TABLE OF CONTENTS · Chinese Medicine (CM) Journal Information SUBSCRIPTIONS The Chinese Medicine (Online at Scientific Research Publishing, ) is published quarterly by Scientific

Chinese Medicine, 2010, 1, 1-29 Published Online June 2010 in SciRes (http://www.SciRP.org/journal/cm/)

Copyright © 2010 SciRes. CM

TABLE OF CONTENTS

Volume 1 Number 1 June 2010 Bikram Yoga as a Countermeasure of Bone Loss in Women

A. Mukherjee, P. Mukherjee, R. R. Rude…………………………………………………………………………………………1

Effects of Indole-3-carbinol and Flavonoids Administered Separately and in Combination on Nitric Oxide Production and iNOS Expression in Rats

E. Rostoka, L. Baumane, S. Isajevs, A. Line, K. Silina, M. Dzintare, D. Svirina, J. Sharipova, I. Kalvinsh, N. Sjakste……………5

Antihepatotoxic Effect of Feronia Limonia Fruit against Carbon Tetrachloride Induced Hepatic Damage in Albino Rats

R. Upadhyay, N. D. Pandey, S. S. Narvi, A. Verma, B. Ahmed…………………………………………………………………18

Determination of Cichoric Acid as a Biomarker in Echinacea Purpurea Cultivated in Iran Using High Performance Liquid Chromatography

J. Zolgharnein, A. Niazi, S. Afiuni-Zadeh, K. Zamani……………………………………………………………………………23

Non-Exclusion Effects in Aqueous Size-Exclusion Chromatography of Polysaccharides

A. S. Boymirzaev, A. S. Turaev…………………………………………………………………………………………………28

Chinese Medicine (CM)

Journal Information SUBSCRIPTIONS

The Chinese Medicine (Online at Scientific Research Publishing, www.SciRP.org) is published quarterly by Scientific Research Publishing, Inc., USA.

Subscription rates: Print: $50 per issue. Electronic: free, available on www.SciPR.org. To subscribe, please contact Journals Subscriptions Department, E-mail: [email protected]

SERVICES

Advertisements Advertisement Sales Department, E-mail: [email protected]

Reprints (minimum quantity 100 copies) Reprints Co-ordinator, Scientific Research Publishing, Inc., USA. E-mail: [email protected]

COPYRIGHT

Copyright©2010 Scientific Research Publishing, Inc.

All Rights Reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, scanning or otherwise, except as described below, without the permission in writing of the Publisher.

Copying of articles is not permitted except for personal and internal use, to the extent permitted by national copyright law, or under the terms of a license issued by the national Reproduction Rights Organization.

Requests for permission for other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works or for resale, and other enquiries should be addressed to the Publisher.

Statements and opinions expressed in the articles and communications are those of the individual contributors and not the statements and opinion of Scientific Research Publishing, Inc. We assumes no responsibility or liability for any damage or injury to persons or property arising out of the use of any materials, instructions, methods or ideas contained herein. We expressly disclaim any implied warranties of merchantability or fitness for a particular purpose. If expert assistance is required, the services of a competent professional person should be sought.

PRODUCTION INFORMATION

For manuscripts that have been accepted for publication, please contact: E-mail: [email protected]

Chinese Medicine, 2010, 1, 1-4 doi:10.4236/cm.2010.11001 Published Online June 2010 (http://www.SciRP.org/journal/cm)


Bikram Yoga as a Countermeasure of Bone Loss in Women

Apurba Mukherjee1, Prithwis Mukherjee1, Robert R. Rude2 1Bikram’s Yoga College of India, Los Angeles, USA

2Keck School of Medicine, University of Southern California and Orthopedic Hospital of Los, Los Angeles, USA E-mail: [email protected]

Received May 28, 2010; revised June 10, 2010; accepted June 20, 2010

Abstract The purpose of this pilot study was to observe whether Bikram Yoga training helps bone growth or arrest bone loss in women. In this study, the bone mineral density (BMD) was measured at the hip, spine and whole body for a group of 14 pre-menopausal women (11 Caucasians, 2 Asians and 1 African American). These 14 women had participated in Bikram Yoga classes (26 yoga postures) at least 3 times a week for 3 or more years. DEXA scans were used to measure BMD at the lumbar spine, hip and the whole body. In addi-tion, the Z-scores were calculated for each subject at these three locations. The study results indicate that the BMD at these body areas of this group of women is generally above the mean BMD expected for normal, healthy, women of comparable age and ethnicity. Overall, the study findings suggest that the intervention of Bikram Yoga training may be beneficial for skeletal health and could prevent bone loss. Keywords: Osteoporosis, Bone Mineral Density (BMD)

1. Introduction A pilot study was initiated to examine whether preven-tion/recovery from bone loss is possible through Bikram Yoga postural exercise intervention. Bone loss is a seri-ous medical problem [1], and an estimated 200 million adults (both men/women) have this condition worldwide. The sharp decline of production of the hormone estrogen following menopause contributes greatly to bone loss for the elderly women population. Bone loss also results from joint disuse and reduced movements. The study was aimed to explore the possibility of using Bikram Yoga postural exercise intervention as a possible means of prevention/recovery for bone loss.

In general, the skeleton reaches its peak bone density for people between the ages of 20 and 30, and after age 35, the body begins to lose more bone than the body can rebuild. According to the Surgeon General’s Report (2004) [2], there are over 10 million Americans over the age of 50 with osteoporosis and an additional 34 million with low bone mass or osteopenia. An estimated 1.5 mil-lion Americans suffer from fractures related to bone dis-ease every year. In addition, about two-thirds of the peo-ple suffering from osteoporosis are women, as they abruptly lose the bone-protecting estrogen hormone at menopause [3]. However, men also suffer from osteopo-rosis as they age. In fact, one in four elderly men in

America suffers from osteoporotic fracture in older age. Bones are living tissues and are continually broken

down and rebuilt in human bodies. Consisting of 75% minerals, (like calcium and phosphorous), and 25% gela-tin matrix of water and collagen, bones continue to be reshaped and renewed all the time. While bones appear solid they can be quite flexible and strong at the same time.

There is a delicate balancing act that goes on daily with minerals and human bones. A healthy body reabsorbs bone at the same rate as new bone growth occurs. Special-ized cells called osteoclasts will remove bone while os-teoblasts build bone. This cycle continues over and over.

Bikram Yoga, which is a certified form of exercise by the State of California [4], is suitable for the young as well as the elderly people. Although Bikram Yoga is widely practiced in over 1500 studios in USA, Canada, Europe, Japan, Australia, Thailand, India and many other countries around the world, there have been limited ef-forts to scientifically study and document the far reaching benefits of Bikram Yoga to the mind and body. 2. Study Methods All study subjects were enrolled by Bikram’s Yoga Col- lege of India in Los Angeles and they signed informed-consent forms for Bone Mineral Density (BMD) meas-

A. MUKHERJEE ET AL.


2

urements at the Orthopedic Hospital in Los Angeles. The subjects were Bikram Yoga teachers and they consis-tently practiced and taught Bikram Yoga on a daily basis. The Bikram Yoga exercise intervention is a 90-minute protocol of specially designed sequence of 26 postures and two breathing exercises, which can be practiced by men or women of any age. These are static isometric postural exercises and there are no dynamic movements. These exercises, which involve both the mind and the body, collectively stimulate all the vital organs of the body. The details of the different 26 postures are de-scribed in reference [5].

Bikram Yoga is a standardized yoga program that is designed to scientifically warm up and stretch muscles, ligaments and tendons. In all parts of the world, Bikram Yoga teachers train participants to perform the same 26 postures, in the same sequence, at 105 degrees Fahren-heit. Bikram Yoga is a standardized system of yoga and no deviation is allowed between classes. In fact, all Bik-ram Yoga teachers are trained and certified by the Bik-ram’s Yoga College of India. All certified teachers also undergo periodic evaluations and their teaching certifi-cates are revalidated every three years. The 26 exercises systematically move fresh, oxygenated blood to each organ [6].

Bone mineral density (BMD) was determined utilizing a Hologic bone densitometer. Bone mass is commonly expressed as Z-scores as defined below.

Z-score: The Z-scores are calculated by comparing pa-tient’s BMD with the corresponding BMD of women of the same age. sex and race. Z-scores are calculated as

follows: Z-score = (BMD-Expected BMD)/S. D. BMD = patient’s BMD Expected BMD = BMD of someone of the same age,

sex and race. S. D = Standard Deviation corresponding to the above

“expected BMD”. Z-score is a statistical concept. The Z-score compares the BMD to age, gender and

ethnicity of the individual. Data presented here therefore are the Z-scores.

The Z-score value lower than –2.0 is a warning sign that the patient has less bone mass (and/or may be losing more rapidly) than expected for someone of correspond-ing age, sex and race. 3. Results The bone mass density (BMD) for each subject was cal-culated for the lumbar spine, hip, and whole body. Table 1 shows the BMD data, in addition to basic demographic data, for each of the 14 study participants.

Figure 1 demonstrates the standard interpretation of Z-score for the whole body and Figure 2 demonstrates the Z-scores for the study population.

Figure 2 demonstrates that whole body calcium of all the 14 subjects are in the above average calcium group of population. In addition, Figures 1 and 2 show that 6 out of 14 subjects have bone density higher than 84% (or in the top 16%) of the population.

Table 1. Age, yoga experience and DEXA scan BMD data.

Age (years) Total # of yoga classes Years of Yoga practice Z-score (spine) Z-score (hip) Z-score (whole body)

Subject 1 46 450 3 2.0 1.7 2.1

Subject 2 47 800 4 1.2 0.6 1.2

Subject 3 49 600 4 0.6 –0.5 0.2

Subject 4 39 975 4 –0.3 0 –0.3

Subject 5 41 700 4 –0.8 –0.5 –0.7

Subject 6 47 675 3 –0.4 0.4 0.7

Subject 7 31 975 4 2.1 1.5 2.6

Subject 8 50 950 4 1.4 0.9 3.7

Subject 9 47 950 4 –1.3 –0.9 –1.1

Subject 10 45 900 4 –0.5 –0.4 0.3

Subject 11 41 525 3 –0.8 –0.1 –0.3

Subject 12 49 525 3 1.0 0.5 1.9

Subject 13 45 700 4 –0.5 –0.2 0

Subject 14 44 600 3 –0.6 0.6 1.6

Mean 44 738 3.6 0.22 0.26 0.85

A. MUKHERJEE ET AL.


3

Figure 1. Standard interpretation of Z-score (whole body).

Figure 2. Z-score (whole body) by the number of Bikram Yoga classes.

Figure 3. Photo of a DEXA scan (lumbar spine).

A. MUKHERJEE ET AL.


4

Figure 3 shows a typical DEXA scan photo from the HOLOGIC bone densitometer at the Orthopedic Hospital in Los Angeles.

4. Discussion

The results demonstrate that the mean bone mineral den-sity of all 14 subjects at the lumber spine, total hip, and the whole body are above average considering their ages and ethnicities. This is most notable for the total body calcium where the Z-score (whole body) is nearly one standard deviation above the mean. The data suggests that an intervention trial of Bikram Yoga exercise activ-ity may arrest bone loss in women.

