The influence of the combination of methanolic extract Scurrula atropurpurea (Blume) and Dendrophthoe pentandra on rat liver function and structure

INTRODUCTION / ВВЕДЕНИЕ

Tea mistletoe (Scurrula atropurpurea (Blume)) from Loranthaceae family is a parasitic plant or mistletoe which lives on the tea plant [1]. It has been known to have wide potential as medicine including leaves, stems, and pea-sized berries [2]. Several studies have reported on the in vitro role of methanolic extract Scurrula atropurpurea (MESA) leaves in reducing the contractility of the separated rat tail arteries [1]. It also has the potential to lower blood pressure by improving oxidative stress and endothelial dysfunction [3–8]. Moreover, according to previous research, MESA can be used as a cure for hypertension [6][9–10].

Mango mistletoe (Dendrophthoe pentandra, DP) belongs to one family with tea mistletoe. It is a semi-parasitic plant that grows on mango trees as a host plant. It is widely found in tropical rainforests at lowland plantations including Indonesia. Many previous studies reported that its leaves, stems, and flowers had potential for medicine [9]. Mango mistletoe leaves extract is known as a chemotherapeutic agent for cervical cancer [11] and has anti-inflammatory properties [12]. The study showed that the combination of mango mistletoe and 5-Fluorouracil can increase the number of apoptosis cells and p21 expression as well as reduce the surviving expression of HeLa cells [11]. Mango mistletoe treatment significantly inhibited the population of CD4⁺ T-cells, interleukin (IL) 17 cells and the IL-17 concentration in the supernatant. It decreased Th17-associated cytokines IL-17, whereas the production of IL-10 of regulatory T cells in mesenteric lymph nodes of colonic tissue was enhanced by mango mistletoe extract-treated mice [12].

The tea mistletoe leaves contain several secondary metabolites such as tannins, flavonoids, quercetin, glycosides, alkaloids, saponins, and inulin. These active substances have been reported to have roles in hypertension [6]. On the other hand, mango mistletoe leaves were also reported to be rich in tannins, saponins, flavonoids, alkaloids, acetoacetic acid, citramalic acid, linolenic acid, ethylene glycol, monoacetate, and beta-sitosterin [13]. Recently, in vitro and in vivo studies on the pharmacological activity of mango mistletoe leaves extracts have found various efficacy, namely anticancer, immunomodulatory, cardiac, antidiabetic, antihyperglycemic, hepatoprotective, neuropharmacological, antibacterial, and antifungal [14][15].

Thus, the preparation of tea mistletoe leaves will have commercial opportunities when combined with mango mistletoe leaves. However, the toxicity effects of tea mistletoe when combined with mango mistletoe have never been studied. The current research included toxicity tests, histopathological studies [9], in vitro and in vivo tests for combination of methanolic extract of Scurrula atropurpurea (BI.) and Dendrophthoe pentandra (MESA-DP), and resulted that these extracts are safe and can be used as health drinks to prevent hypertension [6].

Objective: to investigate the effect of MESA-DP combination on rat liver function and structure using serological and histopathological analysis.

MATERIAL AND METHODS / МАТЕРИАЛ И МЕТОДЫ

Study design / Дизайн исследования

The experimental method used in this study was a completely randomized design (CRD). In the true experimental study, 20 female Wistar rats (Rattus norvegicus) were divided into three treatments and one control (Group 1 as a control, while Groups 2, 3, and 4 were given MESA-DP at doses 250, 500, and 1,000 mg/kg of body weight (BW), respectively) based on Frederer formula. Each treatment had five replications. The tea mistletoe and mango mistletoe combination was administered for 28 days. The clinical biochemical examination of the liver function and the histopathological observations of the rat livers were conducted [16].

Experimental animals / Экспериментальные животные

The inclusion criteria were female white rats (R. norvegicus) aged 6–8 weeks with body weight 100–200 g. Each rat except the control was administered with MESA-DP combination 5 times a week for 28 days (sub-chronic study) [9].

