THE UTILIZATION OF GINGER (Zingiber officinale) OLEORESIN AS
ANTI INFLAMMATION ON THE LIVER OF RATS
UNDER STRES CONDITION
(PEMANFAATAN OLEORESIN JAHE SEBAGAI ANTI RADANG HATI PADA MENCIT YANG MENDERITA STRES)

Tutik Wresdiyati
Laboratorium of Histology, Department of Anatomy, Faculty of Veterinary Medicine,
Bogor Agricultural University, Jalan Agatis - IPB Darmaga Bogor 16680, INDONESIA.
Telp: (0251) 626064, Telp/Fax: (0251) 629464, e-mail : [email protected]

ABSTRACT
The ginger (Zingiber officinale) oleoresin is well known to content natural antioxidant. The anti inflammation activities of such oleoresin in the liver of rats under stress condition was histochemically observed in this study. A total of sixty male Wistar rats were used for this study. They were divided into twelve groups; (1) control group, without treatment of both stress and oleoresin, (2) stress group, was treated by stress only, without oleoresin treatment, while (3) and (4) are groups that were treated by stress and then fed standard feed for three and seven days, without oleoresin. Group (5) to group (12) were treated by stress then followed by treatment of oleoresin for three and seven days. The doses of oleoresin were 20, 40, 60, and 80 mg/kgBW/day. Stress condition was done by 5 days fasting and swimming for 5 minutes/day drinking water was provided ad libitum. The inflammatory cell number in the liver of rats was highest in the stress group. The animal treated with oleoresin after fasting stress showed decreased number of inflammatory cells in the tissues. The decrease was higher in the animals treated with higher dose of oleoresin. The effect of oleoresin as anti inflammation was significantly higher in the groups treated with oleoresin at 60 mg/kgBW/day for seven days, and 80 mg/kgBW/day for three and seven days.
Key words : oleoresin, ginger, inflammation, liver, stress

ABSTRAK
Oleoresin jahe (Zingiber officinale) telah diketahui mengandung antioksidan alami. Apakah oleoresin jahe dapat bekerja sebagai anti inflamasi pada hati tikus di bawah kondisi stres telah diteliti secara histokimia pada penelitian ini. Sebanyak 60 ekor tikus jantan galur Wistar telah digunakan pada penelitian ini. Hewan percobaan tersebut dibagi menjadi 12 kelompok perlakuan, yaitu : (1) kelompok kontrol, tanpa diberi perlakuan stres maupun oleoresin, (2) kelompok stres, hanya diberi perlakuan stres saja tanpa pemberian oleoresin. Sedangkan kelompok (3) dan (4) masing-masing diberi perlakuan stres yang dilanjutkan dengan pemberian ransum standar selama 3 dan 7 hari, tanpa pemberian oleoresin. Kelompok (5) sampai dengan kelompok (12) masing-masing diberi perlakuan stres yang dilanjutkan dengan pemberian oleoresin selama 3 dan 7 hari, dengan dosis oleoresin 20, 40, 60, dan 80 mg/kgBB/hari. Perlakuan stres dilakukan dengan puasa selama 5 hari dan berenang selama 5 menit/hari dengan pemberian air minum secara ad libitum. Jumlah sel inflamasi pada jaringan hati tikus paling tinggi terlihat pada kelompok stres. Pemberian oleoresin setelah perlakuan stres menunjukkan adanya penurunan jumlah sel inflamasi pada jaringan tersebut. Penurunan tersebut semakin besar seiring dengan semakin tingginya dosis oleoresin yang diberikan. Efek anti inflamasi oleoresin jahe pada hati tikus terlihat nyata pada kelompok dengan dosis 60 mg/kgBB/hari selama 7 hari, dan 80 mg/kgBB/hari baik selama 3 hari maupun 7 hari.
Kata-kata kunci : oleoresin, jahe, inflamasi, hati, stres