5. Practical Applications

The practice of Bikram Yoga may provide another means to stabilize bone mass and prevent bone loss and osteo-porotic fractures.

6. Acknowledgements

This study was supported by Bikram’s Yoga College of India in Los Angeles. The contribution of Travis Liggett in the early stage of study in obtaining the Institutional

Review Board (IRB) approval is gratefully acknowl-edged. In addition the contribution of Anita Mukherjee (Stanford University) in statistical analysis and develop-ing Figures 1 & 2 from the data of this article are ac-knowledged.

7. References

[1] B. W. Watts, B. Ettinger and M. S. LeBoff, “Perspective, FRAX Facts,” Journal of Bone and Mineral Research, Vol. 24, 2009, pp. 975-979.

[2] U. S. Dept, “Bone Health and Osteoporosis,” Health and Human Services, Washington, D.C., 2004. http://www. surgeongeneral.gov

[3] “Risk and Benefits of Estrogen Plus Progestin in Healthy Post Menopausal Women,” Journal of the American Medical Association, Vol. 288, No. 3, 17 July 2002, pp. 321-333.

[4] State of California License to Bikram Choudhury for Bikram Yoga College of India, 2005.

[5] B. Choudhury, “Bikram’s Beginning Yoga Classes,” Penguin Putnam Inc., New York, 2000.

[6] B. Choudhury, “Bikram Yoga,” Harper Collins Publishers, New York, 2007, p. 84.



Effects of Indole-3-Carbinol and Flavonoids Administered Separately and in Combination on Nitric Oxide

Production and iNOS Expression in Rats

Evita Rostoka1, Larisa Baumane1, Sergejs Isajevs2, Aija Line3, Karina Silina3, Maija Dzintare1, Darja Svirina2, Jelena Sharipova1, Ivars Kalvinsh1, Nikolajs Sjakste1,2

1Latvian Institute of Organic Synthesis, Riga, Latvia 2Faculty of Medicine, University of Latvia, Riga, Latvia

3Latvian Biomedical Research and Study Centre, Riga, Latvia E-mail: [email protected]

Received January 20, 2010; revised April 29, 2010; accepted May 10, 2010

Abstract Beneficial effects of natural compounds are often attributed to modulation of NO production; however ef-fects produced by plant extracts do not correlate with effects of purified components. The goal of our work was to study ability of flavonoids and indole-3-carbinol, as well as their combinations to modify NO produc-tion, iNOS gene and protein expression in rat tissues. Baicalein and luteolin decreased NO concentration in both intact and LPS-treated animals. Baicalein decreased iNOS gene expression. Luteolin decreased NO production in the liver and heart and number of iNOS-positive cells in the liver of LPS-treated animals. Combination of the two substances did not decrease the NO synthesis triggered by LPS, although it de-creased iNOS gene expression. Quercetin decreased NO production in the heart, kidneys and blood of intact rats, but enhanced the LPS effect in testes, spleen and blood on NO production and iNOS protein expression in the liver. Indole-3-carbinol decreased NO concentration in the cerebellum, blood, lungs and skeletal mus-cles. The drug enhanced the LPS-triggered increase of NO production in all rat organs. It increased iNOS protein expression in intact liver; however it decreased the LPS-triggered outburst of the enzyme biosynthe-sis. Combination of indole3-carbinol with quercetin decreased NO production in LPS-treated animals how-ever it slightly increased iNOS gene expression. Taken together our results suggest that modifications of NO level in tissues by a natural compound cannot be predicted from data about its effects on NOS expression or activity. Combination of substances can produce an effect differing from that of individual substances. This stresses importance of direct measurements of NO in the tissues. Keywords: Nitric Oxide, Baicalein, Luteolin, Indole-3-Carbinol, Quercetin, Inducible Nitric Oxide Synthase

1. Introduction Natural biologically active compounds of plant origin including flavonoids are main active substances of tradi-tional Chinese medicines: herbal extracts and similar preparations. Nowadays some of these substances are used in purified form as drugs. Anti-inflammatory activ-ity, antioxidant activities, anticancer activity of phyto-genic antineoplastic agents, and neuroprotective effects of Chinese herbal drugs are in focus of interest of many researchers worldwide [1]. Chinese traditional medicines are known to influence also nitric oxide enzymatic pro-duction and NO synthase activity [2]. It is supposed that

flavonoid intake influences mortality from nitric ox-ide-dependent processes: ischemic heart disease, stroke, diabetes mellitus, and cancer [3], NO production is also modified by chemicals of plant origin [4]. This implies significance of flavonoid and other natural compound uptake for functions of cardiovascular, immune and nervous systems. However biological activity of poly-phenol-rich food product does not correlate with effects that could be deduced from effects of individual com-pounds on NO synthase activity. For example, red wine is known as vasodilator [5], however purified quercetin, that is abundant in red wine inhibits iNOS gene expres-sion [6,7]. It also destabilizes eNOS mRNA [8] and is

E. ROSTOKA ET AL. 6 even considered to be inhibitor of the NOS enzymatic activity [9]. Nevertheless the compound produces vasorelaxing effects [10] despite the fact that nitric oxide re-lease in rat aorta is not detected after quercetin admini-stration [11]. In this work we have studied the ability of several natural compounds of plant origin administered separately and in combinations to modify NO production in rat tissues monitored by ESR spectroscopy of Fe (DETC)2-NO complexes conducted in parallel to evalua-tion of iNOS gene expression assayed by real-time RT- PCR technique. Combination of direct NO detection in tissues with other approaches characterizing NO produc-tion at different levels enabled us to reveal unforeseen effects of presumable NO-donors, anaesthetics and an anti-ischemic drug [12-14]. The same approach was ap-plied this time to natural compounds. Flavonoids luteolin, baicalein and quercetin as well as simple phenolic com-pound indole-3-carbinol were chosen among many other compounds after a piloting study.

Luteolin, 3’, 4’, 5, 7-tetrahydroxyflavone is abundant in vegetables: roots of celery, rutabaga, red pepper, spin- ach and flowering plants: Ajuga decumbens, Taraxacum officinale (dandelion), Medicago sativa (alfalfa). Luteo-lin is known as dietary compound with antioxidant activ-ity [15]. Baicalein (5, 6, 7-trihydroxyflavone) is found in Scutellaria baicalensis Georgi roots. Quercetin (penta-hydroxyflavonol) is found in numerous higher plants. This flavonol is abundant in onions, apples, leaf vegeta-bles, beans, tea, red wine, clover, pollen. Indole-3-carbinol (3-indolmetanol) is found in Mustard family plants: (Brassica sp.): cabbage, broccoli, Brussels sprouts. The compound is widely studied as chemotherapeutic agent for cancer treatment [16]. Chemical structures of the compounds are given in Figure 1. Literature data indi-cated possible impact of all the three substances on NOS expression and/or NO production [15-22]. The chosen compounds are active substances in several drugs used in Chinese medicine. Quercetin is in important component of Shaofu Zhuyu decoction active extract [23], Saururus chinensis, a herb used traditionally in Chinese medicine for treatment of urological diseases [24], together with luteolin it is found in tree peony yellow flowers also widely used in Chinese medicine [25]. Baicalein is an active component of numerous Chinese medicines in-cluding Niu Huang Jie Du Pill [26].

The aims of the present work were: 1) To study effects NO production in several organs of intact rats and in LPS model of sepsis; 2) To reveal modifications of NO pro-duction by luteolin, baicalein, quercetin and indole-3- carbinol given separately and in combinations in both intact and LPS-treated animals; 3) To study contribution of changes in iNOS gene and protein expression in modifications of NO production by natural compounds and their modifications.

Figure 1. Chemical structures of luteolin, baicalein, quercetin and indole-3-carbinole. 2. Material and Methods 2.1. Chemicals Indole-3-carbinol, quercetin, baicalein and luteolin were purchased from Dayang Chemical Co., LTD (Hangzhou, China). Lipopolysaccharide, diethylthiocarbamate, fer-rous sulfate, sodium citrate, TRI reagent and all other chemicals were from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany). 2.2. Experiment Design and Drug

Administration Animals were obtained from the Laboratory of Experi-mental Animals, Riga Stradins University, Riga, Latvia. All experimental procedures were carried out in accor-dance with guidelines of the Directive 86/609/EEC “European Convention for the Protection of Vertebrate Animals Used for Experimental and other Scientific Purposes” (1986) and were approved by the Animal Eth-ics Committee of the Food and Veterinary Service (Riga, Latvia).

Wistar male rats, each weighing 215.00 ± 5.63 g at the beginning of the experiments, were used in all the work. The environment was maintained at a temperature of 22 ± 0.5˚C with a 12-h light/dark cycle. The animals were fed a standard laboratory diet. Description of the experi-mental groups is given in Table 1. In NO production experiments substances were administered per os in con-centrations indicated in the Table 1. 3.5 hours after sub-stance administration spin trap was injected, after 30 min


E. ROSTOKA ET AL.


7

Table 1. Description of groups and experiment design.

Group number

Number of animals Substance/dose LPS Parameter studied Organs studied

1. 24 - - NO production Brain cortex, liver, heart, kidney, blood, lungs

2. 9 Indole-3-carbinol (50 mg/kg) - NO production Brain cortex, liver, heart, kidney, blood, lungs

3. 5 Luteolin (50 mg/kg) - NO production Brain cortex, liver, heart, kidney, blood, lungs

4. 6 Quercetin (50 mg/kg) - NO production Brain cortex, liver, heart, kidney, blood, lungs

5. 5 Baicalein (50 mg/kg) NO production Brain cortex, liver, heart, kidney, blood, lungs

6. 28 - 10 mg/kg NO production Brain cortex, cerebellum, liver, heart, kidney, blood, lungs, testes, spleen, skeletal muscles

7. 12 Indole-3-carbinol (50 mg/kg) 10 mg/kg NO production Brain cortex, cerebellum, liver, heart, kidney, blood, lungs, testes, spleen, skeletal muscles

8. 6 Luteolin (30 mg/kg) 10 mg/kg NO production Brain cortex, cerebellum, liver, heart, kidney, blood, lungs, testes, spleen, skeletal muscles

9. 6 Quercetin (50 mg/kg) 10 mg/kg NO production Brain cortex, cerebellum, liver, heart, kidney, blood, lungs, testes, spleen, skeletal muscles

10. 6 Baicalein (30 mg/kg) 10 mg/kg NO production Brain cortex, cerebellum, liver, heart, kidney, blood, lungs, testes, spleen, skeletal muscles

11. 8 Baicalein (30 mg/kg) Luteolin (30 mg/kg) 10 mg/kg NO production Brain cortex, cerebellum, liver, heart, kidney, blood, lungs, testes, spleen, skeletal muscles

12. 6 Quercetin (50 mg/kg) Indole-3-carbinol (50 mg/kg) 10 mg/kg NO production Brain cortex, cerebellum, liver, heart, kidney, blood, lungs, testes, spleen, skeletal muscles

