Hepato-protective Effects of Ocimum Canum Sims Hydro-alcoholic Leaf Extract in I/R Induced Hepatic Injury in Rats

Saiprasanna Behera, S Manohar Babu, Y Roja Ramani, Prasanta Kumar Choudhury

Saiprasanna Behera, Department of Pharmacology, Royal College of Pharmacy and Health Sciences, Berhampur, Odisha- 760002
Prasanta Kumar Choudhury, Department of Pharmaceutics. Royal College of Pharmacy and Health Sciences, Berhampur, Odisha- 760002, India
S Manohar Babu, Y Roja Ramani, Department of Pharmacology, SIMS College of Pharmacy, Mangaldas Nagar, Guntur- 522001, India

Correspondence to: Saiprasanna Behera, Department of Pharmacology, Royal College of Pharmacy and Health Sciences, Berhampur, Odisha- 760002, India.
Telephone: +09861252518
Received: June 17, 2012
Revised: July 31, 2012
Accepted: August 6, 2012
Published online: November 21, 2012


AIM: The plant Ocimum canum Sims (Family: Lamiaceae) is rich in antioxidants, particularly in flavonoids. It has been highly useful in treating various types of diseases and in lowering blood glucose, especially in managing diabetes mellitus. The aim of our work is to evaluate the effect of Ocimum canum on a model of hepatic ischemia-reperfusion in Rat.

METHOD: Wistar albino rats were subjected to 45 min of hepatic ischemia, followed by a 60 min reperfusion period. Ocimum canum (OC) hydro-alcoholic leaf extract were administered in doses of 100, 200 and 400 mg/kg/day, orally for 15 days before I/R injury respectively and repeated before the reperfusion period. Liver samples were taken for histological examination or determination of hepatic malondialdehyde (MDA), super oxide dismutase (SOD), catalase and glutathione (GSH) activity. Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT,) alkaline phosphatase (ALP) and total bilirubin levels were determined to assess liver functions. Lactate dehydrogenase (LDH) was assayed in serum samples for the evaluation of generalized tissue damage.

RESULTS: Ischemia/reperfusion caused a significant decrease in hepatic SOD, catalase and GSH, and significant increase in MDA levels. Serum AST, ALT, ALP and bilirubin levels, as well as LDH activity levels were also elevated in the I/R group. Treatment with OC hydro-alcoholic leaf extract reversed all these biochemical parameters as well as histological alterations induced by I/R. In all the testing, a significant correlation existed between concentrations of the extract and alteration in the biochemical and histological parameters.

CONCLUSION: OC hydro-alcoholic leaf extract at the dose of 400 mg/kg/day reduced I/R-induced organ injury through its ability to balance the oxidant–antioxidant status.

Key words: Ischemia/reperfusion; Ocimum canum (OC) hydro-alcoholic leaf extract; Antioxidant

© 2012 The Authors. Published by Thomson research Group Ltd.

Behera S, Babu SM, Ramani YR, Choudhury PK. Hepato-Protective Effects of Ocimum Canum Hydro-alcoholic Leaf Extract, in I/R Induced Hepatic Injury in Rats. Journal of Gastroenterology and Hepatology Research 2012; 1(10): 266-273 Available from: URL: http://www.ghrnet.org/index./joghr/


Most surgical procedures involve a period of ischemia followed by reperfusion, as do many disease states such as shock, sepsis and pancreatitis. In the liver, ischemia/reperfusion (I/R) injury can occur in several clinical settings such as hepatic trauma, resection of large intrahepatic tumors and liver transplantation[1] (Serracino-Inglott et al, 2001). Ischemia is a state of tissue oxygen deprivation accompanied by a reduced washout of the resulting metabolites[2]. Reperfusion is the restoration of blood flow to the ischemic tissue. Despite the unequivocal benefit of reperfusion of blood to an ischemic tissue, reperfusion itself can elicit a cascade of adverse reactions that paradoxically injure tissue[3]. Deprivation of oxygen to the liver during ischemia induces severe damage; however, much more damaging reactive oxygen species (ROS) are generated during the reperfusion period[4]. The organ dysfunction that accompanies this condition is generally associated with increased microvascular permeability, interstitial edema, impaired vasoregulation, inflammatory cell infiltration and parenchymal cell dysfunction and necrosis[5,6].

