Ayurveda

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R esearch study Bacopa monniera extract plays a signicantroleinlipofuscin scavenging in cultured human vascular endothelial cells
SI Almelkar1*, AM Patwardhan2

Abstract
Introduction We investigated the generation of lipofuscin granules in human umbilical vein endothelial cells that were exposed to hydrogen peroxide (H2O2, 100 M) for 1 h. Lipofuscin granule scavenging was studied using the Bacopa monniera (Brahmi) extract (100 g) for 2 h, 4 h and 6 h exposures. We used an endothelial cell culture model to investigate the antioxidant effects of the B. monniera extract on lipofuscin granules scavenging in cultured human umbilical vein endothelial cells. Materials and methods To understand the protective role of the B. monniera extract in postoxidative stressed human umbilical vein endothelial cells, an experiment was set-up with the following exposure regimen: pre-B. monniera extract (100 g) for 2 h plus postH2O2 (100 M) for 1 h. To analyse whether pre-oxidative stressed human umbilical vein endothelial cells could be protected with exposure to post-B. monniera extract and whether the lipofuscin granules could be scavenged by post-B. monniera extract treatment, the following exposure regimen was provided: pre-H2O2 (100 M) for 1 h plus post-B. monniera extract (100 g) for 2 h.

Material and methods

This work conforms to the values laid down in the Declaration of Helsinki (1964). The protocol of this study has been approved by the relevant ethical committee related to our institution in which it was performed. All subjects gave full informed consent to participate in this study. BME preparation The dried herb was macerated in 95% methanol and the crude extract of B. monniera was prepared and filtered with Whatman no.1 filter paper2. The extract was stored at 30C5. Harvest and expansion of HUVECs6 The isolation protocol for HUVECs was adapted from Baudin et al. 20077. The umbilical cord was collected immediately and the cord was rinsed

Introduction
Medicinal plants that have been useful in history are obvious choices as sources with significant pharmacological and biological activities1. One such medicinal plant is Bacopa monniera, commonly termed as Brahmi2. Studies in the past have focused on B. monnieras cognitive enhancing effects that specifically include memory, learning and concentration. Post-mitotic cells accumulate a nondegradable, intralysosomal substance called lipofuscin which is formed

* Corresponding author Email: shahdabalmelkar@gmail.com Department of Cardiovascular and Thoracic Surgery, Seth G.S. Medical College and KEM Hospital, Parel, Mumbai 400012, Maharashtra, India 2 Department of Cardiovascular and Thoracic Surgery, JNMC, Acharya Vinoba Bhave Rural Hospital, Sawangi (Meghe), Wardha 442004, Maharashtra, India
1

Licensee OA Publishing London 2013. Creative Commons Attribution Licence (CC-BY)

F : Almelkar SI, Patwardhan AM. Bacopa monniera extract plays a significant role in lipofuscin scavenging in cultured human vascular endothelial cells. OA Alternative Medicine 2013 Feb 02;1(1):5.

Compeng interests: none declared. Conict of Interests: none declared. All authors contributed to the concepon, design, and preparaon of the manuscript, as well as read and approved the nal manuscript. All authors abide by the Associaon for Medical Ethics (AME) ethical rules of disclosure.

Results The presence of lipofuscin granule formation was confirmed by the Ziehl-Neelsons staining method and microscopic examinat-ion. Exposures to H2O2 (100 M) showed the abundant generation and formation of lipofuscin granules. B. monniera extract exposures of 2 h, 4 h and 6h (100 g), showed the abundant generation and formation of lipofuscin granules. B. monniera extract exposures of 2 h, 4 h and 6h (100 g), showed a complete absence of lipofuscin granules. Pre-B. monniera extract (100 g) exposure for 2 h plus postH2O2 (100 M) exposure for 1 h, resulted in extremely reduced lipofuscin granules. H2O2 exposure for 1 h and pre-H2O2 (100 M) exposure for 1 h plus post-B. monniera extract (100 g) exposure for 2 h, resulted in the moderate appearance of lipofuscin granules. Conclusion This study concluded that the B. monniera extract has potential antioxidant properties, which play a pivotal role in the analysis of lipofuscin granules scavenging in cultured human umbilical vein endothelial cells.

due to the iron-catalysed oxidation/polymerisation of protein and lipid residues3. It is believed that these changes occur not only due to continuous oxidative stress (causing oxidation of mitochondrial constituents and autophagocytosed material) but also because of the inherent inability of cells to completely remove oxidatively damaged structures (biological garbage)4. Accumulation of lipofuscin granules is mostly observed in various neurological disorders2. There are no reported studies of B. monniera and its antioxidant activities, which play a significant role in lipofuscin scavenging in cultured human umbilical vein endothelial cells (HUVECs). Therefore, this study is the first to show that the B. monniera extract (BME) plays a pivotal role by strongly supporting the scavenging of lipo-fuscin granules in cultured HUVECs.