13. 20 - - iNOS mRNA and protein expression Liver, brain cortex

14. 3 Indole-3-carbinol (50 mg/kg) iNOS mRNA and protein expression Liver, brain cortex

15. 3 Luteolin (50 mg/kg) iNOS mRNA and protein expression Liver, brain cortex

16. 3 Quercetin (50 mg/kg) iNOS mRNA and protein expression Liver, brain cortex

17. 3 Baicalein (50 mg/kg) iNOS mRNA and protein expression Liver, brain cortex

18. 3 Quercetin (50 mg/kg) Indole-3-carbinol (50 mg/kg) iNOS mRNA and protein expression Liver, brain cortex

19. 3 Baicalein (50 mg/kg) Luteolin (50 mg/kg) iNOS mRNA and protein expression Liver, brain cortex

20. 21 - 10 mg/kg iNOS mRNA and protein expression Liver, brain cortex

21. 6 Indole-3-carbinol (50 mg/kg) 10 mg/kg iNOS mRNA and protein expression Liver, brain cortex

22. 6 Luteolin (50 mg/kg) 10 mg/kg iNOS mRNA and protein expression Liver, brain cortex

23. 6 Quercetin (50 mg/kg) 10 mg/kg iNOS mRNA and protein expression Liver, brain cortex

24. 6 Baicalein (50 mg/kg) 10 mg/kg iNOS mRNA and protein expression Liver, brain cortex

25. 6 Quercetin (50 mg/kg) Indole-3-carbinol (50 mg/kg) 10 mg/kg iNOS mRNA and protein expression Liver, brain cortex

26. 6 Baicalein (50 mg/kg) Luteolin (50 mg/kg) 10 mg/kg iNOS mRNA and protein expression Liver, brain cortex

E. ROSTOKA ET AL.


8

rats were decapitated under slight ether narcosis. In sev-eral groups lipopolysaccharide (10 mg/kg) was intrap-eritoneally injected to rats, substances or their combina-tions were administered per os in the same time, spin traps were administered 3.5 hours later, 30 minutes after spin trap injection rats were decapitated under slight ether narcosis. In additional piloting series of experi-ments (not shown) iNOS inhibitor AMT (2 mg/kg), was administered intraperitoneally shortly before spin-trap administration, 30 minutes later rats were decapitated. For real-time PCR and immunochemistry rats were de-capitated under slight ether narcosis; liver tissue was taken for RNA extraction and immunohistochemical examination. Brain cortex tissue was also taken from some animals for immunohistochemistry. Natural com-pounds and LPS were administered following the above time schedule.

2.3. Administration of Spin Trap Agents

To determine production level of nitric oxide in the tis-sues we used ESR spectroscopy of paramagnetic Fe- diethylthiocarbamate–nitric oxide complex (Fe (DETC)2- NO) [27]. Spin traps were administered 30 minutes be-fore the sacrifice. Rats were administered 400 mg/kg of the nitric oxide scavenger diethylthiocarbamate via in-traperitoneal injection and ferrous citrate subcutaneously (40 mg/kg ferrous sulphate + 200 mg/kg sodium citrate). Diethylthiocarbamate binds ferrous ion, the resulting complex traps nitric oxide converting to the paramag-netic Fe (DETC)2-NO complex that is detected by ESR spectroscopy.

2.4. Sacrifice, Organ Dissection and Sample Preparation for Electron Paramagnetic Resonance Spectroscopy

Following the drug and spin trap administration the rats were decapitated under slight ether anesthesia, samples of brain cortex, cerebellum, myocardium tissue, liver, kidney, testes, skeletal muscles, lungs and blood were compacted in a glass tube 30 mm in length with inner diameter 4 mm and immediately frozen in liquid nitrogen. Before recording the ESR spectra, the specimen was placed in a quartz finger Dewar flask ER 167 FDS-Q (Bruker, Karlsruhe, Germany) filled with liquid nitrogen.

2.5. ESR Measurements

ESR spectra were recorded in liquid nitrogen using an ESR spectrometer “Radiopan” SE/X2544 (Radiopan, Poznan, Poland). The conditions of the electron paramag-netic resonance measurements were: operation at X-band, 25 mW microwave power, 100 kHz modulation frequency, 5 G modulation amplitude, receiver gain 0.5 × 104, and

time constant 1 s. Spectra were recorded for 4 minutes. The nitric oxide content in the samples was evaluated from the height of the third component of the NO signal at g = 2.031.

The NO concentration (ng/g of tissue) was calculated on the basis of calibration curves as described previously. Briefly, different quantities of NaNO2 (final concentra-tions 10, 20, 30, 40, 60, 100 M) were mixed with DETC (33 mg/mL) and FeSO4·7H2O (3.3 mmol/L), an excess of Na2S2O4 (2 mol/L) was added to the mixture. The EPR spectra were taken as described above.

Further details are given in our previous publications [12-14,28-30]. 2.6. RNA Extraction and cDNA Preparation Total RNA was isolated from liver and brain cortex us-ing TRI reagent (Sigma Aldrich, Taufkirchen, Germany). DNA contaminations were removed with RNA-free kit (Ambion, Austin, TX, USA). The resulting RNA quan-tity and purity were determined by spectrophotometry, integrity of RNA molecules was monitored by gel elec-trophoresis, and only specimens with well-pronounced rRNA bands were taken for reactions. RNA (2 μg) was reverse-transcribed using a random hexamer primer (RevertAid™ First Strand cDNA Synthesis Kit, Fermen-tas, Vilnius, Lithuania) to obtain cDNA. 2.7. Real Time RT-PCR The mRNA expression rates of the brain cortex, liver iNOS and reference gene were determined using the SYBR® Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) according to the instructions pro-vided by the manufacturer. Amplification and detection of specific products were performed on a StepOne™ Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using the following temperature-time profile: one cycle of 95˚C for 10.00 min; and 40 cycles of 95˚C for 0.15 min, 60˚C for 1.00 min. To check speci-ficity of amplification products, the dissociation curve mode was used (one cycle at 95˚C for 0.15 min, 60˚C for 1 min and 95˚C for 0.15 min). To evaluate the suitability of candidates as reference genes, we applied the GeNorm program [31]. Primers were designed using Primer3 software. The primers were supplied by Metabion inter-national AG, Germany. The 2–∆∆CT method was applied for analysis of the results. Primer sequences for iNOS gene were 5’-GCTACACTTCCAACGCAACA-3’ for forward and 5’-CATGGTGAACACGTTCTTGG for reverse primer, the expected size of the product was 116 bp. RNA-polymerase II [32] was used as reference gene (5’- GCCAGAGTCTCCCATGTGTT-3’and 5’-GTCGGTGG GACTCTGTTTGT-3’, 135 bp).

E. ROSTOKA ET AL. 9 2.8. Histological and Immunohistochemical

Examination Paraffin-embedded tissue was cut in 4-micron-thick sec-tions and stained with haematoxylin and eosin for mor-phological examination. Infiltration of inflammatory cells in brain tissue was assessed in subcortical perivascular, subcortical parenchymal, and intracortical peri-vascular regions (magnification × 400). Perivascular infiltrates were defined as inflammatory cells, which are located not fur-ther than three cell layers from blood vessels. Inflamma-tory cells further than three layers from a blood vessel wall were defined as parenchymal infiltrates. Infiltration of inflammatory cells was assessed according to four score scale: 0－no infiltration; 1－light infiltration; 2－medium infiltration; 3－marked infiltration; 4－very marked infiltration (more than 25% of the total field of vision).

The morphology of liver tissue was evaluated by evaluating the histological activity index (HAI), as de-scribed [33]: infiltration of inflammatory cells (0-4 scores); necrosis of hepatocytes around a central vein (0-6 scores); necrosis of hepatocytes and apoptosis in periphery lobules (0-4 scores); inflammatory changes of portal tracts (0-4 scores).

Tissue sections were stained for visualization of iNOS positive cells by an immunochemical approach as previ-ously described [34]. Briefly, antigen retrieval was achieved by treatment in a microwave oven for 20 min at 300 W in citrate buffer, pH = 6.0. Endogenous peroxi-dase activity was blocked by 0.5 % H2O2 for 10 min. Nonspecific primary antibody binding was blocked by serum-free protein block for 10 min. Rabbit polyclonal active iNOS antibody Abcam Inc. (Cambridge, MA, USA) was applied in 1:200 dilution and incubated for 1h at room temperature in a humidified chamber. Detection of primary antibody binding was performed using spe-cific peroxidase conjugated polyclonal goat anti-rabbit IgG (1:100 for 30 min) and subsequently peroxidase conjugated polyclonal rabbit anti-goat IgG (1:100 for 30 min). The immunoperoxidase color reaction was devel-oped by incubation with diaminobenzidine (7 min). A negative control without primary antibody was included in each staining run. iNOS positive cells were counted in twenty high-powered fields at magnification × 400. All cell counts were expressed as cells per square millimeter. For morphological examination, at least 3 replicate measurements of iNOS positive cells were performed by the same observer in 10 randomly selected slides, and the intraobserver reproducibility was assessed with the coef-ficient of variation and with the interclass correlation coefficient. The intraobserver coefficient of variation was 4%, and the intraobserver correlation coefficient was 0.94.

2.9. Statistical Analysis Results were expressed as mean ± SD. The significance of differences in NO concentration and iNOS expression between groups was evaluated according to Student's unpaired t-test, the Mann-Whitney U test was used for quantification of immunohistochemical experiments. Re- sults were considered to be significant when P was less than 0.05.

3. Results

3.1. Effects of Natural Compounds and their Combinations on NO Production in Intact and LPS-Treated Rats

In order to test the ability of the natural compounds to modify NO production in animals the radical concentra-tion was monitored in several rat organs and tissues. Data are summarized in Figure 2. ESR spectra of the different organs had a typical Cu-DETC spectrum shape with a superposed Fe(DETC)2-NO peak, spectra were published previously [14]. The NO production reached the highest levels in the brain cortex, liver, lungs, and blood Figure 2. The NO production in heart and kidneys was an order of magnitude lower.

When control group of animals was compared to ani-mals treated with natural substances it turned out that baicalein decreased NO concentration in heart, kidney, liver and lungs (Figure 2). Luteolin decreased NO pro-duction in the liver and heart. Quercetin induced signifi-cant decrease of NO production the heart, kidneys and blood. Indole -3-carbinol caused a significant decrease of NO production in the cerebellum, spleen, blood, lungs and skeletal muscles.

In the following set of experiments the eventual activ-ity of the compounds as modifiers of NO production was tested against the background of the iNOS induction. Intraperitoneal injection of LPS to the animals caused a drastic increase of NO production levels in all tissues studied (Figure 2). The highest production of nitric ox-ide was detected in liver, whereas very strong increases in nitric oxide accumulation (50-100 fold compared to control) were observed in heart, blood and kidney. However, the effects of LPS were less pronounced in brain tissues where nitric oxide increased 4-6 times only. Nitric oxide production increase in testes was of compa-rable magnitude.

