|Year : 2012 | Volume
| Issue : 2 | Page : 71-77
Curative properties of Buchanania lanzan: As evaluated by its anti-oxidant, anti-inflammatory and DNA protective properties
Sekhar Shailasree, Karmakar Ruma, Harishchandra S Prakash
Department of Studies in Biotechnology, University of Mysore, Mysore, Karnataka, India
|Date of Web Publication||20-Oct-2012|
Harishchandra S Prakash
Department of Studies in Biotechnology, University of Mysore, Mysore - 570 006, Karnataka
Background: Parts of B. lanzan is reported to have several medicinal properties. Methods: Buchanania lanzan bark was assessed for its anti-oxidant, anti-inflammatory, and DNA protective properties. Results: Soxhlet solvent extracts exhibited DPPH scavenging capacity to a varied extent. Methanolic extract could scavenge ABTS radicals with IC 50 of 0.25 mg/ml. The anti-inflammatory properties were elucidated by its capacity to inhibit 15-lipoxygenase and human cyclooxygenase-2. As a measure of anti-ageing effect, anti-hyaluronidase and anti-elastase activity was measured. The extract significantly inhibited both 15-LOX and human COX-2 in a dose-dependent manner. The extract abolished elastase activity and inhibited hyaluronidase as observed in zymogram by substrate-gel assay. In addition, the methanolic extract could prevent damage to DNA from the hydroxyl radicals produced during Fenton reaction. Further, an absence of hemolytic activity for this extract suggests the non-toxic nature. Conclusion: Studies on unexplored medicinal plants may lead to identification of potential drug candidates.
Keywords: 15- Lipoxygenase, DNA nicking assay, elastase, human cyclooxygenase-2, hyaluronidase
|How to cite this article:|
Shailasree S, Ruma K, Prakash HS. Curative properties of Buchanania lanzan: As evaluated by its anti-oxidant, anti-inflammatory and DNA protective properties. J Nat Pharm 2012;3:71-7
|How to cite this URL:|
Shailasree S, Ruma K, Prakash HS. Curative properties of Buchanania lanzan: As evaluated by its anti-oxidant, anti-inflammatory and DNA protective properties. J Nat Pharm [serial online] 2012 [cited 2013 Dec 9];3:71-7. Available from: http://www.jnatpharm.org/text.asp?2012/3/2/71/102748
| Introduction|| |
The skin is generally considered as protective barrier against the physical and biological threats from the external environment. Loss of function of the skin results in an itchy inflammatory atopic eczema and other associated diseases. The pathophysiology of an eczema involves systemic, cutaneous immune and epidermal dysfunction.  In normal physiological conditions, skin generates reactive oxygen species (ROS), such as superoxide anion (O 2 - ) and H 2 O 2 in small quantities to maintain homeostasis. But, the levels of ROS augment in skin when exposed to xenobiotics and UV rays, leadings to lipid peroxidation. ROS generation triggers the NFκB and upregulate the pro-inflammatory genes. In addition, augmented oxidative stress activates phospholipase A2-mediated arachidonic acid (AA) formation. AA is oxidatively metabolized to produce pro-oxidant metabolites classified as eicosanoids [prostaglandins (PGE2) and hydroxyeicosatetraenoic acids] by cyclooxygenase (COX) and lipoxygenases (LOX). ,
Skin elasticity is due to the elastin fiber network with a primary role in providing an elastic stretch and recoil to the skin.  Elastase, a member of the chymotrypsin family of proteases, is responsible primarily for the breakdown of elastin, an important protein found within the extracellular membrane (ECM). , A proportional relationship between degeneration of elastic fibers and wrinkle formation leading to loss of skin elasticity/ function put forth the study of importance of inhibition of elastases. Other enzymes of interest for reinforcement of skin function include hyaluronidase, an endoglycosidase hydrolyze hyaluronic acid (HA). HA is a megadalton, non-sulfated glycosaminoglycan participate in different pathophysiological conditions ranging from embryogenesis to aging. The high molecular weight HA is considered to be anti-inflammatory, used as an anti-adhesion and anti-scar drug in general surgery. In contrast, the enzyme or free radical-mediated degradative products of HA are involved in scar formation.  Taken together, in the arena of skin disorders, hydrolytic enzymes significantly induce inflammation and their inhibitors bring down the cutaneous inflammation.