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and washed with sterile phosphate buffered saline (PBS). One end of the umbilical vein was clamped using artery forceps and an enzyme cocktail of 0.15% collagenase type IV (Sigma Aldrich, St. Louis, Missouri, USA) and dispase II (Roche, Nutley, New Jersey, USA) was filled into the lumen cavity of the vein for dislodging the endothelial cells (ECs). The ECs were incubated for 20 min at 37C. After completion of incubation, the vein was flushed with M199 medium (Invitrogen, Carlsbad, California, USA). The cells in suspension were centrifuged at 1500 rpm for 10 min. Cell pellets were refreshed twice by centrifugation and the final pellet was again resuspended in an endothelial cell growth medium (ECGM) (PromoCell GmbH, Germany) containing 20% foetal bovine serum (FBS) (Invitrogen, Carlsbad, California, USA); isolated HUVECs were plated in tissue culture flasks. After 12 h of incubation, the HUVECs were fed with complete ECGM containing 20% FBS, 2 mM L-glutamine, 10 g/ mL heparin and penicillin 5 unit/mL and incubated at 37C with 5% CO2. Lipofuscin granules stained by ZiehlNeelson method for control cells (without exposure), oxidative stress (H2O2) for 1h8 HUVECs were cultured onto cover slips until resembling a cobble stone appearance. HUVECs without

any exposure served as the control. The HUVECs were exposed to oxidative stress (H2O2) for 1 h (100 M) and were fixed with 4% paraformaldehyde and refreshed with PBS 1 (5 min 3 washes). The HUVECs were washed with 70% isopropanol (10 min 2 washes) and stained with carbolfuscin for 30 min at 60oC. The HUVECs were further rinsed in acidalcohol (1% HCl in 70% ethanol) until they turned light pink. They were washed in distilled water for 5 min, counter stained (0.5% toluidene blue) for 10 min and rinsed again with distilled water for 5 min. Isopropyl alcohol (70%) was used to dehydrate the cells. The cover slips were mounted with dibutyl phthalate xylene (DPX). Stained HUVECs were observed under a bright field microscope for lipofuscin granules. Exposure to BME for 2h, 4h and 6 h, pre-BME exposure for 2hpluspostH2O2 for 1h and pre-H2O2 exposure for 1hpluspost-BME for 2h to cultured HUVECs to study the eff ect of BME on lipofuscin granules8 HUVECs were cultured on cover slips until they reached a cobble stone morphology. HUVECs were further processed as follows: BME exposure for 2 h, 4 h and 6 h, preBME expo-sure for 2 h plus post-H2O2 for 1 h and pre-H2O2 exposure for 1 h plus post-BME for 2 h.

All exposed HUVECs were fixed with 4% paraformaldehyde and refreshed with PBS 1 (5 min 3 washes). The HUVECs were washed with 70% isopropanol (10 min 2 washes) and stained with carbolfuscin for 30 min at 60oC. The HUVECs were rinsed in acid-alcohol (1% HCl in 70% ethanol) until they turned light pink. The HUVECs were washed with distilled water for 5 min, counter stained (0.5% toluidene blue) for 10 min and rinsed again with distilled water for 5 min. Isopropyl alcohol (70%) was used to dehydrate the cells. The cover slips were mounted with DPX. Stained HUVECs were observed under a bright field microscope for lipofuscin granules.

Results
B. monniera crude extract A 12% yield of sticky, crude extract was obtained from B. monniera and further used for experimental work. Isolation and culture of HUVECs HUVECs were isolated in clusters of 30. HUVECs were attached to the plate surface immediately after seeding for 2 h. HUVECs started expanding in clusters and reached confluence on the 10th day (Figure 1). On 70% confluence, the HUVECs resembled the perfect cobblestone morphology, and the cell density was 3 105 cells/cm2.

Licensee OA Publishing London 2013. Creative Commons Attribution Licence (CC-BY)

F : Almelkar SI, Patwardhan AM. Bacopa monniera extract plays a significant role in lipofuscin scavenging in cultured human vascular endothelial cells. OA Alternative Medicine 2013 Feb 02;1(1):5.

Compeng interests: none declared. Conict of Interests: none declared. All authors contributed to the concepon, design, and preparaon of the manuscript, as well as read and approved the nal manuscript. All authors abide by the Associaon for Medical Ethics (AME) ethical rules of disclosure.