Free radical ablation for the treatment of reperfusion injury found its first clinical application in the prevention of postischemic tissue injury after organ transplantation[7,8]. Thus, free radical scavengers and antioxidant agents are thought to be useful in the clinical setting of hepatic I/R damage[9].

Flavonoids exert their antioxidant effects (countering inflammatory, bacterial, viral, microbial, hormonal, carcinogenic, neoplastic and allergic disorders) by neutralizing all types of oxidizing radicals including the super oxide and hydroxyl radicals and by chelation. Chemically flavonoids are polyphenolic compounds possessing fifteen carbon atoms, two benzene rings joined by a linear three carbon chain. The six subgroups of flavonoids are Chalcones, Flavone, Flavonol, Flavonone, Anthocyanins, Isoflavonoid. Flavonoids also act as powerful chain breaking antioxidants due to the electron donating capacity of their phenolic groups[10]. Two types of flavonoids are present in Ocimum species. Lipophilic flavonoid aglycones (external flavonoids), often highly methylated, are found in glandular hairs on the surface of the leaves, stems and inflorescences. These have been the subject of two recent studies[11,12]. The second type of flavonoids is polar flavonoid glycosides, which are stored in the vacuoles of aerial plant parts. Nevadensin and salvigenin are found in O. canum Sims[13] (Figure 1).

Ocimum canum Sims. (Hairy Basil) synonymously known as O. americanum Linn. is a traditional medicinal plant distributes throughout Odisha and it is commonly known as Kala Tulasi in Odiya has an unusual mint-like flavor. The plant branches out from its base, with angle stems and open foliage. It is not often used as a culinary herb, unlike the related basil species O. basilicum, but more often as a medicinal plant. The essential oils found in this species have strong fungicidal activity against certain plant pathogens[14-16]. In Africa, leaves of O. americanum have been used as an insecticide for the protection against postharvest insect damage especially that by bruchid beetles[17]. Medicinal properties may be associated with the external flavonoids, as some specimens produce very high levels of these compounds, especially nevadensin[12], which has antioxidant activity[18].

is used specially for treating various types of diseases and lowering blood glucose and also treats cold, fever, parasitic infestations on the body and inflammation of joints and headaches[19]. Essential oil from the leaves of O. canum possesses antibacterial and insecticidal properties[20]. The present study, therefore, investigated the protective effect of Ocimum canum hydro-alcoholic leaf extract against oxidative stress during I/R injury of the liver, by measuring biochemical values and conducting histological examinations.


The experimental protocols were conducted with the approval of the Animal Research Committee at Royal College of Pharmacy and Health Sciences, Berhampur. Odisha. All animals were maintained in accordance with the recommendations of the CPCSEA.


Male Wistar albino rats (200-250 g) were obtained from the animal house of R.C.P.H.S. and were housed in an air-conditioned room with 12 h light and dark cycles, with constant temperature (22±2 °C) and relative humidity (65-70%) levels. All experimental protocols were approved by the Institutional Animal Ethical Committee (Approval No-07/IAEC/2011) of Royal College of Pharmacy and Health Sciences, Berhampur. Odisha. India. The rats were anesthetized by intraperitoneal injection of sodium pentobarbital (30 mg/kg). All surgical procedures were conducted with clean but not sterile instruments.

Plant collection

Leaves of Ocimum canum were collected in the month of December 2011 from its natural habitat from nearby Mohuda village, Berhampur, Ganjam district of Odisha. India. The plant was authenticated from Department of Botany, Khalikote College, Berhampur, Odisha. India. The leaves were cleaned and dried under the shade to avoid degradation of volatile oil. The leaves were dried in hot air woven at 55°C for 3 d and at 40°C for the next 4 d (Figure 2).

Preparation of Plant Extracts

The dried leaves were coarsely powdered and extracted with a mixture of methanol: water (7:3, v/v) by a Soxhlet apparatus at 50°C. The solvent was completely removed and obtained dried crude extract which was used for investigation. Further the extracts were subjected for the antioxidant study as well as pharmacological screening.