INTRODUCTION
Indonesian Rhizomes is well known to have potency as antioxidant. It was reported that ginger extract from methanol extraction has the highest antioxidative effect compared to other rhizomes (Dewi et al., 2000). Dyatmiko et al. (2000) reported that in vitro, that extract of Zingiber officinale using methanol has free-radical scavenger activity more than other species of Zingiber.
Ginger in Indonesia is well known and used in many purposes since long time ago. The rhizome contains certain phenolic compound. It was reported in vitro, ginger oleoresin has antioxidative effect that more than a-tocopherol . However, there is only a few report on antioxidative effect of ginger oleoresin in the liver tissue, in vivo, especially under tress condition.
Stress condition was altered morphologly and increased the number of peroxisomes in the kidney of Japanese monkeys (Wresdiyati and Makita, 1995). It was also reported that stress condition caused inflammation and decreased intracellular antioxidant- copper,zinc-superoxide dismutase (Cu,Zn-SOD) in the liver and kidney of rats (Wresdiyati et al., 1999; Wresdiyati et al., 2002).
Increased level of the active oxygen species, free radical, created a situation known as oxidative stress (Halliwell and Gutteridge, 1989; Langseth, 1995). Oxidative stress can be induced by certain factors such as antioxidant deficiency or over production of free radical. The condition can lead to a variety of biochemical and physiological lesions often resulting in metabolic impairment and cell death. These highly reactive oxygens can readily react with various biological macromolecules such as DNA, proteins, lipids, and caused protein destruction. The lesions in turn lead to various diseases and degenerative processes such as aging and carcinogenesis in human and animals (Ames and Shigenaga, 1992).
Antioxidants, free-radical scavenger, play an important role in protection of cells against oxidative stress and maintain a balance between the various toxic oxygen species (Touati, 1992). The protection can be done by several ways such as prevention, stopping or decreasing of oxidations (Schuler, 1990), as well as catalyzing free radicals by intracellular antioxidant enzymes (Mates et al. 1999).
The intracellular antioxidant enzymes comprise catalase, glutathione peroxidase, and three isoforms of superoxide dismutase (SOD); copper, zinc (Cu,Zn)-SOD, manganese (Mn)-SOD, and iron (Fe)-SOD. The SOD provides a primary defence against superoxide anion radical generated intracellularly. Wresdiyati and Makita (1998) reported that Cu,Zn-SOD was cytochemically localized in the renal tubule of rats kidney.
The present study was conducted to observe the role of antioxidant of ginger oleoresin (Zingiber officinale) on inflammation in the liver of rats under stress condition, as an anti inflammation.