Baicalein (30 mg/kg) decreased NO concentration in brain cortex, liver, heart and kidneys. Luteolin (30 mg/kg) decreased NO outburst in all organs except skeletal mus-cles. In contrast, administration of the indole-3-carbinol (50 mg/kg) enhanced the LPS-induced increase of NO production in all organs except spleen and testes, Figure 2. Quercetin (50 mg/kg) produced similar effect: NO


E. ROSTOKA ET AL. 10

(a)

(b)

(c)

Figure 2. Effects of indole-3-carbinol, luteolin, baicalein quercetin and combinations of indole-3-carbinole + quercetin, baicalein + luteolin on NO production in rat organs of intact (a) and LPS-treated rats (b, c). Stars indicate statis-tically significant differences (P < 0.05) with control (a) or LPS (b, c) groups. production increased in testes blood and spleen (Figure 2).

The observed ability of individual compounds to mod-ify the LPS-triggered NO production raised the question about maintenance of the effects in presence of other substances. This approach modeled to some extent ad-ministration of flavonoid-containing herbal extracts. Baicalein as NO down-regulating and indole-3-carbinol as NO up-regulating substances were supplemented by luteolin and quercetin that produced NO-decreasing and NO-increasing effects correspondingly. Synergism of the effects was anticipated. Surprisingly, the results indi-

cated adverse effects in both cases. Combination of in-dole3-carbinol with quercetin (50/50) decreased NO concentration up-regulated by LPS in many tissues. Similarly, combination of baicalein and luteolin lost the NO-decreasing activity characteristic of individual sub-stances (Figure 2).

To test possible involvement of iNOS in the observed modifications natural substances were combined with iNOS inhibitor AMT (not shown). In intact animals the inhibitor markedly decreased NO production in all or-gans except the heart, AMT inhibiting action was attenu-ated by indole-3-carbinol in skeletal muscles. Quercetin also attenuated AMT effects in brain cortex, testes, blood and muscles. Luteolin slightly interfered with AMT ac-tion. These results indicated possible involvement of iNOS in effects of the compounds; this encouraged us to test ability of the substances to modify iNOS gene ex-pression in rat liver. 3.2. Effects of Natural Compounds on iNOS

Gene mRNA Expression in Intact and LPS-Treated Rats

No influence of indole-3-carbinol on iNOS expression in liver was observed (Figure 3). Surprisingly, luteolin up- regulated the gene expression. Baicalein decreased level of the gene expression. Level of transcription was still decreased when baicalein was given in combination with luteolin. Quercetin did not produce any significant effect on iNOS gene transcription, a tendency for increase was observed when it was given in combination with in-dole-3-carbinol (Figure 3(a)).

The LPS effect on the gene expression in the liver was drastic (Figure 3(b)) as it could be predicted from the increase of NO production (3442.82 761.24). However the effect was not well-reproducible between individual animals. Quercetin (50 mg/kg) significantly decreased the LPS-triggered iNOS mRNA expression. Enhance-ment of the iNOS mRNA expression by indole-3-carbinol was observed (7715.01 1877.35% compared to control, the result is not statistically significant). The same trend was observed when indole-3-carbinol was supplemented by quercetin (50 mg/kg). Baicalein somewhat decreased the gene expression triggered by LPS, the effect was better pronounced if it was combined with luteolin (Fig-ure 3(b)).

3.3. Effects of Natural Compounds on iNOS

Protein Expression in Intact and LPS-Treated Rats

Data on effects of the tested compounds on number of iNOS positive cells in rat liver and brain cortex are given in Figures 4 and 5. Interestingly, indole-3-carbinol pro-



*

0

500

1000

1500

2000

Control BaicaleinQuercetinIndole- 3-carbinol

Luteolin LuteolinBaicalein

Indole- 3-carbinol

Quercetin

iNO

S ge

ne ex

pres

sion

vs co

ntro

l A

(a)

##

0

500

1000

1500

2000

LPS BaicaleinLPS

QuercetinLPS

Indole- 3-carbinol

LPS

LuteolinLPS

Luteolin,Baicalein

LPS

Indole- 3-carbinol,Quercetin

LPS

iNO

S ge

ne ex

pres

sion

vs co

ntro

l B

(b)

Figure 2. Effects of indole-3-carbinol, luteolin, baicalein quercetin and combinations of indole-3-carbinole + quercetin, baicalein + luteolin on iNOS gene expression in rat liver. Results are presented as percentage vs average of the control. (a) intact animals; (b) LPS-treated animals. All compounds were administered in dose 50 mg/kg. *–P < 0.05 versus control group, #–P < 0.05 versus LPS group. duced statistically significant increase of the protein ex-pression; an effect is coherent with ESR data (Figure 2), the substance increased iNOS expression also in brain cortex (Figure 5(f)). Quercetin (50 mg/kg) and luteolin did not modify the protein expression level. Baicalein manifested a tendency to decrease number of iNOS- positive cells in the liver.

LPS significantly increased the number of iNOS posi-tive cells in liver and brain cortex tissue. Indole-3-carbinol decreased outburst of iNOS protein translation triggered by LPS both in liver tissue (predominantly in Kupfer cells) and brain cortex tissue (Figures 5(c) and (g)), in this case immunohistochemistry data are in contradiction with ESR data indicating enhancement of LPS effects by the compound. Baicalein significantly decreased the LPS effect. On the contrary, quercetin enhanced expression of the enzyme in the liver of LPS-treated animals; this ef-fect followed the trend observed in ESR experiments.

*

*

0

100

200

300

400

500

600

700

Control Indole- 3-carbinol

Quercetin Luteolin Baicalein

% o

f iNO

S po

sitiv

e cel

ls

A

(a)

##

#

0

100

200300

400

500

600700

800

900

LPS Indole- 3-carbinol

LPS

QuercetinLPS

LuteolinLPS

BaicaleinLPS

% o

f iNO

S po

sitiv

e cel

ls

B

(b)

Figure 3. Effects of indole-3-carbinol, luteolin, baicalein and quercetin on number of iNOS-positive cells in rat liver. Results are presented as percentage vs average of the con-trol. (a) intact animals; (b) LPS-treated animals. All com-pounds were administered in dose 50 mg/kg. *–P < 0.05 versus control group, #–P < 0.05 versus LPS group.

4. Discussion

4.1 Baicalein and luteolin

According published data baicalein suppresses iNOS gene expression in glia [35-37] and macrophages [38, 39] the inhibiting effect is achieved by decrease of lipoxine synthesis. In blood vessels this compound acts as antago-nist of nitric oxide, it inhibits the soluble guanylate cy-clase [40]. Baicalein depresses also the smooth muscle iNOS [41]. Luteolin is also is known as a weak inhibitor of iNOS expression, but it is not capable to inhibit the enzyme activity [17-20]. Some authors find that luteolin stimulates eNOS gene expression [21]. Apparently our results about decrease of NO concentration in some tissues after luteolin and baicalein administration are in good agreement with literature data, as this effect was observed both in intact and LPS-treated animals. Unexpect-edly combination of the two compounds did not produce


E. ROSTOKA ET AL.


12

(a) (b) (c) (d)

(e) (f) (g) (h)

Figure 5. Photomicrographs of in rat liver tissue stained immunohistochemically with iNOS antibody (a-d), and brain cortex tissue (e-h). (a,e)-control, (b,f)–indole-3-carbinol, (c,g)–LPS at dose 10 mg/kg; (d,h)–simultaneous administration of in-dole-3-carbinol and LPS. Arrows indicate iNOS positively stained cells. Magnification at ×100 (a-d) and at × 400 (e-h). this effect. iNOS gene and protein expression was de-creased by baicalein, this effect persisted when luteolin was administered to the same animal. Interestingly, Scutellaria baicalensis extract, i.e. combination of bai-calein with other compounds increases NO synthesis in induced macrophages [42]. Interesting explanation can be proposed on the basis of hypothesis about nitrite pro-tonation in acidic medium with generation of nitric acid. In this case flavonoids could reduce the acid to NO and stomach could turn into NO generator [43]. Simpler ex-planations can be also proposed. We have observed that higher dose of baicalein (100 mg/kg, not shown) pro-duced less pronounced NO-lowering effect compared to a lower dose (50 mg/kg). Probably cytotoxic effect of the substances interferes with NO-modulating activity. Both baicalein and luteolin are inducers of apoptosis, the sub-stances intercalate into DNA, inhibit topoisomerases I and II and DNA polymerase, induce TNF [44]. Thus apoptosis process can be triggered in many cells, NO level would increase as protection reaction against apop-tosis [45]. 4.2. Indole-3-Carbinol and Quercetin Indole-3-carbinol is a weak iNOS induction inhibitor [22]. The substance interferes with several signaling pathways [46]. Decrease of NO concentration in blood, lungs and skeletal muscles caused by the substance can also indirectly indicate iNOS-inhibiting activity of the compound. Surprisingly, inverse effect was observed when we assessed number of iNOS-positive cells in the

liver of intact rats. The compound enhanced increase of the gene transcription triggered by LPS in the liver, NO production in liver of intact animals and the LPS-induced increase of NO production in all the organs studied, amazingly, number of iNOS-positive cells in the liver of LPS-treated animals dropped down after administration of the substance. Thus the substance can either increase or decrease NO production in rat tissues. The effect de-pends on the tissue and physiological state of the animal. The NO decreasing activity of indole-3-carbinol can be explained by its capability to reduce NF-kB DNA bind-ing activity [16,46]. Moreover the substance was shown to inhibit Akt-kinase activity, this leads to decrease of NF-kB expression [16,46]. Ability of the compound to increase the NO production in some organs, especially in LPS-treated animals could be rather ascribed to antioxi-dant activity of indole-3-carbinol [22]. Scavenging of reactive oxygen species prevents involvement of NO in interaction with these radicals increasing its bioavailabil-ity. This effect is produced by several natural compounds including cocoa polyphenols [47] and resveratrol [48]. Increase of enzyme expression by the drug indicates ex-istence of some mechanism for regulation on transcrip-tion level. Enhancement by the substance of nitric oxide production induced by lypopolysaccharide upregulation of protein kinase C is also quite possible [49,50].

