**1. Introduction**

Since the publication of the first vanilla book by Bouriquet (1954c) and the more recent review on vanilla biodiversity (Bory et al., 2008b), there has been a world regain of interest for this genus, as witnessed by the recently published vanilla books (Cameron, 2011a; Havkin-Frenkel & Belanger, 2011; Odoux & Grisoni, 2010). A large amount of new data regarding the genus biodiversity and its evolution has also been obtained. These will be reviewed in the present paper and new data will also be presented.


**4. Volatile allelochemicals**

Weeds promote two basic types of interference in agricultural crops: allelospoly and allelopathy. Allelospoly is the type of interference promoted by competition for essential factors to the species survival, such as water, nutrients and physical space. Allelopathy involves the production of allelochemicals and subsequent release into the environment [26]. Almost all allelochemicals exist in conjugated, non-toxic forms. The toxic fragment can be released after exposure to stress or after tissue death [27].

The use of allelopathy for weed control may be an ecologically viable alternative [28]. Thus, the use of essential oils with phytotoxic potential is becoming widespread, since the allelochemicals present in these oils generally have low cytotoxicity. For example, [29] evaluated the effect of *Carum carvis* essential oils rich in carvone (71.08%) and limonene (25.42%), and verified that this oil has a strong phytotoxic activity on seed germination and radicle elongation of *Linum usitatissimum*, *Phalaris canariensis* and *Triticum aestivum*.

Another example is the eucalypt essential oil that has a rich chemical composition in 1,8-cineole (58.3%), α-pinene (17.3%) and α-thujene (15.5%), which significantly inhibited seed germination of *Sinapis arvensis*, *Diplotaxis harra* and *Trifolium campestre*, in different intensities according to the recipient species, demonstrating that each species has a different specificity. In addition, the application of post-emergence oil causes inhibition of chlorophyll production, leading to injuries such as chlorosis, necrosis and even complete wilting of plants [30].

Plant species such as *Origanum onites L.* and *Rosmarinus officinalis L*. also show strong allelopathic activity on species of *Poaceae* and invasive plants, by suppressing germination rate and elongation of radicle and hypocotyl [31]. The phytotoxic effects related to these two species of aromatic plants may be related to their rich chemical composition in the oxygenated monoterpenes 1,8-cineole, linalool, camphor and carvacrol and the monoterpene hydrocarbon p-cymene [32–35], however, compounds found in lower concentrations as methyl phenylpropanoids have also demonstrated good allelopathic activity [36].

10-(tetrahydro-pyran-2-yloxy)-tricyclo[4.2.1(2,5)]decan-9-ol, (−)-caryophyllene oxide, dihydroβ-ionone, viridiflorol, cubenol, caryophyllene, α-bisabolol oxide-b, tetracosane and *n*-hexadecane can be found in *Anisomeles indica* essential oil and also present good phytotoxic activity against invasive plants [42]. As well as *P. heyneanus Benth* essential oils, rich in patchouli alcohol, α-bulnesene, α-guaiene, seichelene and α-patchulene, and *P. hispidinervium* C. DC

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The monoterpenes have presented good phytotoxic activity, and reports of the use of these compounds to control plants refer to the 1960s [44]. This activity depends on the structural

oils, rich in safrole, terpinolene, (E)-β-ocimene, δ-3-carene and pentadecane [43].

**Figure 2.** Chemical structures of oxygenated and non-oxygenated monoterpenes with bioherbicidal action.

**4.1. Monoterpenes**

In the case of essential oils for the control of invasive plants, it is usually analyzed the effects of individual form, attributing the phytotoxic activity to only one component [37, 38]. However, the effects of volatile oils can also be related to the mixture of compounds, such as *Artemisia scoparia* oil which has a mixture of compounds such as monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpene hydrocarbons, oxygenated sesquiterpenes, aliphatic compounds and other aromatic compounds [39]. The chemical composition of the essential oils depends on the biosynthetic path of the different classes of compounds, as can be observed in **Figure 1**, which brings the biosynthesis of some classes of volatile compounds.

Compounds such as eucalyptol, β-phellandrene, hexyl butanoate, *p*-cymene, α-ionone, (z)-3-octen-1-ol, theaspirane a, vitispirane, dihydro-(−)-neoclovene, β-caryophyllene, (e)-2-octen-1-ol, a-terpineol, dehydro-ar-ionene, methyl salicylate, (z)-b-damascenone, (z)-dehydro-ar-ionene,

**Figure 1.** Biosynthesis of plant volatiles. Overview of biosynthetic pathways leading to the emission of plant volatile organic compounds. The plant precursors originate from primary metabolism. Abbreviations: DTS: Diterpene synthase; FPP: farnesyldiphosphate; GGPP: geranylgeranyldiphosphate; GLVs: green-leaf volatiles; GPP: geranyldiphosphate; IPP: isopentenyl pyrophosphate; MTS: Monoterpene synthase; STS: Sesquiterpene synthase; DAHP: 3-deoxy-Darabinoheptulosonate-7 phosphate; E4P: erythrose 4-phosphate; PEP: phosphoenolpyruvate; Phe: phenylalanine. This flowchart was adapted from [40] and [41].

