BLDE University Journal of Health Sciences

ORIGINAL ARTICLE
Year
: 2016  |  Volume : 1  |  Issue : 2  |  Page : 113--119

Evaluation of endophytic fractions of Boerhaavia diffusa Linn. roots for hepatoprotective activity in rats


Smita D Madagundi1, Poonam Kothli1, Prasanna V Habbu1, Venkatrao H Kulkarni2,  
1 Department of Pharmacognosy, SET's College of Pharmacy, Dharwad, Karnataka, India
2 Department of Pharmacology, SET's College of Pharmacy, Dharwad, Karnataka, India

Correspondence Address:
Smita D Madagundi
Department of Pharmacognosy, SET«SQ»s College of Pharmacy, S. R. Nagar, Dharwad - 580 002, Karnataka
India

Abstract

Objectives: Endophytes are major originators of new bioactive compounds with fascinating pharmacological activities. In this study, a perusal was done for isolation, characterization, and screening of endophytic bacteria of Boerhaavia diffusa Linn (BDEF) root for antioxidant and hepatoprotective activities. Methods: BDEF was isolated from the roots and grown in nutrient agar media aseptically. The grown bacteria was further fermented in nutrient broth and extracted using chloroform (CBD) and ethyl acetate (EABD). CBD and EABD were assayed for free radical scavenging properties against 2, 2 diphenyl 1 picryl hydrazyl (DPPH), hydroxyl radical, and reducing power. Findings: The highest inhibition was exhibited in EABD with IC50 level of 22.86 μg/ml for DPPH and 82.78 μg/ml for hydroxyl radical, respectively. Further, CBD and EABD (100 and 200 mg/kg) were evaluated for antihepatotoxic activity against CCl4 induced hepatotoxicity. The results revealed that CBD and EABD (200 mg/kg p.o.) restored the biochemical parameters, against CCl4 induced hepatotoxicity to the normal values. The altered lipid peroxidation, superoxide dismutase, and catalase levels were also restored by EABD (200 mg/kg p.o.). BDEF was studied for rDNA sequencing by polymerase chain reaction technique. The endophytic bacterium was identified as Bacillus cereus based on its morphological and molecular characterization. Conclusions: CBD and EABD fractions have exhibited antioxidant and hepatoprotective activity.



How to cite this article:
Madagundi SD, Kothli P, Habbu PV, Kulkarni VH. Evaluation of endophytic fractions of Boerhaavia diffusa Linn. roots for hepatoprotective activity in rats.BLDE Univ J Health Sci 2016;1:113-119


How to cite this URL:
Madagundi SD, Kothli P, Habbu PV, Kulkarni VH. Evaluation of endophytic fractions of Boerhaavia diffusa Linn. roots for hepatoprotective activity in rats. BLDE Univ J Health Sci [serial online] 2016 [cited 2019 Dec 5 ];1:113-119
Available from: http://www.bldeujournalhs.in/text.asp?2016/1/2/113/196095


Full Text

In human body, liver is the largest and important organ, involved in metabolism and elimination. The liver performs, maintains, and regulates body temperature. Detoxification, bile secretion, and storage of vitamin along with protein, fat, and carbohydrate metabolism are some of the important functions of the liver.[1] Liver diseases such as hepatitis, hepatosis, and cirrhosis may be acute or chronic. Synthetic or conventional drugs are insufficient for treating liver as they may cause adverse effects.[2] Thus, maintenance of liver is precious for healthy being. The efforts in exploiting drugs from the traditional system of medicine "Ayurveda" has led to betterment in drug discovery technology in India. Raw materials obtained from plants have been used for different drug preparations in medicine.[3],[4] Some of the plants used in traditional Indian medicines such as antioxidants and have a protective effect on DNA cleavage[5] are Moringa oleifera,[6] Eclipta alba,[7] Phyllanthus niruri,[8],[9] Picrorhiza kurroa,[10] and Boerhaavia diffusa[11] which also exhibits hepatoprotective activities. Apart from medicinal plant search for novel, source of phytochemicals is continued for the benefits for human health.