Quercetin decreased NO concentration in the heart, kidneys and blood; however it enhanced the LPS effect in testes, blood and spleen. The substance attenuated the LPS effects on the level of iNOS gene expression, how-ever it enhanced these effects on protein expression level. Data about quercetin impact on NO synthesis are rather

E. ROSTOKA ET AL. 13 contradictory. The substance inhibits iNOS induction, the effect is better detectable in in vitro cultured cells [6,7,51]. In vivo quercetin did not decrease NFkB activa-tion in kidney cortex [52]. In systems, where iNOS ex-pression decrease by quercetin was observed, the flavon-oid did not down-regulate NFkB activation [7,53]. Proba-bly, quercetin interferes with tyrosin kinase-mediated pathways [54], and decreases TNFα expression [55]. However other authors observed the quercetin-induced inhibition of IkBα (inhibitor of kappa B alpha) degrada-tion via depression of IkB kinase activity, this leads to inhibition of NFkB [56]. Other reports inform about prevention and/or inhibition of IkB phosphorylation [57, 58], depression of NFkB activation by interleukin [59] and hydrogen peroxide [60] produced by the flavonoid. Inhibition of iNOS expression by quercetin is often as-cribed to inhibition of the NFkB pathway [61,62]. Inter-estingly, some authors [63] find out that quercetin does not inhibit iNOS gene expression; however it inhibits iNOS enzyme expression. We came up to quite an oppo-site conclusion; however both the cited and our data in-dicate separate mechanisms for regulation of the iNOS gene and protein expression by this substance. Quercetin also destabilizes eNOS mRNA [8]. There are even data about ability of the substance to inhibit NOS enzymatic activity [9]. In the same time quercetin protects epithe-lium against lesions produced by NOS inhibitors [64] and stimulates NO synthesis in leukemia cells as protec-tion reaction against induction of apoptosis [45]. Quercetin is considered to be the main active compound of red wine; some authors have observed NO-dependent vaso- relaxation induced by quercetin [10]. Supplementation of diet with quercetin favors NO production in endothelium [5]. However the nitric oxide release from endothelium was not detected in special studies [11]. Quercetin is also one of the active substances of the Gingko biloba ex-tracts, both purified quercetin and Gingko biloba extracts (i.e. mixture of quercetin with other natural compounds) decrease LPS-induced iNOS expression, however the extract acts via NF-kB inhibition, but quercetin inhibits TNFa pathway [65,66]. In our studies quercetin in-creased the LPS-triggered NO outburst in lungs, testes and lungs. Apparently, the NOS-inhibiting effect was not detectable on organism level. Our data contradict for-merly published reports about decrease of nitrite produc-tion in brain [67] and blood plasma of LPS-treated ani-mals [68] or streptozotocin-treated animals [69] by quercetin. However nitrite and NO production levels do not always correlate. When NO production was assessed by an approach similar to ours the quercetin-induced increase of NO production in rat brain was also observed [70]. Moreover the substance did not modify much iNOS gene expression in both healthy and LPS-treated animals. This effect can be associated with apoptosis-promoting activity of quercetin [71]. Probably simultaneous ad-ministration of indole-3-carbinol and quercetin favored

manifestation of NOS-inhibiting activity of quercetin and abolished increase of NO bioavailability produced by indole-3-carbinol, as no decrease of iNOS expression was observed in this case. NO scavenging activity of quercetin [69-73] also could manifest itself in this case.

5. Conclusions

Taken together our results suggest that modifications of NO level in tissues by the studied natural compounds cannot be predicted from data about its effects on NOS expression or activity. Effects of individual compounds are not additive when these are administered in combina-tion. This stresses importance of direct measurements of NO in the tissues using ESR method.

6. Acknowledgements

This study was supported in part by the National Pro-gram “Novel drugs and biocorrection remedies: con-struction, transport forms and mechanisms of action sub-project Study of synergism and resistance phenom-ena of antic-cancer substances and creation of novel anticancer drugs” managed by I. Kalvinsh and the grant 04.1317, Pathological production of nitric oxide, possi-bilities of its pharmacological correction” awarded to N. Sjakste by the Latvian Council of Science. We thank L. Lauberte for technical assistance. Participation of D. Meirena in experiment design and discussion is greatly appreciated.

7. References

[1] H. Y. Li, L. Cui and M. Cui, “Hot Topics in Chinese Herbal Drugs Research Documented in PubMed/Medline by Authors inside China and Outside of China in the Past 10 Years: Based on Co-Word Cluster Analysis,” Journal of Alternative and Complementary Medicine, Vol. 15, No. 7, 2009, pp. 779-785.

[2] H. Zeng, X. Liu, S. Dou, W. Xu, N. Li, X. Liu, W. Zhang, Z. Hu and R. Liu, “Huang-Lian-Jie-Du-Tang Exerts Anti- Inflammatory Effects in Rats through Inhibition of Nitric Oxide Production and Eicosanoid Biosynthesis via the Lipoxygenase Pathway” Journal of Pharmacy and Pharmacology, Vol. 61, No. 2, 2009, pp. 1699-1707.

[3] V. Bayard, F. Chamorro, J. Motta and N. K. Hollenberg, “Does Flavanol Intake Influence Mortality from Nitric Oxide-Dependent Processes? Ischemic Heart Disease, Stroke, Diabetes Mellitus, and Cancer in Panama,” In-ternational Journal of Medical Microbiology, Vol. 4, No. 1, 2007, pp. 53-58.

[4] C. A. Schmitt and V. M. Dirsch, “Modulation of Endothe Lial Nitric Oxide by Plant-Derived Products,” Nitric Ox-ide, Vol. 21, No. 2, 2009, pp. 77-91.

[5] S. Benito, D. Lopez, M. P. Saiz, S. Buxaderas, J. Sanchez, P. Puig-Parellada and M. T. Mitjavila, “A Flavonoid-Rich



Diet Increases Nitric Oxide Production in Rat Aorta,” British Journal of Pharmacology, Vol. 135, No. 4, 2002, pp. 910-916.

[6] R. Olszanecki, A. Gebska, V. I. Kozlovski and R. J. Gry-glewski, “Flavonoids and Nitric Oxide Synthase,” Jour-nal of Physiology and Pharmacology, Vol. 53, No. 4, 2002, pp. 571-584.

[7] B. H. Kim, S. M. Cho, A. M. Reddy, Y. S. Kim, K. R. Min and Y. Kim, “Down-regulatory Effect of Quercitin Gallate on Nuclear Factor-Kappa B-Dependent Inducible Nitric Oxide Synthase Expression in Lipopolysaccharide- Stimulated Macrophages RAW 264.7,” Biochemical Pharmacology, Vol. 69, No. 11, 2005, pp. 1577-1183.

[8] T. Wallerath, H. Li, U. Godtel-Ambrust, P. M. Schwarz and U. Forstermann, “A Blend of Polyphenolic Com-pounds Explains the Stimulatory Effect of Red Wine on Human Endothelial NO Synthase,” Nitric Oxide, Vol. 12, No. 2, 2005, pp. 97-104.

[9] L. Luo, Q. Sun, Y. Y. Mao, Y. H. Lu and R. X. Tan, “In-hibitory Effects of Flavonoids from Hypericum Perfora-tum on Nitric Oxide Synthase,” Journal of Ethnophar-macology, Vol. 93, No. 2-3, 2004, pp. 221-225.

[10] C. K. Chen and C. R. Pace-Asciak, “Vasorelaxing Activ-ity of Resveratrol and Quercetin in Isolated Rat Aorta,” General Pharmacology, Vol. 27, No. 2, 1996, pp. 363- 366.

[11] J. C. Stoclet, A. Kleschyov, E. Andriambeloson, M. Die-bolt and R. Andriantsitohaina, “Endothelial NO Release Caused by Red Wine Polyphenols,” Journal of Physiol-ogy and Pharmacology, Vol. 50, No. 4, 1999, pp. 535- 540.

[12] N. Sjakste, A. L. Kleschyov, J. L. Boucher, L. Baumane, M. Dzintare, D. Meirena, J. Sjakste, K. Sydow, T. Mün-zel and I. Kalvinsh, “Endothelium- and Nitric Oxide- Dependent Vasorelaxing Activities of Gamma-Butyrobetaine Esters: Possible Link to the Antiischemic Activities of Mildronate,” European Journal of Pharmacology, Vol. 495, No. 1, 2004, pp. 67-73.

[13] N. Sjakste, J. Sjakste, J. L. Boucher, L. Baumane, T. Sjakste, M. Dzintare, D. Meirena, J. Sharipova and I. Kalvinsh, “Putative Role of Nitric Oxide Synthase Iso-forms in the Changes of Nitric Oxide Concentration in Rat Brain Cortex and Cerebellum Following Sevoflurane and Isoflurane Anaesthesia,” European Journal of Phar-macology, Vol. 513, No. 3, 2005, pp. 193-205.

[14] N. Sjakste, V. G. Andrianov, J. L. Boucher, I. Shestakova, L. Baumane, M. Dzintare, D. Meirena and I. Kalvins, “Paradoxical Effects of Two Oximes on Nitric Oxide Production by Purified NO Synthases, in Cell Culture and in Animals,” Nitric Oxide, Vol. 17, No. 3-4, 2007, pp. 107-114.

[15] M. E. van Meeteren, J. J. Hendriks, C. D. Dijkstra and E. A. van Tol, “Dietary Compounds Prevent Oxidative Damage and Nitric Oxide Production by Cells Involved in Demyelinating Disease,” Biochemical Pharmacology, Vol. 67, No. 5, 2004, pp. 967-975.

[16] K. M. Rahman, S. Ali, A. Aboukameel, S. H. Sarkar, Z. Wang, P. A. Philip, W. A. Sakr and A. Raz, “Inactivation

of NF-kappaB by 3,3'-diindolylmethane Contributes to Increased Apoptosis Induced by Chemotherapeutic Agent in Breast Cancer Cells,” Molecular Cancer Therapeutics, Vol. 6, 2007, pp. 2757-2765.

[17] S. J. Kim, H. Park and H. P. Kim, “Inhibition of Nitric Oxide Production from Lipopolysaccharide-Treated RAW 264.7 Cells by Synthetic Flavones: Structure- Activity Relationship and Action Mechanism,” Archives of Pharmacal Research, Vol. 27, No. 9, 2004, pp. 937- 943.

[18] J. S. Kim, H. J. Lee, M. H. Lee, J. Kim, C. Jin and J. H. Ryu “Luteolin Inhibits LPS-Stimulated Inducible Nitric Oxide Synthase Expression in BV-2 Microglial Cells,” Planta Medica, Vol. 72, No. 1, 2006, pp. 65-68.

[19] C. Hu and D. D. Kitts, “Luteolin and Luteolin-7-O-gluco- side from Dandelion Flower Suppress iNOS and COX-2 in RAW264.7 Cells,” Molecular and Cellular Biochem-istry, Vol. 265, 2004, pp. 107-113.

[20] L. S. Scuro, P. U. Simioni, D. L. Grabriel, E. E. Saviani, L. V. Modolo, W. M. Tamashiro and I. Salgado, “Sup-pression of Nitric Oxide Production in Mouse Macro-phages by Soybean Flavonoids Accumulated in Response to Nitroprusside and Fungal Elicitation,” BMC Medicine, Vol. 5, 2004, p. 5.

[21] H. Li, N. Xia, I. Brausch, Y. Yao and U. Forstermann, “Flavonoids from Artichoke (Cynara scolymus L.) up- Regulate Endothelial-Type Nitric-Oxide Synthase Gene Expression in Human Endothelial Cells,” Journal of Pharmacology and Experimental Therapeutics, Vol. 310, No. 3, 2004, pp. 926-932.

[22] C. Gerhäuser, K. Klimo, E. Heiss, I. Neumann, A. Ga-mal-Eldeen, J. Knauft, G. Y. Liu, S. Sitthimonchai and N. Frank, “Mechanism-based in Vitro Screening of Potential Cancer Chemopreventive Agents,” Mutation Research, Vol. 523-524, 2003, pp. 163-172.