10-(tetrahydro-pyran-2-yloxy)-tricyclo[4.2.1(2,5)]decan-9-ol, (−)-caryophyllene oxide, dihydroβ-ionone, viridiflorol, cubenol, caryophyllene, α-bisabolol oxide-b, tetracosane and *n*-hexadecane can be found in *Anisomeles indica* essential oil and also present good phytotoxic activity against invasive plants [42]. As well as *P. heyneanus Benth* essential oils, rich in patchouli alcohol, α-bulnesene, α-guaiene, seichelene and α-patchulene, and *P. hispidinervium* C. DC oils, rich in safrole, terpinolene, (E)-β-ocimene, δ-3-carene and pentadecane [43].

#### **4.1. Monoterpenes**

Plant species such as *Origanum onites L.* and *Rosmarinus officinalis L*. also show strong allelopathic activity on species of *Poaceae* and invasive plants, by suppressing germination rate and elongation of radicle and hypocotyl [31]. The phytotoxic effects related to these two species of aromatic plants may be related to their rich chemical composition in the oxygenated monoterpenes 1,8-cineole, linalool, camphor and carvacrol and the monoterpene hydrocarbon p-cymene [32–35], however, compounds found in lower concentrations as methyl phen-

In the case of essential oils for the control of invasive plants, it is usually analyzed the effects of individual form, attributing the phytotoxic activity to only one component [37, 38]. However, the effects of volatile oils can also be related to the mixture of compounds, such as *Artemisia scoparia* oil which has a mixture of compounds such as monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpene hydrocarbons, oxygenated sesquiterpenes, aliphatic compounds and other aromatic compounds [39]. The chemical composition of the essential oils depends on the biosynthetic path of the different classes of compounds, as can be observed in **Figure 1**, which brings the biosynthesis of some classes of volatile

Compounds such as eucalyptol, β-phellandrene, hexyl butanoate, *p*-cymene, α-ionone, (z)-3-octen-1-ol, theaspirane a, vitispirane, dihydro-(−)-neoclovene, β-caryophyllene, (e)-2-octen-1-ol, a-terpineol, dehydro-ar-ionene, methyl salicylate, (z)-b-damascenone, (z)-dehydro-ar-ionene,

**Figure 1.** Biosynthesis of plant volatiles. Overview of biosynthetic pathways leading to the emission of plant volatile organic compounds. The plant precursors originate from primary metabolism. Abbreviations: DTS: Diterpene synthase; FPP: farnesyldiphosphate; GGPP: geranylgeranyldiphosphate; GLVs: green-leaf volatiles; GPP: geranyldiphosphate; IPP: isopentenyl pyrophosphate; MTS: Monoterpene synthase; STS: Sesquiterpene synthase; DAHP: 3-deoxy-Darabinoheptulosonate-7 phosphate; E4P: erythrose 4-phosphate; PEP: phosphoenolpyruvate; Phe: phenylalanine. This

ylpropanoids have also demonstrated good allelopathic activity [36].

compounds.

52 Biological Approaches for Controlling Weeds

flowchart was adapted from [40] and [41].

The monoterpenes have presented good phytotoxic activity, and reports of the use of these compounds to control plants refer to the 1960s [44]. This activity depends on the structural

**Figure 2.** Chemical structures of oxygenated and non-oxygenated monoterpenes with bioherbicidal action.

characteristics of the molecules; for example, oxygenated monoterpenes exhibit different effects on germination and seedling development, and also alter cellular respiration, which impairs energetic metabolism [33, 34]. However, these phytotoxic effects promoted by a chemical species depend on its concentration, for example, *Lactuca sativa* essential oil composed essentially of α-pinene (16.00%), 1,8-cineole (66.93%) and pimonene (10.04%) presents different rates of germination inhibition [45].

In general, oxygenated monoterpenes have the highest phytotoxic effects over non-oxygenated [46]. However, there are non-oxygenated volatile molecules such as limonene which also have good phytotoxic activity [47]. Some monoterpenes had high inhibitory activity on germination and radicle elongation, and this may be related to the anatomical and physiological changes in the host plants, as well as to the reduction in some organelles such as mitochondria, and accumulation of lipid globules in the cytoplasm [48]. In **Figure 2**, the chemical structures of some monoterpenes with phytotoxic activity can be observed.

#### **4.2. Sesquiterpenes**

Bioassays have demonstrated that the sesquiterpenic allelochemicals β-cariofilene, β-copaene, spathulenol, germacrene B, bicyclogermacrene, globulol, viridiflorol, a-guaiene, and g-elemene have presented phytotoxity against various invasive plants and, in some cases, promote inhibition of other plants development, when they are close to species that produce these secondary metabolites [49–51]. Authors compared the effects of essential oils rich in sesquiterpenes and others rich in monoterpenes and found that the effects presented by sesquiterpenes, in some cases, may be smaller in relation to the affections exhibited by monoterpenes [52]. **Figure 3** shows the chemical structures of oxygenated and non-oxygenated sesquiterpenes with phytotoxic action.

