Journal of Pharmacy and Pharmacology

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Journal of Pharmacy and Pharmacology Volume 2, Number 4, April 2014 (Serial Number 5)

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Publication Information Journal of Pharmacy and Pharmacology is published monthly in hard copy (ISSN 2328-2150) by David Publishing Company located at 240 Nagle Avenue #15C, New York, NY 10034, USA. Aims and Scope Journal of Pharmacy and Pharmacology, a monthly professional academic journal, covers all sorts of researches on Pharmacokinetics, Biopharmaceutics, Pharmaceutical Analysis, Pharmaceutical Biotechnology and Drug Delivery, Pharmaceutical Outcomes and Policy, Pharmacy Administration, Advanced Pharmacology, Experimental Method and Technique of Pharmacology, Clinical Pharmacology, Medical Statistics, Pathophysiology, and Medicinal Chemistry, as well as other issues related to Pharmacy and Pharmacology. Editorial Board Members Dr. Jinhua Zhang (Canada), Dr. Preetpal Singh Sidhu (USA), Dr. Xiaoming Xie (China), Dr. Young Jin Chun (Korea), Dr. Sumio Chono (Japan), Dr. İnci Selin (Zorkun) Dogan (Turkey), Dr. Katarzyna Kieć-Kononowicz (Poland), Dr. Horng-Jyh Harn (Taiwan), Dr. Michele Navarra (Italy), Dr. Jordi Caballé Serrano (Spain), Dr. Leonardo Luiz Gomes Ferreira (Brazil), Dr. Qiliang Cai (China), Dr. Susruta Majumdar (India), Dr. Swati Misra (India), Dr. Junyan Liu (China), Dr. Andre Filipe de Barros Vieira (Portugal), Dr. Beom-Jin Lee (Korea), Dr. Farzin Roohvand (France), Dr. Yuanye (Vickie) Zhang (China), Dr. Shayli Varasteh Moradi (Iran), Dr. Haibin Zhou (China). Manuscripts and correspondence are invited for publication. You can submit your papers via E-mail to [email protected] or [email protected]. Submission guidelines are available at http://www.davidpublishing.com. Editorial Office 240 Nagle Avenue #15C, New York, NY 10034, USA Tel: 1-323-984-7526, 323-410-1082; Fax: 1-323-984-7374, 323-908-0457 E-mail: [email protected], [email protected] Copyright©2014 by David Publishing Company and individual contributors. All rights reserved. David Publishing Company holds the exclusive copyright of all the contents of this journal. In accordance with the international convention, no part of this journal may be reproduced or transmitted by any media or publishing organs (including various websites) without the written permission of the copyright holder. Otherwise, any conduct would be considered as the violation of the copyright. The contents of this journal are available for any citation. However, all the citations should be clearly indicated with the title of this journal, serial number and the name of the author. Abstracted/Indexed in Database of EBSCO, Massachusetts, USA Universe Digital Library S/B, ProQuest Summon Serials Solutions, USA Google Scholar (scholar.google.com) Chinese Database of CEPS, American Federal Computer Library Center (OCLC), USA Universe Digital Library Sdn Bhd (UDLSB), Malaysia China National Knowledge Infrastructure (CNKI), China Subscription Information Price (per year): Print $520, Online $320, Print and Online $600. David Publishing Company 240 Nagle Avenue #15C, New York, NY 10034, USA Tel: 1-323-984-7526, 323-410-1082; Fax: 1-323-984-7374, 323-908-0457 E-mail: [email protected] Digital Cooperative Company: www.bookan.com.cn

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DAVID PUBLISHING

David Publishing Company www.davidpublishing.com

Journal of

Pharmacy and Pharmacology Volume 2, Number 4, April 2014 (Serial Number 5)

Contents Reviews 231

Novel Multimodal Evaluation Technology for Symptom Characterization in GI Functional Diseases and Pharmacological Testing of Drugs Hans Gregersen and Asbjørn Mohr Drewes

Original Articles 243

Inhibition of Embryonic Stem Cell Differentiation by Gold Nanoparticles Sumi Dinda, Sarah Rasul Chaudhry, Naimisha Reddy Beeravolu, Christina McKee and Ghulam Rasul Chaudhry

257

Antioxidant and Antifungal Activities of the Essential Oils of Ocimum gratissimum from Yaoundé and Dschang (Cameroon) Jean Baptiste Hzounda Fokou, Pierre Michel Jazet Dongmo, Fabrice Fekam Boyom, Elizabeth Zeuko’o Menkem, Issakou Bakargna-Via, Ide Flavie Kenfack Tsague, Marguerite Simo Kamdem, Paul Henri Amvam Zollo and Chantal Menut

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Central Sensitization of HPA Axis in Modulation of Neuropathic Pain in Diabetic Rats Vandana Sharma, Rohit Goyal, Shaila Khah and Babita Thakur

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Study of Immunomodulating Activity of Rectal Suppositories with an Extract of Licorice Root Larisa V. Yakovleva, Tatiana G. Yarnykh, Elena Yu. Koshevaya, Olga A. Rukhmakova and Galina N. Melnik

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Journal of Pharmacy and Pharmacology 2 (2014) 231-242