In recent years, non-steroidal anti-inflammatory drugs (NSAIDs) are being used to treat skin disorders, but the drugs carry adverse effects.  Therefore, medical practitioners and researchers are in continuous quest for natural medicine with no/less side effects. Ayurveda, a traditional Indian system of medicine, has about 25% resource derived from plants and more than 80% people are dependent on it. World-wide research on the effectiveness of herbal medicine is on rise for chronic diseases putting forth scientific evidence for traditional beliefs. Buchanania lanzan, Spr. (Family: Anacardiaceae) is commonly known as Chironji. All parts of the tree is used for treating ailments viz., seed oil to reduce granular swelling; kernel to relieve an itch and prickly heat, bark gum for treating diarrhea and intercostals pain, leaves as anti-diarrheal, anti-rheumatism, wound healing and as anti-ophidian. , On this background, for the first time, we made an attempt to validate and provide a scientific platform for this medicinal plant.
| Materials and Methods|| |
Chemicals and reagents: Linoleic acid, 1,1-Diphenyl-2-picrylhydrazyl (DPPH), 15-lipoxigenase (soybean), quercetin and 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) were purchased from Sigma-Aldrich (St. Louis, MO). COX-2 inhibition kit was obtained from Cayman Chemicals, Ann Arbor, USA. Ascorbic acid (AA) and butylated hydroxyl toluene (BHT) were purchased from HiMedia (India). Plasmid (pBR322) was obtained from Merck Biosciences, Bangalore (India).
Plants: The Buchanania lanzan tree barks were collected from Kigga region of Western Ghats in Chikmagalore District of Karnataka State. Herbarium specimen has been deposited at the Department of Studies in Biotechnology (B. lanzan # IOE LP0004).
Plant extract preparation: Fresh bark pieces were thoroughly washed and dried under shade. They were ground to a coarse powder. Ground powder (100 g) was extracted sequentially using 500 mL of non-polar and polar solvents in increasing polarity hexane < chloroform < methanol < water in Soxhlet apparatus. The solvent was evaporated to yield dry extracts in SpeedVac (Savant SPD 2010, Thermo Scientific). The extracts were stored under dark at room temperature (RT).
Free Radical-Scavenging Activity
DPPH assay: The traditional 2,2-diphenyl-1-picrylhydrazyl free radical (DPPH) method was used in this study.  The DPPH radical (DPPH . ) solution (300 μM) was prepared in methanol. The Soxhlet extracts were assayed at different concentrations. To DPPH solution (95 μL; absorbance of 0.68 ± 0.005 at 517 nm) 5 μL of samples (extracts and reference compounds) were added. Scavenging of DPPH . was carried out at RT in the dark for 30 min, and the reduction in absorbance was recorded at 517 nm using Spectra Max 340PC (Molecular Devices). The results were expressed as DPPH scavenging activity. The values are mean ± SD of 3 independent experiments.
ABTS assay: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) scavenging activity of methanolic extract and reference standards viz., AA, quercetin and BHT at different concentrations was measured using a UV-vis spectrophotometer (Beckman Coulter, DU 730 Life Sciences) as per the Re et al. The results were expressed in terms of ABTS .+ scavenging activity. The values are mean ± SD of 3 independent experiments.
Phytochemical analysis: Phytochemical screening was carried out as per reported procedure. 
