\*

\*\*

##

\*

vs. normal group, ##: *P*<0.05 vs. control group, \*\*: *P*<0.05

\*\* \*\*

\*

\*

\*\*

vs. normal group, ##: *P*<0.05 vs. control group, \*\*: *P*<0.05

Fig. 6. Fuzheng Huayu Recipe and SA-B could decrease the avtivity of serum ALT and AST, and serum level of TBiL, but increase serum concentration of Alb in DMN model rats normal group, n=14; control group, n=9; FZHY group, n=13; SA-B group, n=14.

normal controls FZHY SA-B

**<IP_ADDRESS>.4 The concentration of Hyp in the liver tissue of cirrhotic rats was decreased by**

Compared with normal group, concentration of Hyp in the liver tissue was significantly increased at the end of experiment in controls (*P*<0.01). The concentration of Hyp, however, was significantly lower both in FZHY group and SA-B group than in controls (*P*<0.05). (Fig. 7)

Fig. 5. Fuzheng Huayu Recipe and SA-B could relieve collagen fiber deposition in the liver tissue of DMN model rats A: normal group, B: control group, C: FZHY group, and D: SA-B group. The staining was by Sirius red (200)

Fig. 5. Fuzheng Huayu Recipe and SA-B could relieve collagen fiber deposition in the liver tissue of DMN model rats A: normal group, B: control group, C: FZHY group, and D: SA-B

normal controls FZHY SA-B

\*

\*\*\*\*

\*

## vs. control group, \*\*: *P*<0.01

\*

\*\*

vs. normal group, ##: *P*<0.01

\*

\*\*

#

##

#

ALT(U/L) AST(U/L)

group. The staining was by Sirius red (200)

serum activity of transaminase(U/L)

**A B**

**C D**

Fig. 6. Fuzheng Huayu Recipe and SA-B could decrease the avtivity of serum ALT and AST, and serum level of TBiL, but increase serum concentration of Alb in DMN model rats normal group, n=14; control group, n=9; FZHY group, n=13; SA-B group, n=14.

#### **<IP_ADDRESS>.4 The concentration of Hyp in the liver tissue of cirrhotic rats was decreased by Chinese herb medicine**

Compared with normal group, concentration of Hyp in the liver tissue was significantly increased at the end of experiment in controls (*P*<0.01). The concentration of Hyp, however, was significantly lower both in FZHY group and SA-B group than in controls (*P*<0.05). (Fig. 7)

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Portal vein pressure (mmHg)

n=14.

**3.1.4 Summary**

ET-1(pg/mg)

Can Improve Liver Microcirculation and Reduce Portal Hypertension in Liver Cirrhosis 103

##

vs. normal group, ##: *P*<0.01 vs. control group, \*: *P*<0.05, \*\*: *P*<0.01

\* \*\*

\* \*

vs. normal group, ##: *P*<0.01 vs. control group, \*: *P*<0.05

Fig. 8. Fuzheng Huayu Recipe and SA-B could decrease portal vein pressure in DMN model

normal controls FZHY SA-B

Fig. 9. Fuzheng Huayu Recipe and SA-B could decrease content of ET-1 in the liver tissue of DMN model rats normal group, n=14; control group, n=9; FZHY group, n=13; SA-B group,

normal controls FZHY SA-B

The elevation of portal vein pressure is positively correlated with the increase of ET-1 concentration in the liver tissue during the process of liver cirrhosis. FZHY Recipe and SA-B

rats normal group, n=12; control group, n=9; FZHY group, n=13; SA-B group, n=14.

##

#### **<IP_ADDRESS>.5 Portal vein pressure of the cirrhotic rats was declined and the tissue concentration of ET-1 was dropped by Chinese herb medicine**

The portal vein pressure of control rats was significantly increased in the end of experiment compared with normal rats (*P*<0.01). The increased range was up to 8mmHg. The portal pressure,however, was significantly lower in the rats treated with FZHY Recipe or SA-B than controls (*P*<0.05). (Fig. 8) The hepatic tissue concentration of ET-1 was obviously higher in controls than in normal group (*P*<0.01). But concentration of ET-1 was remarkably declined in FZHY group and SA-B group compared with controls (*P*<0.05). (Fig. 9.)

Fig. 8. Fuzheng Huayu Recipe and SA-B could decrease portal vein pressure in DMN model rats normal group, n=12; control group, n=9; FZHY group, n=13; SA-B group, n=14.

Fig. 9. Fuzheng Huayu Recipe and SA-B could decrease content of ET-1 in the liver tissue of DMN model rats normal group, n=14; control group, n=9; FZHY group, n=13; SA-B group, n=14.

#### **3.1.4 Summary**

102 Portal Hypertension – Causes and Complications

##

\*

\*

vs. normal group, ##: *P*<0.01 vs. control group, \*: *P*<0.05

vs. normal group, ##: *P*<0.01 vs. control group, \*: *P*<0.05

normal controls FZHY

##

Fig. 7. Fuzheng Huayu Recipe and SA-B could decrease concentration of Hyp in the liver tissue of DMN model rats A. normal group, n=12; control group, n=9; FZHY group, n=12. B.

normal controls SA-B

The portal vein pressure of control rats was significantly increased in the end of experiment compared with normal rats (*P*<0.01). The increased range was up to 8mmHg. The portal pressure,however, was significantly lower in the rats treated with FZHY Recipe or SA-B than controls (*P*<0.05). (Fig. 8) The hepatic tissue concentration of ET-1 was obviously higher in controls than in normal group (*P*<0.01). But concentration of ET-1 was remarkably declined in FZHY group and SA-B group compared with controls

**<IP_ADDRESS>.5 Portal vein pressure of the cirrhotic rats was declined and the tissue**

normal group, n=13; control group, n=9; SA-B group, n=12.

**B**

Hyp(ug/g)

**A**

Hyp(ug/g)

(*P*<0.05). (Fig. 9.)

**concentration of ET-1 was dropped by Chinese herb medicine**

The elevation of portal vein pressure is positively correlated with the increase of ET-1 concentration in the liver tissue during the process of liver cirrhosis. FZHY Recipe and SA-B

Traditional Chinese Medicine

Can Improve Liver Microcirculation and Reduce Portal Hypertension in Liver Cirrhosis 105

Fig. 10. Fuzheng Huayu Recipe inhibited hepatic fibrosis The liver sections were stained with Masson and showed hepatic fibrosis. A: Fibrous septa (arrows indicated) and pseudolobule (arrow's head indicated) have formed in controls. B: Fibrous septa (arrow

> **Groups n Hyp(μg/g liver wet weight)**  normal 7 112.37±11.93 controls 11 292.83±36.78\* FZHY 14 215.03±75.46#

**<IP_ADDRESS> Fuzheng Huayu Recipe could effectively inhibit sinusoid capillarization in fibrotic**

It was observed that the structure of sinusoids is clearly and the phenotype of SEC is thin by microscopy in normal group. There were many fenestrae in SEC cytoplasm and no basement membrane covered on SEC in perisinusoid side. In controls, the sinusoids were twisted and narrow, the fenestrae in SEC were reduced or disappeared, and basement membrane was observed. (Fig.11)Factor Ⅷ related antigen (Ⅷ R·Ag), -smooth muscle actin (-SMA) and laminin (LM), the important indexes for hepatic sinusoid capillarization, were positive staining in liver tissure. But collagen type Ⅳ (Col Ⅳ), which is a composition of functional membrane but not a composition of basement membrane, expressed majorly in fibrous septa and HSC but less and discontinuously in perisinusoids in controls. Compared with controls, however, the narrow sinusoids were reduced and some sinusoids returned to normal phenotype, basement membrane looked discontinuous thiner, and the positive degree of Ⅷ R·Ag, -SMA and LM were lower and their positive area was significantly smaller by image analysis in the liver of FZHY group than in that of controls (*P*<0.05). Col Ⅳ expressed still continuously in perisinusoids in FZHY group. It indicated that the normal structure of sinusoids was most kept and it meansed that the alteration of hepatic sinusoid

indicated) is less and pseudolobule is absent in FZHY group (100×).

Note: compared with normal, \*: *P*<0.05; compared with controls, #: *P*<0.05

Table 4. Liver Hydroxyproline level in liver tissues ( *x* ±**s**)

capillarization was reversed. (Fig.12)

**rats**

can dramatically decrease the cirrhosis-induced elevation of portal vein pressure and concentration of ET-1 in the liver tissue through their effect on anti-fibrosis in the liver.

#### **3.2 Experiment 2: Chinese herb medicine can relieve capillarization of sinusoid in cirrhotic rats**

#### **3.2.1 Aims**

To investigate the effects of FZHY Recipe on capillarization of hepatic sinusoids.

#### **3.2.2 Methods**

32 Sprague-Dawley male rats, weighing about 150g, were randomly divided into normal group (n=7) and model group (n=25). The modeling mathod was the same as Experiment 1 for making cirrhosis. The cirrhotic rats were more randomly divided into control group (n=11), and FZHY group (n=14). The drug, dose and method of administration for each group were the same as Experiment 1, too. But the duration of treatment was for 4 weeks.

Rat's blood, under etherization condition, was collected from inferior vena cava, and was separated to serum. The sample of liver (1.0cm×0.8cm×0.3cm) was immediately taken and fixed in neutral formalin, embedded in paraffin, sectioned at 5 m thick. The sections of liver were stained with HE, Masson and immunohistochemistry. Three rats were randomly selected from each group respectively for observation of hepatic microstructure. The each liver sample was cut to a 1 mm3 cube, fixed with 2.5% glutaraldehyde and 1% osmium tetroxide for 2h, embedded with araldite 618, and cut to ultrathin sections. The sections were observed by the electron microscope (H-600). 100mg liver tissue was used to detect Hyp.

#### **3.2.3 Results**

#### **<IP_ADDRESS> Fuzheng Huayu Recipe had the potent effect against hepatic fibrosis of the model rat**

It was observed that hemorrhage, necrosis and extensive connective tissue hyperplasia in the liver tissues of model rats. The fiber septa reached out its branches towards the liver lobule. (Fig. 10 and Tab. 3) The level of hydroxyproline was significantly increased. Compared with controls, liver inflammation, necrosis of hepatocytes and hepatic fibrosis was reduced in FZHY group. The concentration of liver hydroxyproline was significantly lower in FZHY group than in controls (*P*<0.05). (Tab. 4)

Note: compared with controls, \*: U=4.06, *P*<0.01

Table 3. Hyperplasia degree of collagenous fibers in each group

can dramatically decrease the cirrhosis-induced elevation of portal vein pressure and concentration of ET-1 in the liver tissue through their effect on anti-fibrosis in the liver.

32 Sprague-Dawley male rats, weighing about 150g, were randomly divided into normal group (n=7) and model group (n=25). The modeling mathod was the same as Experiment 1 for making cirrhosis. The cirrhotic rats were more randomly divided into control group (n=11), and FZHY group (n=14). The drug, dose and method of administration for each group were the same as Experiment 1, too. But the duration of treatment was for 4 weeks.