Endophytes can be defined as microbes living in internal tissues of host plants without causing any adverse effect.[12] Endophytes being unstudied are now promising outcome of novel drugs for exploitation in area of medicine. The existence of new technologies and development offers unique exploitation in the evaluation of natural products and can be a major source of drug discovery.[13] Endophytes have different relationships such as mutualistic, symbiotic, trophobiotic, and communalistic.

Endophytes are useful to their host by bearing some novel bioactive compounds for utilization of possible use in agriculture, industry, and medicine.[14] Endophytic microorganisms and their metabolites have been isolated by many workers from different medicinal plants and marine sources with antimicrobial,[15],[16] antibacterial,[17] antioxidant,[18] and immunomodulatory[19] activities.

Examples of metabolites from endophytes are used as anticancer are - the first isolated drug from the endophyte Taxomyces andreanae was taxol obtained from the bark of Taxus brevifolia,[20] camptothecin was isolated from endophytic fungi Entrophospora associated with the inner bark of the plant Nothapodytes foetida[21] and so on. Antifungal metabolites such as Pestalachlorides A and B were obtained from fungal endophyte Pestalotiopsis adusta.[22] Scoparasin B was isolated from the endophytic fungus Eutypella scoparia PSU-D44 inhabiting in the leaves of Garcinia dulcis.[23] Antimicrobial metabolites such as Cytosporone B and C were isolated from a mangrove endophytic fungus, Phomopsis species.[24] An endophytic fungus Paecilomyces variotti Bain. was isolated from the roots of Ocimum sanctum L. showed antioxidant and hepatoprotective activity in CCl4 -induced hepatic damage.[25]

B. diffusa Linn. is a herb belonging to the family Nyctaginaceae. It is widely distributed in the tropical and subtropical regions of India. It is reported that B. diffusa is used as antibacterial,[26] antinociceptive,[26] hepatoprotective,[26] hypoglycemic,[26] antiestrogenic,[26] and antimetastatic[27] activities. The major chemical constituents of B. diffusa extraction of roots are identified as rotenoids known as boeravinones,[28] eupalitin,[28] β-sitosterol, α-2-sitosterol, palmitic acid, ester of b-sitosterol, tetracosanoic, hexacosanoic, stearic acid,[29] arachidic acid, urosilic acid, hentriacontane, b-ecdysone, triacontanol,[30] etc. Hence, the present investigation was proposed for isolation, characterization, and pharmacological evaluation of endophytic fraction (s) from the roots of B. diffusa.

 Materials and Methods



Plant material

Roots of B. diffusa Linn. were collected from Dharwad district, Karnataka, India. Authentication of the plant was done by Dr. G. R. Hegde, Karnatak University, Dharwad (India). A specimen is stored in the herbarium, postgraduate Department of Pharmacognosy (SETCPD/Ph. cog/herb/25/12/2014).

Isolation of endophytes

Roots of B. diffusa Linn. were collected from Dharwad without any damage. The roots were washed thoroughly under running tap water to remove any foreign particle adhering to roots and were dried. Epiphytes were removed by washing the sample for 10 min with 30% ethanol.[31] The roots were surface sterilized with 4% sodium hypochlorite for 30 s, following immersion in alcohol for 2 min. Then, they were rinsed with sterile water for 2-3 times. They were taken to sterilized pestle and mortar, and a suspension was prepared using sterile water. The diluted aliquots were placed on to plates containing nutrient agar medium for isolating bacteria. The plates were incubated at 30°C for 4 days and the organisms enumerated. The predominant isolates of bacteria were picked up and purified. The selected isolates were observed for their morphological characteristics. Bacterial colonies were picked randomly from the dilution plates, checked for purity, and grouped according to colony morphology.[32]

Fermentation and extraction

The endophytic bacteria were fermented in 5000 ml reagent bottle containing 3000 ml of nutrient broth (peptic digest of animal tissue 5 g, beef extract 1 g, yeast extract 2 g, sodium chloride 5 g, and pH at 25°C 7.4 ± 0.2 Hi-media) for 7 days at 37°C under static condition. Bacterial mycelia were homogenized and extracted with equal volumes of chloroform and ethyl acetate, dried by flash evaporation. The yield of the extracts ranged from 100 to 200 mg/L fermented medium. The fractions were screened for free radical scavenging and hepatoprotective activity.[33]

Preliminary phytochemical investigations

Chloroform and ethyl acetate extracts of BDEF were subjected to qualitative analysis to identify the presence of group of active components following established procedures.