The rats were acclimatized in the animal house laboratory of the Faculty of Medicine, Islamic University of Malang for 1 week under room temperature ±24 °C with approximately 50–60% humidity, protected from industrial fumes and other pollutants. On the 7^th day of the acclimatization process, the rats were weighed for pre-condition body weight. The weighing continued once every 7 days during the research.

Plants material and extraction / Растительный материал и его экстракция

Scurrula atropurpurea (Blume) and Dendrophthoe pentandra leaves were obtained from Kepanjen, Malang, Indonesia then identified and determined in Balai Materia Medica Laboratory (Batu, East Java, Indonesia). The leaves used were dry simplicia leaves which were clean and not rotten. Each leaf was heated at 40–60 oC in the oven until the water content was lost. Once dried, the leaves were then cut until crushed. Then, they were mashed by a blender to form a powder (simplicia powder) [7].

The mistletoe extraction was conducted using maceration. Extraction began after simplicia powder was formed; each of the tea mistletoe leaves and mango mistletoe leaves was weighed at 100 g and then put into a 1.5-liter bottle. The simplicia powder was soaked in 90% methanol as much as 1 liter and was shaken for 60 minutes until the solution was homogenous. Then, the shaken simplicia powder was allowed to stand and precipitate for 24 hours to break the cell walls of tea mistletoe leaves and mango mistletoe leaves, and the active substances in the leaves could be drawn by methanol. The soaking of simplicia powder for 24 hours result in the formation of two layers in which the top layer was called supernatant while the bottom layer was natant. This supernatant was collected and extracted using a rotary evaporator. The supernatant was the active substance of mistletoe leaves in methanol [5–7].

The administration of extracts / Введение экстрактов

The doses used in this study were three doses of methanolic extracts of the combination of MESA-DP. The doses in Groups 2, 3, 4 were 250, 500, and 1,000 mg/kg BW, respectively [7]. Of the dosage administered to the rats, the maximum volume of the fluid administered at one time depends on the weight of the rats. Administration by oral gavage was carried out according to the given dosage and volume of the fluid to each rat. Before the rats were administered the combination of tea and mango mistletoes, they were not given any food or drink for 14–18 hours (fasting conditions). Food and drink were given again 3–4 hours after oral gavage. The treatment in this study was each dose given 5 times a week for 28 days (sub-chronic toxicity test) orally [7]. Weighing of the rats’ body weight was conducted once a week to determine the volume of the fluid to be administered to the rats, which was 1 ml/100 g BW.

Sample isolation / Изоляция образцов

After 28 days of observation and treatment on the female Wistar rats, the incision was carried out and blood samples were taken according to the order of the treatment [7]. Liver and blood were then isolated. Blood was separated from serum plasma at 3,000 rpm (600 g) for 10 minutes using Heraeus Labofuge 400R (Kendro Laboratory Products GmbH, Germany) to separate sera. The liver was placed in kalium cloride (KCl) and phosphate-buffered saline (PBS) 25 mM, then kept in 40% formaldehyde buffered neutral at room temperature for histological preparations.

Hematoxylin-eosin staining / Окрашивание гематоксилином и эозином

Liver organs were placed in 25 mM KCl and PBS buffer, then stored in 40% neutral buffered formaldehyde at room temperature. Hematoxylin and eosin (~5 μm) parts were prepared for histopathological measurement. The parts were photographed at ×400 magnification using Olympus (Tokyo, Japan). Microscope lighting, focus, and plane selection were optimized to differentiate cell boundaries. The images were opened and, after setting the threshold, they were analyzed.

Blood serum analysis / Анализ сыворотки крови

Blood serum was used to examination the levels of serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), total bilirubin, albumin, cholesterol, triglycerides, globulins, total protein, high-density lipoprotein (HDL), and low-density lipoprotein (LDL). This process was performed at Bromo Clinic (Malang, Indonesia).