[23] S. Su, J. Guo, J. Duan, T. Wang, D. Qian, E. Shang and Y. Tang, “Ultra-performance Liquid Chromatography －Tandem Mass Spectrometry Analysis of the Bioactive Components and their Metabolites of Shaofu Zhuyu De-coction Active Extract in Rat Plasma,” Journal of Chro-matography B Analytical Technologies in the Biomedical and Life Sciences, 2009. (in press)

[24] H. J. Chen, X. Li, J. W. Chen, S. Guo and B. C. Cai, “Simultaneous Determination of Eleven Bioactive Com-pounds in Saururus Chinensis from Different Harvesting Seasons by HPLC-DAD,” Journal of Pharmaceutical and Biomedical Analysis, 2010. (in press)

[25] C. Li, H. Du, L. Wang, Q. Shu, Y. Zheng, Y. Xu, J. Zhang, J. Zhang, R. Yang and Y. Ge, “Flavonoid Com-position and Antioxidant Activity of Tree Peony (Paeonia section moutan) Yellow Flowers,” Journal of Agricul-tural and Food Chemistry, Vol. 57, No. 18, 2009, pp. 8496-8503.

[26] X. Liang, L. Zhang, X. Zhang, W. Dai, H. Li, L. Hu, H. Liu, J. Su and W. Zhang, “Qualitative and Quantitative Analysis of Traditional Chinese Medicine Niu Huang Jie Du Pill Using Ultra Performance Liquid Chromatography Coupled with Tunable UV Detector and Rapid Resolution



Liquid Chromatography Coupled with Time-of-Flight Tandem Mass Spectrometry,” Journal of Pharmaceutical and Biomedical Analysis, Vol. 51, No. 3, 2010, pp. 565- 571.

[27] A. L. Kleschyov, P. Wenzel and T. Münzel, “Electron Paramagnetic Resonance (EPR) Spin Trapping of Bio-logical Nitric Oxide,” Journal of Chromatography B Analytical Technologies in the Biomedical and Life Sci-ences, Vol. 851, No. 1-2, 2007, pp. 12-20.

[28] N. Sjakste, L. Baumane, D. Meirena, L. Lauberte, M. Dzintare and I. Kalvinsh, “Drastic Increase in Nitric Ox-ide Content in Rat Brain under Halothane Anesthesia, Revealed by EPR Method,” Biochemical Pharmacology, Vol. 58, No. 12, 1999, pp. 1955-1959.

[29] L. Baumane, M. Dzintare, L. Zvejniece, D. Meirena, L. Lauberte, V. Sile, I. Kalvinsh and N. Sjakste, “Increased Synthesis of Nitric Oxide in Rat Brain Cortex Due to Halogenated Volatile Anesthetics Confirmed by ESR Spectroscopy,” Acta Anaesthesiologica Scandinavica, Vol. 46, No. 4, 2002, pp. 378-383.

[30] E. Rostoka, L. Baumane, S. Isajevs, A. Line, M. Dzintare, D. Svirina, J. Sharipova, K. Silina, I. Kalvinsh and N. Sjakste, “Effects of Kaempferol and Myricetin on Induc-ible Nitric Oxide Synthase Expression and NO Produc-tion in Rats,” Basic & Clinical Pharmacology & Toxi-cology, 2010. (in press)

[31] J. Vandesompele, K. De Preter, F. Pattyn, B. Poppe, N. Van Roy, A. De Paepe and F. Speleman, “Accurate Nor-malization of Real-Time Quantitative RT-PCR Data by Geometric Averaging of Multiple Internal Control Genes,” Genome Biology, Vol. 18, 2002.

[32] A. Radonić, S. Thulke, I. M. Mackay, O. Landt, W. Siegert and A. Nitsche, “Guideline to Reference Gene Selection for Quantitative Real-Time PCR,” Biochemical and Biophysical Research Communications, Vol. 313, No. 4, 2004, pp. 856-862.

[33] K. Ishak, A. Baptista, L. Bianchi, F. Callea, J. De Groote, J. Gudat, F. Gudat, H. Denk, V. Desmet, G. Korb, R. N. MacSween, “Histological Grading and Staging of Chronic Hepatitis,” Journal of Hepatology, Vol. 22, No. 6, 1995, pp. 696-699.

[34] A. D. Stefano, G. Caramori, T. Oates, A. Capelli, M. Lusuardi, I. Gnemmi, F. Ioli, K. F. Chung, C. F. Donner, P. J. Barnes and I. M. Adcock, “Increased Expression of Nuclear Factor-kB in Bronchial Biopsies from Smokers and Patients with COPD,” European Respiratory Journal, Vol. 20, 2000, pp. 556-563.

[35] J. S. Won, Y. B. Im, M. Khan, A. K. Singh and I. Singh, “Involvement of Phospholipase A2 and Lipoxygenase in Lipopolysaccharide-Induced Inducible Nitric Oxide Syn- Thase Expression in Glial Cells,” Glia, Vol. 51, 2005, pp. 13-21.

[36] C. J. Chen, S. L. Raung, S. L. Liao and S. Y. Chen “Inhi-bition of Inducible Nitric Oxide Synthase Expression by Baicalein in Endotoxin/Cytokine-Stimulated Microglia,” Biochemical Pharmacology, Vol. 67, No. 5, 2004, pp. 957-965.

[37] K. Suk, H. Lee, S. S. Kang, G. J. Cho and W. S. Choi,

“Flavonoid Baicalein Attenuates Activation-Induced Cell Death of Brain Microglia,” Journal of Pharmacology and Experimental Therapeutics, Vol. 305, No. 2, 2003, pp. 638-645.

[38] M. Vivancos and J. J. Moreno, “Role of Ca2+-Independent Phospholipase A(2) and Cyclooxygenase/Lip-Oxygenase Pathways in the Nitric Oxide Production by Murine Macrophages Stimulated by Lipopolysaccharides,” Nitric Oxide, Vol. 6, No. 3, 2002, pp. 255-262.

[39] I. Wakabayashi, “Inhibitory Effects of Baicalein and Wogonin on Lipopolysaccharide-Induced Nitricoxide Production in Macrophages,” Pharmacology & Toxicol-ogy, Vol. 84, 1999, pp. 288-291.

[40] Y. Huang, C. M. Wong, C. W. Lau, X. Yao, S. Y. Tsang, Y. L. Su and Z. Y. Chen, “Inhibition of Nitric Ox-ide/Cyclic GMP-Mediated Relaxation by Purified Fla-vonoids, Baicalin and Baicalein, in Rat Aortic Rings,” Biochemical Pharmacology, Vol. 67, No. 4, 2004, pp. 787-794.

[41] T. Hashimoto, M. Kihara, K. Yokoyama, T. Fujita, S. Kobayashi, K. Matsushita, K. Tamura, N. Hirawa, Y. Toya and S. Umemura, “Lipooxygenase Products Regu-late Nitric Oxide and Inducible Nitric Oxide Synthase Production in Interleukin-1beta Stimulated Vascular Smooth Muscle Cells,” Hypertension Research, Vol. 26, 2003, pp. 177-184.

[42] H. M. Kim, E. J. Moon, E. Li, K. M. Kim, S. Y. Nam and C. K. Chung, “The Nitric Oxide-Producing Activities of Scutellaria Baicalensis,” Toxicology, Vol. 135, No. 2-3, 1999, pp. 109-115.

[43] U. Takahama, T. Oniki and S. Hirota, “Oxidation of Quercetin by Salivary Components. Quercetin-Dependent Reduction of Salivary Nitrite under Acidic Conditions Producing Nitric Oxide,” Journal of Agricultural and Food Chemistry, Vol. 50, 2002, pp. 4317-4322.

[44] R. D. Snyder and P. J. Gillies, “Evaluation of the Clasto-genic, DNA Intercalative, and Topoisomerase II-Interactive Properties of Bioflavonoids in Chinese Hamster V79 Cells,” Environmental and Molecular Mutagenesis, Vol. 40, 2002, pp. 266-276.

[45] C. Kellner and S. J Zunino, “Nitric Oxide Is Synthesized in Acute Leukemia Cells after Exposure to Phenolic Anti- Oxidants and Initially Protects against Mitochondrial Membrane Depolarization,” Cancer Letter, Vol. 215, No. 1, 2004, pp. 43-52.

[46] F. H. Sarkar and Y. Li, “Cell Signaling Pathways Altered by Natural Chemopreventive Agents,” Mutation Re-search, Vol. 555, No. 1-2, 2004, pp. 53-64.

[47] H. Sies, T. Schewe, C. Heiss and M. Kelm, “Cocoa Polyphenols and Inflammatory Mediators,” American Journal of Clinical Nutrition, Vol. 81, 2005, pp. 304S- 312S.

[48] F. Orallo, E. Alvarez, M. Camiña, J. M. Leiro, E. Gómez and P. Fernández, “The Possible Implication of Trans- Resveratrol in the Cardioprotective Effects of Long-Term Moderate Wine Consumption,” Molecular Pharmacology, Vol. 61, No. 2, 2002, pp. 294-302.

[49] K. Fukuzawa, K. Kogure, M. Morita, S. Hama, S. Ma-



nabe and A. Tokumura, “Enhancement of Nitric Oxide and Superoxide Generations by Alpha-Tocopheryl Suc-cinate and its Apoptotic and Anticancer Effects,” Bio-chemistry, Vol. 69, 2004, pp. 50-57.

[50] K. Kogure, M. Morita, S. Hama, S. Nakashima, A. To- kumura and K. Fukuzawa, “Enhancement by Alpha- to-Copheryl Hemisuccinate of Nitric Oxide Production Induced by Lypopolysaccharide and Interferon-Gamma through the Upregulation of Protein Kinase C in Rat Vas-cular Smooth Muscle Cells,” European Journal of Bio-chemistry, Vol. 269, 2002, pp. 2367-2372.

[51] G. M. Raso, R. Meli, G. Di Carlo, M. Pacilio and R. D. Carlo, “Inhibition of Inducible Nitric Oxide Synthase and Cyclooxygenase-2 Expression by Flavonoids in Macro-phage J774A.1,” Life Sciences, Vol. 68, 2001, pp. 921-931.

[52] G. K. Rangan, Y. Wang and D. C. Harris, “Dietary Quercetin Augments Activator Protein-1 and Does Not Reduce Nuclear Factor-Kappa B in the Renal Cortex of Rats with Established Chronic Glomerular Disease,” Nephron, Vol. 90, No. 3, 2002, pp. 313-319.

[53] T. L. Wadsworth and D. R. Koop, “Effects of the Wine Polyphenolics Quercetin and Resveratrol on Pro- inflammatory Cytokine Expression in RAW 264.7 Macro-phages,” Biochemical Pharmacology, Vol. 57, No. 8, 1999, pp. 941-949.

[63]

[54] S. Wang, V. L. DeGroff and S. K. Clinton, “Tomato and Soy Polyphenols Reduce Insulin-Like Growth Factor-I- Stimulated Rat Prostate Cancer Cell Proliferation and Apoptotic Resistance in Vitro via Inhibition of Intracel-lular Signaling Pathways Involving Tyrosine Kinase,” Journal of Nutrition, Vol. 133, 2003, pp. 2367-2376.

