December 2010, Volume 1, No.

2 International Journal of Chemical and Environmental Engineering

Evaluation of the hepatoprotective activity of Citrus microcarpa Bunge (Family Rutaceae) fruit peel against acetaminophen-induced liver damage in male BFAD- Sprague Dawley rats
Casimiro,Marifel Franchesca 1 Margarita Gutierrez 2 Danice Romagne Leano 1 Judilynn N. Solidum3
1 2

BS Industrial Pharmacy Graduate, College of Pharmacy, University of the Philippines, Manila Faculty member, College of Pharmacy University of the Philippines, Manila 3 Associate Professor, College of Pharmacy University of the Philippines, Manila

Abstract

The Philippine Department of Health stated that liver cancer is the third common forms of cancer for both males and females, hence the need for more hepatoprotective agents. Silymarin, from milk thistle is the most well known hepatoprotective agent (Prahan and Girish, 2006) but due to availability and economic concerns with the use of milk thistle other sources were explored. Fruit peels constitute a bulk in Philippine wastes. If such wastes can be used as hepatoprotective agents, then wastes will be decreased and new sources of important products may be discovered. This study aimed to evaluate the hepatoprotective activity of Citrus microcarpa Bunge fruit peel extract relative to the commercially available Silymarin preparations. The chemical components of the fruit peels were analyzed to ascertain pharmacologic value. The study used an experimental research design using BFAD- Sprague Dawley rats as subjects. The hepatoprotective activity was evaluated based on changes in the liver morphology- gross examination and differences in serum liver enzyme levels- bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (AP) within and among the groups of rats. There was a significant decrease in ALT, AST and AP levels among rats administered with the fruit peel extract. Silymarin significantly decreased bilirubin levels. These suggest a comparable hepatoprotective activity between Silymarin and the fruit peel extract tested. Phytochemical analysis showed that the fruit peel extract contained flavonoids, tannins, and glycosides. Quantitative analysis on the chemical components of the fruit peel extract is suggested to facilitate the study of its exact mechanism of action. Research on the protective ability of the fruit peel extract on other organ systems is recommended. It is also suggested that other chemical liver toxicity inducers be used to observe the range of hepatoprotective activity of the fruit peel extract studied. Keywords: hepatoprotective, Citrus microcarpa Bunge (Family Rutaceae), ALT,AST,AP

1. Introduction
1.1. Background and significance of the study Hepatotoxicity is a growing concern of today’s modern society. The increasing incidence of alcoholism, cigarette smoking, substance abuse and other unhealthy lifestyle options, like eating fatty foods, have contributed to the morbidity and mortality due to liver disease. Many researchers have investigated plants for use in a wide variety of liver disorders [1]. Recent studies have shown that plants under Family Rutaceae like Murraya koenigii, Citrus limon, Citrus aurantium and Tephrosia purpure have been reported to possess hepatoprotective activity [2, 3, 4, 5].

Citrus microcarpa Bunge plant, locally known as kalamansi, is a member of Family Rutaceae that is native and widely cultivated in the Philippines. This species is a native of the Philippines and does not occur naturally outside of the archipelago. Kalamansi is a common fruit vegetable often used by Filipinos in seasoning dishes or as dipping sauce to other viands. It is a smooth and slightly spiny plant, growing to a height of 3 to 5 meters. Leaflets are elliptic to oblong-elliptic, 4 to 8 cm long. Petioles are narrowly and scarcely winged, about a cm long. Flowers are axillary, solitary, rarely in pairs, white, and short-stalked. Fruit is yellow when ripe, nearly spherical, 2 to 3.5 cm diameter, 6- to 7-celled, and thin-

skinned. The skin or peel is green to yellowish green or yellow, loosely adhering to the flesh. The flesh contains a few seeds. The fruit contains volatile oil at 0.9 to 1.06% and its rind contains aldehydes, sesquiterpenes, betapinene, linalool, linalyl acetate, tannin, glucoside and cyanogenetic substances [6]. The present study was undertaken to study the possible hepatoprotective property of ethanolic extract of rind or peel from kalamansi. It was also the intention of the researchers to address the foul odor emited by decomposing kalamansi rind in household garbage that attracts flies. At pharmacological doses, acetaminophen is considered relatively safe as antipyretic and analgesic. At high doses, acetaminophen induceds acute liver damage, which is attributed largely to a by-product of its metabolism, N-acetyl-p-benzoquinone imine (NAPQI). NAPQI promotes depletion of glutathione sources, thereby increasing the risk of liver damage by oxidizing tissue macromolecules such as lipid or SH group of protein and alter homeostasis of calcium [7, 8]. According to Barnes and Denz [9], rats have been widely used for evaluating hepatic toxicity and the criteria of the toxic action in rats are reduction in the rate of bodyweight gain, detection of gross and histologic abnormalities in the organs, increased mortality, and changes in organ weights. Should the fruit peels of kalamansi be proven to be hepatoprotective, a new source for drug discovery will be presented which will help make medicinal formulations more cost effective. Further the innovation will help alleviate problems of wastes in the environment. 1.2. Conceptual framework INPUT
I.Collection of kalamansi fruit peels II. Preparation of kalamansi peels, animal models, drugs for lab analyses