Data analysis / Анализ данных

The data were expressed as mean ± standard error. The mean values and standard errors were calculated using Excel 2007 (Microsoft, USA). Data were analyzed using a one-way ANOVA analysis (ANalysis Of VAriance) and performed via SPSS Statistics 17.0 (IBM, USA). Histopathological analysis of the liver was carried out descriptively.

RESULTS / РЕЗУЛЬТАТЫ

Blood biochemical profile / Биохимический профиль крови

In this study, the three doses of the herbal combination of MESA-DP were safe, hence they did not cause an increase in total bilirubin level compared to the control group (Table 1). The administration of the herbal combination at the third dose (Group 4) did not cause erythrocyte abnormality, which was indicated by an increase in bilirubin level.

Furthermore, there were also no significant differences in the effect of the administration of the three doses of the MESA-DP on the SGOT and SGPT serum levels compared to the control group (see Table 1). The first possibility might be because the three doses of this herbal combination were safe, hence they did not cause liver dysfunction, which was indicated by an increase in hepatic transaminase serum levels due to hepatic enzyme leakage. The secondary metabolites here are those of tea mistletoe and mango mistletoe.

The interaction between the components in the two herbs might be mutually neutralizing or antagonistic, thereby reducing their toxic effects. The second possibility was that the deviations of the SGOT and SGPT levels were almost above 20%, resulting in being not significantly different when the data were analyzed statistically (p>0.05) (see Table 1).

The effects of MESA-DP administration at three doses did not show any significant difference in the serum albumin level compared to the control group but were different for globulin level and total protein, which tend to decrease (p>0.05) (see Table 1). The three doses of the herbal combination were safe. Hence, they did not cause liver dysfunction, which was indicated by the decrease in serum albumin level.

The three doses of the herbal combination were safe. Therefore, they did not cause an increase in the levels of total cholesterol and serum triglyceride compared to the control group (p>0.05). The administration of the three doses did not cause disruption of lipid metabolism in the liver, which was indicated by an increase in the levels of total cholesterol and triglyceride. The level of serum LDL decreased significantly compared to the control group due to the administration of the herbal combination (p<0.05), hence further research could be carried out on this herbal combination as an anti-hyperlipidemia (see Table 1). It was presumed that the herbal combination decreased serum LDL levels through the mechanism of increasing the number of LDL receptors. On the other hand, there were a significant decrease in HDL levels compared to the control group.

Histopathology / Гистопатологическое исследование

The histopathological examination was conducted by counting the damaged liver cells. The liver cells were observed by the microscope under ×400 magnification (Fig. 1). The normal structure of liver cell tissue will have clear nuclei. On the other hand, abnormal liver tissues have necrotic nuclei which are pyknotic, karyorrhectic, or karyolysis. Based on the microscopic observation of the liver, administration of excessive MESA-DP could affect the structure of liver cell tissues in the form of pyknotic, karyorrhectic, and karyolysis cells.

Damaged liver cells in the female rats treated by MESA-DP at a dose 250 mg/kg BW showed fewer necrotic cells compared to those treated using doses 500 and 1,000 mg/kg BW (Table 2). This dose was applied with the expectation of not causing toxicity to the liver cells.

DISCUSSION / ОБСУЖДЕНИЕ

The liver is one of the organs which has an important role as a toxin neutralizer and is responsible for the biotransformation of toxic substances into non-toxic substances. Therefore, this process causes liver cells to easily experience damage both in the form of damage to the cell structure as well as liver dysfunction [17]. Several herbal compounds which enter the body will undergo absorption, distribution, metabolism, and excretion processes [18]. The liver is the main organ of metabolism, which is often damaged by the compound itself or the accumulation of metabolites. Compounds will be metabolized in the liver and a chemical structure change will occur which is catalyzed by an enzyme produced by the microsome of hepatocyte cell and called biotransformation. The medicinal or herbal compounds will be transformed into metabolites that are usually less active than the original medicines. The process of medicine metabolism is not always a process of medical detoxification or elimination of the compounds; sometimes the transformation of medicine results in the formation of intermediate compounds that are reactive and toxic to the liver. Acute liver damage will cause metabolic changes which, in turn, will result in structural and functional changes.

