**:621-639. DOI: 10.1111/j.1365-313X.2008.03492.x

2012;**30**(6):1655-1661. DOI: 10.1016/j.biotechadv.2012.07.003

2013;**167**:201-214. DOI: 10.1016/j.jbiotec.2013.07.020

## **Bioactive Molecules Profile from Natural Compounds**

Adina‐Elena Segneanu, Silvia Maria Velciov, Sorin Olariu, Florentina Cziple, Daniel Damian and Ioan Grozescu

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.68643

#### **Abstract**

[18] Perez-Garcia O, Escalante FM, de-Bashan LE, Bashan Y. Heterotrophic cultures of microalgae: Metabolism and potential products. Water Research. 2011;**45**(1):11-36. DOI:

[19] Commichau FM, Forchhammer K, Stulke J. Regulatory links between carbon and nitrogen metabolism. Current Opinion on Microbiology. 2006;**9**(2):167-172. DOI: 10.1016/j.

[20] Brown MR. The amino-acid and sugar composition of 16 species of microalgae used in mariculture. Journal of Experimental Marine Biology and Ecology. 1991;**145**(1):79-99.

[21] Macko SA, Fogel ML, Hare PE, Hoering TC. Isotopic fractionation of nitrogen and carbon in the synthesis of amino acids by microorganisms. Chemical Geology. 1987;**65**(1):79-92.

10.1016/j.watres.2010.08.037

208 Amino Acid - New Insights and Roles in Plant and Animal

DOI: 10.1016/0022-0981(91)90007-J

DOI: 10.1016/0168-9622(87)90064-9

mib.2006.01.001

Currently, wide world research is focused on sustainable development and the demand for innovative clean technologies, nevertheless natural potential reconsideration could represent a viable solution for the identification and design of new pharmacological agents from renewable resources. The main reason consists of special properties of these natural derivates: immunomodulating activity with continuously perfectible selectiv‐ ity and efficiency. Plants and herb extracts have been used for centuries as traditional medicines, throughout the entire world. Romanian phytotherapy represents practically a very important part of our traditional knowledge and heritage. Therapeutic properties of plant active principles still continue to be the subject of many researches. In this chapter, an overview of plant bioactive molecules from the perspective of modern phytochemis‐ try is presented. A special part is devoted to a very special medicinal plant, Viscum *album,* in particular identification of amino acids and thionins from mistletoe.

**Keywords:** phytochemicals, secondary metabolites, analytic methods

## **1. Introduction**

Since ancient times, people have searched and found in nature remedies for various dis‐ eases [1, 2]. Romanian tradition pays a special attention to plants which attributes them the properties of living beings (soul, feeling, hearing and sight). Also there is an extraordinary relation between human beings and nature, an almost mystical interdependence. Most often the healing herbs were considered sacred. Phytotherapy origins are lost in the mists of time. In Romania, the traditional medicine has a very long history. Platon, Herodot and Pedanos Dioscoride have mentioned about the herbal medical system from Dacia and medicinal plants

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

used by our ancestors [1]. In Romanian tradition, there is a ritual harvesting these herbs which requires strict compliance with the optimal schedule at a specified date and time. Such is the case of belladonna (Atropa *belladonna)* that is harvested on full moon only from April–May period, before Pentecost. Medicago *falcate* known as earth vortex must be collected only on harvest time. Melilotus *officinali* is plucked only on Sanziene holiday and on Cross day, two important Romanian holidays. It is believed that after this period the plant loses its proper‐ ties. Romanian traditional medicine involves a very large number of heal plants: twigs, buds, bark and leaves of trees (alder, sambucus), flowers, seeds, stems or roots from plants. Some of the healing herbs were specific to Romanian herbal medicine: Salicornia *herbacea*, Anchusa *officinalis*, Actaea *spicata*, Symphytum *officinale*, Verbascum *thapsus*, Urtica *dioica*, Cicuta *virosa*, Typha *angustifolia,* Chelidonium *majus*, Bryonia *alba L.,* Thymus *vulgaris L., Alisma plantago‐ aquatica L.,* Hyoscyamus *niger L.,* Verbascum *phlomoides L.,* Achillea *millefolium L.,* Veratrum *album,* Clemantis *vitalba L.,* Potentilla *reptans L.,* Lappa *maior Gartn.,* Datura *stramonium L.,* Dipsacus *pilosus L.,* Erythraea *centaurium Pers.,* Mentha *piperita L.,* Cynoglossum *officinale L.,* Lithospermum *arvense L.* and Galim *verum* [3]. But then their use was spread throughout Balkan areal and Europe. Currently, it is widely used for *Symphytum officinale for its* anti‐inflammatory and wound healing activity. Withal, this plant has a high content of allantoin, one of the active principles of the plant it became more important as an ingredient in cosmetics [4–7].

Recent studies on medicinal plants assigned the therapeutic capacity of medicinal plants to their complex structure composed mainly from highly bioactive compounds, minerals, vita‐ mins, etc. [2].

Generally, medicines contain just one active substance, synthetically, whereas medicinal plants are practically a mixture of over dozens or even hundreds of chemicals that act syn‐ ergistically [2–3]. Moreover, medicinal plants contain a large amount of vitamins and miner‐ als, easily assimilated by human body. Many recent studies demonstrate that vitamins and minerals obtained through chemical synthesis have not the same beneficial effect as similar natural products. It may be due to the fact that in natural products there is a synergistic and complementary action between vitamins, minerals and enzymes, while synthetic compounds (vitamins or minerals) are isolated and even obtained as a different enantiomeric form [8–10]. On the other hand, drugs present other major disadvantages compared with medicinal plants: (i) various side effects; (ii) contraindications; (iii) interactions with other substances; (iv) drug resistance (drug dependence); (v) expensive and (vi) long time consuming research [8]. In comparison, natural compounds present a superior structural diversity, complex structure and multiple stereocenters [10–12]. These are just few arguments that may tilt the scales in favor of herbal medicines. Moreover, World Health Organization (WHO) aims to increase the integration of traditional medicine in order to improve health care system [13].

#### **2. Plant metabolite**

Paramount importance of botanic products for humanity is due mainly to their phytocom‐ pounds, active principles with therapeutic properties. Several studies have investigated these plant‐derived compounds [14–19]. Depending on the role they hold in living organisms, natural substances are divided in the next major categories: (i) *primary metabolites*, molecules common to all biological systems (proteins, fats, sugars) and (ii) *secondary metabolites*, com‐ pounds that could be specific for different species as a direct result of the evolution process of a particular phylogenetic group [16, 18–20]. **Figure 1** shows a schematic representation of plant metabolites [16–20].

used by our ancestors [1]. In Romanian tradition, there is a ritual harvesting these herbs which requires strict compliance with the optimal schedule at a specified date and time. Such is the case of belladonna (Atropa *belladonna)* that is harvested on full moon only from April–May period, before Pentecost. Medicago *falcate* known as earth vortex must be collected only on harvest time. Melilotus *officinali* is plucked only on Sanziene holiday and on Cross day, two important Romanian holidays. It is believed that after this period the plant loses its proper‐ ties. Romanian traditional medicine involves a very large number of heal plants: twigs, buds, bark and leaves of trees (alder, sambucus), flowers, seeds, stems or roots from plants. Some of the healing herbs were specific to Romanian herbal medicine: Salicornia *herbacea*, Anchusa *officinalis*, Actaea *spicata*, Symphytum *officinale*, Verbascum *thapsus*, Urtica *dioica*, Cicuta *virosa*, Typha *angustifolia,* Chelidonium *majus*, Bryonia *alba L.,* Thymus *vulgaris L., Alisma plantago‐ aquatica L.,* Hyoscyamus *niger L.,* Verbascum *phlomoides L.,* Achillea *millefolium L.,* Veratrum *album,* Clemantis *vitalba L.,* Potentilla *reptans L.,* Lappa *maior Gartn.,* Datura *stramonium L.,* Dipsacus *pilosus L.,* Erythraea *centaurium Pers.,* Mentha *piperita L.,* Cynoglossum *officinale L.,* Lithospermum *arvense L.* and Galim *verum* [3]. But then their use was spread throughout Balkan areal and Europe. Currently, it is widely used for *Symphytum officinale for its* anti‐inflammatory and wound healing activity. Withal, this plant has a high content of allantoin, one of the active

principles of the plant it became more important as an ingredient in cosmetics [4–7].

integration of traditional medicine in order to improve health care system [13].

Paramount importance of botanic products for humanity is due mainly to their phytocom‐ pounds, active principles with therapeutic properties. Several studies have investigated these plant‐derived compounds [14–19]. Depending on the role they hold in living organisms,

mins, etc. [2].