Rat's blood, under etherization condition, was collected from inferior vena cava, and was separated to serum. The sample of liver (1.0cm×0.8cm×0.3cm) was immediately taken and fixed in neutral formalin, embedded in paraffin, sectioned at 5 m thick. The sections of liver were stained with HE, Masson and immunohistochemistry. Three rats were randomly selected from each group respectively for observation of hepatic microstructure. The each liver sample was cut to a 1 mm3 cube, fixed with 2.5% glutaraldehyde and 1% osmium tetroxide for 2h, embedded with araldite 618, and cut to ultrathin sections. The sections were observed by the electron microscope (H-600). 100mg liver tissue was used to detect Hyp.

**<IP_ADDRESS> Fuzheng Huayu Recipe had the potent effect against hepatic fibrosis of the model**

It was observed that hemorrhage, necrosis and extensive connective tissue hyperplasia in the liver tissues of model rats. The fiber septa reached out its branches towards the liver lobule. (Fig. 10 and Tab. 3) The level of hydroxyproline was significantly increased. Compared with controls, liver inflammation, necrosis of hepatocytes and hepatic fibrosis was reduced in FZHY group. The concentration of liver hydroxyproline was significantly

**Groups n Hyperplasia degree of collagenous fibers**

normal 7 7 0 0 0 0 controls 11 0 1 1 4 5 FZHY \* 14 0 0 8 4 2

0 + ++ +++ ++++

lower in FZHY group than in controls (*P*<0.05). (Tab. 4)

Table 3. Hyperplasia degree of collagenous fibers in each group

Note: compared with controls, \*: U=4.06, *P*<0.01

**3.2 Experiment 2: Chinese herb medicine can relieve capillarization of sinusoid in**

To investigate the effects of FZHY Recipe on capillarization of hepatic sinusoids.

**cirrhotic rats 3.2.1 Aims**

**3.2.2 Methods**

**3.2.3 Results**

**rat**

Fig. 10. Fuzheng Huayu Recipe inhibited hepatic fibrosis The liver sections were stained with Masson and showed hepatic fibrosis. A: Fibrous septa (arrows indicated) and pseudolobule (arrow's head indicated) have formed in controls. B: Fibrous septa (arrow indicated) is less and pseudolobule is absent in FZHY group (100×).

Note: compared with normal, \*: *P*<0.05; compared with controls, #: *P*<0.05

Table 4. Liver Hydroxyproline level in liver tissues ( *x* ±**s**)

#### **<IP_ADDRESS> Fuzheng Huayu Recipe could effectively inhibit sinusoid capillarization in fibrotic rats**

It was observed that the structure of sinusoids is clearly and the phenotype of SEC is thin by microscopy in normal group. There were many fenestrae in SEC cytoplasm and no basement membrane covered on SEC in perisinusoid side. In controls, the sinusoids were twisted and narrow, the fenestrae in SEC were reduced or disappeared, and basement membrane was observed. (Fig.11)Factor Ⅷ related antigen (Ⅷ R·Ag), -smooth muscle actin (-SMA) and laminin (LM), the important indexes for hepatic sinusoid capillarization, were positive staining in liver tissure. But collagen type Ⅳ (Col Ⅳ), which is a composition of functional membrane but not a composition of basement membrane, expressed majorly in fibrous septa and HSC but less and discontinuously in perisinusoids in controls. Compared with controls, however, the narrow sinusoids were reduced and some sinusoids returned to normal phenotype, basement membrane looked discontinuous thiner, and the positive degree of Ⅷ R·Ag, -SMA and LM were lower and their positive area was significantly smaller by image analysis in the liver of FZHY group than in that of controls (*P*<0.05). Col Ⅳ expressed still continuously in perisinusoids in FZHY group. It indicated that the normal structure of sinusoids was most kept and it meansed that the alteration of hepatic sinusoid capillarization was reversed. (Fig.12)

Traditional Chinese Medicine

Can Improve Liver Microcirculation and Reduce Portal Hypertension in Liver Cirrhosis 107

Fig. 11. Fuzheng Huayu Recipe could inhibit sinusoid capillarization by observation of electron micrographs S: sinusoid; C: collagen. A: Hepatic sinusoid appeare as a typical capillary surrounded by continuous basement membrane. (arrow's heads indicated) in the liver of fibrotic rat. The perisinusoid is stenosis (arrows indicated) and microvilli of hepatocyte are almost absent in the space (15000×). B: Sinusoid capillarization is not formed in the liver of rat treated with Fuzheng Huayu Recipe. The fenestrae are observed (arrow's heads indicated) in cytoplasm of SEC. A mass of microvilli of hepatocyte stretchs into perisinusoid is exist (arrow indicated) but basement membrane is absent (10000×).

Fig. 11. Fuzheng Huayu Recipe could inhibit sinusoid capillarization by observation of electron micrographs S: sinusoid; C: collagen. A: Hepatic sinusoid appeare as a typical capillary surrounded by continuous basement membrane. (arrow's heads indicated) in the liver of fibrotic rat. The perisinusoid is stenosis (arrows indicated) and microvilli of

hepatocyte are almost absent in the space (15000×). B: Sinusoid capillarization is not formed in the liver of rat treated with Fuzheng Huayu Recipe. The fenestrae are observed (arrow's heads indicated) in cytoplasm of SEC. A mass of microvilli of hepatocyte stretchs into perisinusoid is exist (arrow indicated) but basement membrane is absent (10000×).

Traditional Chinese Medicine

30min after injection.

injection of ET-1.

**3.3.3 Results**

**injection of ET-1**

Can Improve Liver Microcirculation and Reduce Portal Hypertension in Liver Cirrhosis 109

(1.0g/kg body weight) and the ET-1 high-dose group (3.0g/kg body weight). Rats were injected with 200l NS solution into rat via femoral vein by a pump with a rate of 80μl/min in NS solution group. As the same method, rats were injected with 200ml ET-1 solution with the dose of 0.3g/kg body weight, 1.0g/kg body weight or 3.0g/kg body weight respectively in 3 ET-1 groups. Then, monitored the changes of carotid pressure and portal vein pressure in each group at 2min, 4min, 6min, 8min, 10min, 15min, 20min, 25min and

20 Sprague-Dawley male rats with about 330g body weight were randomly divided into four groups: normal group, SA-B group, BQ-123 (ETAR antagonist) group and BQ-788 (ETBR antagonist) group (n=5 in each group). In normal group, BQ-123 group and BQ-788 group, rats were gavaged with water at a dose of 1ml/100g body weight and rats in SA-B group were gavaged with SA-B solution (containing 125mg/100ml) at the same dose. The gavage was performed twice per day for 5 days. The experiment started at 1 hour after the final gavage. First, via rat's femoral vein, rats was injected with BQ-123 (12.5µg/kg body weight) in BQ-123 group and with BQ-788 (15µg/kg body weight) in BQ-788 group. 30min later, rats were injected with a dose (3µg/kg body weight) of ET-1 solution by a pump with rate of 80μl/min in each group. Then, the portal vein pressure was measured before and after

A piece of PE-50 tube, which full with heparin solution, was inserted into the left carotid artery of the rat. Following that, connect the tube to a pressure transducer in order to measure carotid pressure and heart rate. Then a piece of PE-10 tube, which full with heparin solution, was inserted from superior mesenteric vein to the middle of portal vein. Connect

**<IP_ADDRESS> A kind of model of rat with portal hypertension was successfully established by**

There was no obvious effect of solution volume on carotid pressure and portal vein pressure after injection of NS solution. ET-1 solution with equal volume of NS solution was injected into the rats in low- and medium-dose groups. The carotid pressure was not noticeable rise or fall, only minor fluctuations within 20 min after injection. The carotid pressure, however, was increased slightly after injection of ET-1 in high-dose group but the alteration was not significant (Fig.13). Portal vein pressure was increased in short time after injection of ET-1 although the alteration was not same in three different dose groups. The alteration of pressure was most remarkable in high-dose group than in others. After reaching to peak level, portal vein pressure was stable for 4min, 4min and 10min in low-, medium- and highdose groups, respectively (Fig.14 and Tab. 5). The results indicated that the increase of portal vein pressure was solely caused by the injection of ET-1 and without related with the volume of solution in this kind of animal model. This inducing method would not cause a significant increase of systemic blood pressure. Compared with the existing portal vein perfusion model, this kind of portal hypertension model is more similar to the "living" state. It was also avoided that increase of portal vein pressure caused by sudden increase of partial

**<IP_ADDRESS> To treat rats with drugs in portal hypertension models**

**<IP_ADDRESS> To measure carotid pressure, heart rate and portal vein pressure**

the tube to a pressure transducer for measurement of portal vein pressure.

Fig. 12. Fuzheng Huayu Recipe could alter the express of indexes for hepatic sinusoid capillarization by immunohistochemistry staining in fibrotic liver of rats Arrows show the positive area. A: Ⅷ R·Ag is around the vessels with strong staining in controls. B: The positive area of Ⅷ R·Ag is decreased in FZHY group. C: Col Ⅳ expresses majorly in fibrous septa and HSC but less and discontinuously in perisinusoids in controls. D: Col Ⅳ expresses almost continuously in perisinusoids in FZHY group. E: The express of LM is observed on the wall of vessels and sinusoids in the liver of controls. F: The express of LM is only observed on the wall of vessels in the liver of FZHY group. G: -SMA expresses strongly in perisinusoids, HSC and fibous septa in controls. H: -SMA expresses dramaticlly less in FZHY group (100×).

#### **3.2.4 Summary**

Fuzheng Huayu Recipe had the potent effect on against liver fibrosis. The one of mechanisms of the recipe perhaps is associated with inhibition of hepatic sinusoid capillarization.

#### **3.3 Experiment 3: SA-B could decrease portal vein pressure in rats induced by ET-1**

#### **3.3.1 Aims**

To establish a rat model of portal hypertension induced by ET-1 and investigate the effect of SA-B on decreasing portal vein pressure in the model.

#### **3.3.2 Methods**

#### **<IP_ADDRESS> To establish portal hypertension model of rats induced by ET-1**

48 Sprague-Dawley male rats were randomly divided into four groups: the NS solution group, the ET-1 low-dose group (0.3g/kg body weight), the ET-1 medium-dose group (1.0g/kg body weight) and the ET-1 high-dose group (3.0g/kg body weight). Rats were injected with 200l NS solution into rat via femoral vein by a pump with a rate of 80μl/min in NS solution group. As the same method, rats were injected with 200ml ET-1 solution with the dose of 0.3g/kg body weight, 1.0g/kg body weight or 3.0g/kg body weight respectively in 3 ET-1 groups. Then, monitored the changes of carotid pressure and portal vein pressure in each group at 2min, 4min, 6min, 8min, 10min, 15min, 20min, 25min and 30min after injection.