However, this depends largely on the presence of oxygenated and non-oxygenated, cyclic or acyclic molecules, because depending on the molecular conformation the allelopathic effect may be higher or lower [53, 54]. This justifies the results obtained by other authors [55], who analyzed the effects of fractions of essential oils of *E. adenophorum,* of the inflorescence region, rich in sesquiterpenes, and its root rich in monterpenes. When the oils were tested at the same concentration (1 μL/mL), they inhibited germination and seedling elongation at the same ratio.

#### **4.3. Phenylpropanoids**

Phenylpropanoids are a class of secondary metabolites that are also naturally present in plants, and have exhibited strong phytotoxic activity against invasive plants. In 2016, [9] demonstrated that eugenol is the main active ingredient of clove essential oil and is also the agent possibly promoting phytotoxic activity against the invasive plants *Mimosa pudica* and *Senna obtusifolia*. Other authors also report the potentially allelopathic activity

of clove essential oil *Syzygium aromaticum* [56–58]. In addition to eugenol, other phenylpropanoids present in essential oils with phytotoxic activity are eugenyl acetate, safrole, methyl eugenol, anethole, myristicin, estragole, anethole and trans-anethole [36, 59–64]. **Figure 4**

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**Figure 3.** Chemical structures of oxygenated and non-oxygenated sesquiterpenes with bioherbicidal action.

Potentially Phytotoxic of Chemical Compounds Present in Essential Oil for Invasive Plants… http://dx.doi.org/10.5772/intechopen.74346 55

characteristics of the molecules; for example, oxygenated monoterpenes exhibit different effects on germination and seedling development, and also alter cellular respiration, which impairs energetic metabolism [33, 34]. However, these phytotoxic effects promoted by a chemical species depend on its concentration, for example, *Lactuca sativa* essential oil composed essentially of α-pinene (16.00%), 1,8-cineole (66.93%) and pimonene (10.04%) presents

In general, oxygenated monoterpenes have the highest phytotoxic effects over non-oxygenated [46]. However, there are non-oxygenated volatile molecules such as limonene which also have good phytotoxic activity [47]. Some monoterpenes had high inhibitory activity on germination and radicle elongation, and this may be related to the anatomical and physiological changes in the host plants, as well as to the reduction in some organelles such as mitochondria, and accumulation of lipid globules in the cytoplasm [48]. In **Figure 2**, the chemical structures of some monoterpenes with phytotoxic activity can be

Bioassays have demonstrated that the sesquiterpenic allelochemicals β-cariofilene, β-copaene, spathulenol, germacrene B, bicyclogermacrene, globulol, viridiflorol, a-guaiene, and g-elemene have presented phytotoxity against various invasive plants and, in some cases, promote inhibition of other plants development, when they are close to species that produce these secondary metabolites [49–51]. Authors compared the effects of essential oils rich in sesquiterpenes and others rich in monoterpenes and found that the effects presented by sesquiterpenes, in some cases, may be smaller in relation to the affections exhibited by monoterpenes [52]. **Figure 3** shows the chemical structures of oxygenated and non-oxygenated sesquiterpenes with phy-

However, this depends largely on the presence of oxygenated and non-oxygenated, cyclic or acyclic molecules, because depending on the molecular conformation the allelopathic effect may be higher or lower [53, 54]. This justifies the results obtained by other authors [55], who analyzed the effects of fractions of essential oils of *E. adenophorum,* of the inflorescence region, rich in sesquiterpenes, and its root rich in monterpenes. When the oils were tested at the same concentration (1 μL/mL), they inhibited germination and seedling elongation at the same

Phenylpropanoids are a class of secondary metabolites that are also naturally present in plants, and have exhibited strong phytotoxic activity against invasive plants. In 2016, [9] demonstrated that eugenol is the main active ingredient of clove essential oil and is also the agent possibly promoting phytotoxic activity against the invasive plants *Mimosa pudica* and *Senna obtusifolia*. Other authors also report the potentially allelopathic activity

different rates of germination inhibition [45].

54 Biological Approaches for Controlling Weeds

observed.

**4.2. Sesquiterpenes**

totoxic action.

ratio.

**4.3. Phenylpropanoids**

**Figure 3.** Chemical structures of oxygenated and non-oxygenated sesquiterpenes with bioherbicidal action.

of clove essential oil *Syzygium aromaticum* [56–58]. In addition to eugenol, other phenylpropanoids present in essential oils with phytotoxic activity are eugenyl acetate, safrole, methyl eugenol, anethole, myristicin, estragole, anethole and trans-anethole [36, 59–64]. **Figure 4**

**Author details**

Belém, Pará, Brazil

**References**

Mozaniel Santana de Oliveira1

Antonio Pedro da Silva Souza Filho3

e Desenvolv. 2003;**30**:1-17

Abastecimento; 2008

vier.com/retrieve/pii/S026121940000123X

\*, Wanessa Almeida da Costa2

1 LABEX/FEA (Faculty of Food Engineering), Program of Post-Graduation in Food Science

2 Program of Post-Graduation in Natural Resources Engineering, Federal University of Para,

[1] Poletti M, Omoto C. Resistência de Inimigos Naturais a Pesticidas. Rev Biotecnol Ciência