Experimental protocol

Under anesthesia, a midline laparotomy was made using minimal dissection. The abdomen was shaved and a transverse incision was performed. The bowel loops were covered with saline-soaked gauze. Total hepatic ischemia was induced for 45 min by clamping the hepatic artery, the portal vein and the bile duct using a vascular clamp and the rats were then allowed to reperfuse for 60 min. Abdominal incision was closed in layers with 4-0 dexon and 2-0 nylon during reperfusion stage in order to prevent the loss of body fluid and quantity of heat.

Animals were divided into seven groups consisting of six rats each. Ocimum canum (OC) hydro-alcoholic leaf extract was dissolved in water and administered to the animals. Ocimum canum (OC) hydro-alcoholic leaf extract used in this study contains external flavonoids, especially nevadensin.

Group-I: -NAIVE-Normal control-rats in this group did not undergo ischemia or reperfusion and served as the control group.

Group-II: -SHAM-Sham-operated (animals subjected to the identical procedure of surgery without ischemia-reperfusion injury) plus physiologic saline treatment.

Group-III: -I/R-Animals subjected 45 min of total hepatic ischemia, followed by reperfusion for 60 mins and served as untreated experimental control.

Group-IV: -OC control- Sham operated plus Ocimum canum control (400 mg/kg body wt. treatment up to 15 d).

Group-V: -OC 100 mg/kg+I/R-Hepatic I/R plus Ocimum canum hydro-alcoholic leaf extract 100 mg/kg body wt. treatment up to 15 d.

Group-VI: -OC 200 mg/k +I/R-Hepatic I/R plus Ocimum canum hydro-alcoholic leaf extract 200 mg/kg body wt. up to 15 d.

Group-VII: -OC 400 mg/kg+I/R-Hepatic I/R plus Ocimum canum hydro-alcoholic leaf extract 400 mg/kg body wt. up to15 d.

None of the animals died during these procedures. At the end of the reperfusion period, animals were decapitated and trunk blood samples were collected to determine serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), bilirubin and lactate dehydrogenase (LDH) activity, the indicators of liver functions and generalized tissue damage, respectively. The hepatic tissue samples were stored at -20°C. Afterwards, tissue malondialdehyde (MDA) levels, an end product of lipid peroxidation, superoxide dismutase (SOD), Catalase and glutathione (GSH), key endogenous antioxidants, were measured in these samples. The hepatic tissue samples were also placed in formaldehyde (10%) for histological evaluation.

Measurement of serum index of hepatotoxicity

Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), bilirubin and lactate dehydrogenase (LDH) activity was measured using commercial kits (Crest Biosystems, Bambolim Complex. Goa, India) and the results are expressed in international units per liter[21].