#### **<IP_ADDRESS> To treat rats with drugs in portal hypertension models**

20 Sprague-Dawley male rats with about 330g body weight were randomly divided into four groups: normal group, SA-B group, BQ-123 (ETAR antagonist) group and BQ-788 (ETBR antagonist) group (n=5 in each group). In normal group, BQ-123 group and BQ-788 group, rats were gavaged with water at a dose of 1ml/100g body weight and rats in SA-B group were gavaged with SA-B solution (containing 125mg/100ml) at the same dose. The gavage was performed twice per day for 5 days. The experiment started at 1 hour after the final gavage. First, via rat's femoral vein, rats was injected with BQ-123 (12.5µg/kg body weight) in BQ-123 group and with BQ-788 (15µg/kg body weight) in BQ-788 group. 30min later, rats were injected with a dose (3µg/kg body weight) of ET-1 solution by a pump with rate of 80μl/min in each group. Then, the portal vein pressure was measured before and after injection of ET-1.

#### **<IP_ADDRESS> To measure carotid pressure, heart rate and portal vein pressure**

A piece of PE-50 tube, which full with heparin solution, was inserted into the left carotid artery of the rat. Following that, connect the tube to a pressure transducer in order to measure carotid pressure and heart rate. Then a piece of PE-10 tube, which full with heparin solution, was inserted from superior mesenteric vein to the middle of portal vein. Connect the tube to a pressure transducer for measurement of portal vein pressure.

#### **3.3.3 Results**

108 Portal Hypertension – Causes and Complications

Fig. 12. Fuzheng Huayu Recipe could alter the express of indexes for hepatic sinusoid capillarization by immunohistochemistry staining in fibrotic liver of rats Arrows show the positive area. A: Ⅷ R·Ag is around the vessels with strong staining in controls. B: The positive area of Ⅷ R·Ag is decreased in FZHY group. C: Col Ⅳ expresses majorly in fibrous septa and HSC but less and discontinuously in perisinusoids in controls. D: Col Ⅳ expresses almost continuously in perisinusoids in FZHY group. E: The express of LM is observed on the wall of vessels and sinusoids in the liver of controls. F: The express of LM is only observed on the wall of vessels in the liver of FZHY group. G: -SMA expresses strongly in perisinusoids, HSC and

fibous septa in controls. H: -SMA expresses dramaticlly less in FZHY group (100×).

Fuzheng Huayu Recipe had the potent effect on against liver fibrosis. The one of mechanisms of the recipe perhaps is associated with inhibition of hepatic sinusoid

**3.3 Experiment 3: SA-B could decrease portal vein pressure in rats induced by ET-1**

To establish a rat model of portal hypertension induced by ET-1 and investigate the effect of

48 Sprague-Dawley male rats were randomly divided into four groups: the NS solution group, the ET-1 low-dose group (0.3g/kg body weight), the ET-1 medium-dose group

SA-B on decreasing portal vein pressure in the model.

**<IP_ADDRESS> To establish portal hypertension model of rats induced by ET-1**

**3.2.4 Summary**

capillarization.

**3.3.1 Aims**

**3.3.2 Methods**

#### **<IP_ADDRESS> A kind of model of rat with portal hypertension was successfully established by injection of ET-1**

There was no obvious effect of solution volume on carotid pressure and portal vein pressure after injection of NS solution. ET-1 solution with equal volume of NS solution was injected into the rats in low- and medium-dose groups. The carotid pressure was not noticeable rise or fall, only minor fluctuations within 20 min after injection. The carotid pressure, however, was increased slightly after injection of ET-1 in high-dose group but the alteration was not significant (Fig.13). Portal vein pressure was increased in short time after injection of ET-1 although the alteration was not same in three different dose groups. The alteration of pressure was most remarkable in high-dose group than in others. After reaching to peak level, portal vein pressure was stable for 4min, 4min and 10min in low-, medium- and highdose groups, respectively (Fig.14 and Tab. 5). The results indicated that the increase of portal vein pressure was solely caused by the injection of ET-1 and without related with the volume of solution in this kind of animal model. This inducing method would not cause a significant increase of systemic blood pressure. Compared with the existing portal vein perfusion model, this kind of portal hypertension model is more similar to the "living" state. It was also avoided that increase of portal vein pressure caused by sudden increase of partial

Traditional Chinese Medicine

Portal vein pressure

(mmHg)

*P*<0.01

controls, ##: *P*<0.01, ###: *P*<0.001

by ET-1 (X±S)

Can Improve Liver Microcirculation and Reduce Portal Hypertension in Liver Cirrhosis 111

medium-dose group high-dose group

**Groups n Initial pressure Highest**

0 5 7 9 11 13 15 20 25 30 35

Time(min)

Fig. 14. Effect of ET-1 with different doses on rat portal vein pressure After detecting portal vein pressure for 5min, NS solution and ET-1 with same volume but different doses were constantly injected and portal vein pressure was detected for 30min. There was no obvious effect of NS solution on portal vein pressure. The pressure was increased at 5min when starting injection of ET-1 and kept high level for 4min, 4min and 10min in low-, mediumand high-dose groups after the pressure reaching to a peak level at 9 min, respectively.

Portal vein pressure

(mmHg)

 (mmHg) (mmHg) (mmHg) NS 8 6.6±0.3 6.5±0.4 -0.1±0.2 low-dose 10 6.1±0.8 7.5±0.8\*\* 1.3±0.3 medium-dose 10 6.3±0.5 8.1±0.6\*\* 1.8±0.5## high-dose 9 6.0±0.6 8.5±0.9\*\* 2.5±0.5##☆☆

Note: compared with the initial pressure in the interior-group, \*\*: *P*<0.01; compared with NS group, *: P*<0.001; compared with low-dose group, ##*: P*<0.01; compared with medium-dose group, ☆☆:

**Groups n Initial pressure Highest pressure Increased range**  (mmHg) (mmHg) (mmHg) control 5 6.1±0.7 8.6±1.1\*\*\* 2.5±0.4 SA-B 5 6.1±0.7 7.8±1.6\*\* 1.6±0.5## BQ-123 5 5.9±1.1 7.1±1.0 1.2±0.2### BQ-788 5 5.9±0.9 6.9±1.1 0.98±0.5###

Table 5. Effect of ET-1 with different doses on portal vein pressure of rats (X±S)

Note: compared with initial pressure interior-group, \*\*: *P*<0.01, \*\*\*: *P*<0.001; compared with the

**<IP_ADDRESS> SA-B could inhibit the rise of portal vein pressure in rats induced by ET-1**

Table 6. Effect of SA-B, BQ-123 or BQ-788 on reducining portal vein pressure of rats induced

Portal vein pressur significantly increased after ET-1 solution (3μg/kg body weight) was injected to rats pretreated with water by gavage. Although portal vein pressure of the rats,

**pressure Increased range**

0 5 7 9 11 13 15 20 25 30 35

Time (min)

blood volume by quickly direct injection of ET-1 in portal vein. This kind of model would better reflect the true effect of ET-1 compared with other kind of model. Therefore, this kind of model could use not only for investigation of efficacy and mechanism of drug, but also provided an ideal experimental subject for screening drugs to treat portal hypertension.

medium-dose group high-dose group

Fig. 13. Effect of ET-1 with different doses on rat carotid pressures After detecting carotic pressure for 5min, NS solution or ET-1 with same volume but different doses were constantly injected and carotic pressure was detected for 30min. The pressure was not increased during detected time in NS group, and low- and medium-dose groups. But the carotid pressure was increased slightly in high-dose group.

medium-dose group high-dose group

blood volume by quickly direct injection of ET-1 in portal vein. This kind of model would better reflect the true effect of ET-1 compared with other kind of model. Therefore, this kind of model could use not only for investigation of efficacy and mechanism of drug, but also provided an ideal experimental subject for screening drugs to treat portal hypertension.

Portal vein pressure

Carotid pressure

(mmHg)

(mmHg)

Portal vein pressure

(mmHg)

0 5 7 9 11 13 15 20 25 30 35

Time(min)

0 5 7 9 11 13 15 20 25 30 35

Time(min)

0 5 7 9 11 13 15 20 25 30 35

Time(min)

NS group low-dose group

medium-dose group high-dose group

NS group low-dose group

carotid pressure was increased slightly in high-dose group.

0 5 7 9 11 13 15 20 25 30 35

Time (min)

0 5 7 9 11 13 15 20 25 30 35

Time (min)

0 5 7 9 11 13 15 20 25 30 35

Time (min)

Portal vein pressure

(mmHg)

Carotid pressure

Carotid pressure

(mmHg)

(mmHg)

Fig. 13. Effect of ET-1 with different doses on rat carotid pressures After detecting carotic pressure for 5min, NS solution or ET-1 with same volume but different doses were constantly injected and carotic pressure was detected for 30min. The pressure was not increased during detected time in NS group, and low- and medium-dose groups. But the

Fig. 14. Effect of ET-1 with different doses on rat portal vein pressure After detecting portal vein pressure for 5min, NS solution and ET-1 with same volume but different doses were constantly injected and portal vein pressure was detected for 30min. There was no obvious effect of NS solution on portal vein pressure. The pressure was increased at 5min when starting injection of ET-1 and kept high level for 4min, 4min and 10min in low-, mediumand high-dose groups after the pressure reaching to a peak level at 9 min, respectively.

Note: compared with the initial pressure in the interior-group, \*\*: *P*<0.01; compared with NS group, *: P*<0.001; compared with low-dose group, ##*: P*<0.01; compared with medium-dose group, ☆☆: *P*<0.01

Note: compared with initial pressure interior-group, \*\*: *P*<0.01, \*\*\*: *P*<0.001; compared with the controls, ##: *P*<0.01, ###: *P*<0.001

Table 6. Effect of SA-B, BQ-123 or BQ-788 on reducining portal vein pressure of rats induced by ET-1 (X±S)

#### **<IP_ADDRESS> SA-B could inhibit the rise of portal vein pressure in rats induced by ET-1**

Portal vein pressur significantly increased after ET-1 solution (3μg/kg body weight) was injected to rats pretreated with water by gavage. Although portal vein pressure of the rats,

Traditional Chinese Medicine

**3.4.1 Aims**

**3.4.2 Methods**

blocker group.

after injection of ET-1.

**microcirculation**

3 groups.