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PUBLISHING

Novel Multimodal Evaluation Technology for Symptom Characterization in GI Functional Diseases and Pharmacological Testing of Drugs Hans Gregersen1, 2 and Asbjørn Mohr Drewes3 1. GIOME Center, College of Bioengineering, Chongqing University, Chongqing 400030, China 2. GIOME Institute, GIOME Free Zone Enterprise, Ras Al Khaimah 10055, United Arab Emirates 3. Department of Gastroenterology, Aalborg University Hospital, Aalborg DK 9000, Denmark Abstract: Characterizations of pain and other sensory symptoms are very important for diagnosis and assessment of patient with gastrointestinal disorders. Methods to evoke and assess experimental pain have developed into a new area for multimodal assessment with electrical, mechanical, thermal and chemical stimulation of pain pathways in the human gut. Stimulation with this technology mimics to a large degree the pain experienced by patients seeking the health system for treatment. Multimodal methods have increased our understanding of peripheral receptors in the gut in health and disease. Combined with advanced muscle analysis, the methods have increased our understanding of receptors sensitive to mechanical, chemical and temperature stimuli in diseases such as systemic sclerosis and diabetes. Multimodal technology can also be used to unravel central pain mechanisms involved in allodynia, hyperalgesia and referred pain. Abnormalities in central pain mechanisms are often found in patients with chronic gut pain. Hence methods relying on multimodal pain stimulation may help to understand symptoms in these patients. In addition, multimodal methods have been used to gain more insight into the drug effects against pain. It is expected that multimodal methods represent a major step forward in the future characterization and treatment of patients with various gastrointestinal diseases. Key words: Pain, cut, experimental, allodynia, hyperalgesia, neurophysiology.

1. Introduction Abdominal pain is a frequent symptom in the general population [1] and pain is the most prevalent symptom in the gastroenterological clinic [2]. As a consequence, it is an important issue for the clinician to characterize gut pain for the diagnosis and assessment of organ dysfunction. Unfortunately, different symptoms of the underlying diseases often confound the characterization of pain. The confounders include systemic reactions such as fever and general malaise as well as complaints relating to psychological, cognitive and social aspects of the illness [3]. Furthermore, treatment with analgesics confounds by causing sedation and other side effects.

Corresponding author: Hans Gregersen, M.D., professor, research field: biomechanics. E-mail: [email protected].

This may bias the clinical evaluation of the pain related symptoms. The patients tend to interpret other effects of the medication, e.g., an effect on the anxiety and depression relating to the disease, as a relief of pain [4]. Due to the existence of these biases, there is a need for standardized experimental pain models. Using these models, the investigator can control the experimentally induced pain (including the nature, localization, intensity, frequency and duration of the stimulus) without confounding and provide quantitative measures of the psychophysical, behavioral and neurophysiological responses [3, 5-6]. Experimental models have been used in different animal species. With such models scientists can study neuronal activity in anesthetized or spinalized animals directly with invasive techniques or with assessment of behavior [7]. However, due to inter-species

232 Novel Multimodal Evaluation Technology for Symptom Characterization in GI Functional Diseases and Pharmacological Testing of Drugs

variation for pain neurobiology, the interpolation of findings from animal studies to man is limited. Pain is a product of net effects of complex multidimensional mechanisms including intensity coding, affective, behavioral and cognitive components involving most parts of the central nervous system. Furthermore, pain is closely related to linguistic terms and expressions in humans. Hence, pain is a very complex sensory experience that is difficult to quantify with simple neurophysiological or behavioral methods. For those reasons, animal experiments can only to some degree reflect the experience of clinical pain in humans. Consequently, the interest in “human experimental pain studies” has increased rapidly during the last decades [3, 8]. The advantages of experimental pain models are that the stimulus can be controlled, delivered repeatedly and modulated. Furthermore, the responses can be assessed quantitatively with psychophysical and/or neurophysiological methods (Fig. 1). Depending on the model, different central mechanisms and conditions mimicking pathological pain can be studied. These are for example increased sensation to normal physiologic/non-painful and painful stimuli such as allodynia and hyperalgesia. Experimental models can be used in the laboratory for basic studies in healthy subjects and in patient groups or used for preliminary screening of drug efficacy [9].

The methods can also be used in hospitals to characterize patients with sensory dysfunction and pain in organic and functional diseases [5, 10-11]. Somewhat similar methods have often been used in skin and muscle [6]. However, due to the difficulties with access to the organs in the GI (gastrointestinal) tract, experimental GI pain testing is much more difficult than stimulation of the skin. Most previous studies have relied on relative simple mechanical or electrical stimuli. These methods are readily applicable but have several limitations [3]. Most importantly, as pain is a complex and multidimensional perception, it is evident that the reaction to a single stimulus of a given modality can represent only a limited fraction of the entire pain experience. However, combination of different methods to stimulate the GI tract and evoke hyperalgesia will approximate the clinical situation and give more comprehensive and differentiated information about the nociceptive system [3]. Multimodal models have clearly shown their value in testing of analgesics where single stimuli have been inadequate assessing effects of specific drugs. In that regard, it was demonstrated that tricyclic antidepressants, valuable in treatment of functional pain disorders of the GI tract, increased the pain threshold to electrical stimuli, but did not reduce cold pressor pain [12]. More advanced methods based on multiple

Modulation of spinal and supraspinal mechanisms

Exact stimulation •Localization •Time •Frequency •Modality

Fig. 1

Pain system

Assessment of evoked responses •Psychophysical •Neurophysiological •Imaging

Concept for experimental induction, assessment and modulation of experimental gastrointestinal pain in man.