[2] Norsworthy JK, Ward SM, Shaw DR, Llewellyn RS, Nichols RL, Webster TM, Bradley KW, Frisvold G, Powles SB, Burgos NR, Witt WW, Barrett M. Reducing the risks of herbicide resistance: Best management practices and recommendations. Weed Science [Internet]. Jan 20, 2012;**60**(SP1):31-62. DOI: http://www.bioone.org/doi/full/10.1614/WS-D-11-00155.1

[3] Devine MD, Shukla A. Altered target sites as a mechanism of herbicide resistance. Crop Protection [Internet]. Sep 2000;**19**(8-10):881-889. Available from: http://linkinghub.else-

[4] Souza Filho APS. In: da Silva Sousa Filho AP, editor.  In: de Miranda FVC, do Nascimento FE, de Oliveira JF, editors. Daninhas do Café. 1st ed. Planaltina, DF- Brazil: Empresa Brasileira de Pesquisa Agropecuária Embrapa Cerrados Ministério da Agricultura, Pecuária e

[6] Filho AJC, Santos LS, Guilhon GMSP, Moraes RPC, dos Santos RA, Filho AP da SS, Felizzola JF. Identified substances from the leaves of *Tephrosia cinerea* (Leguminoseae) crude extracts and their phytotoxic effects. International Journal of Life-Sciences Scientific Research [Internet]. Jul 6, 2017;**3**(4):1137-1141. Available from: http://ijlssr.com/currentissue/ Tephrosia cinerea (Leguminoseae) Crude Leaves Extracts and their Phytotoxic Effects.pdf

[7] Pereira SG, Soares AM dos S, Guilhon G, Santos LS, Pacheco LC, Souza Filho AP da S. Phytotoxic potential of piperine and extracts from fruits of *Piper tuberculatum* Jaq. on Senna obtusifolia and *Mimosa pudica* plants. Allelopathy Journal 2016;**38**(1):91-102

\*Address all correspondence to<EMAIL_ADDRESS>and<EMAIL_ADDRESS>
3 Laboratory of Agro-industry, Embrapa Eastern Amazon, Belém, Pará, Brazil

and Technology, Federal University of Para, Belém, Pará, Brazil

and Raul Nunes de Carvalho Junior1,2\*

Potentially Phytotoxic of Chemical Compounds Present in Essential Oil for Invasive Plants…

, Priscila Nascimento Bezerra1

http://dx.doi.org/10.5772/intechopen.74346

,

57

**Figure 4.** Chemical structures of phenylpropanoids with bioherbicidal action.

shows the chemical structures of the phenylpropanoids with potential use for control of invasive plants.

#### **5. Conclusion**

For essential oils to have good phytotoxic activity, some factors such as chemical composition, concentration and host plants may be taken into account. Among the monoterpene allelochemicals we can highlight the 1,8 cineole, among the sesquiterpenes or β-caryophyllene and among phenylpropanoids, eugenol. On the other hand, one of the difficulties that can appear for the use in large scale of essential oils is the volatility of their components.

#### **Acknowledgements**

Oliveira MS (Process Number: 1662230) and Costa WA (Process Number: 1427204) thank CAPES for the doctorate scholarship.


**Author details**

Adrián Gutiérrez-González1,2\*, José Iván Robles-Torres1,2 and Daniel García-Sánchez1,2

1 Hospital Universitario "Dr. José Eleuterio González", Universidad Autonoma de Nuevo León, Monterrey, Mexico

2 UroContinent, Monterrey, Mexico

\*Address all correspondence to<EMAIL_ADDRESS>
© 2020 The Author(s). Licensee IntechOpen. 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.


**4. Discussion**

166 Liver Regeneration

increase in the quantity of binucleated hepatocytes in the hypertrophic liver lobes in the TGF-β1 group (see figure 14). We observed a larger distribution of the number of binucleated hepatocytes per field in the hypertrophic lobe from TGF-β1 group (1-4 binucleated hepatocytes per field) in contrast to the control group (2-3,5 binucleated hepatocytes per field). The test of normality demonstrated a normal distribution of this parameter in each animal in both experimental groups. The size of hepatocytes was also not proved to be a statistically significant parameter for differences between the TGF-β1 group and the control group (see figure 15). Proliferative activity in both groups was

In case of comparison of histological findings at IL-6 and control groups the differences in lobulus length were not statisticaly significant. The statistical analysis proved significant increased amount of binucleated hepatocytes in hypertrophic liver lobes in IL-6 group (fig. 16). The length of hepatocytes was not proved also as statistically significant parameter. The

Fig. 14. Comparison of the concentration of binucleated hepatocytes in hypertrophic lobes between TGF-β1 and control groups. The binucleated hepatocytes were detected in 20

Fig. 15. Comparison of size of hepatocytes in hypertrophic lobes between TGF-β1 and

proliferative activity in both groups was very decreased (Liska, 2009).

greatly reduced.

microscopic fields.

control groups.