Can Improve Liver Microcirculation and Reduce Portal Hypertension in Liver Cirrhosis 113

To establish portal hypertension model of mice induced by ET-1 and investigate the effect of

36 male Kunming mices, 30±5g body weight, were randomly divided into three groups: control group (n=12), SA-B group (n=12) and blocker group (n=12). Mice were gavaged with SA-B (1mg/ml) in SA-B group and gavaged with water in control group and blocker group. The volume of gvage was 0.5 ml per mouse once a day for 3days. Mice were injected with BQ-123(2g/kg body weight)by tail vein at 0.5h before start of below experiment in

Mice were injected with ET-1(1.6g/kg body weight)at constant velocity by tail vein. 6 mice were randomly taken out from each group to measure blood flow volume in liver microcirculation using a laser-Doppler flow instrument before and after injection of ET-1. Other 6 mice in each group were measured the blood flow rate in liver microcirculation by an inverted fluorescence microscope with microscopic live video technology before and

Each mouse was injected with 2% pentobarbital sodium solution at a dose of 2ml/kg body weight for anesthesia. After opening abdomen the liver was put on a detector connected with a laser-Doppler flow instrument to record the blood flow volum in the perfusion of liver microcirculation for 5min. After a pause, ET-1 was injected by a syringe pump at a constant rate of 80l/ min through tail vein. Then, the blood flow volum was recorded again for 5min. The average blood flow volume was calculated from the stable datas during the

Each mouse was injected with 2% pentobarbital sodium solution at a dose of 2ml/kg body weight for anesthesia.  After 0.05ml pre-labeled erythrocytes with FITC was injected into the inferior vena cava, a suitable site was focused to observe the liver microcirculation. Some FITC-RBCs were observed to be moving quickly in microvasculature or sinusoids and recorded with digital video. ET-1(1.6g /kg body weight) was injected by a syringe pump at a constant rate of 80l/ min through tail vein of mouse then moving FITC-RBCs were immediately recorded again with digital video. The moving range of FITC-RBCs in record was calculated by the analytic software to compare the differences of blood flow rate in liver microcirculation in

**3.4 Experiment 4: SA-B can decrease portal vein pressure in mice induced by ET-1**

**<IP_ADDRESS> To establish a kind of portal hypertension model mice induced by ET-1**

**<IP_ADDRESS> To measure the average blood flow volume in the perfusion of liver**

former 5min record and late 5min record, respectively.

**<IP_ADDRESS> To measure the blood flow rate in liver microcirculation**

SA-B on decreasing portal vein pressure in this kind of model.

which were pretreated with SA-B solution by gavage or with BQ-123 or BQ-788 by injection, was also increased after ET-1 injection, however, the ranget-increased was obviously less than that in controls (*P*<0.01, *P*<0.001) (Tab.6). Furthermore, the stable time, when portal vein pressure kept high level after reaching peak level, was short in SA-B group. It was just 6 min in SA-B group but was 10min in controls. (Fig. 15)

Fig. 15. SA-B could inhibit portal vein pressure of rats induced by ET-1 Portal vein pressure began increase at 5min when starting injection of ET-1. The peak level of pressure was at 9min in SA-B group. Then the pressure kept high level until to at 15min. In controls, however, the pressure continuously increased until to highest peak level at 15min and began to decrease. The pressure level was still hinger (a little bit higher than a peak level in SA-B group) until to at 25min.

Table 7. Compare of the carotid artery pressure and heart rate in SA-B group and controls

#### **<IP_ADDRESS> SA-B did not affect on carotid pressure and heart rate in normal rat**

Rats were pretreated with SA-B solution by gavage for 5d, but the carotid pressure and heart rate were no significant different between in SA-B group and controls. (Tab.7)

#### **3.3.4 Summary**

A kind of rat model of portal hypertension is successfully established by injection of ET-1. SA-B can down regulate the raised portal vein pressure by antagonizing the effect of extraneous ET-1.

#### **3.4 Experiment 4: SA-B can decrease portal vein pressure in mice induced by ET-1**

#### **3.4.1 Aims**

112 Portal Hypertension – Causes and Complications

which were pretreated with SA-B solution by gavage or with BQ-123 or BQ-788 by injection, was also increased after ET-1 injection, however, the ranget-increased was obviously less than that in controls (*P*<0.01, *P*<0.001) (Tab.6). Furthermore, the stable time, when portal vein pressure kept high level after reaching peak level, was short in SA-B group. It was just

Fig. 15. SA-B could inhibit portal vein pressure of rats induced by ET-1 Portal vein pressure began increase at 5min when starting injection of ET-1. The peak level of pressure was at 9min in SA-B group. Then the pressure kept high level until to at 15min. In controls, however, the pressure continuously increased until to highest peak level at 15min and began to decrease. The pressure level was still hinger (a little bit higher than a peak level in

Time

13min

15min

20min

25min

30min

35min

**pressure**

 (mmHg) (Beats/min) control 3 130.9±13.2 439±21 SA-B 5 145.6±10.1 439±21 Table 7. Compare of the carotid artery pressure and heart rate in SA-B group and controls

Rats were pretreated with SA-B solution by gavage for 5d, but the carotid pressure and

A kind of rat model of portal hypertension is successfully established by injection of ET-1. SA-B can down regulate the raised portal vein pressure by antagonizing the effect of

**Heart rate**

Control SA-B

**Groups n Carotid artery**

**<IP_ADDRESS> SA-B did not affect on carotid pressure and heart rate in normal rat**

heart rate were no significant different between in SA-B group and controls. (Tab.7)

6 min in SA-B group but was 10min in controls. (Fig. 15)

SA-B group) until to at 25min.

0.0 2.0 4.0 6.0 8.0 10.0

Portal vein pressure(mmHg)

0min

5min

7min

9min

11min

**3.3.4 Summary**

extraneous ET-1.

To establish portal hypertension model of mice induced by ET-1 and investigate the effect of SA-B on decreasing portal vein pressure in this kind of model.

#### **3.4.2 Methods**

#### **<IP_ADDRESS> To establish a kind of portal hypertension model mice induced by ET-1**

36 male Kunming mices, 30±5g body weight, were randomly divided into three groups: control group (n=12), SA-B group (n=12) and blocker group (n=12). Mice were gavaged with SA-B (1mg/ml) in SA-B group and gavaged with water in control group and blocker group. The volume of gvage was 0.5 ml per mouse once a day for 3days. Mice were injected with BQ-123(2g/kg body weight)by tail vein at 0.5h before start of below experiment in blocker group.

Mice were injected with ET-1(1.6g/kg body weight)at constant velocity by tail vein. 6 mice were randomly taken out from each group to measure blood flow volume in liver microcirculation using a laser-Doppler flow instrument before and after injection of ET-1. Other 6 mice in each group were measured the blood flow rate in liver microcirculation by an inverted fluorescence microscope with microscopic live video technology before and after injection of ET-1.

#### **<IP_ADDRESS> To measure the average blood flow volume in the perfusion of liver microcirculation**

Each mouse was injected with 2% pentobarbital sodium solution at a dose of 2ml/kg body weight for anesthesia. After opening abdomen the liver was put on a detector connected with a laser-Doppler flow instrument to record the blood flow volum in the perfusion of liver microcirculation for 5min. After a pause, ET-1 was injected by a syringe pump at a constant rate of 80l/ min through tail vein. Then, the blood flow volum was recorded again for 5min. The average blood flow volume was calculated from the stable datas during the former 5min record and late 5min record, respectively.

#### **<IP_ADDRESS> To measure the blood flow rate in liver microcirculation**

Each mouse was injected with 2% pentobarbital sodium solution at a dose of 2ml/kg body weight for anesthesia.  After 0.05ml pre-labeled erythrocytes with FITC was injected into the inferior vena cava, a suitable site was focused to observe the liver microcirculation. Some FITC-RBCs were observed to be moving quickly in microvasculature or sinusoids and recorded with digital video. ET-1(1.6g /kg body weight) was injected by a syringe pump at a constant rate of 80l/ min through tail vein of mouse then moving FITC-RBCs were immediately recorded again with digital video. The moving range of FITC-RBCs in record was calculated by the analytic software to compare the differences of blood flow rate in liver microcirculation in 3 groups.

Traditional Chinese Medicine

**4.2.1 To observe contraction of HSC**

experiment was repeated for 3 times.

**4.2.2 To detect the concentration of free calcium ([Ca2+]i) in HSC**

**4.3.1 SA-B inhibited contraction of HSC induced by ET-1**

group was between the low- and high-dose groups. (Fig. 17)

**4.1 Aims**

**4.2 Methods**

repeated 2 times.

**4.3 Results**

Can Improve Liver Microcirculation and Reduce Portal Hypertension in Liver Cirrhosis 115

To investigate the effects of SA-B on inhibiting contraction of human HSC induced by ET-1.

Human HSC was isolated from the normal section of transplant patients'liver by enzymatic digestion and density-gradient centrifugation with Nycodenz solution.14. Passaged HSC was planted on the collagen gel which was pre-poured into 12-wells plates. After cells had adhered, HSC was divided into 5 groups: 1) serum-free group in which HSC was cultured with serum-free M199; 2) ET group in which HSC was treated with ET-1(10 -8 mol/L); 3) low-dose group in which HSC was treated with ET-1 and 10-7mol/L SA-B; 4) medium-dose group in which HSC was treated with ET-1 and 10-6mol/L SA-B; 5) high-dose group in which HSC was treated with ET-1 and 10-5mol/L SA-B. The edge of the gel was scraped out wall of the well by a syringe needle after adding drugs. The diameter of gel was observed at 2h, 4h, 6h, and 12 h during HSC culture and a gel image analysis system was used to calculate the area of gel. The area of gel can indicate the contractibility of HSC. The

HSC was planted in 6-wells plates. Flu-3/AM was added into media with final concentration of 5μmol/L. Then HSC was in a incubator with 37℃, 5%CO2-95% humid air and dark for 30min. HSC was washed twice with serum-free M199 for moving the dye and divided into 5 groups as above. HSC in each group was observed under a laser confocal microscope before and after adding relevant drugs. The observation conditions were Kr/Ar laser, excitation wavelength 488nm, emission wavelength 505-530nm. The experiment was

Area of gel was significantly bigger in low-dose group (2209.02±177.96μm2), medium-dose group (2164.95±111.84μm2) and high-dose group (2374.73±218.38μm2) than in ET group (156.23±102.16m2). The statistical difference was dramatically (*P*<0.01). But there was no obviously difference among in 3 SA-B treatment groups. (Fig. 16) The morphological changes of HSC showed dose-dependent after incubation with SA-B. The phenotype of HSC in low-dose group was similar to that in ET group. The phenotype of HSC in high-dose group was similar to that in serum-free group. The phenotype of HSC in medium-dose

**4.3.2 SA-B suppressed the intracellular concentration of [Ca2+]i in HSC induced by ET-1**  Compared with ET group in which fluorescence of HSC was very strong, [Ca2+]i fluorescence image appeared the opposite phenomenon in 3 SA-B treatment groups. Only a few cells appeared slightly enhanced fluorescent and the intracellular concentration of

#### **3.4.3 Results**

#### **<IP_ADDRESS> SA-B reduced average blood flow volume in liver microcirculation**

The blood flow volume in liver microcirculation was decreased in 3 groups after injection of ET-1. The reduced range was smaller in SA-B group and in block group than in controls (*P*<0.01). (Tab. 8)

#### **<IP_ADDRESS> SA-B reduced average blood flow rate in liver microcirculation**

The blood flow rate in liver microcirculation was significantly decreased after injection of ET-1 (*P*<0.01). But the reduced range was smaller in SA-B group and in blocker group than in controls (*P*<0.05 and *P*<0.01). (Tab. 9)

Note: compared with the controls, \*\*: *P*<0.01

Table 8. SA-B and BQ-123 could inhibit reduction of average blood flow volume in mice induced by ET-1 (X ± S)

Note: compared with initial blood flow rate,☆☆: *P*<0.01; compared with controls, \*: *P*<0.05,\*\*: *P*<0.01

Table 9. SA-B and BQ-123 could inhibit reduction of the liver blood flow rate in mice induced by ET-1 (X ± S).