MATERIAL AND METHODS
Ginger oleoresin. Ten month old small ginger (Zingeber officinale) was used for this study. The ginger was cleaned and dried in oven blower (40-60°C) for 30-36 hours to obtain dried ginger with 8-11% water content. Dried ginger was then blended and filtered to obtain ginger powder. A total of 250 gr of ginger powder was extracted 4 times using two kinds of solvents (500ml), methanol and ethanol. Resulting extract then was filtered at vacuum condition, and then distilled using rotaryvacum-evaporator. Resulting oleoresin from distilling process was light to dark brown solid materials. The oleoresin was then weighed in rotavapor.
Total phenol of ginger oleoresin analysis. Total phenol (AOAC, 1984) was analysed to both ginger oleoresin resulted from methanol and ethanol extraction. A total of 0.1 ml oleoresin added 75 ml aquadest, 10 ml Folin-ciocalteu and 10 ml Na2CO3. The solution then was added distilled water to 100 ml, mixed properly, and leaved at room temperature fo 30 miniutes. The solution then observed using spectrofotometer at 760 nm. For control negatif, the 0.1 ml oleoresin was changed with 0.1 ml distilled water. The standard of total phenol is tanic acid in concentration of 1.0, 2.0, 2.5, 3.0, 3.5, 4.0 and 4.5 mg/ml.
Analysis of antioxidant activity of ginger oleoresin. A total of 200 ppm ginger oleoresin was added to 2 ml linoleat acid (50 mM) in 99.8% ethanol, 2 ml phosphate buffer (0.1 M pH 7.0), and 1 ml dionized water. The solution then was incubated at 37°C. Every second day for 14 days, 50 ml of the solution was added to 2.35 ml ethanol (75%), 50 ml amonium tiosianat (30%), and 50 ml FeCl2.4H2O (20 mM in HCl 3.5%). After 3 minutes, the solution was then observed using spectrofotometer at 500 nm (Chen et al., 1996). Distilled water and alfa tocopherol were also observed for comparison to the ginger oleoresin.
Characterization of ginger oleoresin. GF-254 plate was heated at 110º C for 4 hours. Put ginger oleoresin on the start line of the plate, then stored at development media that contain eluent hexan and diethylether in ratio 3:7. Then let eluen run to the final line. The plate then was taken from the media and shown some separate fractions which have different Retardation Factor (RF) from each others. In order to clean and detailed seen of the fractions, reagen Folin-ciocalteu was sprayed to the fractions. RF is ratio distance of oleoresin (start spot to final spot) to the distance of mobile phase.
Treatment of Animals. A total of 60 male Wistar rats (250 ± 5 gr BW) were used for this study. The animals were adapted to situation and condition of animal laboratory for 2 weeks, and then they were divided into 12 treatment groups. The 12 treatment groups were showe in Table 1. Group in stress condition was treated by fasting stress with only drinking water for 5 days and swimming 5 minutes per day. The oleoresin was orally administrated by using sonde. While treated by oleoresin the animals were also fed ad libitum.
Tissues preparation, observation and data analysis. Tissue sampling was done at the end of treatments. Cervical dislocation was done to the animals before removing the liver tissues. Fixation in Bouin solution was done to the tissues for 24 hours. After fixation, the tissues were then dehydrated in a series of alcohol and cleared in xylol. Finally, the tissues were embedded in paraffin before sectioning using microtome (4 mm thickness). Sectioned tissues were then histochemically stained for inflammatory cells (hematoxylinand eosin staining).
The stained tissues were then observed under a light microscope. Inflammatory cells were observed and counted under 400 magnification. The number of inflammatory cells of each treatment group was statistically analyzed.

Table 1. Animal treatment groups based on stress, dose and length of oleoresin treatments
Group Stress (5 days) Dose of Oleoresin(mg/kgBW/day) Length of oleoresin treatment (day)
1 - - -
2 + - -
3 +* - -
4 +** - -
5 + 20 3
6 + 20 7
7 + 40 3
8 + 40 7
9 + 60 3
10 + 60 7
11 + 80 3
12 + 80 7

* : after stress were fed for 3 days
** : after stress were fed for 7 days
+ : treated
- : no treatment

RESULTS AND DISCUSSION
Oleoresin and total phenol of ginger extract. Total amount of oleoresin and it’s phenol from ginger extraction, using methanol and ethanol, were showen in Table 2. It was showen in the Table that methanol extraction yielded more oleoresin (6.38%) than ethanol extraction (4.10%). However, statistically there was no significant difference between metanhol and ethanol extraction in yielding oleoresio.
Phenolic coumpond in oleoresin resulted from methanol extraction differed from the oleoresin that ectracted from ethanol. Methanol extraction yielded total phenol: 647.22 mg/mL, while ethanol extraction yielded total phenol: 522.22 mg/mL. Methanol extraction is better than ethanol extraction in yielding phenol coumpond, which gives the antioxidative effect, of ginger.

Table 2. Total amount of oleoresin and total phenol of ginger extract using methanol and ethanol
Coumpond analyzed Solvent
Methanol Ethanol
Oleoresin (%) 6.38 a 4.10 a
Phenol (mg/ml) 647.22 p 522.22 q
Different superscript letters at the same line mean significantly different (P