#### **4.1 Functional liver remnant volume**

In the presented chapter the authors want to review all experiences with influence of experimental portal vein ligation with cytokines, monoclonal antibody against growth factor and mesenchymal stem cells. This model closely mimics the situation of human oncological liver surgery, in which portal vein embolization can be utilized to create a larger future liver remnant volume before resection. The clinical goal of portal vein embolization is to increase the number of patients that can undergo extensive liver resections. This porcine experimental model of liver regeneration is especially applicable to the clinical situation since pigs closely resemble human liver anatomy and fysiology (Liska, 2009, Liska, 2009).

After portal venous infusion of MSC in this model, augmented and accelerated regeneration of the non-occluded liver lobes is outlined. This effect was strongest between the third and seventh postoperative day and slowly weaned off thereafter. However, there was no overall volume difference of statistical significance between both groups at the end of the observation period. This result might have been expected, though, since the body weight to liver weight ratio is tightly controlled in most species (Liska, 2009).

Previous studies have tried to clarify the potential contribution of MSC to liver regeneration in rodent models. Most of these approaches used toxic models, in which the intrinsic selfrenewing capacity of the recipient liver was suppressed (Dahlke, 2003, Kang, 2005). Di Bonzo observed only 0,2% human hepatocytes after transplantation of human MSC into immunodeficient mice with chronic liver injury (Di Bonzo, 2008). Overall, whether MSC can significantly contribute to liver regeneration in vivo remains controversial (Dahlke, 2004, Lysy, 2008, Popp, 2006), although MSC can show some features of hepatic differentiation in culture or under immunoprivileged conditions (Najimi, 2007, Ong, 2006). Chamberlain observed differentiation of human MSC into hepatocytes after infusion of MSC into foetal sheep (Chamberlain, 2007) and transplantable hepatocytes were also obtained from heavily growth factor-treated MSC (Banas, 2007, Yamamoto, 2008). Despite the fact that it was not possible to identify transplanted MSC within recipient livers in this model after 14 days, liver hypertrophy was augmented. It is therefore likely that bystander effects of the transplanted

Liver Parenchyma Regeneration in Connection

**4.2 Biochemistry**

periphery (Liska, 2009).

with Extended Surgical Procedure – Experiment on Large Animal 169

human medicine. The achieved acceleration of growth of the hypertrophic liver lobes after application of monoclonal antibody against TGF-β1between 3rd and 7th postoperative days confirmed the key role of the studied cytokine in terminating the regeneration of liver parenchyma after portal vein ligation. The experimental results could be settings for a clinical study in patients with a low response regeneration of future liver remnant volume

All biochemical parameters, including cytokine levels, that were assessed during the observation period, did not outline any significant differences between the MSC-infused and the control group, indicating that both groups were systemically stable throughout the regenerative period. Concerning the role of cytokines it may also be assumed that these act on a more local level within the liver parenchym and differences cannot be observed in the

On the contrary application of TNF-α brought the tendency to differences in serum level of AST, ALT, GGT and creatinine in postoperative period, which were not statistically significant. All these biochemical parameters could be due to operative stress and also to occlusion of portal branches of right liver lobes, which represent significant liver function reserves. The postoperative elevation of these parameters could be hypothesized also to atrophic changes in occluded lobes. This reduction of expected increased serum level of discussed biochemical parameters after application of studied cytokine in non occluded portal vein could present also one of its pleiotropic functions – hepatoprotection agains changes after operative stress and reduction of functional parenchyma (Teoh, 2006, Baier, 2006). This hypothesis should be probably examined on non-resection experimental model (Liska, 2012). We did not prove any statistically significant differences between serum levels of studied biochemical parameters in particular time points between IL-6 and control groups. It demonstrates also no unsuitable influence of applied cytokine on the liver function. The changes in serum levels of studied cytokines, growth factors were also not observed as

different between IL-6 and control group in separate time points (Liska, 2009).

**4.3 Histology and immunohistochemistry**

In case of comparison of TGF-β1 and control group no statistically significant differences were shown between serum levels of the studied biochemical parameters at particular points in time. This also demonstrates that there was no unsuitable influence of the applied monoclonal antibody on the liver function. This results support our ideas to choose monoclonal antibody against TGF-β1 for future clinical studies in human liver (Liska, 2012).

No histological differences were observed after 14 days between the groups (number of mitotic figures, binucleated hepatocytes, length of lobuli, hepatocyte size), indicating that the initial phase of liver regeneration had been completed in both groups and there was no influence of the infused MSC on liver architecture. Using BrdU staining, only very few of the infused MSC could be detected. Thus, injected MSC do not contribute to liver regeneration through proliferation, but most likely help to establish a micormilieu supportive for intrinsic proliferation of resident hepatocytes. Results acquired by this experimental study confirm the experiences of Furst and Esch, who have previously shown that cell grafts of bone marrow

after portal vein embolization that does not allow surgical treatment (Liska, 2012).