#### **3.4.4 Summary**

The average blood flow volume and blood flow rate in mice liver microcirculation were reduced by extraneous ET-1. SA-B could inhibit the effect of ET-1 to improve liver microcirculation.

#### **4. The experiment** *in vitro*

It is believed that recipe of herb medicine is not suitable for research of mechanism *in vitro*. For more studying the mechanism of Fuzheng Huayu Recipe on reducing portal hypertension, we can only select SA-B as investigated subject *in vitro*.

#### **4.1 Aims**

114 Portal Hypertension – Causes and Complications

The blood flow volume in liver microcirculation was decreased in 3 groups after injection of ET-1. The reduced range was smaller in SA-B group and in block group than in controls

The blood flow rate in liver microcirculation was significantly decreased after injection of ET-1 (*P*<0.01). But the reduced range was smaller in SA-B group and in blocker group than

**Average blood flow volume in liver microcirculation**(**BPU**)

**Before ET-1 injection**

Table 8. SA-B and BQ-123 could inhibit reduction of average blood flow volume in mice

**Blood flow rate in liver microcirculation**(**mm/s**)

**injection**

Table 9. SA-B and BQ-123 could inhibit reduction of the liver blood flow rate in mice

Control 6 0.60±0.01 0.36±0.02☆☆ 0.23±0.02 SA-B 6 0.62±0.04 0.39±0.01☆☆ 0.18±0.04 \* blocker 6 0.60±0.05 0.43±0.01☆☆ 0.17±0.05 \*\* Note: compared with initial blood flow rate,☆☆: *P*<0.01; compared with controls, \*: *P*<0.05,\*\*: *P*<0.01

The average blood flow volume and blood flow rate in mice liver microcirculation were reduced by extraneous ET-1. SA-B could inhibit the effect of ET-1 to improve liver

It is believed that recipe of herb medicine is not suitable for research of mechanism *in vitro*. For more studying the mechanism of Fuzheng Huayu Recipe on reducing portal

hypertension, we can only select SA-B as investigated subject *in vitro*.

control 6 1186.83±41.14 1060.50±18.33 126.33±27.51 SA-B 6 1269.50±59.90 1189.33±40.33 80.17±20.30 \*\* blocker 6 202.00±36.54 1131.00±23.67 71.00±12.82 \*\*

**After ET-1**

**After ET-1**

**injection Reduced range**

**injection Reduced range**

**<IP_ADDRESS> SA-B reduced average blood flow volume in liver microcirculation**

**<IP_ADDRESS> SA-B reduced average blood flow rate in liver microcirculation**

**3.4.3 Results**

(*P*<0.01). (Tab. 8)

in controls (*P*<0.05 and *P*<0.01). (Tab. 9)

Note: compared with the controls, \*\*: *P*<0.01

**Groups n Before ET-1**

**Groups n**

induced by ET-1 (X ± S)

induced by ET-1 (X ± S).

**4. The experiment** *in vitro*

**3.4.4 Summary**

microcirculation.

To investigate the effects of SA-B on inhibiting contraction of human HSC induced by ET-1.

#### **4.2 Methods**

#### **4.2.1 To observe contraction of HSC**

Human HSC was isolated from the normal section of transplant patients'liver by enzymatic digestion and density-gradient centrifugation with Nycodenz solution.14. Passaged HSC was planted on the collagen gel which was pre-poured into 12-wells plates. After cells had adhered, HSC was divided into 5 groups: 1) serum-free group in which HSC was cultured with serum-free M199; 2) ET group in which HSC was treated with ET-1(10 -8 mol/L); 3) low-dose group in which HSC was treated with ET-1 and 10-7mol/L SA-B; 4) medium-dose group in which HSC was treated with ET-1 and 10-6mol/L SA-B; 5) high-dose group in which HSC was treated with ET-1 and 10-5mol/L SA-B. The edge of the gel was scraped out wall of the well by a syringe needle after adding drugs. The diameter of gel was observed at 2h, 4h, 6h, and 12 h during HSC culture and a gel image analysis system was used to calculate the area of gel. The area of gel can indicate the contractibility of HSC. The experiment was repeated for 3 times.

#### **4.2.2 To detect the concentration of free calcium ([Ca2+]i) in HSC**

HSC was planted in 6-wells plates. Flu-3/AM was added into media with final concentration of 5μmol/L. Then HSC was in a incubator with 37℃, 5%CO2-95% humid air and dark for 30min. HSC was washed twice with serum-free M199 for moving the dye and divided into 5 groups as above. HSC in each group was observed under a laser confocal microscope before and after adding relevant drugs. The observation conditions were Kr/Ar laser, excitation wavelength 488nm, emission wavelength 505-530nm. The experiment was repeated 2 times.

#### **4.3 Results**

#### **4.3.1 SA-B inhibited contraction of HSC induced by ET-1**

Area of gel was significantly bigger in low-dose group (2209.02±177.96μm2), medium-dose group (2164.95±111.84μm2) and high-dose group (2374.73±218.38μm2) than in ET group (156.23±102.16m2). The statistical difference was dramatically (*P*<0.01). But there was no obviously difference among in 3 SA-B treatment groups. (Fig. 16) The morphological changes of HSC showed dose-dependent after incubation with SA-B. The phenotype of HSC in low-dose group was similar to that in ET group. The phenotype of HSC in high-dose group was similar to that in serum-free group. The phenotype of HSC in medium-dose group was between the low- and high-dose groups. (Fig. 17)

#### **4.3.2 SA-B suppressed the intracellular concentration of [Ca2+]i in HSC induced by ET-1**

Compared with ET group in which fluorescence of HSC was very strong, [Ca2+]i fluorescence image appeared the opposite phenomenon in 3 SA-B treatment groups. Only a few cells appeared slightly enhanced fluorescent and the intracellular concentration of

Traditional Chinese Medicine

**A B**

**C D**

**E**

system microscope, Japan, 200×).

Can Improve Liver Microcirculation and Reduce Portal Hypertension in Liver Cirrhosis 117

Fig. 17. The morphological changes of HSC in each group a: serum-free group; b: ET group; c: low-dose group; d: medium-dose group; e: high-dose group. HSC grew exuberantly on gel and the pseudopodium of HSC was a lot, thin and long in serum-free group. After induced by ET-1 for 12h, however, HSC became obviously small with contraction. The quantity and the pseudopodium of HSC was decreased in ET group. The phenotype of HSC in low-dose group was similar to that in ET group. The phenotype of HSC in high-dose group was similar to that in serum-free group. The phenotype of HSC in medium-dose group was between the low- and high-dose groups (By OLYMPUS I × 50 / I × 70 inverted

[Ca2+]i was obviously lower in 3 SA-B treatment groups than in ET group. The intracellular concentration of [Ca2+]i was weak and only two relative low-lying peaks were in low-dose group within 120sec. The alteration of intracellular concentration of [Ca2+]i was smaller in medium-dose group and high-dose group during observation. (Fig.18). The results indicated that one of mechanisms of SA-B on inhibiting contraction of HSC may be related with that SA-B suppressed the intracellular concentration of [Ca2+]i in HSC.

★★ VS model group *P*<0.01 ☆☆ VS serum-free group *P*<0.01

Fig. 16. SA-B could inhibit contraction of human HSC a: serum-free group; b: ET group; c: low-dose group; d: medium-dose group; e: high-dose group. Human HSC was cultured on collangen gel. Actived HSC are able to contract to lead to area of gel becoming small. In serum-free group, contraction capacity of HSC was weak so that the area of gel was nearly invariant. Active HSC induced by ET-1 contracted strongly. The area of gel was significantly smaller in ET group than in serum-free groups. SA-B could inhibit contraction of HSC induced by ET-1. The area of gel was bigger in 3 SA-B treatment groups than in ET group.

[Ca2+]i was obviously lower in 3 SA-B treatment groups than in ET group. The intracellular concentration of [Ca2+]i was weak and only two relative low-lying peaks were in low-dose group within 120sec. The alteration of intracellular concentration of [Ca2+]i was smaller in medium-dose group and high-dose group during observation. (Fig.18). The results indicated that one of mechanisms of SA-B on inhibiting contraction of HSC may be related

d

e

Fig. 16. SA-B could inhibit contraction of human HSC a: serum-free group; b: ET group; c: low-dose group; d: medium-dose group; e: high-dose group. Human HSC was cultured on collangen gel. Actived HSC are able to contract to lead to area of gel becoming small. In serum-free group, contraction capacity of HSC was weak so that the area of gel was nearly invariant. Active HSC induced by ET-1 contracted strongly. The area of gel was significantly smaller in ET group than in serum-free groups. SA-B could inhibit contraction of HSC induced by ET-1. The area of gel was bigger in 3 SA-B treatment groups than in ET group.

☆☆

Groups

★★ ★★ ★★

medium-dose

★★ VS model group *P*<0.01 ☆☆ VS serum-free group *P*<0.01

with that SA-B suppressed the intracellular concentration of [Ca2+]i in HSC.

a

b

c

Fig. 17. The morphological changes of HSC in each group a: serum-free group; b: ET group; c: low-dose group; d: medium-dose group; e: high-dose group. HSC grew exuberantly on gel and the pseudopodium of HSC was a lot, thin and long in serum-free group. After induced by ET-1 for 12h, however, HSC became obviously small with contraction. The quantity and the pseudopodium of HSC was decreased in ET group. The phenotype of HSC in low-dose group was similar to that in ET group. The phenotype of HSC in high-dose group was similar to that in serum-free group. The phenotype of HSC in medium-dose group was between the low- and high-dose groups (By OLYMPUS I × 50 / I × 70 inverted system microscope, Japan, 200×).

Traditional Chinese Medicine

**5. Conclusion**

liver diseases.

**7. Reference**

**6. Acknowledgements**

Yuanwei is gratefully acknowledged.

1996,110:534-548.

Anat Rec (Hoboken). 2008,291(6):661-71.

hepatitis. J Hepatol. 2009,50(3):631-3.

Hepatol 1995, 22: 700-706.

Hepatopancreatobiliary Surgery. 2000,12(1):53-54.