Novel Multimodal Evaluation Technology for Symptom Characterization in GI Functional Diseases and Pharmacological Testing of Drugs

tests will make possible selection of the best test procedures to explore different basic aspects of pain as well as pharmacological modulations [9]. The purpose of this review is to reveal recent developments in test systems allowing standardized multimodal stimulations of the GI tract and their applications, including the use in pharmacological research.

2. Rationale for Gastrointestinal Multimodal Stimulations The ideal experimental stimulus to elicit gut pain in man must be natural, minimally invasive, reliable in test-retest experiments and quantifiable [13]. The response to the stimulus must increase with increasing stimulus intensity and the pain must mimic the observations in diseased organs by evoking phenomena such as allodynia and hyperalgesia [8]. The applicable methods for GI pain stimulation are electrical, mechanical, chemical, thermal and ischemic stimulation [3]. Ischemic stimulation is difficult to quantify in man and is normally not used as a direct stimulus. One of the major limitations of the different models is that they may not mimic clinical pain. For comprehensive experimental studies mimicking the clinical situation, a multimodal testing approach must be used. Multimodal stimulation will increase the probability for activation of a range of relevant Table 1

nervous mechanisms. Especially if the stimulation is relative long lasting and includes modalities known to evoke peripheral as well as central sensitization, the likelihood that the model will mimic clinical pain is high despite the non-harmful nature of the stimulation. In the GI tract technical limitations of the currently available models have until now made a multimodal stimulation approach difficult. Some authors have combined mechanical and electrical stimuli [14-15] or used electrical stimuli combined with sensitization to acid [16]. Several years ago, we introduced a multimodal pain model where mechanical, electrical, cold and warmth stimuli were combined. Table 1 shows the different stimulation modalities and Table 2 lists findings in healthy volunteers in esophagus. For more detailed summaries of findings in all GI parts in healthy subjects and patients, we refer to previous reviews by Drewes, Gregersen or coworkers [17-20]. Thermal stimulation is achieved by re-circulating fluid inside the bag with concomitant measurement of temperature. Chemical and electrical stimulations are done using side-holes placed proximal to the bag and by electrodes on the outside of the bag [17]. Sensitization with acid was added to the protocol to evoke allodynia and hyperalgesia together with referred pain areas (Fig. 2) [18]. In the multimodal model, mechanical stimulation is achieved with bag distension. Quantification of the bag pressure and

Advantages and limitations with different stimulation modalities.

Stimulation modality Mechanical Thermal

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Stimulated structures

Advantages

Limitations

Mechanoreceptors located in different layers Thermal sensitive receptors primarily located in the superficial layers

Imitates a bolus, reproducible stimulus

Problems with estimating the transmural pressure and change in circumference Temperature stimuli in the range that can be felt are normally only relevant for sensation in the upper GI tract

Activation of unmyelinated afferents in the mucosa selectively Resembles clinical inflammation, chemical stimuli activate predominantly unmyelinated C-fibers

Chemical

Chemo-sensitive receptors, primarily located in the mucosa

Electrical

Nerve fibers primarily located in Excellent for repeated stimulation, mucosa (not specific activation of suitable for neurophysiologic the nociceptors) assessments of the pain

Require a relative long latency time to the onset of effects. They are often not reproducible when repeated The electrical threshold depends on the fiber diameter, i.e., small-diameter nerves cannot be excited without exciting others. May induce arrhythmias in areas near the heart

234 Novel Multimodal Evaluation Technology for Symptom Characterization in GI Functional Diseases and Pharmacological Testing of Drugs Table 2

Clinical experimental multimodal data obtained in esophagus from healthy volunteers.

Group

Mechanical stimuli

Heat stimuli

Cold stimuli

Electrical stimuli

Basic data

Differences between the sensations and referred pain areas evoked by the stimulus modalities [17-18] Reliability demonstrated [18] Sensation to mechanical stimulations unaffected by relaxation of the smooth muscle [20] Evidence for low and high threshold mechanoreceptors [21]

Reliability demonstrated. Stimulus-response functions obtained [17-18]

Reliability demonstrated. Stimulusresponse functions obtained [17-18]

Reliability demonstrated. Stimulus-response functions obtained [17-18]

Oxycodone better Oxycodone better than than morphine Pharmacologic morphine (and placebo) (and placebo) in Not done modulation in attenuating attenuating heat mechanical pain [22] pain [22]

Channel for acid perfusion

Sensitization with acid Allodynia and hyperalgesia evoked [18, 23], though not consistent for mechanical stimuli [23, 24] Increased referred pain and amplitude of the nociceptive reflex indicating central hyperexcitability [18, 23, 25] Acid perfusion sensitizes the oesophagus to heat but not cold indicating sensitization of peripheral TRPV1 receptors [25, 26] Remote hyperalgesia was found in rectum after acid perfusion of the esophagus [27] Hyperreactivity of contractions in esophagus, but tone unaffected [23, 27]