Polymerase chain reaction amplification of BDEF

Genomic DNA was prepared using standard methods with a cell lysis performed at 68°C for 30 min. The extracted DNA was dissolved in 2 μl 10X reaction buffer and used as the template for the polymerase chain reaction (PCR) reactions. PCR amplifications were performed in a total volume of 50 μl by mixing 20 mg of the template DNA with 2.5 mM concentration of each deoxynucleotide triphosphate, 1 μM concentration of each primer of pA (5'- AGA GTT TGA TCC TGG CTC AG-3'), and pH (5'-ACG GCTACCTTGTTACGACTT-3') and 2.5 U/μl of Taq DNA polymerase in 10X Reaction Buffer (Genei, Bengaluru). The amplification was carried out in a Master cycler® Thermocycler (DNA-AMP Bhat Biotech) using the following program. Initial denaturation was carried out at 94°C for 2 min followed by forty cycles of denaturation at 94°C for a minute, annealing at 55°C for a minute, and extension at 72°C for a minute. Final extension was carried out at 72°C for 10 min. The ~1500 bp PCR product was purified to remove unincorporated dNTPS and primers before sequencing using PCR purification kit (Geneasy PCR Product Purification Kit Bangalore, India.). Both strands of the 16 s RNA region amplified by PCR were sequenced by automated DNA sequence −3037xl DNA analyzer from Applied Biosystems using BigDye® Terminator version 3.1 cycle sequencing Kit (Applied Biosystems). Cluster analysis was performed by an unweighted paired-group method for the arithmetic average. Neighbor-joining method (NJM) was used to estimate the phylogram based on the idea of parsimony, and the tree is usually close to the true phylogenetic tree.[33],[34] Sequence data were aligned, and dendrograms were generated using sequence analysis software version 5.2 from Applied Biosystems. The sequences obtained for plus and minus strands were aligned using appropriate software before performing bioinformatics. Sequences were compared to the nonredundant NCBI database using BLASTN, with the default settings used to find sequences closest to each other. The expected value and e values were noted for the most similar sequences. Ten similar neighbors were aligned using CLUSTAL W2. The multiple alignment file thus obtained was then used to create a Phylogram using the MEGA5 software.[35],[36]

In vitro free radical scavenging activity

2,2-diphenyl-1-picrylhydrazyl assay

The scavenging effect of EABD (20-500 μg/ml) against 2,2-diphenyl-1-picrylhydrazyl (DPPH) stable radical was determined using ascorbic acid (ASC, 1-5 μg/ml) as standard. The percentage of DPPH scavenging against ASC gave the standard curve.[37]

Hydroxyl radical scavenging assay

The hydroxyl radical scavenging activity of EABD (20-500 μg/ml) was measured by degradation of deoxy-D-ribose method. Standard drug mannitol was used at different concentrations (0.5-5.0 μg/ml) for comparison. The percentage inhibition of × OH scavenging against mannitol gave the standard curve.[37]

Reducing power assay

As per the method reported,[38] reducing power of EABD (25-500 μg/ml) was determined. The antioxidant compound formed a colored complex with potassium ferricyanide, trichloroacetic acid, and ferric chloride, the absorbance measured at 700 nm. Ascorbic acid was used as standard and phosphate buffer (pH 6.6) was used as blank solution. The absorbance of the final reaction mixture was taken and expressed as mean ± standard error of mean (SEM).

Animals

Albino Wister rats weighing 150-200 g were taken for the present study. The animals were maintained under temperature (23 ± 2°C), humidity (50% ±5%), and 12 h light-dark cycles. All the animals were acclimatized for 7 days before the study. The animals were divided into experimental and control groups and housed individually in sanitized polypropylene cages containing sterile paddy husk as bedding. They had free access to standard pellets as basal diet and water ad libitum. All the studies conducted were approved by the Institutional Animal Ethical Committee of SET's College of Pharmacy, Dharwad, Karnataka (Reg. No. 112/PO/Re/1999/CPCSEA dated June 02, 2015), according to Committee for the Purpose of Control and Supervision of Experiments on Animals, Government of India guidelines.