Hemolytic activity assay: Indirect hemolytic activity of methanolic extract was determined according to the method of Boman and Kaletta  using packed human erythrocytes (blood group B). It was washed several times with phosphate buffered saline (PBS) (0.15 mM, pH 7.2) and was sedimented by gentle centrifugation (1000 X g) until a clear supernatant was obtained. For the assay, the stock was prepared by mixing packed erythrocytes (1 ml), egg yolk (1 ml), and PBS (8 ml). 1 ml of suspension from stock was incubated with various concentrations of extract (0 - 1000 μg) for 30 min at 37°C. The reaction was terminated by adding 10 ml ice cold PBS and centrifuged (800 x g) at 4°C. The amount of hemoglobin released in the supernatant was measured at 540 nm. Erythrocytes stock (1 ml) with 10 ml ice cold PBS is considered as 0% lysis and the lysis (%) with water as 100% lysis.
15-Lipoxygenase (15- LOX) inhibition assay: A spectrophotometric assay for determination of soybean 15-LOX (5 μg) activity with 0.2 μM linoleic acid (substrate) in buffer [(0.2M borate buffer (pH 9.0)] was carried out.  The increase in absorbance was monitored at 234 nm using UV-Vis spectrophotometer (Beckman Coulter, DU 730 Life Sciences). Inhibition experiments were run by measuring the loss of 15-LOX activity in presence of various concentrations of methanolic extracts and quercetin. Values of hydroperoxide content and LOX activity were calculated as per Rackova et al.  IC 50 , indicating the concentration required to inhibit 50% LOX activity, was also calculated.
Human Cycloxygenase (COX)-2 Inhibition: COX-2 inhibition was measured using a colorimetric human COX-2 inhibitor screening assay kit (Cayman, Ann Arbor, USA). The methanolic extract (10, 25, 50 μg) were used for inhibition studies as per manufacturer's protocol. The absorbance at 415 nm was read by using a microtitre plate reader Varioskan Flash with SkanIt Software 2.4.3 RE.
Elastase inhibition assay: The proteolytic activity was determined according to the method of Feinstein et al.,  using SApNA (N-succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide) as the substrate. The reaction mixture containing 0.5 ml (60 μg) of the enzyme pre-incubated with Tris-HCl buffer (50 mM, pH 8.2, with 20 mM CaCl 2 ) for 15 min at 37°C. The enzyme activity was initiated by the addition of substrate (1.25 ml, Tris-HCl buffer) dissolved in DMSO. It was incubated for 10 min at 37°C and terminated by adding 0.25 ml of 30% acetic acid. The p-nitroanilide liberated was measured at 410 nm. For inhibition studies, enzyme (60 μg) was incubated with different concentrations of methanolic extract (0.6, 1.5, 3 mg with ratio 1:10; 1:25; 1:50 w/w enzyme: extract) for 10 min at 37°C. Further increase in concentration of extracts results in color interference.
Hyaluronidase inhibition (Zymogram assay): Substrate gel assay for hyaluronidase was performed as described  with slight modifications. Briefly, hyaluronic acid was incorporated at a final concentration of 0.17 mg/ml into the SDS-polyacrylamide gel matrix (10%). Ovine testicular hyaluronidase (OTH; 3U; 2 μg) was preincubated with different concentrations of methanolic extract for 10 min at 37°C. Further, samples were prepared under non-reducing condition and separated. After electrophoresis, the gel was washed thrice in 50 ml of 2.5% Triton X-100 in sodium formate buffer pH 5.5 with 0.15 M sodium chloride for 1h facilitating replacement of SDS in the gel by Triton X-100. The gel was equilibrated in 0.1 M sodium formate buffer pH 5.5 containing 0.15 M sodium chloride for 20h at 37°C. Later, the gel was washed and stained with Alcian blue stain (0.5%) and documented using XR+ Molecular Imager Gel documentation system (Bio Rad, USA).