Both morphine and oxycodone attenuated the electrically evoked Not done pain but no differences between the opioids [22]

Acid perfusion hole (●)

Circulation channels

Stimulation electrodes

Impedance electrodes

Temperature sensor connector Pressure channel

Excitation and detection electrodes

Electrical stimulation wires Bag and holes ( ) for water perfusion inside the catheter Fig. 2 Schematic illustration of the multimodal probe used for electrical, mechanical, cold and warmth stimuli of the esophagus.

cross-sectional area are typically done by means of manometry and impedance planimetry or ultrasonography. The multimodal approach provides differentiated stimulation of receptors in the superficial and deep layers of the gut. Induction of

hyperalgesia and evoking central phenomena such as summation, allodynia and referred pain make the models clinically relevant with respect to studying peripheral and central pain mechanisms. The model has mainly been used in the esophagus, duodenum and

Novel Multimodal Evaluation Technology for Symptom Characterization in GI Functional Diseases and Pharmacological Testing of Drugs

rectum. Pain assessment should ideally also be multimodal and for example include quantitative and qualitative sensations, assessment of referred pain and neurophysiological measurements [18].

3. Stimulation of Peripheral Receptors With the multimodal model, thermal stimulation activates preferentially the receptors in the mucosa, electrical stimulation penetrates into deeper layers of the gut and mechanical stimulations affect predominantly receptors in the muscle layers (Fig. 3 and Table 1). Most visceral afferents are polymodal and respond to a wide range of stimuli [28]. Receptor sub-populations exist. Combined controlled distension with statistical modeling was used to demonstrate the existence of low and high threshold mechano-receptors in the human esophagus [21]. Most data on the sensation to thermal stimuli of the human viscera relate to few and relatively old studies [29-31], although some newer studies have recently been published [17, 18, 23, 26, 32, 33]. These

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studies point towards the existence of sensory pathways for thermal stimuli in the human GI tract. The thermal energy spreads from the superficial layers into the deeper layers of the GI tract depending on the temperature difference and conductance of the tissue [4]. However, thermal stimuli is rather short-lasting in man and therefore mainly receptors in the mucosa are believed to be activated. Chemical stimulation with acid or capsaicin also activates receptors in the mucosa. A multimodal model was used to combine acid and heat stimuli of the esophagus [26] where sensitization with acid resulted in a significant increase in the sensation to heat stimuli. The TRPV1 receptor is a polymodal detector of potential harmful stimuli including noxious heat and protons [34]. It was suggested that TRPV1 receptors or receptors with similar characteristics were sensitized with acid which resulted in increased firing of the afferents to heat stimulation. The role of this receptor system was addressed in a clinical study where selective hyperalgesia to heat was found in patients with reflux

Thermal Sensitisation with acid Electrical

Mechanical

Fig. 3 Schematic illustration of the gut layers which are preferentially affected with: (1) thermal stimuli (mucosa—dark grey and submucosa—light grey); (2) mechanical stimuli (circular muscle layer—hatched grey and longitudinal muscle layer—prickled grey); (3) electrical stimuli (all layers depending on stimulus intensity). Perfusion of the esophagus with acid (curved arrows) induces peripheral and (mainly) central sensitization (illustrated with stars).

236 Novel Multimodal Evaluation Technology for Symptom Characterization in GI Functional Diseases and Pharmacological Testing of Drugs

and grade B esophagitis [35]. The evidence for receptor-specific activation patterns in the experimental studies was confirmed in another study where TRPV1 receptors were demonstrated in the human esophagus, including up-regulation in patients with esophagitis [36]. In conclusion, the multimodal approach gave valuable quantitative information on receptor characteristics and pain mechanisms in healthy subjects as well as in patients with acid-evoked inflammation.

4. The Effect on Primary Afferents Electrical stimuli bypass the receptors and all fiber populations (nociceptive as well as fibers mediating physiologic/non-nociceptive sensations) are excited by electrical stimuli. With normal bipolar electrical stimulation, the current is believed to activate receptors in the mucosa and submucosa first whereas the deeper layers are activated with increasing current [3]. The depth of activation also depends on the stimulation method and frequency [4]. In the GI tract electrical stimulation is thought preferentially to activate thinly myelinated (Aδ) fibers [37]. Chemical and thermal stimuli on the other hand activate mainly non-myelinated (C) fibers and terminals of mechanosensitive fibers are mainly localized in the muscle layers or have intraganglionic nerve endings [38-39]. Essentially the different modalities may activate different fiber populations. However, the difference between the stimulation paradigms may be of minor importance as it was shown that evoked brain potentials to mechanical and electrical stimulation of the gut were similar, reflecting that the same pathways were activated [40]. The mechanical stimulation protocol may likewise be important. For example in the human rectum phasic distensions preferentially stimulated spinal pathways thought to mediate pain whereas slow tonic stimuli mainly affect parasympathetic nerves [41]. In general slow ramp distension is recommendable since it is more physiological and allows subjects to assess the pain