Figure 1: HUVECs showed a cobble stone morphology in culture.

Figure 2: HUVECs in control (without treatment) showed a very low appearance of lipofuscin granules.

Figure 3: HUVECs treated with H2O2 (100 M) showed abundant lipofuscin granules.

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resulted in the moderate appearance of lipofuscin granule formation with little nuclear and cell membrane damage (Figure 8).

Discussion
Cells are constantly subjected to free-radical attack. Free radicals are scavenged in the cells by the antioxidant system present in the cell. The unscavenged free radicals exert their cytotoxic action on cell membrane lipids. These peroxidised membranes are engulfed by lysosomes which may bring about damage to the lysosomal enzymes. The residual body of lysosomes that are unable to digest cell membranes turns into lipofuscin granules. In advanced aging, the rate of accumulation of lipofuscin granules increases in tissues like the heart and brain2. B. monniera is a herb, and a variety of studies have been carried out to study its antioxidative properties. Past research investigating BME exposure in neurons, established the lipofuscin scavenging property of B. monniera2. We tried to investigate the role of BME in lipofuscin scavenging in cultured HUVECs. HUVECs without any exposure served as the controls and showed very little presence of lipofuscin granules. Exposure to 1 h of oxidative stress (H2O2) at a concentration of 100 M resulted in

Figure 7: HUVECs treated with pre-BME (100 g) for 2 h plus postH2O2 (100 M) for 1 h showed reduced lipofuscin granules. Lipofuscin granules in the control cells and in cells exposed to oxidative stress (H2O2 for 1h) The current investigation reveals the appearance and occurrence of lipofuscin granules in cultured HUVECs. HUVECs without any exposure served as controls (normal) and showed a much lower presence of lipofuscin granules (Figure 2). Oxidant exposures (H2O2 for 1 h) to cultured HUVECs, led to lipofuscin granule generation. Exposure of H2O2 for 1 h at a concentration of 100 M resulted in maximum damage to the cultured HUVECs; the cells showed distorted cell and nuclear membranes with copious formations of lipofuscin granules in the cytosol region (Figure 3).

Figure 8: HUVECs treated with preH2O2 (100 M) for 1 h plus postBME (100 g) for 2 h showed moderate lipofuscin granules. Exposure to BME for 2h, 4h and 6 h, pre-BME exposure for 2h plus postH2O2 for 1h and pre-H2O2 exposure for 1h plus post-BME for 2h to cultured HUVECs to study the effect of BME on lipofuscin granules Exposure to BME (100 g) treatment for 2 h (Figure 4), 4 h (Figure 5) and 6 h (Figure 6), showed the absence of or the lack of lipofuscin granule formation and an intact cell and nuclear membrane. Pre-BME (100 g) exposure for 2 h plus post-H2O2 (100 M) for 1 h, showed the presence of very reduced amounts of lipofuscin granule formation with intact nuclear membranes and very little injury to the cell membranes (Figure 7). Pre-H2O2 (100 M) exposure for 1 h plus post-BME (100 g) for 2 h,

Licensee OA Publishing London 2013. Creative Commons Attribution Licence (CC-BY)

F : Almelkar SI, Patwardhan AM. Bacopa monniera extract plays a significant role in lipofuscin scavenging in cultured human vascular endothelial cells. OA Alternative Medicine 2013 Feb 02;1(1):5.

Compeng interests: none declared. Conict of Interests: none declared. All authors contributed to the concepon, design, and preparaon of the manuscript, as well as read and approved the nal manuscript. All authors abide by the Associaon for Medical Ethics (AME) ethical rules of disclosure.

Figure 4: HUVECs treated with BME for 2 h (100 g) showed a complete absence of lipofuscin granules.

Figure 5: HUVECs treated with BME for 4 h (100 g) showed a complete absence of lipofuscin granules.

Figure 6: HUVECs treated with BME for 6 h (100 g) showed a complete absence of lipofuscin granules.