Measurement of hepatic oxidative stress markers

(1) Lipid peroxidation was used as an indirect measure of oxidative damage induced by ROS (free radicalinduced injury). Lipid peroxidation was assayed as the malondialdehyde (MDA) level in liver homogenate by the thiobarbituric acid method using tetraethoxypropane as the standard[22]. A mixture of 8.1% sodium dodecylsulphate (0.2 mL, Merck), 20% acetic acid (1.5 mL), and 0.9% thiobarbituric acid (1.5 mL, Merck) was added to 0.2 mL of 10% tissue homogenate. Distilled water was added to the mixture to bring the total volume to 4 mL. This mixture was incubated (95℃, 1 h). After incubation, the tubes were placed in cold water and 1 mL of distilled water plus 5 mL of n-butanol/ pyridine (15:1, v/v) was added, followed by mixing. The samples were centrifuged (4000 rpm, 10 min). The organic phase (supernatant) was removed, and absorbances were measured with respect to a blank at 532 nm. 1, 1, 3, 3-Tetraethoxypropane was used as the standard. Lipid peroxide levels were expressed as nmol MDA/g of wet tissue; (2)The hepatic antioxidant activity, superoxide dismutase (SOD), was assessed in the homogenized liver by the method of Sun et al[23]. [0.3 mM xanthine, 0.6 mM Na2EDTA, 0.15 mM nitroblue tetrazolium (NBT), 0.4 M Na2CO3, and 1 g/L bovine serum albumin (BSA)] was added 100 µL of the tissue supernatant. Xanthine oxidase (50 µL, 167 U/L) was added to initiate the reaction and the reduction of NBT by superoxide anion radicals, which are produced by the xanthine-xanthine oxidase system, was determined by measuring the absorbance at 560 nm. Cu, Zn-SOD activity was expressed as units of SOD/mg of tissue protein, where 1 U is defined as that amount of enzyme causing half-maximal inhibition of NBT reduction To 2.45 mL of assay reagent in addition, the total protein content in the liver tissues was determined according to the Lowry’s method[24]; (3) CAT activity was measured by the method by Aebi[25]. Supernatant (10 µL) was placed in a quartz cuvette and the reaction initiated by adding 2.99 mL of freshly prepared 30 mM H2O2 n phosphate buffer (50 mM, pH 7.0). After rapid mixing, the rate of H2O2 decomposition was determined from absorbance changes at 15 and 30 s at 240 nm. CAT activity was expressed as k/mg of tissue protein, where k is the first order rate constant; And (4) GSH in liver tissue was assayed by the method of Tietze and Anderson[26,27]. Briefly, 100 µL of tissue supernatant was placed in a 3 mL cuvette; 750 µL of 10 mM 5-5’-dithio-bis-2nitrobenzoic acid (DTNB) solution (100 mM KH2PO4KH plus 5 mM Na2EDTA, pH 7.5 and GSHRD, 625 U/L) was added and the mixture was incubated (3 min, room temperature). Then 150 µL of 1.47 mM β-NADPH was added, mixed rapidly by inversion, and the rate of 5-thio-2-nitrobenzoic acid formation (proportional to the sum of reduced and oxidized glutathione) was measured spectrophotometrically for 2 min at 412 nm. The reference cuvette contained equal concentrations of DTNB and NADPH, but no sample; results were expressed as nmoL/mg of wet tissue.

Histological procedures

Liver specimens from all groups were rapidly taken and fixed in Bouin’s solution and processed for light microscopic study using hematoxylin and eosin stain[28]. For light microscopic investigations, hepatic tissue specimens were fixed in 10% formaldehyde, dehydrated in alcohol series, clearing in toluene and embedding in paraffin. Paraffin sections (5 μm) were stained with hematoxylin and eosin (H&E) and examined under a photomicroscope. All tissue sections were examined microscopically for the characterization of histopathological changes by an experienced histologist in blind fashion (Procedures were carried out in NIDAN diagnostics, Berhampur, Odisha, India).

Statistical Analysis

Results are presented as the mean±SEM. All statistical analyses were performed using Graph Pad Prism Software program (version 5)[29]. Data were analyzed using analysis of variance followed by Bonferroni’s post-test. The Kruskal-Wallis 1-way analysis of variance by ranks was used to simultaneously test the pathologic score for the I/R and I/R±Ocimum canum groups. A P value of <0.05 was considered statistically significant.


This study showed no significant difference between the biochemical measurements of group 1 and group 2. Hepatic I/R rats (group 3) showed a significant elevation of serum index of hepatotoxicity (ALT). ALT, AST and ALP levels were significantly higher in the I/R group when compared with those of the control group (p<0.001). OC treatment reversed these values significantly.

Similarly, in the I/R group, increased lactate dehydrogenase activity, as an index of generalized tissue damage, was reversed significantly by OC treatment (p<0.01) (Figure 4).

The liver MDA, which is an index of tissue lipid peroxidation, was found to be significantly higher in the I/R group (27.70±2.51 nmoL/g), however treatment with OC decreased the elevated MDA level significantly back to the control level (17.28±2.87nmoL/g protein) (Figure 5A). The levels of liver SOD lowered significantly after hepatic I/R compared with the sham group (SOD: 30.45±3.19 U/g protein vs 20.43±2.44 U/g protein, P=0.000), after administration of Ocimum canum hydro-alcoholic leaf extract 100, 200 and 400 mg/kg, SOD activity in liver was elevated (20.43±2.44 U/g protein vs 25.37±2.48 U/g protein, 27.68±2.86 U/g protein, 31.75±2.37 U/g protein, P=0.706, P=0.014, P=0.014) (Figure 5B). Figure 5C shows the plasma Catalase levels of the hepatic injured rat treated with Ocimum canum hydro-alcoholic leaf extract 100, 200 and 400 mg/kg. Administration of OC 100, OC 200 and OC 400, showed excellent effects in suppressing the hepatic injury. Among them, OC 400 showed the greatest inhibitory effect against hepatic reperfusion injury. The endogenous antioxidant, GSH, level in the hepatic tissue was decreased significantly after I/R (20.43±2.44 U/g protein). On the other hand OC treatment significantly reversed this I/R-induced GSH reduction (0.35±0.01 U/g protein) (Figure 5D).