The test of SGOT and SGPT levels is one of the indicators which can be used to determine the presence or absence of liver function damage which is caused by increases in SGOT and SGPT levels found mainly in liver cells. Both enzymes are active in serum, which is used to measure indications of liver diseases. In this study, MESA-DP leaf extract showed no toxicity in experimental doses in animals indicating the safety of the bioactive phytochemicals present in the extract. The MESA-DP extract treatment group that was given had no significant effect on the levels of total bilirubin, SGOT, SGPT, albumin, cholesterol, and triglycerides compared to the control group. Otherwise, the biochemical parameters of globulin, serum total protein, HDL, and LDL experienced a significant decrease when compared to control (see Table 1). The previous study reported that male rats which have been exposed to a methanolic extract of tea mistletoe orally for 28 days (sub-chronic) did not show any abnormality on the histopathology examination and no effect compared to the control group rats on the levels of serum SGOT, SGPT, albumin, globulin and total protein [7]. Similarly, the previous study reported that female rats exposed to a methanolic extract of tea mistletoe orally for 28 days (sub-chronic) did not show any toxicity effect on the SGOT and SGPT levels caused by MESA, hence MESA was considered safe [19].

Quercetin is an antioxidant flavonoid and, thus, able to inhibit free radicals, thereby unable to damage the liver cells. The word antioxidant is originated from the word “anti” (fight) and “oxidant” and known as free radicals. Thus, the antioxidant is a compound that can delay, inhibit or prevent lipid or other molecule oxidations by inhibiting the initiation or propagation of the oxidative chain reaction [22]. Oxidation is a chemical reaction that can produce free radicals that trigger a chain reaction that can damage the liver. Cell life depends on the supply of nutrition and oxygen. Nevertheless, oxygen also potential to cause cell damage through an oxidation process. Oxidation which is initiated by an oxidant will release free radicals, which will continuously attract stability by taking electrons from other atoms. This will result in the production of other free radicals. Every time an electron is released or caught by a free radical, then a new free radical will form. The newly formed free radical will do the same thing. Through this mechanism, the free radical chain is created. If this condition continues for a long time, the body’s cells will be damaged. Toxic substances, as well as free radicals, can damage the liver cells and tissues. Under normal circumstances, free radicals will not cause liver damage because the liver has a better defense system than other organs. However, if there is a part of the liver which has been damaged very extensively, the liver will lose its function immediately.

The liver is an organ that is important for detoxifying useless or harmful chemical substances. The liver has a high ability to bind chemical substances [17]. Most toxicants enter the body through the gastrointestinal tract. After being absorbed, toxicants are carried through the portal vein to the liver. The liver has many binding sites. The levels of enzymes that metabolize xenobiotic compounds in the liver are also high (especially cytochrome P450). This makes some toxins less toxic and more soluble in water and, thus, easier to excrete. But, in some cases, toxicants are activated and able to induce lesions. The liver lesion is centrilobular, which is associated with a higher level of cytochrome P450. In addition, the relatively low glutathione level when compared to the glutathione level in other parts of the liver has a role in activating toxicants [23]. Toxicants can cause various types of toxic effects on various organelles in the liver cells, such as fatty liver (steatosis), necrosis, cholestasis, and cirrhosis [17].