210 Amino Acid - New Insights and Roles in Plant and Animal

**2. Plant metabolite**

Recent studies on medicinal plants assigned the therapeutic capacity of medicinal plants to their complex structure composed mainly from highly bioactive compounds, minerals, vita‐

Generally, medicines contain just one active substance, synthetically, whereas medicinal plants are practically a mixture of over dozens or even hundreds of chemicals that act syn‐ ergistically [2–3]. Moreover, medicinal plants contain a large amount of vitamins and miner‐ als, easily assimilated by human body. Many recent studies demonstrate that vitamins and minerals obtained through chemical synthesis have not the same beneficial effect as similar natural products. It may be due to the fact that in natural products there is a synergistic and complementary action between vitamins, minerals and enzymes, while synthetic compounds (vitamins or minerals) are isolated and even obtained as a different enantiomeric form [8–10]. On the other hand, drugs present other major disadvantages compared with medicinal plants: (i) various side effects; (ii) contraindications; (iii) interactions with other substances; (iv) drug resistance (drug dependence); (v) expensive and (vi) long time consuming research [8]. In comparison, natural compounds present a superior structural diversity, complex structure and multiple stereocenters [10–12]. These are just few arguments that may tilt the scales in favor of herbal medicines. Moreover, World Health Organization (WHO) aims to increase the Bioactive molecules are basically those secondary metabolites exhibiting therapeutic, prevent‐ ing, toxicological and immunostimulating activity [16–20]. The most known plant‐derived bioactive compounds are presented in **Figure 2**.

**Figure 2.** Schematic representation of plant bioactive compounds.

Biological activity of these compounds has been extensively investigated in particular in the last decades [4–32]. Thus, it demonstrated that there is a close connection between the chemi‐ cal structure of the natural active principles (functional group types, number and position related to carbon skeleton, substitution in aromatic ring, stereochemistry, side chain length, saturation, etc.) [17, 20, 22, 25, 27, 34]. The role of metabolites in human organism is briefly presented in **Table 1**. And some examples of these compounds are shown in **Table 2**.

**Table 1.** Biologic activity of main groups of natural compounds.

**Table 2.** Some well‐known examples of plant metabolites.

## **3. Profiling of plant bioactive molecule**

Achievement of the natural plant bioactive molecules profile involves more consecutive stages (**Figure 3**) [14, 17, 18].

#### **3.1. Selection of plant species**

Biological activity of these compounds has been extensively investigated in particular in the last decades [4–32]. Thus, it demonstrated that there is a close connection between the chemi‐ cal structure of the natural active principles (functional group types, number and position related to carbon skeleton, substitution in aromatic ring, stereochemistry, side chain length, saturation, etc.) [17, 20, 22, 25, 27, 34]. The role of metabolites in human organism is briefly

presented in **Table 1**. And some examples of these compounds are shown in **Table 2**.

Terpenoid Antimicrobial, antiviral, antiviral, anthelmintic, antibacterial, anticancer, antimalarial, anti‐

Phenolics acids Anticarcinogenic and antimutagenic, anti‐inflammation and anti‐allergic [16, 20, 25, 31–35] Alkaloids Antispasmodic, antimalarial, analgesic, diuretic activities, local anesthetic, antihypertensive,

Flavonoids Antioxidant activity, cardiovascular protective, anti‐inflammatory, hepatoprotective, antiviral,

antihepatotoxic and hepatoprotective, anticoagulant, neuroprotective, antioxidant [16, 20, 24–27, 34]

Saponins Antitumor, antiviral, antifungal, anti‐inflammatory, immunostimulant, antihypoglycemic,

Tannins Antioxidant, anti‐carcinogenic, diuretics, hemostatic, anti‐mutagenic, metal ion‐chelators,

antiasthma, antimalarials, diuretic, bactericidal [14–16, 20, 21]

**Secondary metabolites Important molecules References**

**Terpenes** *Hemiterpene*: isoprene, isovaleric acid [15, 34] *Monoterpene*: limonele, eucalyptol, menthol, nerol, citral

**Flavonoids** *Flavones:* luteolin, diosmetin, apigenin [15, 22, 23] *Flavonols:* quercetin, myricetin, rutin, kaempferol

*Anthocyanidin:* cyanidin, delphinidin, peonidin, petunidin

nicotine, strychnine, codeine, ephedrine, dopamine, serotonine, vinblastine, vincristine, brucine, capsaicin, solanine, tomatine, choline, [15, 21, 34]

**Alkaloids** Caffeine, piperine, atropine, berberine, morphine, quinine, cocaine,

*Sesquiterpene*: zinziberene, farnesol *Diterpene*: cafestol, retinal, retinol

*Flavanones:* hesperetin, naringenin *Flavanonol:* silymarin, taxifolin *Isoflavones:* daidzin, genistin

*Sesterterpenes*: bulgarene, farnesol, lindarene *Triterpene*: provitamin A, betulin, cymarin *Tetraterpene*: lycopen, α si β carotenoids *Polyterpene*: vitamin E, gutta‐percha

etc.

**Table 1.** Biologic activity of main groups of natural compounds.

**Compound type Pharmacological properties**

212 Amino Acid - New Insights and Roles in Plant and Animal

inflammatory [15, 34]

antibacterial [20, 22–24, 34]

antiseptic, [14, 16, 20, 25, 28–32]

First and foremost stage is required to evaluate the existing ethnomedicinal studies, che‐ motaxonomical data regarding a particular medicinal plant, information collected from different historic documents, traditional knowledge from even local quacks and specialists [14, 37].

**Figure 3.** Flowchart of plant bioactive molecules profiling.

#### **3.2. Collection and identification of plant species**

This represents a key stage required to afford a reliable profile of natural active principles. And involve the next steps:

#### **3.3. Preparation of plant material (drying, micronisation, etc.)**

The botanical material processing is needed to avoid the degradation of plant bioactive com‐ pounds [14]. The drying is recommended to be performed in areas‐controlled atmosphere (absence of humidity, well‐ventilated, constant temperature).

The dried botanic material is subjected to micronization process through mechanical tech‐ niques. The other methods of plant sample preparation involve: (i) botanic material homog‐ enization or (ii) plant maceration [14, 39, 42].

This step aims to minimize the sample particle dimensions and thus to enhance the extraction yield [14].

#### **3.4. Extraction and isolation of bioactive molecules**

This is the key stage in evaluation of natural bioactive compounds.

(a) *Extraction and separation techniques*: In literature, there are many studies on extraction of cer‐ tain groups of plant metabolites. However, the selectivity of conventional extraction meth‐ ods (soxhlet extraction, hydrodistillation, maceration, percolation, steam distillation, etc.)

**Table 3.** Plant contamination: chemical assays.

are at least moderate and economically inefficient (energy, hazardous reagents consump‐ tion, time and temperature) [18, 39–42]. The other main disadvantages of these techniques are (i) not environment friendly; (ii) high possibility of degradation of thermostable active principles and (iii) additional steps (extract concentration, cleanse) [39–42]. Advanced ex‐ traction processes (solid‐phase extraction, ultra‐sound‐assisted extraction, microwave‐as‐ sisted extraction, supercritical fluid extraction, pulsed electric field extraction, pressurized liquid extraction, enzyme‐assisted extraction, surfactant‐mediated extraction) have mini‐ mized many of these shortcomings. Usually, the separation of a particular group of bioac‐ tive compounds from a complex natural product required a selective separation strategy based on phytochemicals partition in several different polarity solvents [43]. Nevertheless, natural product chemistry research concerns the development of new and highly efficient extraction techniques. Recent studies have reported that calixarenes could represent an attractive opportunity in this regard [44].

(b) *Isolation methods*: The physical properties (solubility, molecular weight, stability, dipole moment, etc.) of targeted bioactive compounds are essential for an efficient isolation method [39, 41, 42]. Another important factor is the nature of extraction solvent [39]. Generally, based on existing databases, the plant metabolites isolation are carried out through chromatographic methods: thin chromatography (TLC), flash chromatography, high performance liquid chromatography (HPLC), high‐performance thin‐layer chroma‐ tography (HPTLC), gas chromatography (GC) or Fourier transform infrared spectroscopy (FT‐IR) [14, 39, 41, 42]. A biological material previously uninvestigated and is in demand to develop an appropriate isolation procedure that require following additional steps: (i) phytochemical evaluation; (ii) bioassay (immunoassay (monoclonal antibodies) [14, 39].

#### **3.5. Identification and structural elucidation (chemical screening)**

This is the forefront but also the most difficult step in natural product chemistry. Achievement of the bioactive molecules complete profile requires the cutting‐edge technology and advanced knowledge specialists. Investigation on new natural compounds entails a larger work vol‐ ume determined mainly by the absence of plant scientific data [14, 39, 45–48]. Plant bioactive molecules profiling is based on various spectroscopic techniques, advanced chromatographic (hyphenated techniques) methods and a complete morphostructural characterization pro‐ cedure using X‐ray crystallographic techniques, polarimetry and electronic microscopy (**Table 4**) [14, 39, 45–48]. An optimal strategy based on high‐tech technology provides fast and highly efficient complete structural information about the targeted compounds [39, 42, 47, 48]. **Table 5** shows the main analytical techniques applied in natural bioactive compounds chemical screening [14, 39, 45–48].

#### **3.6. Biological and pharmacological screening**

**3.2. Collection and identification of plant species**

214 Amino Acid - New Insights and Roles in Plant and Animal

**3.3. Preparation of plant material (drying, micronisation, etc.)**

(absence of humidity, well‐ventilated, constant temperature).

enization or (ii) plant maceration [14, 39, 42].

**3.4. Extraction and isolation of bioactive molecules**

This is the key stage in evaluation of natural bioactive compounds.