MSC accelerated regeneration in this model. This effect may be well mediated through paracrine effects of the infused MSC on the microenvironment in the periportal regions, where parenchymal regeneration is initiated. Aldeguer et al. suggested that increased production of IL-6 by bone marrow-derived cells, for example, can stimulate intrinsic liver regeneration (Aldeguer, 2002). In addition to being effective in terms of an accelerated regenerative response, the infusion of syngenic MSC into the portal vein also proved to be safe in the preclinical model. Probable side effects of MSC infusions include immunological problems (hypersensitivity, immune complex reactions), metabolic dysregulation and emboli. None of these problems occured in the presented study cohort (Liska, 2009).

The acceleration of growth of hypertrophic liver lobes after application of TNF-α confirmed results gained at in vitro models and in experiments in small laboratory animals (Cornell, 1990). The selected concentration of applied cytokine initiated acceleration of regeneration of liver parenchyma in non-occluded liver lobes (Heinrich, 2006, Teoh, 2006).

The acceleration of growth of hypertrophic liver lobes after application of IL-6 confirmed results gained at in vitro models and in experiments in small laboratory animals (Cornell,1990, Fukuhara, 2003). The selected concentration of applied cytokine initiated acceleration of regeneration of liver parenchyma in non-occluded liver lobes (Baier,2006, Heinrich, 2006).

We presented possibilities of the application of extrinsic MAB TGF-β1 to increase the required future remnant liver volume after partial portal vein ligation. The absolute volume of hypertrophic lobes (left lateral and medial lobes) grew more rapidly after application of MAB TGF-β1, whereas the control group had a slow but continual growth in the hypertrophic liver lobes during the whole follow-up period. The augmentation of growth of the hypertrophic lobes was maximal between the 3rd and 7th postoperative days in comparison with the control group (p<0.05). Nevertheless this growth accelerating effect was lost during the next ultrasonographic controls, and on the 14th postoperative day there were no statistically significant differences. The results of the presented study confirmed in large animal experiment previous findings gained using in vitro models and in experiments with small laboratory animals (Armendariz-Borunda, 1993, Armendariz-Borunda, 1997, Deneme, 2006, Delgado-Rizo, 1998). The selected concentration and timing of the application of monoclonal antibody prolonged acceleration of liver parenchyma regeneration in non-occluded liver lobes of animals (Armendariz-Borunda, 1993, Deneme, 2006). The secondary effects that could be hypothesized after application of the monoclonal antibody against key pleiotropic growth factor (changes in immune reactions and homeostasis) were not observed either during application or in the whole postoperative period. In previous described experimental groups (IL-6 and TNF-α group) we have shown the importance and usefulness of cytokines of the first phase of liver regeneration that increase priming of the hepatocytes – Interleukine-6 and Tumor necrosis factor – α (Liska, 2009, Liska, 2012), but these cytokines have pleitropic functions, which could be altered or changed by application of these cytokines from an extrinsic source. The choice of the monoclonal antibody against growth factor that terminates this first phase of liver regeneration and stimulates hepatocytes to differentiation, production of extracellular matrix and remodelation of liver tissue structure, imitates the same effect of these named cytokines (Kusaka, 2004).

The present study describes a new usage of a monoclonal antibody against TGF-β1 in large animal experimental model of partial portal vein ligation, which simulates the situation in human medicine. The achieved acceleration of growth of the hypertrophic liver lobes after application of monoclonal antibody against TGF-β1between 3rd and 7th postoperative days confirmed the key role of the studied cytokine in terminating the regeneration of liver parenchyma after portal vein ligation. The experimental results could be settings for a clinical study in patients with a low response regeneration of future liver remnant volume after portal vein embolization that does not allow surgical treatment (Liska, 2012).

#### **4.2 Biochemistry**

168 Liver Regeneration

MSC accelerated regeneration in this model. This effect may be well mediated through paracrine effects of the infused MSC on the microenvironment in the periportal regions, where parenchymal regeneration is initiated. Aldeguer et al. suggested that increased production of IL-6 by bone marrow-derived cells, for example, can stimulate intrinsic liver regeneration (Aldeguer, 2002). In addition to being effective in terms of an accelerated regenerative response, the infusion of syngenic MSC into the portal vein also proved to be safe in the preclinical model. Probable side effects of MSC infusions include immunological problems (hypersensitivity, immune complex reactions), metabolic dysregulation and emboli. None of

The acceleration of growth of hypertrophic liver lobes after application of TNF-α confirmed results gained at in vitro models and in experiments in small laboratory animals (Cornell, 1990). The selected concentration of applied cytokine initiated acceleration of regeneration

The acceleration of growth of hypertrophic liver lobes after application of IL-6 confirmed results gained at in vitro models and in experiments in small laboratory animals (Cornell,1990, Fukuhara, 2003). The selected concentration of applied cytokine initiated acceleration of regeneration of liver parenchyma in non-occluded liver lobes (Baier,2006,