Can Improve Liver Microcirculation and Reduce Portal Hypertension in Liver Cirrhosis 119

The Chinese medicine, Fuzheng Huayu Recipe, is able to reduce portal hypertension in liver cirrhosis both in clinic and *in vivo*. The effect of Fuzheng Huayu Recipe is related with reversing hepatic fibrosis and capillarization, and decreasing concentration of ET-1 in the liver tissue. SA-B is an extracted component of Fuzheng Huayu Recipe. Several studies showed that the effect in SA-B was similar to Fuzheng Huayu Recipe on reversing hepatic fibrosis and decreasing concentration of ET-1 in the liver tissue. SA-B inhibited contraction of HSC through suppressing the intracellular concentration of [Ca2+]i. Based on above effects, intrahepatic vessel resistance was reduced which leaded to improve liver microcirculation. It has distinctively character that Chinese herb medicine is used to treat hepatic portal hypertension which is caused by failure of liver microcirculation in chronic

This work was supported by grants from National Natural Science Foundation of China 30672489; Leading Academic Discipline Project of Shanghai Municipal Education Commission J50307; E-Institute of TCM Internal Medicine, Shanghai Municipal Education Commission E03008; Innovation Research Team in Universities, Shanghai Municipal Education Commission; and Leading Academic Discipline Project of Hepatology, SATCM (2011sh). Translation assistance from Lu Chao, Wang Meifeng, Zhang Jing, Wu Mei and Pan

McCuskey RS. The hepatic microvascular system in health and its response to toxicants.

Wu Zhiyong. Regulation of liver microcirculation and portal hypertension. J.

Pinzani M, Milani S, Defranco R, et al. Endothelin 1 is overexpressed in human cirrhotic

Vollmar B, Menger. The hepatic microcirculation: mechanistic contributions and therapeutic

Spengler U. Hepatic microcirculation: a critical but neglected factor for the outcome of viral

Nishida J, McCuskey RS, McDonnell D. Protective Role of NO in Hepatic Microcirculatory Dysfunction during Endotoxemia. Am J Physiol. 1994,267(6 Pt 1):G1135-41. Ramadori G. The stellate cell of the liver. Virchows Arch B Cell Pathol 1991, 61:147-158. Pinzani M. Hepatic stellate (ITO) cells: expanding roles for a liver-specific pericyte. J

ZHANG Jie, XU Lieming, ZHANG Wenwei. Study of Effect and Mechanism of Salvianolic-

by Endothelin-1. Chinese J Integrative Medicine. 2009,29(1):60-64.

acid B Salt on Inhibiting the Contraction of Human Hepatic Stellate Cells Induced

targets in liver injury and repair. Physiol Rev. 2009,89(4):1269-339.

liver and exerts multiple effects on activated hepatic stellate cells. Gastroenterology

Fig. 18. SA-B suppressed the intracellular concentration of [Ca2+]i in HSC Fluorescent of Flu-3/AM which represents the intracellular concentration of [Ca2+]i was observed. The expression of fluorescent in HSC was very stronger in ET group than in serum-free group. But the expression of fluoresent in HSC was dose-dependent in 3 SA-B treatment groups. The higher the dose of SA-B was, the weaker expression of fluorescent was (By Zeiss510 laser confocal microscope, German, 200×).

#### **5. Conclusion**

118 Portal Hypertension – Causes and Complications

**Groups:**

serum-free ET

high-dose

low-dose medium-dose

Fig. 18. SA-B suppressed the intracellular concentration of [Ca2+]i in HSC Fluorescent of Flu-

3/AM which represents the intracellular concentration of [Ca2+]i was observed. The expression of fluorescent in HSC was very stronger in ET group than in serum-free group. But the expression of fluoresent in HSC was dose-dependent in 3 SA-B treatment groups. The higher the dose of SA-B was, the weaker expression of fluorescent was (By Zeiss510

laser confocal microscope, German, 200×).

The Chinese medicine, Fuzheng Huayu Recipe, is able to reduce portal hypertension in liver cirrhosis both in clinic and *in vivo*. The effect of Fuzheng Huayu Recipe is related with reversing hepatic fibrosis and capillarization, and decreasing concentration of ET-1 in the liver tissue. SA-B is an extracted component of Fuzheng Huayu Recipe. Several studies showed that the effect in SA-B was similar to Fuzheng Huayu Recipe on reversing hepatic fibrosis and decreasing concentration of ET-1 in the liver tissue. SA-B inhibited contraction of HSC through suppressing the intracellular concentration of [Ca2+]i. Based on above effects, intrahepatic vessel resistance was reduced which leaded to improve liver microcirculation. It has distinctively character that Chinese herb medicine is used to treat hepatic portal hypertension which is caused by failure of liver microcirculation in chronic liver diseases.

#### **6. Acknowledgements**

This work was supported by grants from National Natural Science Foundation of China 30672489; Leading Academic Discipline Project of Shanghai Municipal Education Commission J50307; E-Institute of TCM Internal Medicine, Shanghai Municipal Education Commission E03008; Innovation Research Team in Universities, Shanghai Municipal Education Commission; and Leading Academic Discipline Project of Hepatology, SATCM (2011sh). Translation assistance from Lu Chao, Wang Meifeng, Zhang Jing, Wu Mei and Pan Yuanwei is gratefully acknowledged.

#### **7. Reference**

**7**

*Argentina*

**Role of Manganese**

Amalia Delfante2, Pablo Souto2,

 *and Department of Pathophysiology, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, 2Laboratory of Portal Hypertension,*

*University of Buenos Aires, Buenos Aires,*

Juan Pablo Prestifilippo1,2, Silvina Tallis2,

*1Institute of Pharmacological Research (ININFA), National Research Council of Argentina (CONICET)*

Juan Carlos Perazzo2 and Gabriela Beatriz Acosta1,2

*School of Pharmacy and Biochemistry & Hepatic Encephalopathy,*

**as Mediator of Central Nervous System:**

**Alteration in Experimental Portal Hypertension**

Portal hypertension (PH) is a major syndrome that frequently accompany chronic liver diseases such as cirrhosis. Prehepatic PH develops a splanchnic hyperdynamic circulation and hyperemia with increased splanchnic resistance and production of collateral vessels that drive splanchnic blood flow to systemic circulation (Chojkier & Groszmann, 1981). Several substances have been proposed as mediators of this hypodynamic circulatory state including prostacyclins, nitric oxide and endotoxins (Bosch et al., 1992; Reiner & Groszmann, 1999; Palma et al., 2005). PH is found in patients with cirrhosis, and in portal vein thrombosis. It is characterized by an increase in splanchnic blood flow and pressure, among others caused by abdominal blood flow resistance, secondary to important liver

Recent studies have demonstrated that experimental PH in rats is also a sub-clinic model of Minimal Hepatic Encephalopathy (MHE) (Butterworth et al., 2009), since rats with PH develop hyperammonemia, electrophysiology alterations, blood-brain barrier (BBB) breakdown, hippocampal mitochondrial dysfunction and changes in frontal cortex and hippocamus on glutamate uptake (Scorticati et al., 2004; Lores-Arnaiz et al., 2005; Eizayaga

Chronic hepatic encephalopathy (HE) is a complex neuropsychiatric syndrome associated with liver dysfunction, such as cirrhosis. The pathophysiology of HE is poorly understood and there are few high-quality diagnostic tests and markers. As a result, its treatment has

**1. Introduction**

parenchyma alterations (fibrosis or cirrhosis).

et al., 2006; Acosta et al., 2009; Bustamante et al., 2011).


*<IP_ADDRESS> Plumb line method*

The two-dimensional evaluation of posture, using a plumb line, is very common, due to its low-cost and simplicity [23]. Kendall et al. postulated guidelines to evaluate posture in accordance with the alignment of the ideal plumb line for the measurement of the sagittal and frontal planes [24]. Kendall et al. state that the ideal alignment of sagittal plane posture is when the plumb line intersects the ear lobe, through the shoulder joint; then through the greater trochanter of the hip, just in front of the knee joint; and finally slightly in front of the lateral malleolus of the ankle before it reaches the floor. Williams and McClay reported that the plumb line method had a good intra-rater reliability for measuring postural variables with an average ICC of 0.80 in both 10 and 90% of body weight bearing scenarios in standing [10]. The standard error of the mean (SEM) reported was between 2 and 5 mm for the lower limb indices and from 5 to 10 mm for patients with a trunk list or lateral shift. List is defined as 'the lateral displacement, in millimetres, of a surface marking of the spinous processes of T12 from that of S1' (McKenzie and May [25], p. 214). Furthermore, Hickey et al. evaluated the reliability of using the plumb line to measure resting head posture in a large sample size of 122 healthy volunteers (80 women and 42 men, ages 18–60 years) [26]. In this study, all participants were screened for cranial, cervical and/or upper thoracic dysfunction. The results of this study demonstrated the plumb line method to have high intra-rater reliability with ICCs ranging from 0.83 to 0.84 for the measurement of resting head posture. Although the plumb line method has been reported to have good intra-rater reliability and is a useful and easy to use instrument for measuring posture, its limitations include the difficulty of minimising movement error or postural sway [9, 27]. Additionally, this plumb line method only measures one plane.


*4.1. Massively Bedded UK Halite Deposits Available*

Important massively bedded halite deposits are developed in the UK and have been associated with, or identified as potential hosts for, large solution-mined natural gas storage caverns (Figure 1; S2, Table S1). The halite deposits considered extensive and thick enough for cavern construction occur in four main basins (with ages) [37]:

• The Fordon Evaporite Formation, on- and extending offshore Eastern England (Upper Permian, Zechstein [Z2]).

**Figure 2.** Workflow for the estimation of exergy storage provided by solution-mined salt caverns in the main halite-bearing basins of the UK.

These deposits offer important alternative energy storage capacity, and this study has assessed their potential for large-scale exergy storage through CAES. Differing from energy that is always conserved, exergy which takes its basis from the second law of thermodynamics, measures the loss of energy quality in every energy transformation process. Exergy tends to be destroyed during any conversion or storage processes, and therefore, exergy storage capacity quantifies the maximum useful work of the stored air that could be used in subsequent power generation. Exergy analysis is employed in applications with electricity output and power generation processes, and an exergy analysis tool was developed to estimate the exergy losses in energy conversions associated with a salt cavern-based CAES system. This permitted an estimate of the exergy storage capacity of the compressed air stored in a salt cavern for generating electricity during the discharging period [38]. Compared to conventional static thermodynamic exergy analysis, our developed tool also considers time-dependent factors that affect the overall electrical efficiency of a CAES system, such as dynamic internal air responses in the cavern and the coupled thermal effects of surrounding rocks [S3].

The Triassic and Permian bedded halite deposits in Northern Ireland have not been included here, as they are poorly defined and largely identified for UGS purposes [37]. Equally, the available Preesall Halite in NW England has also been identified for UGS and is not included here [37,39]. The Zechstein halite beds extend offshore from eastern England into the Southern North Sea, where due to halokinesis, they may attain great thicknesses. For various reasons, they have not been included in this study: they occur often far offshore and show significant changes in thickness over short distances, with some salt structures rising to shallow depths, even approaching close to sea bed, and are often in association with existing producing gasfields [40]. However, they should not be ruled out as CAES hosts, perhaps linked to the growing number of offshore windfarms. If existing hydrocarbon infrastructure (platforms, pipeline and cable routes, etc.) could be re-purposed, development costs, which are high for proposed gas storage caverns (A. Stacey, pers comm.), might be reduced significantly.