After 60 minutes of continuous ischemia, large confluent areas of tissue lysis with blood congestion in the sinusoids and leukocyte infiltrates were observed (Figure 6C). In the liver treated with OC 100, limited and focal areas of hepatocyte necrosis were also observed (Figure 6D), whereas the parenchyma was almost normal after OC 200 and OC 400 treatment respectively (Figure 6E and 6F).

Light microscopic investigation of the control group (either given saline or OC) revealed a regular morphology of liver parenchyma with intact hepatocytes and sinusoids (Figure 6A). In the I/R group, severe sinusoidal congestion and hemorrhage, dilation of central vein, subendothelial edema and degenerated hepatocytes with perinuclear vacuolization were observed (Figure 6C). In the OC treated I/R groups, histological analysis demonstrated a well-preserved liver parenchyma. Despite the mild sinusoidal dilatation and hemorrhage, which were in localized areas, the usual appearance of the central vein and hepatocytes was observed in most areas (Figure 6D, 6E and 6F).


The current data demonstrate that temporary blockade of hepatic blood supply yielded structural and functional alterations in the liver. The antioxidant, OC, on the other hand, reduced the severity of injury.

I/R injury is a complex process involving numerous intracellular signaling pathways, mediators, cells and pathophysiological disturbances; and its prevention during surgery is of the utmost importance[30]. Considerable evidence suggests that oxygen derived free radicals are involved in the hepatic injury caused by ischemia and reperfusion[31-34]. Thus, therapeutic strategies are designed to reduce free radical induced damage, either by intervening in the process by which free radicals are formed or by scavenging the free radicals that have already been formed. Different degrees of protection were obtained with numerous compounds; however, the structure-activity relationship, bioavailability and therapeutic efficacy of these compounds differ extensively. Thus clinical application of these agents is limited in respect of their side effects, toxicity, solubility, membrane penetration etc.

The biological, pharmacological and medicinal properties of bioflavonoids and proanthocyanidins have been extensively reviewed[35,36]. The structure of flavonoid consists of two hydroxy substituted aromatic rings joined by a three carbon link (a C6-C3-C configuration) which renders them hydrogen and electron donors. Thus, they are effective scavengers of free radicals, which are intermediate products of lipid peroxidation, and they slow down oxidation reactions. The antioxidant activity of flavonoids has been studied in lipid substrates, as well as in relation to human health, in particular with regards to retarding the aging of cells and protecting against certain illnesses such as cardiovascular or coronary disease and cancer[37]. The main phenolics reported in basil are phenolic acids and flavonol-glycosides[38,39]. Nevadensin and salvigenin are the two flavonol-glycosides found in O. canum Sims.

Oxygen radical-initiated lipid peroxidation may contribute to the impaired cellular function and necrosis associated with reperfusion of ischemic tissues[40]. In the present study, I/R caused significant increases in the hepatic malondialdehyde levels, end products of lipid peroxidation. This observation is in agreement with the previous studies, where elevated levels of lipid peroxidation products were increased from 40% to 80% above basal values[31,33-34]. Furthermore, our results demonstrated that OC treatment abolishes the increase in malondialdehyde, probably in part by scavenging the very reactive hydroxyl and peroxyl radicals. Previous studies have justified that flavonoid glycosides of Ocimum basilicum (Labiatae) decreased ulcer index, and inhibited gastric acid and pepsin secretions in aspirin-induced ulcers in rats[41]. P Caturvedi et al in 2008[42] demonstrated the antioxidant properties O. canum exhibiting its hepatoprotective abilities against alcohol-induced oxidative stress.