We presented possibilities of the application of extrinsic MAB TGF-β1 to increase the required future remnant liver volume after partial portal vein ligation. The absolute volume of hypertrophic lobes (left lateral and medial lobes) grew more rapidly after application of MAB TGF-β1, whereas the control group had a slow but continual growth in the hypertrophic liver lobes during the whole follow-up period. The augmentation of growth of the hypertrophic lobes was maximal between the 3rd and 7th postoperative days in comparison with the control group (p<0.05). Nevertheless this growth accelerating effect was lost during the next ultrasonographic controls, and on the 14th postoperative day there were no statistically significant differences. The results of the presented study confirmed in large animal experiment previous findings gained using in vitro models and in experiments with small laboratory animals (Armendariz-Borunda, 1993, Armendariz-Borunda, 1997, Deneme, 2006, Delgado-Rizo, 1998). The selected concentration and timing of the application of monoclonal antibody prolonged acceleration of liver parenchyma regeneration in non-occluded liver lobes of animals (Armendariz-Borunda, 1993, Deneme, 2006). The secondary effects that could be hypothesized after application of the monoclonal antibody against key pleiotropic growth factor (changes in immune reactions and homeostasis) were not observed either during application or in the whole postoperative period. In previous described experimental groups (IL-6 and TNF-α group) we have shown the importance and usefulness of cytokines of the first phase of liver regeneration that increase priming of the hepatocytes – Interleukine-6 and Tumor necrosis factor – α (Liska, 2009, Liska, 2012), but these cytokines have pleitropic functions, which could be altered or changed by application of these cytokines from an extrinsic source. The choice of the monoclonal antibody against growth factor that terminates this first phase of liver regeneration and stimulates hepatocytes to differentiation, production of extracellular matrix and remodelation of liver tissue structure, imitates the same effect of

The present study describes a new usage of a monoclonal antibody against TGF-β1 in large animal experimental model of partial portal vein ligation, which simulates the situation in

these problems occured in the presented study cohort (Liska, 2009).

Heinrich, 2006).

these named cytokines (Kusaka, 2004).

of liver parenchyma in non-occluded liver lobes (Heinrich, 2006, Teoh, 2006).

All biochemical parameters, including cytokine levels, that were assessed during the observation period, did not outline any significant differences between the MSC-infused and the control group, indicating that both groups were systemically stable throughout the regenerative period. Concerning the role of cytokines it may also be assumed that these act on a more local level within the liver parenchym and differences cannot be observed in the periphery (Liska, 2009).

On the contrary application of TNF-α brought the tendency to differences in serum level of AST, ALT, GGT and creatinine in postoperative period, which were not statistically significant. All these biochemical parameters could be due to operative stress and also to occlusion of portal branches of right liver lobes, which represent significant liver function reserves. The postoperative elevation of these parameters could be hypothesized also to atrophic changes in occluded lobes. This reduction of expected increased serum level of discussed biochemical parameters after application of studied cytokine in non occluded portal vein could present also one of its pleiotropic functions – hepatoprotection agains changes after operative stress and reduction of functional parenchyma (Teoh, 2006, Baier, 2006). This hypothesis should be probably examined on non-resection experimental model (Liska, 2012).

We did not prove any statistically significant differences between serum levels of studied biochemical parameters in particular time points between IL-6 and control groups. It demonstrates also no unsuitable influence of applied cytokine on the liver function. The changes in serum levels of studied cytokines, growth factors were also not observed as different between IL-6 and control group in separate time points (Liska, 2009).

In case of comparison of TGF-β1 and control group no statistically significant differences were shown between serum levels of the studied biochemical parameters at particular points in time. This also demonstrates that there was no unsuitable influence of the applied monoclonal antibody on the liver function. This results support our ideas to choose monoclonal antibody against TGF-β1 for future clinical studies in human liver (Liska, 2012).

#### **4.3 Histology and immunohistochemistry**

No histological differences were observed after 14 days between the groups (number of mitotic figures, binucleated hepatocytes, length of lobuli, hepatocyte size), indicating that the initial phase of liver regeneration had been completed in both groups and there was no influence of the infused MSC on liver architecture. Using BrdU staining, only very few of the infused MSC could be detected. Thus, injected MSC do not contribute to liver regeneration through proliferation, but most likely help to establish a micormilieu supportive for intrinsic proliferation of resident hepatocytes. Results acquired by this experimental study confirm the experiences of Furst and Esch, who have previously shown that cell grafts of bone marrow

Liver Parenchyma Regeneration in Connection

**5. Conclusion**

extended liver resection.

**6. Summary**

cells, TGF-β1.

**7. Acknowledgement**

pp. 165-175, ISSN 1365-2168

1247-1256. ISSN: 0959-8049

2002), pp. 40-48, ISSN 1527-3350

*of some vital organs*).