CCl4 -induced hepatoprotective activity

Animals divided into five groups of six animals in each group (n = 6).

Group 1: Normal control treated with 0.9% NaCl (2 ml/kg day p.o.)Group 2: Treatment with CCl4 ( 2 ml/kg i.p. in olive oil)Group 3: Treatment with CBD (100 mg/kg p.o.) +CCl4Group 4: Treatment with CBD (200 mg/kg p.o.) + CCl4Group 5: Treatment with EABD (100 mg/kg p.o.) + CCl4Group 6: Treatment with EABD (200 mg/kg p.o.) + CCl4Group 7: Treatment with standard silymarin (25 mg/kg p.o.) + CCl4 .

As reported earlier, the animals were treated with drugs as mentioned above for 5 days.[39] CCl4 ( 2 ml/kg i.p.) in olive oil was administered orally to all groups other than Group 1 on 2nd day and 3rd day. The standard silymarin (100 mg/kg p.o.) was administered to Group 7 once in a day. During this period, rats were maintained under normal diet and water ad libitum. Animals were sacrificed on the 6th day. Blood collected by retro-orbital bleeding under mild ether anesthesia, centrifuged at 3000 rpm for 15 min, and serum collected was analyzed for biochemical estimations. The dissected liver was then placed in 10% formalin solution for histopathological studies. The liver homogenate prepared was used to determine endogenous enzyme levels.

Endogenous enzymatic and nonenzymatic antioxidant levels

Tissue preparation

Animals were sacrificed, the whole liver was perfused in situ with ice-cold saline, dissected out, blotted dry, and immediately weighed. A 10% liver homogenate was prepared separately with ice-cold saline ethylenediaminetetraacetic acid (EDTA) using Teflon-glass homogenizer (Yamato LSG LH-21, Japan). It was then centrifuged at 10,000 rpm for 10 min and the pellet discarded. The supernatant was again centrifuged at 20,000 rpm for 1 h at 4°C. The liver supernatant obtained was used for the estimation of lipid peroxidation (LPO), superoxide dismutase (SOD), and catalase (CAT).

Lipid peroxidation

Thiobarbituric acid reactive substances (TBARS) in the liver homogenate were estimated by a standard protocol. Briefly, the homogenate was incubated with 15% trichloroacetic acid, 0.375% thiobarbituric acid, and 5N HCl at 95°C for 15 min; the mixture was cooled, centrifuged and the absorbance of the supernatant measured at 532 nm against appropriate blank. The amount of LPO was determined by the formula € = 1.56 × 105M - 1 cm and expressed as TBARS (μ moles) per g of tissue.[40]

Superoxide dismutase assay

Liver homogenate (0.5 ml) was taken, and 1 ml of 50 mM sodium carbonate, 0.4 ml of 24 μm nitroblue tetrazolium (NBT), and 0.2 ml of 0.1 mM EDTA were added. The reaction was initiated by adding 0.4 ml of 1 mM hydroxylamine hydrochloride. Zero time absorbance was taken at 560 nm followed by recording the absorbance after 5 min at 25°C. The control was simultaneously run without liver homogenate. Units of SOD activity were expressed as the amount of enzyme required to inhibit the reduction of NBT by 50%. The specific activity was expressed in terms of units per mg of proteins.[40]

Catalase

As per the previously published method, CAT activity was determined spectrophotometrically. To 1.95 ml of 10 mM H2O2 in 60 mM phosphate buffer (pH = 7.0), 0.05 ml of the liver homogenate was added, and rate of degradation of H2O2 was followed at 240 nm/min. CAT content in terms of U/mg of protein was estimated from the rate of decomposition of H2O2 using the formula:

k = 2.303/Δt × log (A1/A2) s − 1

A unit of CAT is defined as the quantity which decomposes 1.0 μmole of H2O2 per min at pH = 7.0 at 25°C, while H2O2 concentration falls from 10.3 to 9.2 mM.[40]

Histopathological studies

At the end of the experiment, animals were sacrificed by administration of high dose of pentobarbitone sodium (40 mg/kg body weight i.p.). Liver of individual animal was excised quickly, fixed in 10% buffered neutral formalin and in bovine solution. They were further processed for paraffin embedding following standard microtechnique. Sections of liver stained with alum-hematoxylin and eosin were observed photo microscopically for histopathological changes.[41]

Statistical analysis

The data were expressed as mean ± SEM statistical comparisons were performed in Origin 6.0 and by one-way ANOVA followed by Turkey's t-test using Graph Pad Prism version 5.0, USA.