Oxidative stress-induced tissue damage can be prevented or ameliorated by favoring the balance towards a lower oxidative status. Glutathione is an important constituent of intracellular protective mechanisms against various noxious stimuli, including oxidative stress[43]. The results of the present study support the notion that depletion of tissue GSH, as observed in the I/R-induced hepatic injury, is one of the major factors that permit lipid peroxidation and subsequent tissue damage. On the other hand, the decrease in hepatic GSH effect was reversed by the administration of Ocimum canum hydro-alcoholic leaf extract. A possible explanation for this effect is that Ocimum canum functioned as free radical scavenger and therefore increased the available free GSH which detoxifies the reactive intermediary oxygen products of lipid peroxidation induced by I/R.

Superoxide dismutase scavenges the superoxide ions produced as cellular by-products of ethanol metabolism. The reduced SOD activity in the I/R group resulted in the accumulation of superoxide radicals and the production of oxidative stress. The Ocimum canum hydro-alcoholic leaf extracts (i.e. OC 100, OC 200 and OC 400) were found to have good scavenging activity, mainly via superoxide anions, at all concentrations tested. The results show significant differences between the normal control and I/R groups (P≤0.001), but no significant difference from the OC -treated groups.

Catalase acts as a preventive antioxidant and plays an important role in protection against the effects of lipid peroxidation. The inhibition of CAT activity suggests that there is increased synthesis of superoxide ions during ischemic reperfusion because superoxide can be a powerful inhibitor of catalase. In all the experiments conducted in the present study, significant differences were seen between the I/R groups and the normal controls (P≤0.05), but no significant differences in the experimental groups (i.e. OC 100, OC 200 and OC 400).

Liver transaminases (AST/ALT) are not liver function tests, but are biomarkers of liver injury in a patient with some degree of intact liver function. Alkaline phosphatase (ALP) is an enzyme in the cells lining the biliary ducts of the liver. ALP levels in plasma will rise with large bile duct obstruction, intrahepatic cholestasis or infiltrative diseases of the liver. Lactate dehydrogenase is an enzyme found in many body tissues, including the liver. Elevated levels of LDH may indicate liver damage. Bilirubin is a breakdown product of heme (a part of hemoglobin in red blood cells). Increased total bilirubin causes jaundice, and can signal a number of clinical problems. A significant increase in the levels of liver markers (ALT, AST, ALP and Total bilirubin) was observed in I/R groups, and this may be due to changes in membrane phospholipid composition caused by the peroxidation process, which in turn increases membrane permeability. Treatment with Ocimum canum hydro-alcoholic leaf extract helped to prevent this significant increase and appeared to restore the normal condition to some extent.

The results of this study clearly demonstrated that temporary blockade of hepatic blood supply yielded structural and functional alterations in the liver. On the other hand OC treatment improved I/R-induced impairment in the liver functions. It significantly decreased I/R-induced elevations in hepatic lipid peroxidation, lactate dehydrogenase activity and serum ALT, AST, ALP and total bilirubin, while decreased GSH, Catalase and SOD levels were replenished by OC treatment. These protective effects of Ocimum canum hydro-alcoholic leaf extract on reperfusion-induced injury can be attributed, at least in part, to its ability to balance oxidant-antioxidant status suggesting a future role in the treatment of organ failures due to ischemia reperfusion.


In conclusion, it was found that the plant based antioxidant Ocimum canum hydro-alcoholic leaf extract decreased oxygen free radicals during hepatic IR injury in rats. Also, evaluation of liver enzymes and histopathological findings of liver tissue indicated that Ocimum canum had beneficial effects on the liver, so Ocimum canum can be considered a preventive treatment agent in hepatic IR injury. As this study does not contain information about the long-term results of OC treatment of hepatic IR injury, further experimental and clinical studies are needed.


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Peer reviewer: Dhruba Sankar Goswami, M.Pharm (Pharmaceutics)(PhD), Lecturer, S.D.College of Pharmacy, Barnala, Punjab 148101, India; Pooja Maity, Kalinga Institute Of Medical Sciences, Bhubaneswar, India.


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