**Plant contamination assay Analytical method**

Microbial content HPLC‐MS, etc

**Table 3.** Plant contamination: chemical assays.

And involve the next steps:

ecules profile [39].

yield [14].

This represents a key stage required to afford a reliable profile of natural active principles.

(a) Procurement of botanic component only from sources with guaranteed good agriculture and collection practices. An essential step demand to investigate a possible microbial, pes‐ ticide or heavy metals contaminations to avoid adversely affect the results of the chemical screening of bioactive metabolites, increased the time and cost of studies [18, 36, 37]. **Table 3** presents the main analytical techniques used to detect a possible plant contamination. (b) Plant taxonomic or genetic identification [18, 36, 37]. A modern method for authentifica‐ tion the botanic precursor use genomic analysis (DNA barcoding method) [38]. Research has been shown that biodiversity and plant growth environmental conditions (tempera‐ ture, humidity, soil physic and chemical properties) could influence the bioactive mol‐

The botanical material processing is needed to avoid the degradation of plant bioactive com‐ pounds [14]. The drying is recommended to be performed in areas‐controlled atmosphere

The dried botanic material is subjected to micronization process through mechanical tech‐ niques. The other methods of plant sample preparation involve: (i) botanic material homog‐

This step aims to minimize the sample particle dimensions and thus to enhance the extraction

(a) *Extraction and separation techniques*: In literature, there are many studies on extraction of cer‐ tain groups of plant metabolites. However, the selectivity of conventional extraction meth‐ ods (soxhlet extraction, hydrodistillation, maceration, percolation, steam distillation, etc.)

Heavy metals Atomic absorption spectroscopy, ICP‐MS, etc. Pesticide or/and herbicide residues GC‐MS, mass spectrometry, HPLC‐MS, etc.

> There are various methods designed to investigate the biological activity of a targeted natural compounds. An optimal procedure must fulfill several criteria: fast, simple, reli‐ able, high sensibility and selectivity, availability and low cost. Bioactivity evaluation for a

**Table 4.** A brief overview of bioactive molecules profiling tools [39, 42, 47, 48].

**Plant sample Propose structure Abbreviation SIM (selected‐ion monitoring)**

**Fourier transform infrared spectroscopy**

(a) Electron impact mass spectrometry (EIMS) (b) Chemical ionization mass spectrometry (CIMS) (c) Electrospray ionization mass spectrometry (ESIMS) (d) Electrospray ionization mass spectrometry (ESIMS) (e) Fast atom bombardment mass spectrometry (FABMS)

Nuclear Magnetic Resonance **(NMR) spectroscopy:**

H‐1

**Chromatography methods Gas‐chromatography**: GC, GC‐MS, GC‐TOF‐MS; GC‐MS/MS, two‐dimensional GC

H‐1

coupled with mass spectrometry (GC×GC‐MS), GC‐FTIR, GC‐NMR

H ROESY, 1

H COSY, 1

**Liquid chromatography**: LC/UV; LC/MS; LC/UV/MS; LC/MS‐MS; LC/NMR, LC‐

H‐1

H‐1

H TOCSY, 1

HNMR, 13CNMR, 13CDEPT, 13CPENDANT,13C J

H‐13C HMBC, 1

H DQF‐COSY, 1H‐1H COSY‐lr,

H‐13C HMQC,

(a) *One‐dimensional techniques:* 1

(b) *Two‐dimensional techniques:* 1

H‐13C HSQC,HSQCTOCSY

1H‐1H NOESY, 1

UV‐DAD, HPLC‐NMR

mod.

1

**Table 4.** A brief overview of bioactive molecules profiling tools [39, 42, 47, 48].

**Other analytic techniques** XRD; TEM; polarimetry

**Mass spectroscopy:**

Glutamic acid Glu 38, 40 Phenylalanine Phe 56, 57 Ornithine Orn 59,60,61 Histidine His 84, 89 Tyrosine Tyr 61, 63, 94 Glycine Gly 116, 74 Homoserine HSER 102,128, 143 Asparagine Asn 155, 69 Isoleucine Ile 171, 129 Valine Val 158, 116 Threonine Thr 160, 101 β‐Alanine β Ala 158, 98 Valine Val 158,72 β‐Alanine β Ala 129, 158, 98 Homoserine HSER 102, 128, 143 Asparagine Asn 155, 69

 **(hexane)** Cystine C‐C 41, 42

**Spectroscopic methods UV‐Vis spectroscopy**

216 Amino Acid - New Insights and Roles in Plant and Animal

**V**1

**Table 5.** Compounds identified through GC‐MS analysis.

plant extraction (plant fraction) is usually performed through *in vitro* or/and *in vivo* studies [14, 49, 50]. Most often, *in vitro* studies are focused on the evaluation of specific cell biology (cell count, growth rate, metabolic rate, cell function and protein expression). *In vitro* tests are conducted on various animal or human cell cultures, enzymes, depending on targeted natural compound biological activity [14, 49, 50]. For instance, the bioassays for antitu‐ mor activity are conducted on tumor experimental models. Complementary, the immuno‐ logical activity on normal cell culture should be monitored. The cells will be analyzed by fluorescence microscopy and will be quantified to establish the degree of apoptosis and implicitly the cell viability. Also, the time‐lapse video microscopy can be used to evaluate the bioactive phytochemicals [43]. The *in vivo* biotests are applied on animals (mice, rats, pigs, etc.).

Natural compounds bioassay can be demonstrated also using computational chemical meth‐ ods: quantitative structure‐activity relationship (2D or 3D QSAR) and structure‐activity rela‐ tionship (SAR) [75, 76].

Regarding the antioxidant activity of natural compounds, literature demonstrates the existence of a considerable number of studies using two analytical techniques: electron spin resonance (ESR) and chemiluminescence. But the obtained results depend on the type of reactant (specific free radical) used [51]. Electrochemistry, especially by the instrumentality of voltammetry has been shown to be a useful method for the investigation of the antioxidant activity of different targeted compounds [52].

## **4. Natural compounds in Viscum** *album* **as an example of medicinal plant**

One of the most renowned medicinal plants is *Viscum album* L., which has very different appli‐ cations: tonic, cardiotonic, antiviral, cancer, etc. In different European countries, mistletoe extracts are prepared and commercially available (*Iscador, Isorel, Eurixor, Plenesol, Vysorel, Lektinol, Helixor,* etc.) as alternative treatment for cancer therapy [53–58].

First information on the use of this plant for its benefits on the human body dates back to ancient times. The druids and Celts considered as sacred mistletoe that grows on oak. Over time, peoples were attributed a special symbolism to this evergreen plant: immortality, knowl‐ edge, wisdom, universal panacea, love, fortune, fertility, etc. [54, 57]. There are considered that magical properties of mistletoe are kept only if the complied both the collection ceremony: a golden knife in a special moment of day before full moon, on right period (summer or winter solstice) [54].

In traditional medicine, Viscum are used for various health benefits: poison antidote, anti‐age, anti‐inflammatory, fertility, antitumor, headaches, preventing epilepsy, cure for plague, ery‐ sipelas, etc. [53–55].

Many studies have been carried out for determination of the outstanding biological effects: antiproliferative activity, antitumor activity, antiviral activity, cardiovascular, immunostimu‐ lant and antidiabetic [56, 58–65]. But the extremely complex chemical composition of this plant has not been precisely determined yet. Nevertheless, several secondary metabolites such as flavonoids, alkaloids, steroids, terpenoids were detected [66]. However, research has demonstrated that viscum chemical composition varies depending on (i) the type of host tree on which it grows (oak, maples, acacia, robinia, poplar, etc.), (ii) time of harvesting, (iii) envi‐ ronmental conditions and (iv) extraction method [56, 67].

The attempts to establish the compounds responsible for biological, immunomodulating and cytotoxic activity had targeted especially the lectins and viscotoxins as active components [56, 67]. Nevertheless, these compounds represent only a small content of percent from the entire plant peptide content which is not fully understood in terms of chemical structure and biological activity. Relatively recent research had emphasized on the presence of other peptide derivate, viscumamide with antitumor activity [68]. However, there are still many compounds pharmacologically active that can be found. Continuous development of analysis techniques can provide important information about new highly bioactive compounds iso‐ lated from plant extracts.

#### **4.1. Importance of natural small peptide**

plant extraction (plant fraction) is usually performed through *in vitro* or/and *in vivo* studies [14, 49, 50]. Most often, *in vitro* studies are focused on the evaluation of specific cell biology (cell count, growth rate, metabolic rate, cell function and protein expression). *In vitro* tests are conducted on various animal or human cell cultures, enzymes, depending on targeted natural compound biological activity [14, 49, 50]. For instance, the bioassays for antitu‐ mor activity are conducted on tumor experimental models. Complementary, the immuno‐ logical activity on normal cell culture should be monitored. The cells will be analyzed by fluorescence microscopy and will be quantified to establish the degree of apoptosis and implicitly the cell viability. Also, the time‐lapse video microscopy can be used to evaluate the bioactive phytochemicals [43]. The *in vivo* biotests are applied on animals (mice, rats,

**Plant sample Propose structure Abbreviation SIM (selected‐ion monitoring)**

Glutamic acid Glu 38, 40 Phenylalanine Phe 56, 57 β‐Alanine β Ala 129, 158, 98 Ornithine Orn 59,60,61 Glycine Gly 116, 74 Isoleucine Ile 170, 130 Histidine Hys 84, 87 Glutamine Gln 84, 187 Valine Val 158,72 Tyrosine Tyr 61, 63, 94 Lysine Lys 170, 129 Homoserine HSER 102,128, 143 Proline‐hydroxyproline (dipeptide) PHP 156, 186 3‐Methyl‐cysteine 1MHIS 172,259,130 Homocysteine HCYS 142, 203 Glycyl‐glycine (dipeptide) Gly‐Gly 117, 144, 201

 **(acetone)** Cystine C‐C 41,42

Natural compounds bioassay can be demonstrated also using computational chemical meth‐ ods: quantitative structure‐activity relationship (2D or 3D QSAR) and structure‐activity rela‐

Regarding the antioxidant activity of natural compounds, literature demonstrates the existence of a considerable number of studies using two analytical techniques: electron spin resonance (ESR) and chemiluminescence. But the obtained results depend on the type of reactant (specific free radical) used [51]. Electrochemistry, especially by the instrumentality of voltammetry has

pigs, etc.).