commercially-available preparation (eg, silymarin capsule)? What are the chemical components in the rind of kalamansi that may prove to be of value in the fields of medicine, and pharmacy? 1.4. Objectives This study generally aimed to determine the presence of hepatoprotective activity of Citrus microcarpa (kalamansi) fruit peel against Acetaminophen-induced liver toxicity to Sprague Dawley rats. Specifically it aimed to evaluate the hepatoprotective activity of kalamansi fruit peel against acetaminopheninduced liver damage in BFAD: SD rat models; compare the hepatoprotective activity (i.e. efficacy) of the kalamansi fruit peel extract from the commerciallyavailable preparation (silymarin capsules); to identify the chemical components present in the kalamansi peel, which may be of value in the fields of medicine and pharmacy, through phytochemical screening. 1.5. Scope and limitations This study involved the determination of the presence of hepatoprotective activity of the kalamansi peel extract against liver toxicity induced by acetaminophen alone. Furthermore, the basic phytochemical components of the fruit peel extract were also determined.The actual compound essential for hepatoprotective activity was not identified nor quantified. Commercially-available Silymarin was utilized as positive control. The parameters used to measure the hepatoprotective activity of the plant was limited only to the monitoring of mortality, bodyweight, food consumption, water intake, gross examination of the liver, liver weight, and measurement of blood serum levels of total bilirubin, direct and indirect bilirubin, alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase.

THROUGHPUT
Laboratory tests for effects of kalamnsi fruit peels on liver toxicity of BFAD Sprague Dawley rats

2. Materials and Methods
2.1 Research design The study involved the extraction of plant material followed by an evaluation of its hepatoprotective activity. The evaluation of the hepatoprotective activity of Citrus microcarpa Bunge employed a pretest – post test only experimental study design. 2.2. Plant Material Kalamansi fruits were obtained from Mindoro Province, Philippines through the Valenzuela Market, Polo, Valenzuela City, Philippines. Samples were identified by the Philippine National Museum. Kalamansi rind were separated from the flesh of the fruit, air dried and reduced to size. The material was macerated in 95% ethanol for 48 hours and then filtered. The filtrate was evaporated to dryness under steam bath. The resulting residue was weighed and diluted with

OUTPUT
Hepatoprotective activity of Kalamansi fruit peels

1.3. Problems Generally, does the fruit rind extract of Citrus microcarpa Bunge (Fam. Rutaceae), locally known as kalamansi, exhibit hepatoprotective activity against acetaminophen-induced liver damagein male BFAD:SD rat models? Specifically ,how does the hepato-protective activity of the kalamansi peel extract compare with the

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distilled water to obtain a final concentration of 1g/mL (w/v).. 2.3. Drugs and Chemicals The Silymarin capsule (LiveraideTM , bearing FDA registration no. 41322, lot no. 83, manufactured on June 2009 with expiry date on June 2011, manufactured by Capsugel, France, and imported and distributed in the Philippines by Herbs & Nature Corporation, Bagbag, Novaliches, Quezon City), was used in the study. Silymarin is a hepatoprotective drug[10]. Each capsule has a net weight of 636 mg and contains 125 mg of sylimarin from the seeds of Silybum marianum of Compositate Family, lecithin and beeswax. Acetaminophen was obtained from the College of Pharmacy, University of the Philippines, Manila. 2.4.Animals Eight week old Sprague Dawley male rats were obtained from the closed colony of the Philippine FDA. All animals were given drinking water and standard laboratory diet before and during the experiment. All test animals were subjected to a one week acclimatization period. The environmental conditions and husbandry practices were kept constant throughout the experiment. The study was approved by the UP Manila Institutional Animal Ethics Committee. 2.5. Study Design Four groups with 10 rats each were chosen. The entire study took eight days to finish. Pre-test liver function test were conducted to determine homogeneity of the groups of rat. Group I served as the negative control. Group II, III and IV were given acetaminophen (500 mg/kg) by oral gavage on the fourth day to induce liver