Based on the results of extraction using methanol and ethanol, the following study on antioxidative activity of ginger oleoresin, characterization of phenolic coumpond of ginger oleoresin and treatment of oleoresin to animals treatment we used oleoresin that extracted from methanol.
Antioxidative activity of ginger oleoresin. Antioxidative activity of ginger oleoresin was compared to control (aquadest), and a-tocopherol were showen in Figure 1. Ginger oleoresin and a-tocopherol showed antioxidative activities better than control, and antioxidative activity of ginger oleoresin was the best a many others. Antioxidative activity was dicided from how can they stop linoleic acid oxidation to become peroxide radical. The radical then oxidizes Fe2+ to Fe3+ and gives red colour. Intensity of red colour showed the score of absorbancy. If the intensity of colour is low, the score of absorbancy will be small. The small score of absorbancy showed that antioxidative activity is better. It is therefore antioxidative activity of ginger oleoresin is better than that of a-tocopherol and antioxidative activity of a-tocopherol is better than control (Figure 1)

Figure 1. Antioxidative activity of ginger oleoresin, a-tocopherol and control (aquadest)

Characterization of phenolic coumpond of ginger oleoresin. The characterization was done using GF-254 plate. The plate showed several spots of certain active coumpond such as gingerol at Rf 0.24, zingeron at Rf 0.32, and shogaol at Rf 0.38. It indicated that the ginger used in this study contained active coumpound namely: gingerol, shogaol, and zingeron. In this study, all of the active coumpounds contained in ginger oleoresin were used to treat the animals. We did not use active coumpound separately. It was reported that antioxidative activity of oleoresin is bigger than that of each phenolic coumpound in the oleoresin.
Anti Inflammatory Effect of Ginger Oleoresin. Ringler (1996) reported that inflammation can be initiated by sublethal tissue injury and finally it can be repaired. Inflammation was also known as a pathological phenomenon that caused by certain irritants. It was also reported that inflammation is a dynamic process and it is not stable, depends on the tissues.
The signs of inflammation are hyperemia, increase of blood vessle permiabibity, and diapedesis of leucocytes from blood vessels to extracellular areas. The leucocytes will phagocyt anything that cause inflammation. The phagocytosis process will result in free radical.
Fasting stress was reported by Wresdiyati and Makita (1995) to alter peroxisomes. The organelles were altered morphologically and their number increased. It was also reported that fasting stress induced inflammation and decreased intracellular antioxidant (Cu,Zn-SOD) content histochemically in the liver and kidneys of rats (Wresdiyati et al., 1999; 2002). The decrease of the antioxidant account for the over production of free radicals under stress condition.
In the normal physiological condition, cells produce free radicals as a consequence of aerobic biochemical reactions. Free radicals became dangerous because it became reactive and be able to complete their electrons (Mates, 1999). A condition with highly free radicals, oxidative stress, can readily react with various biological macromolecules of cells including protein destruction. The lesions in turn lead to degenerative process (Halliwell and Gutteride, 1990). Under fasting stress, the degenerative process may occurre both in the hepatocytes and endothelial cells of blood vessels. Therefore, it may increase blood vessel permeability and diapedesis of inflammatory cells namely leucocytes. Phagocytosis by inflammatory cells also produced free radical. These condition caused degenerative process and inflammation got worse.
The micrograph of inflammatory cells in the liver tissue of treated rats was showen in Figure 2. The inflammatory cells were counted in the liver tissues of all 12 animal groups and viewed at 400 magnification. Statistical analysis of the number of inflammatory cells was showen in Table 3. It showed that ginger oleoresin has an effect in inflammation that occurred in the liver tissues. The mean number of inflammatory cells in the control group was 8.44 ± 2.46. The inflammatory cells remarkably increased in the groups under fasting stress and followed by group feed ad libitum for 3 and 7 days, without treatment of ginger oleoresin. They were 84.33 ± 15.53; 103.78 ± 27.61 and 141.89 ± 25.02, respectively.
The effect of ginger oleoresin in decreasing the number of inflammatory cells in the liver tissues under stress condition was seen at minimal dose 20 of mg/BW/day. Howeever, the number of inflammatory cells was statistically not significantly different from the number of cells in the stress group. The effect of ginger oleoresin to decrease inflammatory cells was more clearly seen in groups at higher doses and longer time of treatment. Statistically, the ginger oleoresin gave significant effect to decrease the inflammatory cells at dose 60 mg/kgBW/day for 7 days, and 80 mg/kg/BW/day for 3 and 7 days. The number of inflammatory cells at these doses was not significantly different from the number of cells in the control group. These results showed that ginger oleoresin has an anti inflammatory effect on the liver of rats under stress condition.
Table 3. The number of inflammatory cells in the liver tissues of rats under stress condition and treatment of oleoresin per view of 400 magnification
Group Stress (5 days) Oleoresin Number of inflammatory cells
Dose of Oleoresin(mg/kgBW/day)) Length of oleoresin treatment (day)
1 - - - 8.44 ± 2.46a
2 + - - 84.33 ± 15.53d
3 +* - - 103.78 ±27.61e
4 +** - - 141.89 ± 25.02f
5 + 20 3 83.56 ±28.77d
6 + 20 7 77.56 ± 22.96d
7 + 40 3 75.22 ± 17.20d
8 + 40 7 53.67 ± 17.66c
9 + 60 3 50.11 ± 18.19b
10 + 60 7 34.33 ± 9.22a
11 + 80 3 20.78 ± 12.64a
12 + 80 7 11.22 ± 4.84a
* : after stress were fed for 3 days. + : treated
** : after stress were fed for 7 days. - : no treatment
Different superscript letter at the same coloum showed significant different (P