continuously. Preconditioning the tissue by two-three distensions until the stress-strain relationship becomes reproducible is also recommendable [19]. For evaluation together with advanced muscle analysis butylscopolamine may be useful to relax the smooth muscle. Butylscopolamine does not seem to modify the sensation per se [20, 27, 42]. The multimodal approach has been used to compare the response to mechanical stimuli before and after chemical stimulation with acid in patients with non-cardiac chest pain [24]. These patients had a normal sensory response to mechanical stimuli at baseline. However, after acid stimulation, the evoked hyperalgesia resulted in a marked increase of the sensory response in the patients. Although peripheral sensitization likely is important, the findings provided evidence for amplification of central pain mechanisms manifested as allodynia, hyperalgesia, and increased and widespread referred pain areas to the mechanical stimulations. Mechanical stimulation together with advanced muscle analysis has also been used to explain the symptoms in patients with SS (systemic sclerosis). In SS patients, the contraction amplitude was smaller and there was evidence for a stiffer small intestinal wall [43]. Pain evoked by a controlled deformation of the gut was increased which may explain many of the symptoms reported in the clinic. In patients with diabetes, we also found evidence for increased stiffness in the duodenum using the multimodal approach [44-45]. This may reflect the increase in collagen deposition reported in these patients and may (together with autonomic neuropathy) explain the motor abnormalities observed in SS patients.

5. Central Pain Mechanisms Multimodal Stimulations

and

In diseases of the GI tract central sensitization and neuroplastic changes are of major importance to understand the sensory response as manifested by pain and hyperalgesia. Central pain mechanisms may be

Novel Multimodal Evaluation Technology for Symptom Characterization in GI Functional Diseases and Pharmacological Testing of Drugs

evoked by multiple stimuli due to temporal or spatial summation. This results in central amplification of the response. The response is comparable to early phase of the frequency dependent “wind-up” as observed in animal experiments. In practice, central integration can be evoked by repeated electrical stimulation above 0.5 Hz [46-47] resulting in increased local and referred pain. The multimodal probe has been used to give repeated mechanical stimulation in patients with non-cardiac chest pain. The data showed that the number of stimuli tolerated was lower in patients compared with healthy controls, reflecting central hyperexcitability as a key to understanding the symptoms in these patients [24]. Sensitization of the esophagus with acid is another way to evoke central and peripheral sensitization. Previously, it was shown that acid perfusion of the distal esophagus resulted in an amplified response to electrical, mechanical and thermal stimuli [18, 23]. The central changes were documented in experiments with amplification of the nociceptive reflex [18]. The reflex was evoked by stimulation of the sural nerve resulting in activity of the biceps muscle of the thigh.

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The connection from the primary afferents to the motor neurons is a polysynaptic spinal pathway, which can be modulated by other afferent input, spinal neuronal excitability and activity in descending control systems [5]. The reflex was evoked together with painful mechanical stimulation of the esophagus. Amplification of the reflex was found after sensitization of the esophagus with acid reflecting central changes at the spinal cord level. Evidence for central changes to acid perfusion was also demonstrated by Sarkar and coworkers et al. [16] who demonstrated decreased pain threshold to electrical stimulation of the proximal esophagus after acid perfusion of the distal part. Since the proximal esophagus was not affected by acid, only central changes would explain the findings. Referred somatic pain to visceral stimuli is regarded a phenomenon generated by central mechanisms due to visceral nerves terminating in the same area of the spinal cord as somatic afferents [48] (Fig. 4). Assessment of the referred pain area to electrical, mechanical and thermal stimulation can be used to determine the central response to these differentiated

Fig. 4 Referred pain in somatic tissues is believed to be generated by central mechanisms with convergence of visceral and somatic nerves in the same area of the spinal cord or at supraspinal centers. The phenomenon also includes unmasking of latent connections and focal central hyperexcitability of the neurons.

238 Novel Multimodal Evaluation Technology for Symptom Characterization in GI Functional Diseases and Pharmacological Testing of Drugs

modalities [18]. The referred pain area to electrical, mechanical, cold and heat pain differs in size and localization reflecting the different peripheral and central nerves being activated [17]. Increase in referred pain areas after acid perfusion is also evidence for central sensitization caused by the chemical stimulation [23, 25]. Pedersen et al. [26] showed that the referred pain to heat stimulation of the esophagus increased after acid perfusion of the esophagus. This was confirmed in a more recent study [25]. As discussed previously selective sensitization of the TRPV1 receptors by acid can result in an increased afferent barrage after a heat stimulus, which again was manifested as an increase in the referred pain area. However, changes in local and referred pain to mechanical stimulations may be difficult to determine as the acid also evokes secondary contractions that squeeze the bag and influence the stimulus parameters [23]. On the other hand, an increase in the referred pain after acid perfusion is typically a result of mechanical stimulation of the esophagus in healthy subjects [18, 23]. Increased referred pain to mechanical stimulations was also observed in patients with esophagitis and in non-erosive reflux disease reflecting that facilitation of central pain mechanisms are important in the understanding of these diseases [35]. Hypoalgesia to peripheral stimulation was observed in patients with diabetes and chronic pancreatitis whereas the referred pain area increased. Hence, the mechanism may be descending inhibition of the afferent input counterbalancing central hyperexcitability [49, 50]. The multimodal model may be used for detailed evaluation of symptoms and the disease stage in these patients. Central changes may also result in allodynia and hyperalgesia to stimulation of other viscera [51]. Such changes are important in the understanding of functional gut disorders where abnormal sensation to physiologic stimuli (for example as caused by luminal contents) may contribute to the symptoms (allodynia). The multimodal model has been used to assess the