[55] J. L. Pang, D. A. Ricupero, S. Huang, N. Fatma, D. P. Sing, J. S. Romero and N. Chattopadhyay, “Differential Activity of Kaempferol and Quercetin in Attenuating Tumor Necrosis Factor Receptor Family Signaling in Bone Cells,” Biochemical Pharmacology, Vol. 71, No. 6, 2006, pp. 818-886.

[56] V. García-Mediavilla, I. Crespo, P. S. Collado, A. Esteller, S. Sánchez-Campos, M. J. Tuñón and J. González-Gallego, “The Anti-Inflammatory Flavones Quercetin and Kaemp- ferol Cause Inhibition of Inducible Nitric Oxide Synthase, Cyclooxygenase-2 and Reactive C-Protein, and Down- Regulation of the Nuclear Factor Kappab Pathway in Chang Liver Cells,” European Journal of Pharmacology, Vol. 557, No. 2-3, 2007, pp. 221-229.

[57] M. Comalada, D. Camuesco, S. Sierra, I. Ballester, J. Xaus, J. Galvez and A. Zarzuelo, “In Vivo Quercitrin Anti-Inflammatory Effect Involves Releases of Quercetin, Which Inhibits Inflammation through Down-Regulation of NF-Kappab Pathway,” European Journal of Immu-nology, Vol. 35, 2005, pp. 584-592.

[58] M. P. Nair, S. Mahajan, J. L. Reynolds, R. Aalinkeel, H. Nair, S. A. Schwartz and C. Kandaswami, “The Flavon-oid Quercetin Inhibits Proinflammatory Cytokine (Tumor Ne-Crosis Factor Alpha) Gene Expression in Normal Pe-ripheral Mononuclear Cells via Modulation of the NF-ΚB System,” Clinical and Vaccine Immunology, Vol. 13, 2006, pp. 319-328.

[59] K. Muraoka, K. Shimizu, X. Sun, T. Tani, R. Izumumi, K. Miwa and K. Yamamoto, “Flavonoids Exert Diverse In-hibitory Effects on the Activation of NF-kB,” Transplan-tation Proceedings, Vol. 34, 2002, pp. 1335-1340.

[60] C. A. Musonda and J. K. Chipman, “Quercetin Inhibits Hydrogen Peroxide (H2O2)-Induced NF-kappaB DNA Binding Activity and DNA Damage in HepG2 Cells,” Carcinogenesis, Vol. 19, No. 9, 1998, pp. 1583-1589.

[61] S. Martínez-Flórez, M. B. Gutiérrez S. Sánchez-Campos, J. González-Gallego and M. J. Tuñón, “Quercetin Pre-vents Nitric Oxide Production and Nuclear Factor Kappa B Activation in Interleukin-1Β-Activated Rat Hepato-cytes,” Journal of Nutrition, Vol. 135, 2005, pp. 1359- 1365.

[62] J. Chen, F. M. Ho, P. L. Chao, C. P. Chen, K. G. Jeng, H. B. Hsu, S. T. Lee, W. Wen Tung and W. W. Lin, “Inhibi-tion of iNOS Gene Expression by Quercetin Is Mediated By the Inhibition of IΚB Kinase, Nuclear Factor-Kappa B and STAT1, and Depends on Heme Oxygenase-1 Induc-tion in Mouse BV-2 Microglia,” European Journal of Pharmacology, Vol. 521, No. 1-3, 2005, pp. 9-20.

T. Banerjee, A. Van der Vliet and V. A. Ziboh, “Down- Regulation of COX-2 and iNOS by Amentoflavone and Quercetin in A549 Human Lung Adenocarcinoma Cell Line,” Prostaglandins Leukot Essent Fatty Acids, Vol. 66, No. 5-6, 2002, pp. 485-492.

[64] J. Duarte, R. Jiménez, F. O'Valle, M. Galisteo, R. Pérez- Palencia, F. Vargas, F. Pérez-Vizcaíno, A. Zar-Zuelo and J. Tamargo, “Protective Effects of the Flavonoid Quercetin in Chronic Nitric Oxide Deficient Rats,” Journal of Hy-pertension, Vol. 20, No. 9, 2002, pp. 1843-1854.

[65] T. L. Wadsworth and D. R. Koop, “Effects of Ginkgo Biloba Extract (EGb 761) and Quercetin on Lipopoly-saccharide-Induced Release of Nitric Oxide,” Chemico- Biological Interaction, Vol. 137, No. 1, 2001, pp. 43-58.

[66] T. L. Wadsworth, T. L. McDonald and D. R. Koop, “Ef-fects of Ginkgo Biloba Extract (EGb 761) and Quercetin on Lipopolysaccharide-Induced Signaling Pathways In-volved in the Release of Tumor Necrosis Factor-Alpha,” Biochemical Pharmacology, Vol. 62, No. 7, 2001, pp. 963-974.

[67] H. M. Abd El-Gawad and A. E. Khalifa, “Quercetin, Coenzyme Q10, and L-canavanine as Protective Agents against Lipid Peroxidation and Nitric Oxide Generation in Endotoxin-Induced Shock in Rat Brain,” Pharmacol-ogical Research, Vol. 43, No. 3, 2001, pp. 257-263.

[68] S. C. Shen, W. R. Lee, H. Y. Lin, H. C. Huang, C. H. Ko, L. L. Yang and Y. C. Chen, “In Vivo and In Vitro In-hibitory Activities of Rutin, Wogonin and Quercetin on Lipopolysaccharide-Induced Nitric Oxide and Prostaglan- Din E(2) Production,” European Journal of Pharmacol-ogy, Vol. 446, No. 1-3, 2002, pp. 187-194.

[69] O. Coskun, M. Kanter, A. Korkmaz and S. Oter, “Quercetin, a Flavonoid Antioxidant, Prevents and Pro-tects Streptozotocin-Induced Oxidative Stress and Beta- Cell Damage in Rat Pancreas,” Pharmacological Re-search, Vol. 51, No. 2, 2005, pp. 117-123.

[70] Z. Shutenko, Y. Henry, E. Pinard, J. Seylaz, P. Potier, F.


E. ROSTOKA ET AL.


17

Berthet, P. Girard and R. Sercombe, “Influence of the Antioxidant Quercetin in Vivo on the Level of Nitric Oxide Determined by Electron Paramagnetic Resonance in Rat Brain During Global Ischemia and Reperfusion,” Biochemical Pharmacology, Vol. 57, No. 2, 1999, pp. 199-208.

[71] J. Da Silva, S. M. Hermann, V. Heuser, W. Peres, N. Marroni, J. González-Gallego and H. Erdtmann, “Evalua-tion of the Genotoxic Effect of Rutin and Quercetin by Comet Assay and Micronucleus Test,” Food and Chemi-cal Toxicology, Vol. 40, No. 7, 2002, pp. 941-947.

[72] M. Číž, M. Pavelková, L. Gallová, J. Králová, L. Kubala and A. Lojek, “The Influence of Wine Polyphenols on Reactive Oxygen and Nitrogen Species Production by Murine Macrophages RAW 264.7,” Physiological Re-search, Vol. 57, 2008, pp. 393-402.

[73] S. Bastianetto, W. H. Zheng and R. Quirion, “Neuropro-tective Abilities of Resveratrol and Other Red Wine Con-stituents against Nitric Oxide-Related Toxicity in Cul-tured Hippocampal Neurons,” British Journal of Phar-macology, Vol. 131, No. 4, 2000, pp. 711-720.



Antihepatotoxic Effect of Feronia Limonia Fruit against Carbon Tetrachloride Induced Hepatic Damage in

Albino Rats

Rucha Upadhyay1, Narendra Deo Pandey1, Shahid Suhail Narvi1, Amita Verma2, Bahar Ahmed2 1Department of Chemistry Motilal Nehru National Institute of Technology, Allahabad, India

2Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hamdard University, New Delh, India E-mail: [email protected]

Received March 10, 2010; revised April 28, 2010; accepted May 10, 2010

Abstract Feronia limonia Swingle, though mentioned as a high utility medicinal plant in Ayurveda for the treatment of liver disorders, but has no scientific investigations. Therefore, the present study was undertaken to inves-tigate the hepatoprotective activity of extracts of Feronia limonia fruit on albino rat liver, damage induced by single dose of carbon-tetrachloride (1.5 ml/kg, p.o.). The oral administration of extracts (500 mg/kg, p.o.) for 7 days has shown significant hepatoprotective action. There is reduction in the elevated levels of serum en-zymes such SGOT, SGPT, ALP and Serum bilirubin and elevation in level of GSH, GPX,GLY and total pro-tein in Feronia limonia protected rats. Keywords: Anti-Hepatotoxic, Carbon Tetrachloride, Feronia Limonia Swingle

1. Introduction Feronia limonia Swingle (Syn. Feronia elephantum Correa, Fam Rutaceae) are described in ayurvedic litera-ture as Kapittha is a moderate-sized tree with straight sharp strong spines, 1.2-3.7 cm long, which is widely distributed throughout Bangladesh, Srilanka, Java and India [1-3]. The fruits of the plant are edible and consid-ered to be a stomachic, astringent, diuretic, cardiotonic and tonic to the liver and lungs [2]; the bark is occasion-ally used for biliousness [4]; the leaves are aromatic and carminative and are used for the treatment of indigestion and minor bowel infections of children [2]; the roots are sometimes given for the treatment of snake-bites [2]. Previous phytochemical work was mainly focused on the isolation of coumarins [5-7], steroids and volatile com-ponents [8, 9] of the plant.

Feronia limonia has great reputation in Ayurvedic medicine for treatment of liver disorders. No scientific and methodical investigations have so far been reported in literature regarding its action on liver. Hence the pre-sent study is focused to evaluate the hepatoprotective potentials of the Feronia limonia fruits against CCl4 in-duced liver injury in rats.