It was reported that phenolic compounds have antioxidant function, because their ability to netralize free radicals by giving their hydrogen molecules to the free radicals (Nabet, 1996). Phenolic compound is organic compound having one aromatic ring with one or more hydroxil compound. Including the phenolic compounds are zingiberen, zingiberol, shogaol, kurkumin, gingerol , zingeron, and atsiri oil.

Figure 2. Fotomicrographs of inflammatory cells (arrows) in the liver tissues of treatment rats. (a) control, (b) stress, (c) stress and 3 days feeding, (d) stress and 7 days feeding, (e) stress and oleoresin 20 mg/kgBW/day for 3 days, (f) stress and oleoresin 20 mg/kgBW/day for 7 days, (g) stress and oleoresin 40 mg/kgBW/day for 3 days, (h) stress and oleoresin 40 mg/kgBW/day for 7 days, (i) stress and oleoresin 60 mg/kgBW/day for 3 days, (j) stress and oleoresin 60 mg/kgBW/day for 7 days, (k) stress and oleoresin 80 mg/kgBW/day for 3 days, (l) stress and oleoresin 80 mg/kgBW/day for 7 days.

In this study, the mechanism of oleoresin as anti inflammation in the liver tissues of rats under stress condition can be explaned ; that phenolic compound of ginger oleoresin such as gingerol, shogaol and gingeron stop and decrease free radicals by giving their hydrogen molecules (Nabet, 1996). So, ginger oleoresin treatment can decrease the amount of free radical which remarkable increase under fasting stress. Furthermore, oleoresin can minimize the degenerative process including inflammation that occur in the liver tissues.

CONCLUSION
Methanol is better than ethanol for extracting of ginger oleoresin. By using methanol in ginger extraction resulted in oleoresin and phenolic compound: 6.38% and 647.22 mg/dL, while using ethanol resulted in 4.10% and 522.22 mg/dL.
Antioxidative activity of ginger oleoresin in vitro is bigger than that of a-tocopherol. Ginger oleoresin has anti inflammatory effect in the liver tissues of rats under stress condition. The effect showed significant different at dose 60 mg/kgBW/day for 7 days, and 80 mg/kg/BW/day for 3 and 7 days.

ACKNOWLEDGMENT

This study was part of research that funded by grant (Hibah Bersaing X) from Ministry of Education of Indonesia (Direktorat Pembinaan Penelitian dan Pengabdian Pada Masyarakat, Direktorat Jendral Pendidikan Tinggi, Departemen Pendidikan Nasional, No. 103/LII/BPPK-SDM/IV/2002) for TW.

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