damage. From the first day to the seventh day Group III was given silymarin (950 mg/kg bwt) as hepatoprotective agent and Group IV was given kalamansi rind ethanol extract (4,000 mg/kg bwt) as the test sample. On the eight day, fourth day post-acetaminophen toxicity, all rats were sacrificed. Blood serum were obtained, major tissues and organs were observed and liver organs were weighed. 2.6. Animal Performance Body weight gained, as well as 24-hour water and feed intake, were measured 24 hours postacetaminophen toxicity. 2.7. Liver Function Test Blood samples collected and submitted to a DOHaccredited clinical diagnostic laboratory in Marikina City, Philippines. Blood serum liver enzyme levels of bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (AP) were analyzed. 2.8. Statistical Analysis Body weight gain, feed and water intake, and liver function test results, liver weight were analyzed using Student t-test, NCSS statistical software. All data are reported as mean and SD.

3.Results and Discussion
3.1. Pre-Test Baseline Data The data shown in Table 1 shows that there were no differences in bodyweight, total bilirubin, direct bilirubin, indirect bilirubin, AST, ALT and alkaline phosphatase among the groups of rats at the start of the treatment.

Table. 1 Bodyweight And Blood Serum Enzyme For Liver Function Body Oral Treatment Weight gm I 256.1 ± 1.75 256.9 ± 1.77 Total Bilirubin mg/dL 0.15± 0.01 0.15± 0.01 Direct Bilirubin mg/dL 0.13 ± 0.01 0.11± 0.004 Indirect Bilirubin mg/dL 0.04± 0.01 0.04± 0.01 AST ALT Alkaline Phosphatase IU/L 331.15 ± 26.42 325.51± 29.26

IU/L 101.31± 2.64 99.11± 4.70

IU/L 49.67± 1.89 47.37± 2.23

Distilled Water

II

Distilled Water + Acetaminophen

III IV

Silymarin + Acetaminophen

255.4 ± 1.86 256.6 ±1.63

0.17± 0.01 0.16± 0.01

0.12± 0.01 0.12± 0.01

0.05± 0.01 0.05± 0.01

112.03± 2.90 101.20± 4.63

51.15± 2.14 49.60 ± 1.70

307.89± 26.16 314.51± 24.62

Kalamansi Rind Extract + Acetaminophen

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3.2 Test Proper 3.2.1 Mortality On the sixth day, second day post-acetaminophen toxicity, one rat in Group II, rat no. 7, was found dead inside the cage. Rat no. 7 was found emaciated inside the cage. Gross examination of the liver organ revealed hemorrhagic lesions. B.1.1 Body Weight Gained Analysis of the bodyweight loss and gained 24 hours post-acetaminophen toxicity are summarized below in Table 2. The data shows that rats in Groups II, III and IV significantly decreased in bodyweight compared to Group I. However, the table also shows that rats in Group II and Group III has significantly lower body weight loss than Group II. Group IV still managed to gain some weight.
Table 2 Body Weight Gain 24 Hours After Acetaminophen Toxicity Body Weight Gained Group Oral Treatment Gm I II III IV Distilled Water Distilled Water + Acetaminophen Silymarin + Acetaminophen 3.70 ± 0.62 - 1.33 ± 1.16 a - 0.10 ± 1.14 a, b

Table 3 Twenty Four Hour Feed And Water Intake 24 Hours Post Acetaminophen Toxicity Feed Water Intake Intake Group Oral Treatment mL grams 28.9 ± I Distilled Water 29.1 ± 1.13 1.68 II Distilled Water + Acetaminophen 14.1 ± 1.83 a 18.50 ± 2.62 a

III

Silymarin + Acetaminophen

18.8 ± 3.11 a

18.7 +/3.57 a 25.80 ± 2.64

IV

Kalamansi Rind Extract + 26.5 ± Acetaminophen 2.65 a p<0.05 compared with Group I

Kalamansi Rind Extract + 1.20 ± 0.93 a, b Acetaminophen a p<0.05 compared with Group I b p < 0.05 compared with Groups II and III

The results revealed that the mean feed and water intake of rats in Group IV was not significantly reduced compared to rats in Group I. It was seen that food and water intake of groups II and III decreased significantly. The findings are biologically significant since the rats were not trained to refuse feeds and water that are made available and accessible ad libitum. The group of rats which were pre-treated with kalamansi rind extract performed better than the group pre-treated with sylimarin 24-hours after giving large doses of acetaminophen to induce liver damage.