**Orthopedics**

#### **Chapter 8 Provisional chapter**

#### **Patellar Luxation in Small Animals Patellar Luxation in Small Animals**

Cleuza M.F. Rezende, Renato César Sachetto Tôrres, Cleuza M.F. Rezende, Renato César Sachetto Tôrres, Anelise Carvalho Nepomuceno,

Anelise Carvalho Nepomuceno, Juliana Soares Lara and

Juliana Soares Lara and Jessica Alejandra Castro Varón

Jessica Alejandra Castro Varón

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/65764

#### **Abstract**

This study describes lesions that occur in the stifle joints of dogs with patellar luxation. These lesions are associated with the animal's age, body weight and degree of luxation. It also reports on the rate of re-dislocation. The patellar lesions found include articular cartilage erosion, subchondral bone exposure, a flattened or concave patellar surface and enthesophytes. Extrapatellar lesions included synovitis, osteophytes, blunting of the trochlear groove, an absent trochlea, erosion of the condylar margins, capsule thickening, a long digital extensor tendon injury, "joint mice," flap formation, cranial cruciate ligament rupture and meniscal prolapse. Such lesions were frequently found in animals with grade II or III luxation that were aged 24 months or older; they were more severe in dogs weighing more than 15 kg. Patellar luxation causes changes that favour articular degeneration and should be treated surgically. Conservative treatment relieves pain but does not address tissue alterations.

**Keywords:** luxation, patella, dogs, lesions, joint


*5.2. Rethinking the Viability of Smart City Domains and Strategies*

A smart economy tends to possess high value-added industries, so it is proposed that high value-added industrial investment promotion initiatives be reconsidered geographically as the distribution of secondary industry is unbalanced and currently heavily favors the Peninsula and urban areas [119]. Sabah and Sarawak are still heavily dependent on primary products (i.e., timber, oil, and LNG). Targeting the relocation of manufacturing sectors to less-developed areas, which would create new urban growth centers or smart cities, needs far more attention, rather than targeting high-value investment in the already mature urban and metropolitan areas. Furthermore, wages in less-developed areas need to be improved since continuing to invest in high-value industries in urban areas will further exacerbate the urbanization issues. Malaysia could learn from India in promoting balanced and sustainable economic growth and ensuring all economic activities work well at the local level (refer to [20]). Furthermore, Malaysia could also learn from Hong Kong in placing greater focus on sharing economic activities among regions and in the re-industrialization of the necessary supporting primary and secondary sectors (refer to [8]).

Another point to consider is the potential of e-commerce. Online transaction expansion initiatives that are gaining a place in the hearts of consumers can be created in line with the increasingly busy lifestyle of urban citizens. A study of online purchasing practices in Malaysia by [120] found that buying online was chosen because it is a convenient and easy way to shop for necessities while avoiding long queues at the counter. Online shopping is a trend in modern society since internet usage has increased in the last decade. It has accelerated under the stay-at-home new normality caused by the COVID-19 pandemic threat. Thus, considering online shopping initiatives as part of the smart economy initiative should enable improvements in the economic status of urban residents, either as traders or customers, which would facilitate the lives of both parties [25].

For smart living, the experts rejected the understanding that the crime rate in Malaysia remains high compared to other countries. The respondents thought that the crime rate was under control level. This opinion matches the findings of [121], whose local studies in Kuala Lumpur showed that city residents are comfortable with the crime situation. Additionally, Ref. [121] found that the perception that the crime rate was high in Malaysia actually did exist in the foreign discourse. Thus, the authorities could consider all such perspectives, turning their focus to the means of adaptation to the fear of crime, the omnipresence of police in public spaces, and assistance to prevent criminal acts in community areas [47,121].

Furthermore, in terms of smart living, voluntary and more active community involvement initiatives related to the safety, educational, and health aspects of the local community can be added to reduce the extent of the dependence on government resources. According to [122], community involvement ensures that the needs and aspirations of the community are not neglected; the result is that community members will be educated and subsequently empowered. This shows that the role of the community can resolve local issues more effectively.

In terms of smart people, referring to skilled and talented human capital, the government must rethink tertiary education and determine how to actively produce digitally talented innovative graduates to suit the value-added industry in the Industry 4.0 era. More structured and holistic learning opportunities within the areas of IoT devices development, telecommunications, middleware, big data analytics, and artificial intelligence are needed. This is because engineering students currently focus mainly on hardware and connectivity aspects while computer science students learn middleware and big data analytics separately [104]. To adopt Industry 4.0 technologies in Malaysian smart city society, a radical paradigm shift in educating graduates so they transform into talented human capital should be the priority, a notion that was reaffirmed by the Prime Minister [67]. Thus, the 2013 national education policy is somewhat outdated, so rethinking how to enhance it through the Industry 4.0 perspective is crucial. Questions such as how to nurture young people so they master the fundamental Industry 4.0 technologies in stages, from primary, secondary, and tertiary education up to life-long learning for the elderly community members, should become the central aim in formulating a new national education policy.

Next, moral and ethical development, as mentioned in the MSCF, is considered a good move for developing countries like Malaysia, as many Western developed countries have resolved this moral element (refer to [2]). As mentioned in the literature, Ungku Aziz's 1980s ideas are still considered fundamental and remain relevant enough to be adopted in the current smart city development in Malaysia. Although the values of education related to moral development were stated in the National Education Blueprint 2013, the latest education plan could be enhanced based on the five principles of Maqasid Al-Syariah, namely caring for religion, caring for life, caring for intellect, caring for one's offspring, and caring for property. Maqasid Al-Syariah refers to the noble purpose of Islamic law, which is based on the principle of Maslahat and which mankind could universally obtain through the text or authority of Islamic law [123]. This universal concept is seen by all as practicable.

Another aspect to enhance in terms of smart people in Malaysia is the level of civic participation in local authority decision-making and programs [73]. Participation in decisionmaking differs from community empowerment: the former involves the level of citizen power and can influence agenda setting, while the latter refers to the tokenism level of service delivery stages [61]. Furthermore, in the former, people are active in decision-making and co-creating with the authorities, whereas, in the latter, people tend to be in a weaker, beneficiary, or reactive position when they are deemed 'empowered' by the authorities. Contemplating the lower level of participation in decision-making in Malaysia, the authors argue that the implementation of the MSCF could enhance the extent of this form of participation. Although it may face dissensus of opinion, in the long term, this move will help in building more democratic spaces and independent citizenship for Malaysian nation-building [124].

On the governance issue, excessive bureaucracy, delays in approving applications and licenses, as well as a lack of information on new policies and regulations are among the main problems plaguing the government's delivery system [81,82]. Inter-governmental data sharing is another challenge due to the separate departmental practices in Malaysia. To address this, Hong Kong's initiatives can be adopted, such as building a new big data analytics platform; adopting public cloud services, which would enable real-time data transmission and sharing among government departments; and enhancing security features so that government departments can deliver efficient and agile e-services [8]. Future smart city governance should make effective use of their data assets to secure outcomes that are appropriate to citizens' needs. Investment by agencies in system-wide data capture, integration, and analytics capabilities [75] is a crucial aspect to develop.

Apart from data sharing, smart governance ultimately aims to produce public values for citizens, such as from the perspective of asset management and financial and economic sustainability [93]. To realize such public values, e-democracy must be upheld through active e-voting and e-decision making [71], which is a major topic for Malaysian smart development advocates to deliberate. According to the Democracy Index 2020, out of 167 countries, Malaysia (ranked 39) and India (ranked 53) fell into the category of flawed democracies. Meanwhile, Hong Kong ranked 87 due to its hybrid regime of flawed democracy and authoritarian control [125]. In terms of the purpose of building independent citizens [2] within the conception that smart cities are democratic ecologies [126], Malaysia and similar places must actually strive further to achieve higher transparency and open governance. As suggested by [127], 'good enough governance' for smart city societies in Malaysia should consider the cultural context of the Muslim majority, prioritize governance content that allows more scope for political participation and free speech, and cultivate the imagination and unselfishness of children.

Furthermore, it was found that the understanding and acceptance of initiatives in the smart environment is the most critical among all the domains. In this regard, announcements on smart environment initiatives must be intensified and expanded to ensure the sustainability of the existing environment. Most importantly, the authors' view is that environmental accountability initiatives must be added to this component to enable each party to understand the concept and play their respective roles in caring for the environment. In the case of maintaining a clean environment, Ref. [57] found that all stakeholders should take responsibility, not solely the authorities. Efforts to maintain and preserve the environmental space relate to the question of community awareness and attitude, which, if sufficiently high, would ensure that the environment is always clean, healthy, and sustainable.

In terms of preserving parks and green spaces in urban areas, Malaysia's development control guidelines set a minimum of 10% green and open space reservation, which is considered relatively low. In comparison, the city of Wuhan, China, has launched its Wuhan Low-Carbon Urban Development Plan 2013, which reserves 28% for green areas in the city [48]. Therefore, a rethink is suggested that would impose a greater green space allocation in new development plans and, together with agencies such as PLAN-Malaysia, the MSCF could incorporate this higher green space allocation as one of its smart environment initiatives.

In terms of smart mobility and, in particular, electrical vehicles (EVs) in Malaysia, Putrajaya city bought 150 electric buses (each costing RM 1.5 million). They operate in Putrajaya and the vicinity, the aim being to cut carbon emissions, noise pollution, and traffic congestion while improving public transport and parking systems [128]. The operation of the electric buses is calibrated by battery capacity and charging facilities and has been found to outperform conventional bus operations [129]. However, cases of the inefficiency of public transport management were identified, whereby the electric buses were found abandoned at the Depoh Putrajaya. Bus breakdowns are frequent due to lack of maintenance, unreliable and delayed bus arrival times, and reductions in bus routes [130]. Thus, although electric cars are efficient in costs and energy saving with long-term usage [84], the adoption of an EV ecosystem is required, involving features such as efficient management and the availability of efficient power charging stations.

As for privately-owned electric cars, it has been found that the understanding and acceptance of the community is still low. The respondents in this study felt the costs involved in owning and maintaining a private electric vehicle were higher than those of a typical vehicle. It is true that research has shown that the cost of electric vehicle ownership in Malaysia is not yet as competitive as typical internal combustion vehicles [131]. This shows the market and society acceptance of electric cars remains still low. Those involved in the MSCF should rethink the issues of EVs, along with the latest National Automotive Policy 2020, in promoting affordable new technologies. For example, incentives and funding are available under the National Automotive Policy 2020 to develop the technology and engineering required for NxGV (next-generation vehicles), autonomous vehicles, MaaS (mobility-as-a-service), and Industry 4.0 [132]. Thus, MSCF initiatives such as promoting collaboration with the private sector in developing affordable EVs could be implemented.