DNA protection studies: A DNA nicking assay was performed by using supercoiled plasmid pBR 322. Controls consisted of plasmid DNA subjected to reaction with Fenton reagent for 30 min under similar conditions. DNA protection effects against Fenton reagent was assessed by incubating the methanolic extract (10, 25, 50 mg) with plasmid DNA (0.5 μg) for 30 min at 37 o C and further to Fenton reagent for 30 min. The samples were electrophoresed using 1% agarose gel. The results were documented using XR+ Molecular Imager Gel documentation system (Bio Rad, USA).
Statistical analysis: All determinations including anti-oxidant capacity, anti-microbial activity, DNA nicking assay and anti-inflammatory properties were conducted in triplicate. The reported value for each sample was calculated as the mean.
| Results|| |
In the present study, the bark of the medicinal plant Buchanania lanzan was analyzed for potential biological activities. The bark was subjected to solvent fractionation using Soxhlet apparatus with solvent identified based on increasing polarity viz., hexane < chloroform < methanol < water. Anti-oxidant property being considered as an important marker for biological activity, the extracts were first assessed for DPPH radical scavenging activities. The Soxhlet extracts viz., hexane, chloroform and water exhibited limited capacity to scavenge these radicals [Figure 1]. Solvent controls (5 μL) were also maintained alongside during the assay. They showed no DPPH scavenging capacity (results not shown). Methanolic extract exhibited potent DPPH radical scavenging activity in a dose-dependent manner. At 5 mg/ml, the methanolic extract exhibited 95.92 % DPPH . scavenging capacity. This extract was used for further studies. ABTS .+ scavenging, also a recommended method for detecting the anti-oxidant potential for plant extracts, was measured at 734 nm (long wavelength) eliminates color interference by plant extracts (Cai et al. 2004). The methanolic extract subjected to ABTS .+ scavenging recorded IC 50 of 0.25 ± 53 mg.ml -1 [Table 1]. Methanol alone did not exhibit any capacity to scavenge ABTS (results not shown). The phytochemical examination of methanolic extract identified the presence of glycosides, flavonoids, phenolics, saponins, steroids, and tannins. The hemolytic assay carried out recorded non-toxic nature of this extract. Further studies were directed towards assessing the extracts capacity for anti-inflammatory property.
|Figure 1: DPPH scavenging activity of solvent soxhlet extracts of Buchanania lanzan bark. Different concentrations of Soxhlet extracts viz.,0.1, 0.5, 1 and 5 mg/mL were incubated with DPPH. solution for 30 min at RT. The results were expressed as DPPH. scavenging activity(%). The values are mean ± SD of 3 independent experiments|
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In the present study, the 15-lipoxygenase (LOX) activity was monitored as an increase in the absorbance at 234 nm indicating the formation of hydroperoxylinoleic acid. The methanolic extract inhibited LOX in a dose-dependent manner [Table 2]. Amongst the tested concentration, complete 15-LOX inhibition was observed at 200 μg. The IC 50 value was found to be 32 μg. Further, the extract was tested for its capacity to inhibit human cyclooxygenase-2 (COX-2). It inhibited the human COX-2 in a dose-dependent fashion. At 50 μg concentration, 73.58% of inhibition was observed [Table 2]. The IC 50 value was found to be 23.45 μg. Methanol alone was used as solvent control did not exhibit any capacity to inhibit 15-LOX or human COX-2 (results not shown).
The methanolic extract of B. lanzan was assessed for its capacity to inhibit elastase and hyaluronidase enzymes. The extract inhibited the elastase activity in a dose-dependent manner. At an enzyme: extract ratio 1:50, 50% inhibition was observed [Figure 2]. Further, the extract also inhibited the activity of ovine testicular hyaluronidase (OTH) in a concentration-dependent manner as assessed by the in-gel enzyme activity measurement. A clear translucent activity band in substrate gel assay suggests the enzyme activity. However, an absence of clear translucent activity band in a substrate gel against a blue background clearly suggests the inhibition. At 100 μg concentration, complete inhibition of OTH was observed [Figure 3].