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maximum damage to the cultured HUVECs, such as distorted cell and nuclear membranes and abundant formation of lipofuscin granules in the cytosol region. This firmly explains that dose-dependant H2O2 exposure for 1 h brings about cellular damage and is involved in the formation of lipofuscin granules in cultured HUVECs. Exposure to BME (100 g) for 2 h, 4 h and 6 h, showed the absence of lipofuscin granule formation and intact cell and nuclear membranes. Pre-BME (100 g) exposure for 2 h plus post-H2O2 (6.25 M to 100 M) for 1 h, showed negligible/reduced amount of lipofuscin granule formation with intact nuclear membrane and very little injury to the cell membrane. This illustrates that pre-BME exposure for 2 h provides the necessary anti-oxidative protection to cultured HUVECs while the results from the post-H2O2 exposure for 1 h illustrate that there was a lack of or minimal lipofuscin granule formation. Therefore, it is deduced that pre-BME exposure has a protective role against lipofuscin granule formation even after post-H2O2 exposures. Pre-H2O2 (100 M) exposure for 1 h plus post-BME (100 g) exposure for 2 h, resulted in the moderate appearance of lipofuscin granule formation with little nuclear and cell membrane damage. This shows that pre-H2O2 exposures bring about the moderate generation of lipofuscin granules, but after post-BME exposures, the generation of lipofuscin granules is halted due to the BME antioxidative properties.

Conclusion
This investigation shows that BME is an antioxidant which plays a pivotal role in lipofuscin scavenging in stressed and normal HUVECs. H2O2 (100 M) treatment is responsible for oxidative impair and brings about the extreme generation or formation of lipofuscin granules. BME exposures for 2 h, 4 h and 6h (100 g), bring about scavenging of lipofuscin granules at different time intervals. Pre-BME (100 g) exposure for 2 h brings about reduced lipofuscin formation. After post-H2O2 (100 M) exposure for 1 h, there is no further rise in lipofuscin. Pre-H2O2 (100 M) exposure for 1 h plus pos-tBME (100 g) exposure for 2 h, resulted in the moderate appearance of lipofuscin granules.

KEM Hospital, Mumbai, India) for his generosity in helping us and providing his support with the tissue culture laboratory. We especially thank Mrs Seema V Sharma (Senior Scientific Officer, CVTC) Mrs Rajeshri (JSO), Mr Sunil Chaudhary, Mrs Nidhi Taneja, Mr Janardhan and other CVTC staff for their laboratory support.

References
1. Shukia S, Khanna NK, Godhwani JL. Effect of Brahmi Rasayana on the central nervous system. J Ethnopharmacol.1994 SepOct;21(1):6574. 2. Singh HK, Dhawan BN. Drugs affecting learning and memory. In: Tandon PN, Bijiani V, Wadhwa S, editors. Lectures in Neurobiology. Vol. 1. New Delhi: Wiley Eastern; 1992.p189207. 3. Terman A, Brunk UT. Lipofuscin. Int J Biochem Cell Biol. 2004 Aug;36(8):14004. 4. Brunk UT, Terman A. The mitochondrial-lysosomal axis theory of aging: accumulation of damaged mitochondria as a result of imperfect autophagocytosis. Euro J Biochem. 2002 Apr;269(8):1996 2002. 5. Phrompittayarata W, Putalunc W, Tanakad H, Jetiyanone K, Wittayaareekulf S, et al. Naresuan Univ J. 2007;15(1):2934. 6. Freshney RI. Culture of Animal Cells: A Manual of Basic Technique. 5th ed. John Wiley & Sons Inc Hoboken; 2005. 7. Baudin B, Bruneel A, Bosselut N, Vaubourdolle M. A protocol for isolation and culture of human umbilical vein endothelial cells. Nat Protoc. 2007;2(3):4815. 8. Pearse AGE. Histochemistry: Theoretical and Applied. 4th ed. Churchill Livingstone Inc Oxford. 1985.

Abbreviations list
BME, Bacopa monniera extract; ECs, endothelial cells; ECGM, endothelial cell growth medium; FBS, foetal bovine serum; PBS, phosphate buffered saline.

Acknowledgement
We gratefully thank the Indian Council of Medical Research (ICMR), File No: 45/47/2010/BMS/TRM IRIS Cell: 2010-06230, Government of India, for providing a senior research fellowship to our first author. We are also thankful to Dr NB Agrawal for providing research funds through Dr PK Sen, Research Society, GSMC and KEM Hospital, Parel, Mumbai. We thank Dr BM Kandalkar (Professor and Head of Department, Department of Pathology, GSMC and

Licensee OA Publishing London 2013. Creative Commons Attribution Licence (CC-BY)

F : Almelkar SI, Patwardhan AM. Bacopa monniera extract plays a significant role in lipofuscin scavenging in cultured human vascular endothelial cells. OA Alternative Medicine 2013 Feb 02;1(1):5.

Compeng interests: none declared. Conict of Interests: none declared. All authors contributed to the concepon, design, and preparaon of the manuscript, as well as read and approved the nal manuscript. All authors abide by the Associaon for Medical Ethics (AME) ethical rules of disclosure.