sensation of the proximal esophagus, duodenum and rectum after sensitization of the distal esophagus with acid [27] where increased sensitivity to mechanical stretch in the three gut segments was found after acid perfusion. This was mainly due to increased sensitivity in the rectum being very remote from the experimentally inflamed esophagus. Neuroplastic changes at the cortical level can also be demonstrated by multimodal stimulations of the GI tract. Sarkar et al. [52] showed changes in the evoked brain potentials to electrical stimulation of the proximal esophagus after acid perfusion of the distal segment [52]. Afterwards we showed that acid perfusion resulted in neuroplastic changes at the cortical level. Latency reduction and backward shift of the electrical dipole in the anterior cingulate dipole were observed to electrically evoked pain in the esophagus after acid perfusion of the organ [53]. Such changes were also found when the gut was electrically stimulated in patients with irritable bowel syndrome [54]. The backward shift in the cingulate activation after sensitization with acid in healthy subjects may represent central nervous system change corresponding to allodynia and hyperalgesia to gut stimuli in patients with functional disorders of the gut. Most of the work using the multimodal model has been conducted in the esophagus. However, studies have also been done more distally in the digestive system. Accarinos group conducted mechanical and electrical stimulation of the jejunum [14]. The verbal response to electrical stimuli and distension was compared without finding differences in the evoked response. The authors concluded that the differences between the two modalities may probably be of minor importance. Our group used thermal and mechanical stimuli of the duodenum in healthy subjects [27] and in patients with diabetes and autonomic neuropathy [44, 55]. The diabetes patients showed hypoalgesia to mechanical and electrical stimuli whereas no changes were found to heat stimulation compared with controls. Furthermore, the referred

Novel Multimodal Evaluation Technology for Symptom Characterization in GI Functional Diseases and Pharmacological Testing of Drugs

pain area in the abdomen was enlarged in the patients. Such data may enhance our knowledge about peripheral and central pain mechanisms in these patients with implications for the treatment. One of the most used methods in GI research is mechanical stimulation of the rectum. Electrical and thermal stimulations have also been done in the rectum [3], but combination of the methods has not been done yet. However, combinations of mechanical and electrical stimulations have been used in assessment of evoked brain potentials [56] and to assess the effect of viscero-visceral hyperalgesia [27]. The stomach has not yet been studied with multimodal stimulations. Although the complicated anatomy, nervous innervation and function of these organs should be taken into account, multimodal models are obviously highly warranted.

6. Multimodal Research

Stimulations

in

Drug

Experimental models are widely used in research of the effect of analgesics. Differentiated information of drug effects can be used as “proof-of concept”, dose-efficacy analysis, and for designing further clinical trials. The clinical situation can be mimicked by using multimodal tests where different receptor types and mechanisms are activated. Multimodal models have clearly shown their value in somatic pain testing, where single stimuli have been inadequate to test for example pathophysiological changes and effects of specific drugs [9]. Differentiated effects can reflect how the drugs can modify different disease mechanisms. In the esophagus Sarkar et al. [57] used a model where the upper esophagus was stimulated following sensitization of the distal segment with acid . The secondary hyperalgesia in the proximal part was reduced with a prostaglandin inhibitor, demonstrating the preferentially central action of prostaglandins in this model. We used a multimodal and multi-tissue approach to test the effect of opioids. Opioids are widely used in treatment of visceral pain despite the

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many side effects. Opioids preferentially attenuate nociceptive responses produced by central integration (spinally amplified signals) to tonic activation of unmyelinated fibers [4, 58]. Therefore, evaluation of the antinociceptive effects of opioids may be clearer using slow rates of temperature or tonic pressure. For internal organs typically only one modality (pressure) has been used in the testing of analgesics [59]. However, Staahl et al. [22] compared the effects of morphine and placebo on the pain thresholds to multimodal stimulation of the esophagus. A clear effect of morphine attenuating heat, electrical and slow-ramp pressure stimulations was found. Morphine can attenuate GI pain in the clinical situation and the model therefore proved its validity. The model was also used to differentiate between morphine and oxycodone, the latter believed also to affect mu-opioid receptors. In equipotent doses oxycodone was better than morphine in attenuating visceral pain, whereas differences were not found between the drugs on pain evoked in the muscle and skin. The study demonstrated a different pharmacological profile of oxycodone compared to morphine. Therefore, oxycodone may be a useful alternative to morphine in the treatment of visceral pain syndromes. Future studies evaluating analgesics in the GI tract should use a multimodal approach for obtaining insight into visceral pain mechanisms and the effect of drugs in the GI tract. This will facilitate the design of subsequent clinical studies. Hence, a substitution of the current “trial and error design” with a mechanisms based approach will reduce the economic and human burden in the development of new drugs targeted against pain in the GI tract.