**V**4

tionship (SAR) [75, 76].

**Table 5.** Compounds identified through GC‐MS analysis.

218 Amino Acid - New Insights and Roles in Plant and Animal

From the multitude of classes of biomolecules isolated from natural compounds, a special attention has been given to amino acids and small peptides due to their remarkable properties (high solubility, strong antioxidant, reduce high blood pressure, analgesic, anti‐tumor, immu‐ nomodulatory, etc.). In addition, these biologically active compounds have various applica‐ tions in pharmacology, cosmetics, sports and food.

In plants, these biomolecules are involved also in defense mechanisms against various classes of pathogens (bacteria, fungi, parasites, etc.) [69, 70].

Given that cancer is the second leading cause of death in European countries, and one of the most imminent health problems in the developed world [71–73], there is an overwhelming interest for new efficient antitumor agents with high bioavailability and minimal side effects. In this context, research on plant bioactive molecules with putative antitumor activity is even more justified.

Thionins represent a special class of small peptide with multiple disulfide bonds [43, 68, 69]. They have shown cytotoxicity and antitumor activity [69, 70]. Research has reported that mistletoe contains several types of thionins: viscothionin A1, viscothionin A2, viscothionin A3, viscothionin B, viscothionin C1, viscothionin D, viscothionin E, viscothionin P1 [69, 70].

#### **4.2. Determination of amino acids and thionins from Viscum** *album*

In an effort to detect the amino acids and thionins from Viscum *album* a selective partition strategy based on solvents with different polarities (methanol, hexane and carbon tetrachlo‐ ride) was developed [43]. The plant material (Viscum *album* leaves and young leaves from Quercus *robur*) was obtained from a collection taken in December 2015 in Timis, Romania. Plant sample was identified at Victor Babes University of Medicine and Pharmacy Timisoara. The botanical material was dried and then finely ground in a ball mill. A plant sample (3 g) was placed in a 100 mL volumetric flask containing 50 mL of methanol. The result mixture was sonicated for 60 min at 40°C, with a frequency of 50 kHz. Then the solution was filtered through a 0.30 μm pore size *filter* and subsequently extracted with the following organic solvents: *n*‐hexane (V1 ) and carbon tetrachloride and (V2 ). The separation of thionins was car‐ ried on the next experiment: 2 g of sample was extracted successively with petroleum ether (30 mL) and acetone (30 mL) [43]. Identity of the compounds from the obtained viscum frac‐ tions: V1 (hexane), V2 (CCl4 ), V3 (petroleum ether) and respectively, fraction V4 (acetone) was performed using GC‐MS and TOF MS methods.

#### **4.3. GC‐MS analysis**

The GC‐MS chromatograms for mistletoe extract fraction V1 –V5 are presented in **Figure 4(a)**–**(d)**.

The results of design isolation strategy based on different solvent polarity were analyzed through GC‐MS [43]. The identified compounds are presented in **Table 5**; after a careful com‐ parison with spectral database, NIST/NBS was used to compare the results of analysis [43].

#### **4.4. TOF‐MS analysis**

The mass spectra of mistletoe fractions V1 –V4 (acquired in positive ion mode, in a mass range of 100–3000 m/z) are presented in **Figure 5(a)**–**(d)**.

**Figure 4.** TIC of (a) V1 extract, (b) V2 extract, (c) V3 extract and (d) V4 extract.

(high solubility, strong antioxidant, reduce high blood pressure, analgesic, anti‐tumor, immu‐ nomodulatory, etc.). In addition, these biologically active compounds have various applica‐

In plants, these biomolecules are involved also in defense mechanisms against various classes

Given that cancer is the second leading cause of death in European countries, and one of the most imminent health problems in the developed world [71–73], there is an overwhelming interest for new efficient antitumor agents with high bioavailability and minimal side effects. In this context, research on plant bioactive molecules with putative antitumor activity is even

Thionins represent a special class of small peptide with multiple disulfide bonds [43, 68, 69]. They have shown cytotoxicity and antitumor activity [69, 70]. Research has reported that mistletoe contains several types of thionins: viscothionin A1, viscothionin A2, viscothionin A3, viscothionin B, viscothionin C1, viscothionin D, viscothionin E, viscothionin P1 [69, 70].

In an effort to detect the amino acids and thionins from Viscum *album* a selective partition strategy based on solvents with different polarities (methanol, hexane and carbon tetrachlo‐ ride) was developed [43]. The plant material (Viscum *album* leaves and young leaves from Quercus *robur*) was obtained from a collection taken in December 2015 in Timis, Romania. Plant sample was identified at Victor Babes University of Medicine and Pharmacy Timisoara. The botanical material was dried and then finely ground in a ball mill. A plant sample (3 g) was placed in a 100 mL volumetric flask containing 50 mL of methanol. The result mixture was sonicated for 60 min at 40°C, with a frequency of 50 kHz. Then the solution was filtered through a 0.30 μm pore size *filter* and subsequently extracted with the following organic

ried on the next experiment: 2 g of sample was extracted successively with petroleum ether (30 mL) and acetone (30 mL) [43]. Identity of the compounds from the obtained viscum frac‐

The results of design isolation strategy based on different solvent polarity were analyzed through GC‐MS [43]. The identified compounds are presented in **Table 5**; after a careful com‐ parison with spectral database, NIST/NBS was used to compare the results of analysis [43].

–V4

(petroleum ether) and respectively, fraction V4

–V5

). The separation of thionins was car‐

are presented in **Figure 4(a)**–**(d)**.

(acquired in positive ion mode, in a mass range

(acetone) was

**4.2. Determination of amino acids and thionins from Viscum** *album*

) and carbon tetrachloride and (V2

tions in pharmacology, cosmetics, sports and food.

220 Amino Acid - New Insights and Roles in Plant and Animal

of pathogens (bacteria, fungi, parasites, etc.) [69, 70].

more justified.

solvents: *n*‐hexane (V1

**4.3. GC‐MS analysis**

**4.4. TOF‐MS analysis**

(hexane), V2

(CCl4

performed using GC‐MS and TOF MS methods.

The mass spectra of mistletoe fractions V1

of 100–3000 m/z) are presented in **Figure 5(a)**–**(d)**.

), V3

The GC‐MS chromatograms for mistletoe extract fraction V1

tions: V1

**Figure 5.** Positive ion mode TOF‐MS of (a) V1 extract, (b) V2 extract, (c) V3 extract and (d) V4 extract.

#### **4.5. FT‐IR spectroscopy**

The solid (fine grounded) sample of mistletoe was analyzed also through FT‐IR spectroscopy (**Figure 6**). It has been aimed to identify the absorptions bands specific to amino acids and peptides from: (i) 3400 cm−1 (O‐H and N‐H bonds); (ii) 3330–3130 cm−1 (NH3 + groups); (iii) symmetric absorption at 2080–2140 cm−1or 2530–2760 cm−1; (iv) 1500–1600 cm−1 (ammonium group deformation vibrations); (v) 1610–1660 cm−1 (carboxylate group); (vi) 1724–1754 cm−1 (carbonyl vibrations) and (vii) vibrations bands characteristic for thionins (1687, 1675, 1663, 1654, 1644, 1632, 1621, 1611) [45, 74].

The FT‐IR spectra were recorded using a Universal ATR accessory (UATR) and mistletoe samples 20 mg and 30 mg, respectively, mixed with KBr.

From the spectra analysis, the presence of bands specific to amino acids, thionins and pep‐ tides can be noticed.

**Figure 6.** The FT‐IR spectra for the mistletoe sample.

#### **5. Conclusions**

The collective results suggest that chosen separation solvent and analytic strategies are effi‐ cient for isolation and identification of targeted natural compounds from mistletoe sample. Further studies on mistletoe extract are necessary to gain insight into the complete bioactive molecules profile with high antitumor activity.

Continuous development of analysis techniques can provide important information about highly bioactive molecules isolated from natural compounds. Particular importance must be paid to the choice of optimal separation methods which must be simple but highly selective and efficient for separation of a certain class of natural metabolites. A special emphasis has been given to identify the peptides because it was considered that nature of amino acids, their quantity in plant and the ratio to known peptides for their high bioactivity may be relevant to their anticancer action. Research on small peptide with pharmacological activity continues to be a topic of great interest to the current science due to their special high biological activity, chemical stability, bioavailability, etc. From this perspective, further research will allow to predict the formulation of the peptide profile from natural extract with a specific biological effect with application in cancer prevention or therapy.