 Results



B. diffusa L. roots showed various bacteria. One purely endophytic bacterium was isolated and designated as BDEF. The yield of CBD and EABD was found to be 214 and 254 mg/L of fermented medium, respectively. Preliminary phytochemical investigations of CBD and EABD revealed the presence of carbohydrates, proteins, alkaloids, and tannins as important constituents.

Identification of BDEF using 16s RNA sequential analysis

The 16s RNA genes from BDEF showed 99% to 100% similarity with bacillus. Based on colony morphology and by PCR analysis, BDEF was identified as Bacillus cereus strain [Figure 1].{Figure 1}

In vitro free radical scavenging activity

Fraction of EABD showed concentration-dependent of DPPH (100 mM) under static conditions. EABD revealed scavenging activity with IC50 value of 28.86 μg/ml against DPPH whereas standard ASC showed IC50 value of 2.99 μg/ml under similar conditions [Figure 2]a.{Figure 2}

The degradation of Deoxy-D-ribose yielding MDA was measured as TBARS. EABD was able to scavenge the hydroxyl radicals generated through Fenton reaction. EABD fraction exhibited concentration-dependent OH scavenging with IC50 value 82.78 μg/ml. Standard mannitol showed an IC50 value of 4.61 μg/ml under the same experimental conditions [Figure 2]b. In reducing power assay, the concentration of EABD increased the percentage absorbance, so the fraction shows reducing power [Figure 2]c.

Acute toxicity (LD50) studies

Acute toxicity studies were carried out according to OECD guidelines (Up and Down method). No deaths were observed at 2000 mg/kg body weight for CBD and EABD. The doses were selected as 100 mg/kg and 200 mg/kg body weight to assess the hepatoprotective activity for CBD and EABD, respectively.

Hepatoprotective activity of CBD and EABD in CCl4 -induced hepatotoxicity

Determination of biochemical parameters

The levels of serum glutamate pyruvate transaminase (SGPT), serum glutamic oxaloacetic transaminase (SGOT), serum alkaline phosphatase (SALP), total and direct bilirubin, and triglyceride were significantly increased as compared to normal control on administration of CCl4 ( 2 ml/kg i.p.). CBD and EABD (200 mg/kg p.o.) reversed the elevated biochemical parameters as compared to CCl4 -treated group (P < 0.001). The results are shown in [Table 1].{Table 1}

Measurement of enzymatic and nonenzymatic antioxidant levels

Increase in LPO in the CCl4 -treated group was observed as TBARS (P < 0.001) compared to control. CBD and EABD (200 mg/kg.) reversed the altered levels of LPO (P < 0.001) significantly which was compared with silymarin. Rats treated with CCl4 (2 ml/kg i.p.) showed a marked depletion in CAT (P < 0.001) and SOD levels. Groups treated with CBD and EABD (200 mg/kg) each, respectively, have shown rise in CAT and SOD activity significantly (***P < 0.001) [Table 2].{Table 2}

Histopathological studies in CCl4 -induced hepatotoxicity

The normal liver showed hepatic globular structure, no inflammation of central vein, portal tract, and Kupffer cells looked normal [Figure 3]a. In a CCl4 -treated group, hepatic cells showed extensive fatty changes and ballooning degradation, broad infiltration of Kupffer cells around the central vein [Figure 3]b. In CBD (100 mg/kg and 200 mg/kg)-treated group, cells showed slight centrilobular necrosis, no fatty change, and sinusoids with mild recovery. Central vein, sinus showed mild congestion [Figure 3]c and d. In EABD (100 mg/kg and 200 mg/kg)-treated group, cells did show moderate change in central vein and sinus congestion and the presence of ballooning of hepatocytes and spotty necrosis. The presence of focal hemorrhage and moderate to mild changes in centrilobular degeneration, portal triad, and inflammation was also observed [Figure 3]e and f. In silymarin (100 mg/kg p.o.)-treated group, the hepatic globular architecture was normal. Central vein and sinus showed mild congestion. However, mild centrilobular degeneration and portal triad were present. There were no inflammatory cells and fibrosis [Figure 3]g.{Figure 3}