7. Conclusions Multimodal pain stimulation in the human GI tract is an advanced experimental approach that mimics the clinical pain to a higher degree than other models. The method has been used to gain insight into basic peripheral and central pain mechanisms as well as

240 Novel Multimodal Evaluation Technology for Symptom Characterization in GI Functional Diseases and Pharmacological Testing of Drugs

characterizing patients with different diseases of GI tract. Together with pharmacological testing, model represents a major leap forward in experimental characterization and treatment patients with gastroenterological diseases.

the the the of

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D

Journal of Pharmacy and Pharmacology 2 (2014) 243-256

DAVID

PUBLISHING

Inhibition of Embryonic Stem Cell Differentiation by Gold Nanoparticles Sumi Dinda1, 2, Sarah Rasul Chaudhry1, Naimisha Reddy Beeravolu3, Christina McKee2, 3 and Ghulam Rasul Chaudhry2, 3 1. School of Health Sciences, Oakland University, Rochester 48309, USA 2. Oakland University-William Beaumont, Institute for Stem Cell and Regenerative Medicine, Oakland University, Rochester 48309, USA 3. Department of Biological Sciences, Oakland University, Rochester 48309, USA Abstract: Nanomaterials are attractive for use in technological advancements because of their small size and unique properties. As a result, there has been a rapid increase in the production and applications of nanomaterials. Nanoparticles like carbon, cadmium and silver are highly toxic and are known to cause oxidative stress. However, there are conflicting reports regarding the toxicity of gold nanoparticles. We have investigated the effects of gold nanoparticles on the growth and differentiation of ESCs (embryonic stem cells). Analysis of ESCs treated with gold nanoparticles revealed a biphasic growth response. Higher concentrations (> 20 μg/mL) of gold nanoparticles inhibited growth, whereas the lower concentrations (< 10 μg/mL) stimulated ESC proliferation. Interestingly, ESC pluripotency was not affected by gold nanoparticles as demonstrated by the near normal expression of the specific pluripotent marker, Oct 4, and their differentiation potential. Inhibition of differentiation of both ESCs and embryoid bodies by gold nanoparticles suggest that they may pose developmental risks. Further analysis by transmission electron microscopy and atomic absorption spectroscopy revealed that gold nanoparticles were actively taken up by ESCs in a concentration dependent manner. These observations suggest that exposure to gold nanoparticles may cause embryotoxicity or effect early childhood development. Key words: Nanomaterials, embryotoxicity, pluripotency, self-renewal, Oct 4.

1. Introduction Nanomaterials are particles measuring less than 100 nm in diameter, obtained by the breakdown of elements to the nano-sized level, and are distinguished from macro-scaled materials by their emergent and novel properties [1]. It is expected that in 2015, nanomaterials will occupy a significant portion of the industrial market share amounting to over 3 trillion dollars [2]. Because of their extremely small size, nanomaterials are attractive for a wide range of applications in industry and medicine. They are used even in personal care products, such as sunscreen and toothpaste [3, 4]. Nanomaterials are also considered useful due to their high surface area to mass ratio, Corresponding author: Ghulam Rasul Chaudhry, Ph.D., professor, research fields: stem cell biology and regenerative medicine. E-mail: [email protected].

which allows them to act as catalysts in chemical reactions [5]. Although the catalytic properties of nanomaterials appeal to commercial use, the emergent characteristics may also have deleterious effects on the human health. Human exposure to nanomaterials can occur through oral, dermal and inhalation exposure [1]. Inhaled nanoparticles can be deposited in the respiratory tract or taken up into the brain through the olfactory epithelium. Nanoparticles have been reported to be associated with several pulmonary pathologies in humans and mammals, tissue inflammation by oxidative stress and producing tumor-related effects in rats [1, 6]. A potential mechanism for cytotoxic effects is via binding of toxic chemicals to nanomaterials due to their catalytic properties, allowing transportation throughout the human body [6, 7]. As the role of

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nanomaterials in the consumer industry increases, so does the potential exposure and the importance to study their effects on human as well as environmental health. Metallic nanoparticles exhibit exceptional optoelectronic properties with applications in plasmonics, biosensing and nanomedicine [8, 9]. Surface-enhanced fluorescence with metal nanoparticles is gaining attention as a way to detect various molecules [10, 11]. Metal particles, such as gold nanospheres with a diameter 100 nm or smaller, appear red (not gold) and gold nanoparticles of diameter less than 3 nm are no longer unreactive and can catalyze chemical reactions [12]. Gold colloids have been used for years as contrast agents in electron microscopy because gold is very electron-dense [13]. More recently, gold and silver nanoparticles have been used in biological optical imaging and sensing applications [14-20]. In drug therapy, gold nanoparticles have been tested as a delivery vehicle for potential tumor necrosis factor alpha (TNF-). TNF- is extremely toxic if released systemically. However, combined with nanoparticles, TNF- travels to a tumor, accumulates and is released by radiation heating. This yields the drug relatively innocuous to surrounding tissues and allows for localized and more effective cancer therapy [21]. Although efficacy of TNF- was increased in these studies and gold nanoparticles had no apparent effects, toxicological studies are required to determine the safe use of gold nanoparticles. VEGF (vascular endothelial growth factor) antibody conjugated to gold nanoparticles induced apoptosis in -chronic lymphocytic leukemia cells at a significantly higher rate compared to VEGF antibody or gold nanoparticles alone [22]. Furthermore, possible usage of gold nanoparticles in the treatment of ovarian cancer, multiple myeloma and arthritis has also been suggested [23]. Studies have reported conflicting evidence on the effects of gold nanoparticles on fully developed