## **Acknowledgements**

We like to thank Dr. Andrei Bunaciu for his contribution to this chapter.

## **Author details**

**4.5. FT‐IR spectroscopy**

tides can be noticed.

**5. Conclusions**

molecules profile with high antitumor activity.

**Figure 6.** The FT‐IR spectra for the mistletoe sample.

1654, 1644, 1632, 1621, 1611) [45, 74].

222 Amino Acid - New Insights and Roles in Plant and Animal

20 mg and 30 mg, respectively, mixed with KBr.

The solid (fine grounded) sample of mistletoe was analyzed also through FT‐IR spectroscopy (**Figure 6**). It has been aimed to identify the absorptions bands specific to amino acids and

symmetric absorption at 2080–2140 cm−1or 2530–2760 cm−1; (iv) 1500–1600 cm−1 (ammonium group deformation vibrations); (v) 1610–1660 cm−1 (carboxylate group); (vi) 1724–1754 cm−1 (carbonyl vibrations) and (vii) vibrations bands characteristic for thionins (1687, 1675, 1663,

The FT‐IR spectra were recorded using a Universal ATR accessory (UATR) and mistletoe samples

From the spectra analysis, the presence of bands specific to amino acids, thionins and pep‐

The collective results suggest that chosen separation solvent and analytic strategies are effi‐ cient for isolation and identification of targeted natural compounds from mistletoe sample. Further studies on mistletoe extract are necessary to gain insight into the complete bioactive

+ groups); (iii)

peptides from: (i) 3400 cm−1 (O‐H and N‐H bonds); (ii) 3330–3130 cm−1 (NH3

Adina‐Elena Segneanu1 \*, Silvia Maria Velciov2 , Sorin Olariu2 , Florentina Cziple3 , Daniel Damian4 and Ioan Grozescu4

\*Address all correspondence to<EMAIL_ADDRESS>
1 Scient Analytics, SCIENT, Research Center for Instrumental Analysis, Cromatec, Plus, Ilfov, Romania

## **References**

[22] Kumar S, Pandey AK. Chemistry and biological activities of flavonoids: An overview. The Scientific World Journal. 2013:Article ID 162750

[5] Neagu E, Roman GP, Radu GL. Antioxidant capacity of some *Symphytum officinalis* extracts processed by ultrafiltration. Romanian Biotechnological Letters. 2010;**15**(4):5505–5511 [6] Alkan FU, Anlas C, Ustuner O, Bakirel T, Sari AB. Antioxidant and proliferative effects of aqueous and ethanolic extracts of *Symphytum officinale* on 3T3 Swiss albino mouse

fibroblast cell line. Asian Journal of Plant Science and Research. 2014;**4**(4):62–68

Surgery. 2005;**31**:873–880

224 Amino Acid - New Insights and Roles in Plant and Animal

USA. 2005. ISBN: 0‐7570‐0144‐0

Biomedical science. 2006;**2**:85–100

Herbal Medicine. 2016;**4**(4):59‐64

Metabolites. 2012;**2**(2):303–336

John Wiley & Sons. 2001. pp. 1–11

2014;**4**:17–31

org/news/nutri‐con‐truth‐about‐vitamins‐supplements

Research Journal of Phytochemistry. 2015;**9**(3):88–115

[7] Thornfeldt C. Cosmeceuticals containing herbs: Fact, fiction, and future. Dermatologic

[8] Taylor L. The Healing Power of Rainforest Herbs. Square One Publishers Inc, New York,

[9] Clement B. Nutri‐Con: The Truth About Vitamins & Supplements. The Vitamin Myth Exposed. Hippocrates Health Institute & OCA; 2005. https://www.organicconsumers.

[10] Nguyen LA, He H, Pham‐Huy C. Chiral drugs: An overview. International Journal of

[11] Lahlou M. The success of natural products in drug discovery. Pharmacology & Pharmacy.

[12] Phillipson JD. Phytochemistry and medicinal plants. Phytochemistry. 2001;**56**:237–243 [13] World Health Organization (WHO). Traditional Medicine Strategy 2014–2023. Hong‐ Kong, China: World Health Organization; 2013. pp.1‐78 1‐78. ISBN<PHONE_NUMBER>090 [14] Chikezie PC, Ibegbulem CO, Mbagwu FN. Bioactive principles from medicinal plants.

[15] Shakya AK. Medicinal plants: Future source of new drugs. International Journal of

[16] Capasso F, Gaginella TS, Grandolini G. Phytotherapy—A Quick Reference to Herbal Medicine. Berlin Heidelberg: Springer‐Verlag; 2003. ISBN 978‐3‐540‐00052‐5

[17] Soetan KO, Aiyelaagbe OO. The need for bioactivity‐safety evaluation and conservation of medicinal plants—A review. Journal of Medicinal Plants Research. 2009;**3**(5):324‐328

[18] Azmir J, Zaidul ISM, Rahman MM, Sharif KM, Mohamed A, Sahena F, Jahurul MHA, Ghafoor K, Norulaini NAN, Omar AKM. Techniques for extraction of bioactive com‐ pounds from plant materials: A review. Journal of Food Engineering. 2013;**117**:426‐436

[19] Dias DA, Urban S, Roessne U. A historical overview of natural products in drug discovery.

[20] Anulika NP, Ignatius EO, Raymond ES, Osasere OI, Abiola AH. The chemistry of natural product: Plant secondary metabolites. International Journal of Technology Enhancements and Emerging Engineering Research. 2016;**4**(8):1–8. ISSN 2347‐4289 [21] Woolley JG. Plant alkaloids. In: Encyclopedia of Life Sciences. Nature Publishing Group,

Sources Isolation, Characterisation and Biological Properties. London: Taylor & Francis; 2001. ISBN 0‐203‐26972‐1;

[51] Rior RLP, Wu X, Schaich K. Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. Journal of Agricultural and Food Chemistry. 2005;**53**(10):4290–4302

[38] Balachandran KRS. Mohanasundaram S, Sathishkumar R. DNA barcoding: A genomic‐ based tool for authentication of phytomedicinals and its products. Botanics: Targets and

[39] Sarker SD, Latif Z, Gray AI. Natural products isolation. 2nd ed. New Jersey, USA:

[40] Segneanu AE, Macarie CA, Pop RO, Balcu I. Combined microwave–acid pretreatment of the biomass. In: Shaukat SS, editor. Progress in Biomass and Bioenergy Production.

[41] Sasidharan S, Chen Y, Saravanan D, Sundram KM, Yoga Latha L. Extraction, isolation and characterization of bioactive compounds from plants' extracts. African Journal of

[42] Kaufmann B, Christen P. Recent extraction techniques for natural products: Microwave‐ assisted extraction and pressurized solvent extraction. Phytochemical Analysis.

[43] Segneanu AE, Grozescu I, Cziple F, Berki D, Damian D, Niculite CM, Florea A, Leabu M. *Helleborus purpurascens*—Amino acid and peptide analysis linked to the chemical and antiproliferative properties of the extracted compounds. Molecules. 2015;**20**:22170–22187

[44] Segneanu AE, Damian D, Hulka I, Grozescu I, Salifoglou A. A simple and rapid method for calixarene‐based selective extraction of bioactive molecules from natural products.

[45] Neda I, Vlazan P, Pop RO, Sfarloaga P, Grozescu I, Segneanu A‐E. Peptide and amino acids separation and identification from natural products. In: Krull IS, editors. Analytical

[46] Segneanu AE, Gozescu I, Dabici A, Sfirloaga P, Szabadai Z. Organic compounds FT‐IR spectroscopy. In: Uddin J, editor. Macro to Nano Spectroscopy. Croatia: InTech; 2012.

[47] Guo X, Lankmayr E. Hyphenated techniques in gas chromatography. In: Mohd MA, editor. Advanced Gas Chromatography—Progress in Agricultural, Biomedical and

[48] Ibekwe NN, Ameh SJ. Hyphenated techniques in liquid chromatography as current trends in natural products analysis. International Research Journal of Pure &Applied

[49] Wijesekera ROB. The Medicinal Plant Industry. CRC Press; Taylor & Francis Inc, Bosa

[50] Vlietinck AJ, Apers S. Biological screening methods in the search for pharmacologi‐ cally active natural products. In: Tringali C, editor. Bioactive Compounds from Natural

Industrial Applications. InTech; Croatia, 2012. pp.5–26. ISBN: 978‐953‐51‐0298‐4

Chemistry. Croatia: Intech; 2012. pp.135–146. ISBN 978‐953‐51‐0837‐5

Traditional, Complementary and Alternative Medicines. 2011;**8**(1):1–10

Therapy. 2015;**5**:77–84

226 Amino Acid - New Insights and Roles in Plant and Animal

2002;**13**(2):105–113

Humana Press Inc; 2006. ISBN 1‐58829‐447‐1

Amino Acids, Springer. 2016;**48**:849–858

pp.145–164. ISBN 978‐953‐51‐0664‐7

Chemistry. 2015;**7**(3):132‐149

Roca, USA. 1991. ISBN<PHONE_NUMBER>697

Croatia: In Tech; 2011. pp.223–238. ISBN 978‐953‐307‐491‐7

**Role of Amino Acids in Animal**

[63] Valentiner U, Pfuller U, Baum C, Schumacher U. The cytotoxic effect of mistletoe lectins I, II and III on sensitive and multidrug resistant human colon cancer cell lines in vitro.