 Discussion



Endophytic bacteria live inside the tissues of plant without causing any symptom or disease. In the study, an attempt was made to evaluate antioxidant and hepatoprotective activity of CBD and EABD fractions from B. diffusa L. root against CCl4 -induced hepatotoxicity in rats.

Phytochemical studies of CBD and EABD showed the presence of carbohydrate, alkaloids, flavonoids, proteins, and tannins. Further, CBD and EABD were studied for in vitro free radical scavenging activities, namely DPPH assay, hydroxyl free radical, and reducing power assay. Of the two fractions, EABD showed significant IC50 values and were selected for in vivo studies. CBD and EABD were subjected to acute toxicity studies (up and down method) according to OECD guidelines. The two fractions showed neither toxicity nor behavioral changes in mice. Thus, 100 mg/kg and 200 mg/kg doses were selected for activity.

The commonly used hepatotoxic chemical is carbon tetrachloride in the study of liver disorder. The hepatotoxic effects of CCl4 are largely due to its active metabolite, trichloromethyl radical. The development of antioxidant system in human being is for counteracting reactive oxygen species (ROS) and to reduce the damage. CCl4 -induced hepatotoxicity is followed by its free radical metabolites such as CCl4 and CCl3 COO as their interaction is with unsaturated lipid membrane producing cell damage and LPO of cellular membranes.[39] In the presence of oxygen, the free radicals leading to auto-oxidation of fatty acids change the morphological and functional features.[42] Liver function can be evaluated by activities of SGOT, SGPT, and SALP. These enzymes are present in high concentration acting as hepatotoxic. Thus, this study activities of SGOT, SGPT, and SALP increased in CCl4 -induced liver damage. The SGOT and SGPT released from liver in the blood increases to higher level due to necrosis of hepatic cells.[43]

LPO is mechanism where cell get injured by the plant which determines oxidative stress in cells and tissues. The unstable lipid peroxides are formed which deteriorate to form complex structures such as carbonyl compounds. On decomposition, these peroxides produce malondialdehyde, indicator of LPO. Antioxidant enzymes as SOD and CAT have a defensive mechanism in damage by oxidative stress. They act by removing the reactive oxygen radical such as superoxide and hydrogen peroxide thus preventing the formation of more reactive hydroxyl radical. The results indicated that CBD and EABD (100 and 200 mg/kg) decreased the elevated enzyme levels in CCl4 -induced liver damage. These two fractions either enhanced or maintained the activity of enzymes involved in combating ROS.[44]

It can be concluded from the study that CBD and EABD protect the liver from oxidative damage and could be used as an effective protector in CCl4 -induced damage. In future, the secondary metabolites can be isolated responsible for the activity and its mode of action be studied.

The endophytic bacterium was identified as B. cereus strain by PCR method. B. cereus is Gram-positive, found in food and soil, rod-shaped, motile, beta hemolytic bacterium. Some of these strains are dangerous to humans causing foodborne diseases such as nausea, vomiting, and diarrhea, while others can be used as probiotics for animals. It causes "fried rice syndrome." B. cereus bacteria are facultative anaerobes and can produce protective endospores, cereolysin, and phospholipase C which are its virulence factors.[45],[46]

This investigation has numerous opportunities for further research in pharmacological activities referring to endophytes from plants in the development of medicines for prevention and treatment of acute or chronic diseases.

 Conclusions



CBD and EABD fractions have exhibited antioxidant and hepatoprotective activity. This investigation has numerous opportunities for further research in pharmacological activities referring to endophytes from plants in the development of medicines for prevention and treatment of acute or chronic diseases.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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