3.2.2. Feed and Water Intake The 24-hour feed and water intake between the groups were studied 24 hours after acetaminophen toxicity. The results are shown in Table 3.
Table.4 Result Of Blood Serum Enzyme For Liver Function Test Total Bilirubin                                                                      Indirect bilirubin  a  p<0.05 compared with Groups I and III                          a p<0.05 compared  with Groups I and III                                                                                     b p<0.01 compared with Groups I and III                           b p<0.01 compared with Groups I and  III  AST a p<0.05 compared with Groups I, II and III ALT a p<0.05 compared with Groups I, II and III

BILIRUBIN Group Oral Treatment Total Bilirubin mg/dL I Distilled Water 0.09 ± 0.96 Direct Bilirubin mg/dL 0.05± 0.004 Indirect Bilirubin mg/dL 0.04± 0.01 AST (SGOT) ALT (SGPT) Alkaline Phosphatase

IU/L 150.8 ± 9.31

IU/L 51.79± 1.78

IU/L 345.22± 18.96 327.67± 21.24 264.41± 13.96 252.7± 24.03

II

Distilled Water + Acetaminophen Silymarin + Acetaminophen Kalamansi Rind Extract + Acetaminophen

0.20 ± 0.06 a

0.05 ± 0.01

0.15 ± 0.05 a

143.16 ± 9.22

51.66± 2.67

III

0.11± 0.02

0.06 ± 0.004

0.05± 0.01

144.72 ± 2.90

63.56± 5.11 44.17± 2.04 a

IV

0.22± 0.04 b

0.06± 0.01

0.16± 0.01 b

108.20± 9.65 a

3.3 Blood Chemistry Liver Profile

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3.3.1 Bilirubin Group III (silymarin + acetaminophen) has normal level of total bilirubin which is comparable to Group I (negative control). Group IV (kalamansi rind extract + acetaminophen), have significantly elevated total bilirubin compared with Groups I and III which may indicate some form of parenchymal injury. Values obtained are near the upper limit of normal bilirubin value of 0.18mg/dL [11]. 3.3.2 AST and ALT In this study the animals were sacrificed after 80 hours before blood serum for hepatic enzyme function tests were collected. Acetaminophen toxicity significantly increases ALT and AST values immediately upon administration as indication of hepatic injury. As shown in the table, AST and ALT of Groups I, II and III increased after 80 hours post-acetaminophen dosing relative to group IV. In humans, serum ALT (SGPT) biological half-life varies from 48 to 60 hours [12] In Group IV (kalamansi rind extract), after an 80 hour post acetaminophen toxicity, the serum AST (SGOT) and ALT (SGPT) were significantly lower than all the groups, including Groups II and III (sylimarin). The major organs affected by acetaminophen in cases of overdose are liver and kidneys. The protective property of kalamansi rind extract may not be specific to acetaminophen induced damage to liver alone, but to other tissues as well, like the red blood cells, heart and skeletal muscles, pancreatic tissues and kidney tissues, which also contain the AST enzyme. The protective effect of kalamansi rind extract may be the result of stabilization of plasma membrane, thus preserving the structural integrity of cell as well as the repair of tissue damage.         The  24‐hour  feed  and  water  intake  (24  hour  post‐ acetaminophen  toxicity  )  shown  in  Table  III  of  Group  IV  were not statistically different from Group I, the negative  control, as kalamansi rind extract may also be protective  to other organs and tissues.  It was observed that when  protected  by  kalamansi  rind  extract  ,  rats  are  least  affected  when  given  large  doses  of  acetaminophen  (  Table IV).  3.3.3 Liver Weight and Lesions The following table shows the absolute and relative mean weight of liver in all groups. Groups II, III and IV relative liver weights were significantly heavier than Group I after 80 hours post-acetaminophen toxicity.