**8. References**

with Extended Surgical Procedure – Experiment on Large Animal 171

Application of IL-6 and TNF-α augments hypertrophy of FLRV on 7th postoperative day in comparison to control group. In case of application of MAB TGF-β1 we observed maximal increase of FLRV between 3rd and 7th days. Application of MSC augments hypetrophy of FLRV on 3rd day. The biochemical and histological examinations did not prove any important differencies among the groups. The use of TNF-α, IL-6, MAB TGF-β1, MSC could increase the process of liver regeneration after portal vein ligation.These experimental results could be used in clinical practice in patients with risk of acute liver failure after

The aim of presented studies was to influence regeneration of liver parenchyma after portal vein embolization/ligation. Physiological solution (controls, 9 animals) or TNF-α (9) or interleukin-6 (8) or MSC (6) were applied into non-occluded portal vein branches after ligation of portal vein branches for right lobes. Administration of MAB TGF-β1 was performed 24 hours after ligation (7). Compensatory hypertrophy (FLRV) was followed-up in next 14 days by regular ultrasonography, serum level of biochemical parametres and by histological examinations. Application of IL-6 and TNF-α augments hypertrophy of FLRV on 7th postoperative day in comparison to control group. In case of application of MAB TGF-β1 we observed maximal increase of FLRV between 3rd and 7th days. Application of MSC augments hypetrophy of FLRV on 3rd day. The biochemical and histological examinations did not prove any differences among the groups. The use of TNF-α, IL-6, MAB TGF-β1, MSC

**Keywords:** Liver surgery, Liver regeneration, Experimental study, Porcine model, Experimental model, Portal vein embolization, TNF-α, Interleukin-6, Mesenchymal stem

Supported by grants IGA MZ CR 12025 and Research project MSM<PHONE_NUMBER> (*Replacement*

Abdalla, E. K., Hicks M. E. & Vauthey, J. N. (2001). Portal vein embolization: rationale,

Alison, M.R. et al.(2006): Stem cell plasticity and tumour formation. *Eur J Cancer*, 42, pp

Aldeguer, X. et al. (2002). Interleukin-6 from intrahepatic cells of bone marrow origin is

Armendariz-Borunda, J. et al. (1993). Transforming growth factor beta gene expression is

*Laboratory Investigation*, Vol. 69, No. 3, (1993), pp. 283-294, ISSN 0023-6837

technique and future prospects. *British Journal of Surgery,* Vol. 88, No. 7, (July 2001),

required for normal murine liver regeneration. *Hepatology*, Vol. 35, No. 1, (January

transiently enhanced at a critical stage during liver regeneration after CCl4 treatment.

could increase the process of liver regeneration after portal vein ligation.

origin (CD133 positive cells) applied after PVE in human surgery can increase the FLRV (Furst, 2007). From the present experimental data, however, it cannot be concluded with certainty that the application of stem cells, including MSC, does not support the growth of liver malignancies to the same extent that it supports liver regeneration. Assuming a bystander effect of MSC on the micromilieu makes this even more likely (Alison, 2006). Thus, further animal investigation is necessary before optimized MSC therapies can be applied in the setting of human medicine (Alison, 2006). In conclusion, it has been hereby shown that the intraportal infusion of syngeneic porcine MSC after PVE in a setting of liver regeneration led to an accelerated and augmented compensatory liver hypertrophy. This effect is most likely due to bystander effects of the transplanted MSC (Liska, 2009).

The increased number of larger lobulus in the hypertrophic parenchyma of TNF-α group in comparison with control group could be explained by incomplete liver regeneration. Because there are practically no mitotic figures or the amount is of same quantity as in the normal liver parenchyma without any surgical procedures or toxic insult, we could hypothesize, that the first phase of liver regeneration is finished and the next phase of regeneration proceeds. It means the remodelation phase and the phase, when the liver microstructure is restored. Next would be objective to future study – the detection of intracellular or extracelular matrix changes during the process of liver regeneration. No differences in the amount of binucleated hepatocytes could be discussed also by end of proliferative phase of liver parenchyma at the time of sampling. The same size of hepatocytes and no atypical hepatocytes in the biopsies could also be explained by the same reason. This hypothesis is supported by restitution of all liver function monitored by biochemical parametres at the moment of sacrifying of experimental animals (Liska, 2012).

The increased number of binucleated hepatocytes in the hypertrophic parenchyma of IL-6 group in comparison with control group could be explained by incomplete liver regeneration at the end of experiment. Because there are practically no mitotic figures or the amount is of same quantity as in the normal liver parenchyma without any surgical procedures or toxic insult, we could hypothesize, that the first phase of liver regeneration is finished and the next phase of regeneration proceeds. It means the remodelation phase and the phase, when the liver microstructure is restored. Next would be objective to future study – the detection of intracellular or extracellular (matrix) changes during the process of liver regeneration. No significant differences in other histological parameters (diameter of lobulus and hepatocytes) were not proved (Liska, 2009).

The larger distribution of the number of binucleated hepatocytes in the hypertrophic parenchyma of TGF-β1 group in comparison with the control group could be explained by incomplete liver regeneration at the end of experiment. Because there are practically no mitotic figures, or the amount is the same as in the normal liver parenchyma without any surgical procedures or toxic insult, it was possible to hypothesize that the first phase of liver regeneration was finished and the next phase of regeneration was proceeding, namely the remodelling phase and the phase when the liver microstructure is restored (Mangnall, 2003). The size of hepatocytes and the length of lobuli were not proved to be statistically different between study and control group. The same size of hepatocytes and length of lobuli in the bioptical samples from the hypertrophic parenchyma could be also explained in the same way. This hypothesis is supported by the restitution of all liver functions monitored by biochemical parameters and completion of the proliferative phase of liver regeneration at the moment of sacrificing of the experimental animals (Liska, 2012).