[64] Antony S, Kuttan R, Kuttan G. Role of natural killer cells in iscador mediated inhi‐ bition of metastasis by adoptive immunotherapy. Immunological Investigations.

[65] Eno AE, Ibokette UE, Ofem OE, Unoh FB, Nkanu E, Azah N, Ibu JO. The effects of a Nigerian specie of *Viscum album* (Mistletoe) leaf extract on the blood pressure of normo‐ tensive and Doca‐induced hypertensive rats. Nigerian Journal of Physiological Sciences.

[66] Li Y, Zhao YL, Yang YP, Li XL. Chemical constituents of *Viscum album* var. Meridianum.

[67] Luczkiewicz M, Cisowski W, Kaiser P, Ochocka R, Piotrowski A. Comparative analysis of phenolic acids in mistletoe plants from various hosts. Acta Poloniae Pharmaceutica‐

[68] Poojary B, Belagali SL. Synthesis, characterisation and biological evaluation of cyclic peptides: Viscumamide, yunnanin A and evolidine. Zeitschrift fur naturforschung sec‐

[69] Larsson S. Mistletoes and thionins as selection models in natural products drug dis‐ covery.. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy 49. Uppsala: Acta Universitatis Upsaliensis; 2004. 65pp. ISBN

[70] Guzmán‐Rodríguez JJ, Ochoa‐Zarzosa A, López‐Gómez R, López‐Meza JE. Plant anti‐ microbial peptides as potential anticancer agents. BioMed Research International.

[71] Colegate SM, Molyneux RJ. Bioactive Natural Products—Detection, Isolation, and

[72] Simard EP, Engels EA. Cancer as a cause of death among people with AIDS in the United

[73] Tantry MA. Plant natural products and drugs: a comprehensive study. Asian Journal of

[74] Giudici M, Pascual R, de la Canal L, Pfüller K, Pfuller U, Villalain J. Interaction of vis‐ cotoxins A and B with membrane model systems: Implications to their mechanism of

[75] Hemmateenejad B, Javidnia K, Nematollahi M, Elyasi M. QSAR studies on the antiviral compounds of natural origin. Journal of the Iranian Chemical Society. 2009;**6**(2):420–435

[76] Kuramochi K. Synthetic and structure‐activity relationship studies on bioactive natural

products. Bioscience, Biotechnology, and Biochemistry. 2013;**77**(3):446–454

Structural Determination. 2nd ed. Taylor & Francis Group, LLC, USA; 2008

Biochemical Systematics and Ecology. 2011;**39**:849–852

Drug Research. 2001;**58**(5):373–379. ISSN 0001‐6837

tion b‐a journal of chemical sciences, 2005;**60**(12):1313–1320

States. Clinical Infectious Diseases. 2010;**51**(8):957–962

Traditional Medicines. 2009;**4**(6):241–249

action. Biophysical Journal. 2003;**85**:971–981

Toxicology. 2002;**171**(2‐3):187–199

228 Amino Acid - New Insights and Roles in Plant and Animal

2000;**29**(3):219‐231

2004;**19**(1‐2):33–38

978‐91‐554‐6824‐8;

2015:**11**:Article ID 735087

## **Amino Acid for Japanese Quails: Methodologies and Nutritional Requirement**

Danilo V.G. Vieira, Fernando G.P. Costa, Matheus R. Lima, José G.V. Júnior, Talita P. Bonaparte, Danilo T. Cavalcante, Sarah G Pinheiro, Marilu S Sousa, Ana C M Conti and Érika M Figueireido

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.68547

#### **Abstract**

The methodologies applied to chickens and laying hens, to determine the digestibility and requirement of protein and amino acids are used with quails, however, they need a more careful evaluation due to peculiarities inherent to the Coturnix genus, in order to provide consistent results. The nutritional requirements of the birds are determinate using the dose-response and the factorial method. Several mathematical models and techniques of diet formulation are allied to the dose-response method in determining nutritional requirements. The curvilinear (hyperbolic) models better portray population behaviour in response to increasing nutrient doses in diets. The reading model, allow a better estimation of the requirement, in relation to the mathematical models used in the dose-response method. The techniques of comparative slaughter and nitrogen balance are effective in determining the nutritional requirements of quails, however, the latter need to be corrected by the loss of nitrogen in the feathers in determining the requirements of crude protein and amino acids for maintenance. The protein-free diet, coupled with the industrial amino acid supplementation, provides more robust digestibility values, since it more effectively predicts the endogenous excretion pattern.

**Keywords:** amino acids, physiology, Japanese quails, methodologies, requirements

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **1. Introduction**

Created for various purposes (hunting, meat, ornamentation, eggs) the production of quail is a reality worldwide. Countries such as Spain, France, China and the United States stand out for the production of meat, however, when the production is intended to egg production, countries, as China, Japan and Brazil are highlights.

Quail farming in Brazil in the year 2015 reached a total of 21.99 million head, either for meat or for eggs and 447.47 million dozens of eggs [1], which means an increase of 8.1 and 13.9%, respectively, in relation to 2014.

The success of the activity in Brazil is due to the large producing companies that have settled in the territory and to the creation of research groups inserted in Academic Center, with studies directed to the genetic improvement, management and production, and nutrition of quail. The Centers that stand out are: Group of Studies and Poultry Technologies—Federal University of Paraiba, Areia, PB; Nucleus of Fish and Bird Studies—Federal University of Paraiba, Bananeiras, PB; Nucleus of Studies in Poultry Science and Technology—Federal University of Lavras, Lavras, MG. As well as research groups located at the University of Espirito Santo, Alegre—ES, Federal University of Minas Gerais, Belo Horizonte, MG, Maringa State University, Maringa, PR, and Federal University of Viçosa, Viçosa, MG, and the last three groups differ from the firsts, because they also present a breeding program. Worldwide countries such as India, France, Spain and Egypt also stand out with quail research.

[2] World studies on quails date back to 1992, and since 2002 the number of studies in the various research Centers has increased, both in the world and in Brazil. This advance in the Brazilian researches is concomitant with advances in methodologies for food evaluation and nutritional requirements [3, 4], in the knowledge of cellular biochemistry, physiology and animal nutrition, in the development of laboratories in the Research Centers, and in industrial manufacturing of amino acids, premix, etc.

Although of the same family (Phasianidae), commercial poultry, broilers, chickens and quail are of different genres. The latter belong to the genus Coturnix, while the former are of the Gallus genus. Faced with this taxonomic difference, quails have peculiar digestive physiology, and in addition, growth and early reproductive activity, and among others, have low feed intake, which gives them a higher rate of passage in the gastric tract. These differences denote a specific nutritional requirement, mainly protein and amino acid.

Several methodologies applied to chickens and laying hens [4] are effective in quail use; however, they need a more careful evaluation, due to peculiarities inherent to the Coturnix genus, in order to provide consistent results. Another aggravating factor is the lack of quail standard lineages that makes nutrition dynamism even more peculiar with quail. There are few reputable and reputable companies in Brazil that work on quail breeding and provide genetic material for sale. There are few reputable companies in Brazil that work on quail breeding and provide genetic material for sale.

There are two basic methods (dose response and factorial method) for determining the nutritional requirements of birds. However, several mathematical models and techniques for formulating diets that are allied, to the dose-response method, and techniques such as comparative slaughter (CS) and nitrogen balance (NB), used in the factorial model to predict the nutritional requirement values of crude protein and amino acids for birds.

In this chapter, we will discuss the peculiarities of Japanese quails in relation to broilers, laying hens and heavy matrices, and the need to use with criteria, the methodologies to estimate digestibility and requirement, and also review the use of mathematical models, diet formulation and the methodologies of CS and NB.

## **2. Peculiarities and methodologies**

#### **2.1. Peculiarities**

**1. Introduction**

research.

respectively, in relation to 2014.

232 Amino Acid - New Insights and Roles in Plant and Animal

manufacturing of amino acids, premix, etc.

and provide genetic material for sale.

countries, as China, Japan and Brazil are highlights.