2. Experimental 2.1. Plant Collection Fruits of Feronia limonia were collected during Sep-tember-October 2006 from wild area in the vicinity of Allahabad. The fruit of plant was authenticated by Dr. M.P. Sharma, Reader and taxonomist, Department of Botany, Hamdard University, New Delhi. A voucher specimen (No. PD-FP-32) of plant was kept in herbarium of Hamdard University, New Delhi. 2.2. Preparation of Extract Coarsely powdered dry fruits of Feronia limonia (500 g) were extracted to exhaustively with petroleum ether (60-80˚C), chloroform and methanol using a sohxlet ap-paratus. The extracts thus obtained were dried under re-duce pressure yielding 6.9%, 17.4%, 31.8% powder with reference to dry starting material respectively. 2.3. Experimental Animals Male albino rats of wistar strain (150-200 g) were em-

R. UPADHYAY ET AL. 19 ployed for assessing the antihepatotoxic activity. They were procured from the Central Animal House of Jamia Hamdard, New Delhi Sanction Letter No. (173/CPCSEA), after approval under the project proposal number-326. They were maintained under controlled condition of light (12/24 h) and temperature (23 ± 1˚C). Food pellets (Hind- ustan lever Ltd. Mumbai, India) and tap water were pro-vided ad libitum. For experimental purposes animals were kept fasting but were allowed free access to water. 2.4. CCl4-Induced Hepatotoxicity The animals were divided into six groups of six animals each. Group I served as normal control which received normal saline only. Group II served as CCl4 control and received CCl4:liquid paraffin (1:1, 1.5 ml/kg, b.w., p.o.) on first day. Group III served as standard control and re-ceived single dose of CCl4:liquid paraffin (1:1, 1.5 ml/kg, b.w., p.o.) on first day and thereafter received treatment with standard drug silymarin (Silybon-70) (10 mg/kg, b.w., p.o.) for 7 days. Groups IV-VI received single dose of CCl4:liquid paraffin (1:1, 1.5 ml/kg, b.w., p.o.) on first day and thereafter treated with petroleum, chloroform and methanolic extract of fruits of Feronia limonia (500 mg/kg, b.w., p.o.) [10], respectively, for 7 days. 2.5. Assessment of Liver Functions Rats of all groups were anaesthetized with 1.2 g/kg b.w, of a 25% w/v aqueous solution of urethane (Loba-Chemie, Bombay), given on 8th day. The blood collected by puncturing the orbital plexus was allowed to coagulate at ambient temperature for 30 min. and the rats were sacri-ficed by decapitation. Serum was separated by centrifu-gation at 3500 rpm for 10 min. The livers of all animals were removed and processed for histological investiga-tions.

In serum, alanine aminotransferase (ALT), aspartate aminotransferase (AST) [11], alkaline phosphatase (ALP) [12], total serum protein (TSP) [13] and bilirubin [14] were measured. Glutathione (GSH) [15], glycogen (GLY) [16] and glutathione peroxidase (GPX) [17] were esti-mated in liver homogenate. 2.6. Histological Investigations Liver slices fixed for 48 h in 10% formosaline were processed for paraffin embedding following the standard microtechnique [18]. Sections (5/~m) of livers stained with haematoxylin and eosin were evaluated for histopa-thological changes under a light microscope. 2.7. Statistical Analysis The data were expressed as mean ± S.E.M. (n = 6). Re-

sults were analyzed statistically by one-way ANOVA followed by Dunnett’s test. The difference was consid-ered significant if p < 0.05. 3. Results and Discussion 3.1. CCl4 Induced Hepatotoxicity It is well established from the earlier studies that the cleavage of carbon-chlorine bond (C-Cl bond) of carbon tetrachloride leads to the formation of trichloromethyl peroxy radical (CCl3·O2−), which is involved in the pathogenesis of liver injury [19].The abnormal higher levels of serum SGPT, SGOT, ALP and bilirubin; and decrease in total protein observed (Table 1) was the con-sequence of carbon tetrachloride induced liver disfunc-tion and denotes the damage to the hepatic cells [20]. Feeding of Feronia limonia extracts to such carbon tet-rachloride-treated rats reduced the enhanced level of se-rum glutamate oxaloacetate transaminase (SGOT) by 16.74, 32.18, and 42.20%, serum glutamate pyruvate transaminase (SGPT) by 14.46, 31.87 and 34.12%, alka-line phosphatase (ALP) by 20.71, 25.98 and 29.64% and serum bilirubin by 39.38, 40.92, 42.19% which seems to offer the protection and maintain the functional integrity of hepatic cells. Glutathione (GSH) constitutes the first line of defense against free radicals. Reduction in liver GSH and decrease in GPX activity in carbon tetrachlo-ride-treated rats (Table 2) indicated the damage to the hepatic cells, which was confirmed by the earlier reports [20]. Administration of Feronia limonia promoted the conversion of GSSG (oxidised glutathione) into GSH by the reactivation of hepatic glutathione reductase enzyme in carbon tetrachloride-treated animals. The availability of sufficient amount of GSH thus increased the detoxifi-cation of active metabolites of carbon tetrachloride through the involvement of GPX. But the restoration of liver glutathione (GSH) by 13.82, 37.50 and 59.04% and liver glutathione peroxidase (GPX) by 18.71, 21.37and 39.83% after the enduration of the Feronia limonia ex-tracts to such carbon tetrachloride-treated rats account for the protective efficacy of the extract. Decrease in total serum protein (TSP) (Table 1) and liver glycogen (GLY) (Table 2) observed in the rats treated with carbon tetrachloride may be associated with the decrease in the number of hepatocytes, which in turn may result into the decreased hepatic capacity to synthesize protein and GLY and consequently decrease in the liver weight (Ta-ble 2) but when the Feronia limonia extracts were given along with the carbon tetrachloride, the significant in-crease in the glycogen (GLY) by 31.61, 15.92 and 39.94%, total serum albumin (TSA) by 37.76%, 53.64%, 63.51% and total serum protein (TSP) by 40.83, 45.41 and 50.41 % was observed indicating the antihepatotoxic activity of the extracts and also accounting for the increase


R. UPADHYAY ET AL.


20

Table 1. Effect of Feronia limonia extracts on the level of serum SGOT, SGPT, ALP, bilirubin and total protein.

Group SGOT (unit/ml) SGPT (unit/ml) ALP (unit/ml) Total Bilirubin (g/dl) Total albumin (g/dl) Total Protein (g/dl)

Group I (Normal) 59.49 ± 2.40** 48.22 ± 2.87** 47.71 ± 1.62** 2.12 ± 0.20** 4.57 ± 0.28** 7.47 ± 0.32**

Group II (CCl4) 175.54 ± 3.80 134.94 ± 5.02 82.06 ± 2.07 3.91 ± 0.07 2.33 ± 0.21 4.80±0.25

Group III (Silymarin)

95.14 ± 3.27**(45.80%)

76.00 ± 3.04** (43.67%)

51.45 ± 1.44** (37.96%)

2.23 ± 0.18** (42.96%)

3.58 ± 0.15** (53.64%)

7.44 ± 0.18** (55.16%)

Group IV (Petroleum ether)

146.15 ± 2.46**(16.74%)

115.42 ± 4.54** (14.46%)

65.06 ± 4.42** (20.71%)

2.39 ± 0.12** (39.38%)

3.21 ± 0.19* (37.76%)

6.76 ± 0.25* (40.83%)

Group V (Chloroform)

119.05 ± 2.94**(32.18%)

91.93 ± 2.57** (31.87%)

60.74 ± 2.36** (25.98%)

2.31 ± 0.18** (40.92%)

3.58 ± 0.14** (53.64%)

6.98 ± 0.21** (45.41%)

Group VI (Methanol)

101.45 ± 2.70**(42.20%)

88.89 ± 2.91** (34.12%)

57.73 ± 1.53** (29.64%)

2.24 ± 0.23* (42.19%)

3.81 ± 0.17** (63.51%)

7.23 ± 0.17** (50.41%)

Values are mean ± S.E. (n = 6). *P < 0.05 as compared with Group II., **P < 0.01 as compared with Group II Table 2. Effect of Feronia limonia extracts on the level of liver GSH, GPX, GLY, body weight (before and after treatment) and liver weight.

Body weight

Group GSH (g/mg protein) GLY

(mg/gm of wet tissue)

GPX (micrograms of glutathione

utilized/min/mg protein) Before treatment (gm)

After treatment (gm)

Liver weight (gm)

Group I (Normal) 6.15 ± 0.25** 28.17 ± 0.33** 7.89 ± 0.02** 160.9 ± 5 175.1± 4 6.1 ± 0.4

Group II (CCl4) 3.76 ± 0.19 18.60 ± 0.20 4.97 ± 0.06 162.4 ± 2 168.3 ± 3 6.08 ± 0.7

Group III (Silymarin)

6.07 ± 0.32** (61.43%)

26.99 ± 0.65** (45.10%)

7.13 ± 0.34** (43.46%) 170.02 ± 6 186 ± 7 5.96 ± 0.4

Group IV (Petroleum ether)

4.28 ± 0.27* (13.82%)

24.48 ± 0.28** (31.61%)

5.90 ± 0.15* (18.71%) 176 ± 4 191 ± 6 6.75 ± 0.2

Group V (Chloroform)

5.17 ± 0.32* (37.50%)

21.56 ± 0.38** (15.92%)

6.05 ± 0.04** (21.73%) 165 ± 3 178 ± 5 5.80 ± 0.4

Group VI (Methanol)

5.98 ± 0.39** (59.04%)

26.03 ± 0.37** (39.94%)

6.95 ± 0.06** (39.83%) 172 ± 4 188 ± 6 6.54 ± 0.6

Values are mean ± S.E. (n = 6). *P < 0.05 as compared with Group II., **P < 0.01 as compared with Group II in the liver weight most probably through the hepatic cell regeneration. It was conclusively observed that the methanolic extract exhibited most significant result among all the extracts. 3.2. Histopathology Histomorphology of the livers of the untreated control (Figure l(a)), the CCl4 treated (Figure l(b)) and the pe-troleum, chloroform, methanolic extract of Feronia li-

monia plus toxin treated (Figures 1(d, e, f)) respectively. CCl4 administration caused gross necrosis of the centri-lobular hepatocytes characterized by nuclear pyknosis, karyolysis and eosinophilic infiltration (Figure l(b)). Treatment with the extracts of the Feronia limonia re-versed, to a large extent, the hepatic lesions produced by CCl4, as is obvious from the absence of eosinophilia and presence of fewer necrotic zones (Figures 1(d, e, f)). Further, the methanolic extract exhibited most significant recovery of hepatic cells, which is in accordance with the results obtained in biochemical parameters.

R. UPADHYAY ET AL.


21

(a) (b)

(c) (d)

(e) (f)

Figure 1. (a) Histology of the liver of control rat showing normal hepatic cells architecture, (b) Histology of the liver of car-bon tetrachloride-treated rats showing necrosis with the obliteration of architecture in hepatic cells, (c) Histology of the liver treated with silymarin showing recovery of the hepatic cells, (d) Histology of the liver treated with petroleum extract of Fero-nia limonia showing recovery of the hepatic cells, (e) Histology of the liver treated with chloroform extract of Feronia limonia showing recovery of the hepatic cells, (f) Histology of the liver treated with methanol extract of Feronia limonia showing re-c overy of the hepatic cells.

R. UPADHYAY ET AL.


22

4. Conclusions It appears from our results that the mode of action of Feronia limonia fruit extracts in affording the hepato-protective activity against carbon tetrachloride may be due to the cell membrane stabilization, hepatic cell re-generation and activation of antioxidative enzymes such as glutathione reductase, glutathione peroxidase, super-oxide dismutase and catalase. 5. References [1] J. D. Hooker, “The Flora of British India,” L. Reeve &

Co, London, 1875, p. 178. [2] K. R. Kirtikar, B. D. Basu and I. C. S. An, “Indian Me-

dicinal Plants,” Orient Enterprises, Dehradun, India, 1993, Vol. 1, pp. 49

Documents

TABLE OF CONTENTS · Chinese Medicine (CM) Journal Information SUBSCRIPTIONS The Chinese Medicine (Online at Scientific Research Publishing, ) is published quarterly by Scientific