|Figure 2: Elastase inhibition by Buchanania lanzan methanolic extract. Elastase was incubated with different concentrations of methanolic extracts in ratio (1:10; 1:25; 1:50 w/w) for 10 min at 37°C. The inhibition was measure by addition of SApNA (N-succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide as the substrate. The values are mean ± SD of 3 independent experiments|
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|Figure 3: Ovine testicular hyaluronidase-A zymogram showing the inhibition of hyaluronidase activity by Buchanania lanzan methanolic extract. Lane 1- Ovine testicular hyaluronidase (OTH) alone (3U), lane 2 - OTH + BE (10 μg), lane 3 - OTH + BE (50 μg), lane 4 - OTH + BE (100 μg), lane 5 - BE alone (10 μg), lane 6 - BE alone (100 μg)|
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In order to investigate the efficacy of B. lanzan methanolic extract against DNA damage, plasmid DNA (pBR322) was exposed to Fenton reaction for 30 min at 37 o C. Fenton reaction caused a change (super shift) in native double stranded DNA band (Form I) to single-stranded, nicked DNA (Form II) confirmed in agarose gel electrophoresis [Figure 4]. In contrast, DNA preincubated with different concentration viz., 10, 25, 50 μg of the extract prevented the scission when subjected to Fenton reagent under similar conditions as above.
|Figure 4: Free radical (.OH) scavenging capacity of Buchanania lanzan methanolic extract as assessed on plasmid pBR322 DNA treated by Fenton reagent. Lane 1: pBR322 (native plasmid DNA), Lane 2: pBR322 DNA + Fenton reagent, Lane 3: pBR322 + methanolic extract (10 μg) + Fenton reagent, Lane 4: pBR322 + methanolic extract (25 μg) + Fenton reagent, Lane 5: pBR322 + methanolic extract (50 μg) + Fenton reagent|
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| Discussion|| |
Medicinal plant parts (roots, leaves, branches/stems, barks, flowers, and fruits) are commonly rich in secondary metabolites due to their property to quench free radicals and are a major source of new drugs. Besides the investigation of single compounds, there is still a growing interest in the application of standardized extracts, complex phytochemical mixtures with a well-defined content of the bioactive constituents.  The aim of this study was to investigate the anti-oxidant and protective effects of Buchanania lanzan. The in vitro assays were chosen to reflect the types of damage caused by radical scavenging in skin-inflammation and to find activity by plant extracts, which might counteract this damage. Hence, the anti-lipoxygenase (LOX), human cyclooxygenase-2 (COX-2), anti-elastase and anti-hyaluronidase activities of the extracts were reported along with the anti-oxidant efficacies.
Amongst the different Soxhlet solvent extracts of the bark, the methanolic extract exhibited potent DPPH and ABTS radical scavenging activity. This extract could inhibit 15-LOX with IC 50 of 32 μg. LOX inhibition was used to evaluate anti-inflammatory activity of a few medicinal plants used in Limousin country. Filipendula ulmaria (Meadowsweet) recorded LOX inhibition with IC 50 of 60μg/ml and Urtica dioica (Nettle) methanolic extract inhibited LOX with IC 50 of 348 μg/ml.  In another study, 8 methanolic extract out of 18 undomesticated plants of South Africa showed significant inhibition of 5-lipoxygenase (5-LOX) activity. Bidens pilosa extract exhibited IC 50 of 21.8 μg/ml and Emex australis extract recorded IC 50 of 81.4 μg/ml for LOX inhibition. LOXs are sensitive to anti-oxidants as they inhibit lipid hydroperoxide formation due to scavenging of lipidoxy or lipidperoxy-radicals. This could lead to less availability of lipid hydroperoxide substrate required for LOX catalysis.  The methanolic extracts of B. lanzan bark exhibited a significant human COX-2 inhibitory activity in vitro, thereby inhibiting PGE2 production with IC50 of 23.45 μg. Several reports on "dual inhibitors" inhibiting COX-2 and 5-LOX for effective management of metabolic processes underlying osteoarthritis with a balanced arachidonic acid metabolism in the body has been highlighted. ,,,