Table 5 Absolute And Relative Weights Of The Liver At The Termination Of The Study Liver Weight Absolute Relative Group Oral Treatment Grams/100 grams gram bwt 10.34 ± I Distilled Water 3.71 ±- 0.06 0.15 II Distilled Water + Acetaminophen Silymarin + Acetaminophen 10.98 ± 0.28 10.60 ± 0.41 4.15 ± 0.10 a

III IV

4.03 ± 0.17 a 4.07 ± 0.14 a

Kalamansi Rind Extract + 10.88 ± Acetaminophen 0.47 a p<0.05 compared with Group I

Literatures reported that when acetaminophen was given to mice at 1,000 mg/kg, extensive necrosis was associated with hemorrhage and seen in the liver. When dose was reduced to 500 mg/kg punctuate hemorrhage necrosis of liver tissue was observed [13]. This study showed that after 80 hours post-acetaminophen toxicity, although liver weights of Groups II,III and IV increased relative to group I, no punctuate hemorrhage or other appreciable liver lesions, like swelling (grossly by observing and cutting the liver margins), were observed particularly with groups III and IV. This finding indicates possible recovery from the initial acetaminophen toxicity. 4. Conclusion From the results obtained, kalamansi peel extract showed hepatoprotective activity against Acetaminopheninduced liver damage in male SD rats. The hepatoprotection it conferred was reflected by the significant decrease in serum enzyme levels and lack of any gross morphological injury to the rat’s liver. It may also be concluded from the results of the study that the hepatoprotective activity of kalamansi peel extract is comparable to that of the commercially available silymarin preparations. Similarities in mechanism of action between the two plant extracts may be expected but the presence of other medicinally beneficial phytochemicals in the kalamansi peel extract (i.e. tannins and glycosides), aside from flavonoids, may offer additional modes of action. As such, the kalamansi peel extract has also been inferred to confer multi-system or multi-organ protection including that of the kidneys. With the observations of hepatoprotective activity in the kalamansi peel extract (against acetaminopheninduced liver damage), evaluation of the same activity against damage induced by other chemical toxicants and subsequent identification of the possible component/s responsible for the activity is recommended. Determination of the most effective extract concentration must also be employed. Quantitative evaluation of the phytochemical components of the extract should be given priority as well so as to provide means for standardizations for future product formulation Finally, similar with other ‘new’ drugs and substances, researches geared toward toxicological profiling of kalamansi peel extract is suggested.

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REFERENCES
[1] N.D. Scott Luper A review of plants used in the treatment of liver disease: Part-I. Alternative medicine Review. 3 (6):410, 1998. [2] S. Gupta , “. Antioxidant Activity of Murraya Koenigii Linn Leaves” [Internet] Available from <http://www.pharmacologyonline.com> 1: 474-478 (Accessed June 19, 2009) [3] S. Bhavsar, “Investigation into Hepatoprotective Activity of Citrus limon”, International Journal of Pharmacognosy Volume 45, Number 4. 2007. [4] A. Jain, A., “A comparative study of ethanol extract of leaves of Tephrosia purpurea pers and the flavonoid isolated for hepatoprotective activity,”Indian J Pharm Sci V.68, pp.740-3,2006 [5] M. Karaca, “ Evaluation of hepato protective activity of Bergamot orange in rats” Eastern Journal of Medicine, 10 :1- 4,2005. [6] E. Quisumbing,. Medicinal Plants of the Philippines.Quezon City: Katha Publication.454-455. 1978. [7]J.G.Hardman, L.E.Limbird, and A.G. Gilman. . Goodman & Gilman’s The Pharmacological Basis of Therapeutics, 10th edition. NY: The McGraw-Hill Companies, Inc. 703 – 70,2001 [8] M.C.Savides, and F.W.” Oehme Acetaminophen and its toxicity”, J. App Toxicol. 3:95-111,1983. [9] J. M. Bames and F.A. Denz. “ Experimental methods used in determining chronic toxicity; a critical review”, Pharmacol Rev. 191–242,1954. [10] S.C. Pradhan and C.Girish, “ Hepatoprotective herbal drug, silymarin from experimental pharmacology to clinical medicine,” Indian Journal of Medicine,2006. [11] C.I.B.Biares and O.G. Gutierrez, Jr. Reference values for hematology and serum biochemistry in young barrier-reared BFAD:Sprague Dawley rats.BFAD Infromation Bulletin,vol 4, no 1 pp 1-7, Dec. 1998. [12] Alanine Aminotransferase (ALT/SGPT) Slideworld; Anonymous Author [13] C. Girish, B. C. Koner, S. Jayanthi, K.R. Rao, B. Rajesh and S.C. Pradahan, “ Hepatoprotective activitiy of six polyherbal formulations in paracetamol induced liver toxicity in mice,” Indian J Me Res 129, 569-578, May 2009.

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