Based on histopathology examination of the female rats' liver on both pyknotic and karyolysis cells showed more increase between all treatment groups (Groups 2, 3, 4) than the control group (Group 1) (see Table 2). One of the causes of many karyorrhectic liver cells was too many toxic substances exposed to the liver cells. Liver cells play important roles in lipid metabolism. If the liver cells are exposed to the toxic substances continuously, then the metabolic process in the liver will be disrupted and cause damage to the liver cells’ tissue structure in the form of the necrotic liver [17]. Necrosis in the liver cells is usually indicated by shrinkage of liver cell nuclei, their boundaries are irregular, and the colors are dark, which are the characteristics of pyknotic cells. The pyknotic nuclei are shrunk nuclei as a result of cytoplasm homogenization and eosinophilic increase. After becoming pyknotic, the liver nuclei can be destroyed and leave chromatin fragments scattered in the cells; this process is called karyorrhexis. Then, if the nuclei are dead due to losing the ability to be colored, this process is called karyolysis [24]. Pyknotic nuclei are the initial stage of necrosis; necrosis is indicated by changes in the liver cells' nuclei [25].

MESA-DP which enters the body through the digestive tract will undergo the first metabolism in the liver because the liver is the main metabolic site that will detoxify and eliminate all toxic substances, both endogenous and exogenous. Therefore, the liver is a potential organ to be damaged by countless various types of pharmaceutical and environmental chemical compounds. Injury is the result of various things, such as direct toxins, liver convention against a xenobiotic becoming an active toxin, and immune mechanism [17].

One of the causes of liver necrosis is too many free radicals. Free radicals are molecules that have unpaired electrons and are reactive. The presence of these electrons in the body tends to attract other electrons belonging to other molecules, thus changing the molecules into free radicals. These molecules are called free radicals because there are reduced or added electrons. The increase of oxidative stress in the liver is caused by free radicals, which can damage the liver cells. In the present study, necrotic cells were observed in the control group. This might be due to no examination of the rats’ liver before sampling. Hence, it might be that the rats taken as samples already had liver damage. Other possible reasons might be because the cage condition was less than ideal, administration of the food and drink was less varied and following the standard, stress factor, the effects of other substances or diseases, immunities, and susceptibilities of the rats. Nevertheless, there was another factor caused by MESA-DP preparations, which was the drying process, that will affect the antioxidant level in the mistletoes because the duration of the drying and storage process also affects the antioxidant power [26]. There was a tendency for hepatocytes to undergo increases in the pyknotic, karyorrhectic, and karyolysis cells count due to the administration of the mistletoe’s combination.

The present study not only examined the clinical biochemistry but also observed the histopathology. The liver can have several changes such as irreversible and reversible damage. Degeneration is reversible damage in which the normal structure of cells changes. The cause of degeneration is a biochemical disorder caused by anemia, abnormal metabolism, and toxic chemical substances. The degeneration that occurs continuously will cause irreversible cell death. Cell death may happen through necrosis and apoptosis. Necrosis is cell death caused by acute cell damage, while apoptosis is a type of programmed cell death used by multicellular organisms to remove cells that are no longer needed by the body [27]. Necrosis can be focal (central, intermediate, peripheral) or massive. Usually, necrosis is acute [17]. The characteristics of necrosis are the appearance of necrotic heart muscle fragments or cells without nuclear outlay or no cells with an inflammatory reaction. Whether or not the remaining liver cells appear depends on the duration and type of necrosis [28].

CONCLUSION / ЗАКЛЮЧЕНИЕ

The outcomes of the current study demonstrated that serological studies for liver function tests proved the protective consequences of MESA-DP in mice and did not cause changes in the levels of total bilirubin, SGOT, SGPT, albumin, cholesterol, and triglycerides, indicating the safe effect of MESA-DP. However, administration of MESA-DP caused a significant increase in necrotic liver cells compared to the control group. The observed hepatoprotective effects may be due to the presence of phytochemicals as well as various biologically active secondary metabolites such as quercetin and flavonoids identified as the main compounds. The protective mechanism is through modulation of oxidative stress and augmentation of antioxidant enzymes and quercetin is known to protect against liver damage by inhibiting inflammatory biomarkers. In future, it is necessary to maintain the availability of natural materials wisely so that conventional and in vitro propagation and cultivation are needed. Because the tea and mango mistletoe are beneficial for the liver health of experimental animals, it is advisable to perform clinical testing on humans.