In the terms of anti-ageing, finding inhibitors of elastase finds increasing interest to prevent loss of skin elasticity and thus skin sagging. Many of the worldwide cultures have relied historically on medicinal plants for treatment of wounds or skin disorders. Healing of wounds and burn injuries by Aloe vera, anti-fungal, anti-viral, anti-bacterial, and acaricidal activity against skin infections by tea tree (Melaleuca alternifolia) oil could be cited as beneficial aspects of medicinal plants.  In the present study, the methanolic extract of B. lanzan was assessed for its capacity to inhibit elastase and hyaluronidase enzymes. The extract inhibited the elastase activity in a dose-dependent manner. Further, the extract also inhibited the hyaluronolytic activity of OTH in a concentration-dependent manner as assessed by the in-gel enzyme activity measurement. Similar studies have reported the use of in vitro enzyme inhibition studies to assess the potential of a plant for skin-related problems. A panel of 23 plant extracts was screened for anti-ageing properties. Very high anti-elastase activities were exhibited by white tea and cleavers extracts with 89% and 57.9% inhibition of enzyme activity, respectively. Burdock root (50.9%), bladderwrack (50.2%), anise (31.9%), and angelica (31.6%) also exhibited good anti-elastase activity. Moderate anti-elastase activities by rose aqueous (24.15%), rose tincture (22.08%), pomegranate (14.64%), and green tea (9.99%) was recorded.  In Okinawa, the aerial roots and bark of Ficus microcarpa have been used as folk herbs for perspiration, alleviating fever, and relieving pain. The methanol extract of its bark showed high anti-oxidant and potential inhibitory activity against hyaluronidase.  Ethyl acetate fraction of Garcinia indica at concentrations as low as 25 μg mL-1 showed significant hyaluronidase inhibition while water fraction proved to be good elastase and hyaluronidase inhibitor at 90 μg mL-1 . 
The ROS generated by ionizing or by UV-radiation and mutagens may interact with the DNA, leading to the base modification and potentially serious consequences for the cell. Numerous reports evidenced that ROS play a vital role in aging, skin damage, and neurodegenerative diseases.  In order to investigate the efficacy of B. lanzan methanolic extract against DNA damage, plasmid DNA (pBR322) was exposed to Fenton reaction for 30 min at 37 o C. Fenton reaction caused a change (super shift) in native double-stranded DNA band (Form I) to single-stranded, nicked DNA (Form II). In contrast, DNA preincubated with the extract prevented the scission. An absence or reduction in Form II DNA indicated a notable protection offered by the extract. Fe 2+ , the important catalyst generating free radical-driven ROS by Fenton reaction, has the potential to directly interact with DNA bases and damage DNA.  Most anti-cancer agents are believed to act mainly by quenching the free radicals or by direct interaction with DNA.  The DNA nicking assay suggests the capacity of the extract to quench the free radicals harmful for DNA.
Taken together, it could be speculated that B. lanzan has wide spectrum of therapeutic values ranging from anti-oxidant, anti-inflammatory to DNA protection. It could find application in skin-related products. This it would be reasonable to indicate that some of these unexplored medicinal plants viz., B. lanzan and their products may lead to the development of potential drugs.
| Acknowledgements|| |
The authors acknowledge the recognition of University of Mysore as an Institution of Excellence by the Government of India and financial support from the Ministry of Human Resource Development and the University Grants Commission, New Delhi, India. SS acknowledge Senior Research Associateship (Scientists' Pool Scheme) from Council of Scientific and Industrial Research, New Delhi, India. Authors acknowledge Dr. Girish, Department of Studies in Biochemistry for elastase and hyaluronidase inhibition assay.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]