Created for various purposes (hunting, meat, ornamentation, eggs) the production of quail is a reality worldwide. Countries such as Spain, France, China and the United States stand out for the production of meat, however, when the production is intended to egg production,

Quail farming in Brazil in the year 2015 reached a total of 21.99 million head, either for meat or for eggs and 447.47 million dozens of eggs [1], which means an increase of 8.1 and 13.9%,

The success of the activity in Brazil is due to the large producing companies that have settled in the territory and to the creation of research groups inserted in Academic Center, with studies directed to the genetic improvement, management and production, and nutrition of quail. The Centers that stand out are: Group of Studies and Poultry Technologies—Federal University of Paraiba, Areia, PB; Nucleus of Fish and Bird Studies—Federal University of Paraiba, Bananeiras, PB; Nucleus of Studies in Poultry Science and Technology—Federal University of Lavras, Lavras, MG. As well as research groups located at the University of Espirito Santo, Alegre—ES, Federal University of Minas Gerais, Belo Horizonte, MG, Maringa State University, Maringa, PR, and Federal University of Viçosa, Viçosa, MG, and the last three groups differ from the firsts, because they also present a breeding program. Worldwide countries such as India, France, Spain and Egypt also stand out with quail

[2] World studies on quails date back to 1992, and since 2002 the number of studies in the various research Centers has increased, both in the world and in Brazil. This advance in the Brazilian researches is concomitant with advances in methodologies for food evaluation and nutritional requirements [3, 4], in the knowledge of cellular biochemistry, physiology and animal nutrition, in the development of laboratories in the Research Centers, and in industrial

Although of the same family (Phasianidae), commercial poultry, broilers, chickens and quail are of different genres. The latter belong to the genus Coturnix, while the former are of the Gallus genus. Faced with this taxonomic difference, quails have peculiar digestive physiology, and in addition, growth and early reproductive activity, and among others, have low feed intake, which gives them a higher rate of passage in the gastric tract. These differences

Several methodologies applied to chickens and laying hens [4] are effective in quail use; however, they need a more careful evaluation, due to peculiarities inherent to the Coturnix genus, in order to provide consistent results. Another aggravating factor is the lack of quail standard lineages that makes nutrition dynamism even more peculiar with quail. There are few reputable and reputable companies in Brazil that work on quail breeding and provide genetic material for sale. There are few reputable companies in Brazil that work on quail breeding

denote a specific nutritional requirement, mainly protein and amino acid.

Part of the requirements for amino acids and protein for maintenance (laying hens and quail) is directly related to precocity, intestinal gastric tract size (IGT), feather production, development of the reproductive tract, and part of the gain requirement is related to egg weight (inside the same species), and also, rate of muscle deposition (maturity).

Quails, whether intended for laying or cutting, have early maturity and are related to growth rate, and also to size of animals [5, 6], and thus, smaller animals have higher growth rates and lower age to maturity.

Precocity in growth is related to the time the animal takes to achieve sexual maturity, is a guiding parameter in breeding programs, and also denotes different requirements for animals. In this sense, the models that describe growth curves [7–10] validate the premise that each species/lineages and animal category have different nutritional requirements.

Comparing the Gompertz growth curves for Japanese quails [9], meat quails [7, 9], light and semi‐heavy laying hens [11] and broilers [8, 10], it is worth mentioning, that between maturity rates (0.720, 0.0594 and 0.0694, 0.0245 and 0.0230, 0.0373 and 0.0411), respectively, and Japanese quails have the highest maturity rate, which refers to higher nutritional needs, protein and amino acids.

Japanese quails have a lower weight of IGT than chickens, laying hens and heavy matrices, but, a higher relative weight in relation to body weight and this factor predisposes a higher rate of passage of the digest by IGT [12–14].

Japanese quail [15] presented weight absolute and relative oviduct of 10.18 g and 3.05%, and ovary of 6.36 g and 2.16%, that are lower in relative to laying hens [16] that presented absolute and relative oviduct weight of 76.98 g and 6.58%, and absolute and relative ovary weight of 36.04 g and 3.08%. However, the relative weight of quail eggs is higher, and may reach 10% of body weight. The weight of eggs of quails has a mean value of 12 g [17–20] and eggs of laying hens around 65 g [21–24].

#### **2.2. Crude protein: methodologies and requirement**

The protein and amino acids requirements for quails can be defined by the method of dose and factorial method. The most common is dose-response method and has generated a lot of information's. However, considering the more accurate method, in predicting the requirement of amino acids and crude protein, in this topic of proteins, we will approach a subject only on the factorial model. The approach of the dose-response method will be in the topic about amino acids.

Some studies have been carried out to estimate crude protein (CP) requirements for commercial bird keeping, gain and production using CS and NB techniques.

There studying the requirements of CP for maintenance and gain with Japanese quails in production using the CS technique, [18] obtained the following equation: CP (g/bird/day) = 6.71 × body weight0.75 + 0.615 × weight gain + 0.258 × egg mass.

The requirement of CP for maintenance and gain for growing Japanese quails was estimated in the period from 01 to 32 days of age, through the CS technique. The predicted equations were: CP (g/bird/day) = 2.845 × body weight0.75 + 0.461 × weight gain for quails aged 01–12 days of age and CP (g/bird/day) = 4.752 × body weight0.75 + 0.843 × weight gain for quails in the period from 15 to 32 days of age [25, 26].

The CP requirement for maintenance and gain using the BN technique was determined by the following equation for the 52 week old Lohmann LSL® laying hens: CP (g/bird/day) = 1.94 × body weight0.75 + 0.480 × weight gain + 0.301 × egg mass [27].

Using the NB technique to determine the maintenance and gaining needs of Ross® broilers, at 7 days of age of 56, [28] the following equation was obtained: CP (g/bird/day) = 1.323 × body weight0.75 + 0.272 × weight gain for males and the following equation for females: CP (g/bird/ day) = 1.748 × body weight0.75 + 0.277 × weight gain.

Working with 5-week-old Hubbard® matrices, [29] determined the CP (g/bird/day) values for maintenance using the CS and NB techniques, and values their obtained, respectively, were 3.77 and 2.02 × body weight0.75, and the mean value for CP requirement for gain was 0.406 × weight gain, for techniques CS.

Working with light replacement pullets, Lohmann LSL®, from the age of 42–63 days, using the CS technique, [30] found CP (g/bird/day) values for maintenance and gain of: 4.7625 × body weight0.75 and 0.313 × weight gain.

When evaluating laying hens Hubbard® at age 36–46 weeks of age, [31] estimated the following equation to predict protein requirements: CP (g/bird/day) = 2.282 × body weight0.75 + 0.356 × weight gain + 0.262 × egg mass.

It is known that nutritional needs are changed according to species, animal category, room temperature, diet composition and animal density. However, another important factor that changes the nutritional needs is the methodologies used [18, 25–31], such as the CS and NB techniques used in the elaboration of prediction equations.

In an attempt to elucidate the effects of the two techniques in determining PB requirements for maintenance and gain, the values predicted by these two techniques will be compared.

**2.2. Crude protein: methodologies and requirement**

234 Amino Acid - New Insights and Roles in Plant and Animal

cial bird keeping, gain and production using CS and NB techniques.

× body weight0.75 + 0.615 × weight gain + 0.258 × egg mass.

body weight0.75 + 0.480 × weight gain + 0.301 × egg mass [27].

techniques used in the elaboration of prediction equations.

day) = 1.748 × body weight0.75 + 0.277 × weight gain.

period from 15 to 32 days of age [25, 26].

weight gain, for techniques CS.

weight0.75 and 0.313 × weight gain.

× weight gain + 0.262 × egg mass.

The protein and amino acids requirements for quails can be defined by the method of dose and factorial method. The most common is dose-response method and has generated a lot of information's. However, considering the more accurate method, in predicting the requirement of amino acids and crude protein, in this topic of proteins, we will approach a subject only on the factorial model. The approach of the dose-response method will be in the topic about amino acids.

Some studies have been carried out to estimate crude protein (CP) requirements for commer-

There studying the requirements of CP for maintenance and gain with Japanese quails in production using the CS technique, [18] obtained the following equation: CP (g/bird/day) = 6.71

The requirement of CP for maintenance and gain for growing Japanese quails was estimated in the period from 01 to 32 days of age, through the CS technique. The predicted equations were: CP (g/bird/day) = 2.845 × body weight0.75 + 0.461 × weight gain for quails aged 01–12 days of age and CP (g/bird/day) = 4.752 × body weight0.75 + 0.843 × weight gain for quails in the

The CP requirement for maintenance and gain using the BN technique was determined by the following equation for the 52 week old Lohmann LSL® laying hens: CP (g/bird/day) = 1.94 ×

Using the NB technique to determine the maintenance and gaining needs of Ross® broilers, at 7 days of age of 56, [28] the following equation was obtained: CP (g/bird/day) = 1.323 × body weight0.75 + 0.272 × weight gain for males and the following equation for females: CP (g/bird/

Working with 5-week-old Hubbard® matrices, [29] determined the CP (g/bird/day) values for maintenance using the CS and NB techniques, and values their obtained, respectively, were 3.77 and 2.02 × body weight0.75, and the mean value for CP requirement for gain was 0.406 ×

Working with light replacement pullets, Lohmann LSL®, from the age of 42–63 days, using the CS technique, [30] found CP (g/bird/day) values for maintenance and gain of: 4.7625 × body

When evaluating laying hens Hubbard® at age 36–46 weeks of age, [31] estimated the following equation to predict protein requirements: CP (g/bird/day) = 2.282 × body weight0.75 + 0.356

It is known that nutritional needs are changed according to species, animal category, room temperature, diet composition and animal density. However, another important factor that changes the nutritional needs is the methodologies used [18, 25–31], such as the CS and NB The CP (g/bird/day) requirements for maintenance were predicted by the CS technique, with growing animals, in the studies [26, 29, 30], which, respectively, used: pullets (42–63 days of age), heavy matrices (3–20 weeks of age) and Japanese quails (15–32 days of age). The values are similar between the species, 4.765 and 4.752 × body weight0.75 for pullets and quails; however, they are discrepant when compared to heavy matrices 3.77 × body weight0.75.