Last but not least, for the smart digital infrastructure, in terms of the cybersecurity and personal data protection issues, it seems that the MSCF did not provide clear direction on how to strengthen the necessary cybersecurity and personal data protection. The ranking of Malaysia as eighth out of 194 countries in the Global Cybersecurity Index 2021 [133] seems to contradict the results of this study's finding. Recently reported cyber intrusion cases [103] were over the targeted 9000 to 10,000 per year [9], and the assessment of the National IoT Strategic Roadmap was also ambiguous [104]. All the supporting agencies, such as Cybersecurity Malaysia, the Department of Personal Data Protection, and the Malaysia Administrative Modernization and Management Planning Unit must work more closely together and actively provide improvements or amendments to the policies, especially more strict enforcement of the Malaysian Personal Data Protection Act 2010.

As for the issues of low internet speed and digital infrastructure coverage in lessdeveloped states and rural areas, more MSCF initiatives could be planned in conjunction with the latest National Digital Infrastructure Plan (JENDELA), the Malaysian Industry 4.0 Policy, and the Malaysia Digital Economy Blueprint (MyDigital). For instance, the current wireless broadband coverage in Malaysia is 96.7% for 2G, 95.3% for 3G, and 91.8% for 4G coverage in populated areas, with 25 Mbps speed [134]. Therefore, MSCF initiatives could plan to achieve 100% 4G coverage in populated areas and a speed of 100 Mbps by adopting 5G.


*Edited by Mattia Bartoli, Mauro Giorcelli and Alberto Tagliaferro*

Biochar is the carbonaceous residue produced from the pyrolytic conversion of biomass. It is generally used for agricultural applications as a soil amendment but has far wider potential. This book presents the use of biochar as a platform for the development of new intriguing solutions in several cutting-edge fields. The book is a useful reference volume for any reader with a strong scientific and technological background, ranging from scientific advisors in private companies to academic researchers promoting the spread of knowledge about biochar to anyone not already working with it.

ISBN 978-1-80356-251-3

Biochar - Productive Technologies, Properties and Applications

Published in London, UK © 2023 IntechOpen © taviphoto / iStock


**4. Antimicrobial activity of EOs**

The antimicrobial effect of essential oils is attributed to actions including alteration of the permeability, and disruption of lipophilic cell membrane. The antimicrobial potential of essential oils can be completely associated with their constituents. Phenolic compounds with their hydrophobicity inherent, breakdown the lipid of cell membrane and mitochondria and enhance the permeability [31, 32]. The inherent of cellular energy generation system (ATP) and damage of proton motive force is a result of changing cell and cytoplasmic membrane permeability [33]. In addition, leakage of internal contents of the cell during the disruption of the membrane is another mechanism which causes cell death [32]. It is generally believed that Gram-negative bacteria are more resistance to essential oils because of their outer hydrophilic cell wall which exhibits inhibitory activity against the penetration of phenolic components [34].

Algae extracts and their components have displayed antimicrobial activity against organisms found in foods. Algae extracts and their components have displayed antimicrobial activity against organisms found in foods. The antibacterial activities of essential oils from two red seaweed *Laurencia obtuse* and *L. obtusa* var. *Pyramidata* showed that the essential oils from Laurencia obtuse exhibited the strong antimicrobial effect against Gram-positive (*Bacillus subtilis*, *Staphylococcus aureus*, methicillin-oxacillin resistant *Staphylococcus aureus*, *Enterococcus faecalis* and *Staphylococcus epidermidis*), Gram-negative bacteria (*Enterobacter cloaceae*) and yeast (*Candida albicans*) [16].

**197**

life of chilled stored fish [37].

*Application of Essential Oils for Shelf-Life Extension of Seafood Products*

Terpenoids, thymol, carvacrol, β-cubebene, β-eudesmol, β-ionone, dactylol and pachydictol A are the usual volatile compounds in seaweeds and it is known that there is correlation between β-ionone and antibacterial and antifungal activity of seaweeds [17]. However, two known sesquiterpenes (1R\*,2S\*,3R\*,5S\*,8S\*,9R\*)- 2,3,5,9-tetramethyltricyclo(<IP_ADDRESS>,5)undecan-2-ol and (1S\*,2S\*,3S\*,5S\*,8S\*,9S\*)- 2,3,5,9-tetramethyltricyclo-(<IP_ADDRESS>,5)undecan-2-ol were isolated from the red macroalgae *Laurencia dendroidea* had no antibacterial effect on eight bacteria strains (*Staphylococcus aureus*, *Bacillus subtilis*, *Enterococcus faecalis*, *Streptococcus*

The short shelf-life of fresh seafood is a practical issue in the industries and distribution chain systems. Short shelf-life caused by chemical and microbial spoilage reactions can be stopped by traditional preservation methods but there is increasing interest in natural preservation methods. EOs are natural antioxidants and antimicrobials by which the shelf-life of seafood can be extended alone or in combination with other techniques. However, the reduction of antimicrobial effect of EOs in a food system due to some components of food and also the reverse action of EOs as antioxidant agents in some cases, has slowed down the use of them in

Combination of EOs exhibit the synergistic antimicrobial activity. Thus, using of EOs into packaging can be the safe approach for food preservation technology [35]. The antimicrobial activity of gelatin-chitosan films incorporated with organo essential oil exhibited the great inhibitory effect through reducing the *E. coli*, *S. aureus*, *B. subtilis* and *B. entritidis* growth. Its inhibition zone was larger for Grampositive bacteria compared with Gram-negative bacteria. Furthermore, the lower total aerobic plate count and total volatile basic nitrogen values that can extend the shelf life of grass carp muscle was recorded in fish muscle packed with film containing the 4% organo essential oil. It seems the high percentage of carvacrol, eugenol, and thymol as phenolic components are responsible for this antimicrobial activity by damaging the cell membrane or interfere the enzyme functionality located on the cell wall. Moreover, the TVB-N value of sample packaged with gelatinchitosan-EOs film was lower compared with control and the shelf life of grass carp muscle packaged with the film containing EOs was extended to 12 days [36]. The same observation was gained from the gelatin-chitosan film incorporated with other EOs including clove, fennel, cypress, lavender, thyme, herb-of-cross, pine and rosemary for cod fillet preservation. Among all EOs, the high antimicrobial effect was obtained from clove against a wide range of food pathogen and spoilage bacteria such as *Salmonella*, *Lactobacillus*, *Listeria*, *Citrobacter*, *Escherichia*, *Yersinia*, *Brochothrix*, *Staphylococcus*, *Bacillus*, *Listeria*, *Clostridium*, *Aeromonas*, *Shewanella*, *Vibrio* and *Photobacterium*. In addition, the film containing the clove essential oil used for preservation of cod fillets, lowered the microorganisms in particular, *Entrobacteria*. Further, by delaying the formation of TVB-N, can extend the shelf

Immersion of salmon in marinade solution containing 1 w/w% essential oil from organo, cinnamon and thyme revealed that the antimicrobial effect, however, organo and cinnamon essential oil caused to enhance the shelf life of salmon and scampi. In addition, reduction of yeast and mold was observed by cinnamon (1%) addition in marinade for 6 days. Moreover, salmon treated with marinade containing 1% essential oil, shoed appropriate sensorial properties and high hedonic score

*pneumonia*, *Klebsiella pneumoniae*, *Salmonella typhi*, *Escherichia coli* and

*Pseudomonas aeruginosa*) and the yeast *Candida albicans* [5].

**5. Shelf-life extension of seafood products**

practical systems.

*DOI: http://dx.doi.org/10.5772/intechopen.86574*

*Application of Essential Oils for Shelf-Life Extension of Seafood Products DOI: http://dx.doi.org/10.5772/intechopen.86574*

*Essential Oils - Oils of Nature*

activities, respectively [30].

**Figure 3.**

*et al. [42]).*

**4. Antimicrobial activity of EOs**

penetration of phenolic components [34].

yeast (*Candida albicans*) [16].

The lipid oxidation is one of the most important limiting factors for the shelflife seafood products. For this purpose the antioxidant activity of the EOs of five Mediterranean spices (*Origanum vulgare*, *Thymus vulgaris*, *Rosmarinus officinalis*, *Salvia officinalis*, and *Syzygium aromaticum*) was analyzed. The *S. aromaticum* essential oil, which comprised the highest level of total phenols (898.89 mg/l GAE), demonstrated the highest antioxidant activity (98.74% for DPPH radical inhibition and 1.47 TEAC for FRAP value). The EOs extracted from *T. vulgaris* and *R. officinalis* showed the highest TBARS inhibition (89.84%) and iron (II) chelating (76.06%)

*The effect of pectin-CEO coating on the bream fillets at day 1 and day 15 of the storage (adopted from Nisar*

The antimicrobial effect of essential oils is attributed to actions including alteration of the permeability, and disruption of lipophilic cell membrane. The antimicrobial potential of essential oils can be completely associated with their constituents. Phenolic compounds with their hydrophobicity inherent, breakdown the lipid of cell membrane and mitochondria and enhance the permeability [31, 32]. The inherent of cellular energy generation system (ATP) and damage of proton motive force is a result of changing cell and cytoplasmic membrane permeability [33]. In addition, leakage of internal contents of the cell during the disruption of the membrane is another mechanism which causes cell death [32]. It is generally believed that Gram-negative bacteria are more resistance to essential oils because of their outer hydrophilic cell wall which exhibits inhibitory activity against the

Algae extracts and their components have displayed antimicrobial activity against organisms found in foods. Algae extracts and their components have displayed antimicrobial activity against organisms found in foods. The antibacterial activities of essential oils from two red seaweed *Laurencia obtuse* and *L. obtusa* var. *Pyramidata* showed that the essential oils from Laurencia obtuse exhibited the strong antimicrobial effect against Gram-positive (*Bacillus subtilis*, *Staphylococcus aureus*, methicillin-oxacillin resistant *Staphylococcus aureus*, *Enterococcus faecalis* and *Staphylococcus epidermidis*), Gram-negative bacteria (*Enterobacter cloaceae*) and

**196**

Terpenoids, thymol, carvacrol, β-cubebene, β-eudesmol, β-ionone, dactylol and pachydictol A are the usual volatile compounds in seaweeds and it is known that there is correlation between β-ionone and antibacterial and antifungal activity of seaweeds [17]. However, two known sesquiterpenes (1R\*,2S\*,3R\*,5S\*,8S\*,9R\*)- 2,3,5,9-tetramethyltricyclo(<IP_ADDRESS>,5)undecan-2-ol and (1S\*,2S\*,3S\*,5S\*,8S\*,9S\*)- 2,3,5,9-tetramethyltricyclo-(<IP_ADDRESS>,5)undecan-2-ol were isolated from the red macroalgae *Laurencia dendroidea* had no antibacterial effect on eight bacteria strains (*Staphylococcus aureus*, *Bacillus subtilis*, *Enterococcus faecalis*, *Streptococcus pneumonia*, *Klebsiella pneumoniae*, *Salmonella typhi*, *Escherichia coli* and *Pseudomonas aeruginosa*) and the yeast *Candida albicans* [5].