Using the NB technique to determine CP (g/bird/day) requirements for maintenance, the values predicted by the authors, [27, 28, 31], who, respectively, worked with broiler chickens (7–56 days of age), laying hens, and heavy matrices, were: 1.323; 1.94 and 2.28 × body weight0.75. It can be observed that there is no similarity between the all determined values. However, the values are consistent when analyzing animals in the same category [27, 31] which were: 1.94 and 2.28 × body weight0.75.

It can be observed that the net requirement of CP (g/bird/day) for gain, determined by the two techniques (CS and NB) and reported in the works of [18, 25–31], is, respectively: 0.615; 0.461; 0.843; 0.480; 0.272; 0.406; 0.313 and 0.356 × weight gain. Comparing the requirements of CP (g/bird/day) to gain, with the CS technique, the values are: 0.406; 0.461; 0.615 and 0.843 × weight gain. Those predicted in the NB technique are: 0.272; 0.356 and 0.480 × weight gain.

A relevant comparison is to analyze the same technique and animal's age, growth and posture. Within the CS technique, with growing animals, the values were: 0.313; 0.461 and 0.843 × weight gain, respectively, pullets (42–63 days of age), quails (01–12 days) and quails (15– 32 days of age). In NB technique for growing animals, the values were, respectively: 0.272 × weight gain, for broilers; and for the animals in posture were: 0.356 and 0.480 × weight gain, respectively for, laying hens and heavy matrices.

The values of requirement of CP (g/bird/day) estimated for egg mass production in laying hens and Japanese quails were, respectively, 0.301 × egg mass [26], and 0.258 × egg mass [18]. However, the first one presents a lower requirement of amino acids and CP, evidencing that the greater requirement of quails is related to the higher maturity rate, that is, higher precocity [9–11]. Corroborating the findings of [18, 25–27, 29], where these authors found a requirement of CP for greater maintenance and gain for Japanese quails in relation to laying hens and heavy matrices.

It is clear from the aforementioned works that the CS and NB techniques used to determine PB requirements for maintenance and gain provide conflicting, inter and intraspecific values, which makes comparison difficult. In an attempt to elucidate this difference between the methodologies, [32] described the potential of nitrogen retention in laying pullets by analyzing the two techniques: CS and NB. The authors describe that excreted nitrogen measured in the BN technique does not seem to contain all possible physiological effects, except for amino acid oxidation, in relation to the CS technique. This factor suggested by the authors seems to be the accounting for the nitrogen lost in feathers (NF), which is not measured when used in the NB technique, and with that, the CP requirement values for maintenance between the two techniques are more discrepant.

For [33], the use of the BN technique is even more aggravating, because in this technique, diets are formulated with different levels of protein to generate deficiency and excess CP in the animals' diet. In this sense, the relationship between the protein level and the loss of NF was established, described by the equation NF = 0.3007 + 0.0086 N, where, for each gram of increase in the nitrogen concentration of the diet, there was a loss of 8.6 mg of nitrogen in feathers, that is, the deficiency in proteins leads to less deposition of amino acids in the feathers, thus, changes the requirement of maintenance the animals. [34] Also verified the influence of nitrogen losses on feathers on the need for maintenance, using the BN technique.

Using the correction value of nitrogen losses in the feathers found by [33], [32] in their work using this correction could conclude that differences between CP needs for maintenance between the two techniques, CS and NB, decreased fell from of 1.56: 1 for 1.28: 1, comparing CS: NB.

#### **2.3. Amino acids: methodologies, digestibility and requirement**

The requirements of amino acids have been described by two methodologies: empirical and factorial method [4]. To evaluate the nutritional requirements in the dose-response or empirical method, the diets are formulated with increasing levels amino acid, gradually, and observed the response of the animals through polynomials (linear and quadratic), broken line and hyperbolic and analyzed in order to estimate the requirements of birds. In the factorial method, the requirements are described in function of the maintenance, growth and production, and relate to the metabolic weight, weight gain and eggs mass production. This method was described in the topic of proteins.

In addition to the methodologies, there are also techniques for formulating diets that also change the requirements. One of the techniques consists of gradual increases of the nutrient tests [35], the other prioritizes the dilution of the diets, which consists of formulating a diet free of the test nutrient and another diet with the same nutrient in excess, and the nutrient levels studied will be obtained by the dilution of the two diets [36].

The success in determining the requirements is a thin line, that is, the robustness of the proposed models and determined requirement are allied to the knowledge and interpretation of each physiological factor of the animals, and mathematical model, in order to promote satisfactory performance to birds.

#### *2.3.1. Amino acids methodologies: techniques for preparing diets*

In the technique proposed by [35], the supplementation of a single amino acid generates imbalances in the relations between amino acids and amino acids/lysine. This point is crucial, since it refers to the ideal protein concept proposed by [37], where the diet needs to have an optimal balance of amino acids to provide maximum performance to the animals with lower nitrogen excretion.

The supplementation technique is widely used [38–45] and has generated a large number of discrepant nutritional information. The main factor is the imbalance between amino acids. The main antagonist relationships between amino acids are: arginine and lysine and the relationship between leucine, isoleucine and valine.

the NB technique, and with that, the CP requirement values for maintenance between the two

For [33], the use of the BN technique is even more aggravating, because in this technique, diets are formulated with different levels of protein to generate deficiency and excess CP in the animals' diet. In this sense, the relationship between the protein level and the loss of NF was established, described by the equation NF = 0.3007 + 0.0086 N, where, for each gram of increase in the nitrogen concentration of the diet, there was a loss of 8.6 mg of nitrogen in feathers, that is, the deficiency in proteins leads to less deposition of amino acids in the feathers, thus, changes the requirement of maintenance the animals. [34] Also verified the influence

Using the correction value of nitrogen losses in the feathers found by [33], [32] in their work using this correction could conclude that differences between CP needs for maintenance between the two techniques, CS and NB, decreased fell from of 1.56: 1 for 1.28: 1, comparing

The requirements of amino acids have been described by two methodologies: empirical and factorial method [4]. To evaluate the nutritional requirements in the dose-response or empirical method, the diets are formulated with increasing levels amino acid, gradually, and observed the response of the animals through polynomials (linear and quadratic), broken line and hyperbolic and analyzed in order to estimate the requirements of birds. In the factorial method, the requirements are described in function of the maintenance, growth and production, and relate to the metabolic weight, weight gain and eggs mass production. This method

In addition to the methodologies, there are also techniques for formulating diets that also change the requirements. One of the techniques consists of gradual increases of the nutrient tests [35], the other prioritizes the dilution of the diets, which consists of formulating a diet free of the test nutrient and another diet with the same nutrient in excess, and the nutrient

The success in determining the requirements is a thin line, that is, the robustness of the proposed models and determined requirement are allied to the knowledge and interpretation of each physiological factor of the animals, and mathematical model, in order to promote

In the technique proposed by [35], the supplementation of a single amino acid generates imbalances in the relations between amino acids and amino acids/lysine. This point is crucial, since it refers to the ideal protein concept proposed by [37], where the diet needs to have an optimal balance of amino acids to provide maximum performance to the animals with lower

of nitrogen losses on feathers on the need for maintenance, using the BN technique.

**2.3. Amino acids: methodologies, digestibility and requirement**

levels studied will be obtained by the dilution of the two diets [36].

*2.3.1. Amino acids methodologies: techniques for preparing diets*

techniques are more discrepant.

236 Amino Acid - New Insights and Roles in Plant and Animal

was described in the topic of proteins.

satisfactory performance to birds.

nitrogen excretion.

CS: NB.

The excess of lysine in the diets, when using the technique proposed by [35], to assessing the lysine requirements, promotes an increase in serum lysine levels, and consequently, a greater loss of arginine by renal catabolism due to the increase in enzyme arginase [46], and generating confusion in the determination of the optimal levels of lysine.

Ref. [45] evaluated different levels of digestible arginine in the diet of Japanese quails, with diets formulated by the supplementation technique, estimated an ideal dietary arginine level of 1.148% in diets with 1.083% digestible lysine and relation arginine/lysine of 1.06.

Refs. [47, 48] evaluated the requirement of digestible lysine with Japanese laying quails, using the supplementation technique, but these authors maintained the relationships between the amino acids of the diets. The authors found digestible lysine levels of 1.117 and 1.120%, respectively, in diets with arginine/lysine ratios of 1.26 and 1.16, respectively.

Ref. [17] found levels of digestible lysine for Japanese quails in production of 1.030% in diets formulated by the supplementation technique and without correction of the arginine/lysine ratio.

The Brazilian Poultry and Swine Table [49] and the Table for Japanese and European Quails [50] present, respectively, digestible lysine values of 1.083 and 1.030% and digestible arginine of 1.256 and 1.260%, respectively, with arginine/lysine ratios of 1.16 and 1.22.

Looking at the data, mentioned above, it is evident that the imbalance of the diets promotes different results among the authors [17, 47, 48]. However, [47, 48] found values equal, but maintained the relationship between the major amino acids; however, this practice of supplementing all amino acids to maintain relationships raises the cost of formulating diets.