primary human cells [24-29]. Gold nanoparticles have been found to adversely affect cell adhesion, cell growth and protein synthesis of human dermal fibroblasts [28]. Conversely, other investigations have found no adverse reactions on human cells from gold nanoparticles [25, 27]. Another report has shown that gold nanoparticles size ranging from 10-30 nm failed to cross the perfused human placenta in detectable amounts into fetal circulation [30]. Studies using primary human cells are limited in their applicability to mature cells because they do not mimic in vivo growth and development. Most current data suggest that the effects of gold nanoparticles are not universal and may be tissue specific [31]. For example, gold nanoparticles have been shown to induce a death response in human carcinoma lung cell line (A549), but have not induced any response from BHK21 (baby hamster kidney) or human HepG2 (hepatocellular liver carcinoma) cells lines [31]. More recently, Cui et al. [32] have shown that small gold nanoparticles endocytosed by HeLa cells resulted in cytotoxicity whereas large aggregates of gold nanoparticles adhering to the cell surface stimulated the proliferation of these cells. Other studies indicate that the gold nanoparticles tend to accumulate in mouse liver cells and the clearance rate from the liver is very slow [33]. Concentrations of gold nanoparticles greater than 10 ppm have shown to cause cytotoxicity with the proliferation of kerantinocytes, whereas concentrations less than 5 ppm had a positive effect on the growth rate of these cells [33]. Since the effects of gold nanoparticles on the developmental process and undifferentiated cells are largely unknown, we have investigated the effects of gold nanoparticles on the growth and differentiation of ESCs (embryonic stem cells), which mimic the developing embryo. In the present study, we determined that gold nanoparticles positively affected the self-renewal of ESC by restraining the differentiation of both ESCs and EBs (embryoid bodies).

Inhibition of Embryonic Stem Cell Differentiation by Gold Nanoparticles

2. Materials and Methods 2.1 Stem Cell Culture mESC (mouse ESC) lines, D3 and 7AC5/EYFP were obtained from Dr. Sue O’Shea, University of Michigan and ATCC (American Type Culture Collection), Manassas, VA, respectively. The D3 line was cultured on 0.1% gelatin (Sigma, St Louis, MO) coated dishes without feeder cells using mESC medium at 37 °C in 5% CO2. mESC medium contained Dulbecco’s modified Eagle’s medium (Invitrogen, Carisbad, CA) supplemented with 10% FBS (fetal bovine serum, Atlanta Biologicals, Atlanta, GA), 0.1 mM 2-mercaptoethanol (Sigma), 0.1 mM nonessential amino acids (Invitrogen), 1 mM sodium pyruvate (Sigma) and 1,000 U/mL of LIF (leukemia inhibitory factor; Chemicon International Inc, Temecula, CA). The 7AC5/EYFP cells, labeled with yellow fluorescent protein [34, 35], were maintained in the same way as the D3 cells, except cultured on a feeder layer of gamma irradiated MEFs (mouse embryonic fibroblasts). The cultures were passaged after dissociating with 0.04% (v/v) trypsin-EDTA (Invitrogen). hESC (human ESC) line H1 (WA01) was obtained from the WiCell WISC Bank (Madison, WI). hESCs were maintained on a feeder layer of gamma irradiated MEFs in a medium which included DMEM/F12, 20% KnockOut SR medium (Invitrogen), 0.1 mM β-mercaptoethanol and 4 ng/mL human bFGF (basic fibroblast growth factor; Invitrogen). The cultures were passaged after being treated with 1 mg/mL collagenase IV (Invitrogen). 2.2 Formation of Embryoid Bodies mESCs and hESCs were dissociated with trypsin and collagenase, respectively. For hanging drop method, ESCs were suspended in ESC medium and 20 µL droplets with 1000 ESCs were transferred on the lids of the low attachment 100 mm culture plates (Corning Inc., Corning, NY) as previously described [36]. Three days after treatment, the efficiency to form EBs

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was evaluated by the percentage of the number of EBs formed to total number of hanging drops containing ESCs. They were then transferred into plates containing differentiation medium (80% KO-DMEM, 20% FBS, 0.5% L-glutamine, 0.2% β-mercaptoethanol, 1% nonessential amino acids and 50 U/mL:50 µg/mL penicillin-streptomycin solution) and incubated at 37 °C, 5% CO2 in a humidified atmosphere. 2.3 Characteristics of Gold Nanoparticles The gold nanoparticles (1-100 nm mixture, product #09818) were purchased from Meliorum Technologies, Ithaca, NY and had the following characteristics: mean particle diameter: range 1 nm to 100 nm, emphasis on products of 8 nm, 13 nm and 17 nm mean diameter with ca. 10% monodispersity. Approximate surface area, average: based on mean diameter, ranges from ca. 17 m2/g to 38 m2/g. Particle purity: 99.9% (metals basis). The gold nanoparticle preparation as purchased was further analyzed by TEM (transmission electron microscopy). Diluted samples (10 µL) of gold nanoparticles were placed on a formvar grid, air dried and analyzed using TEM (Philips FEI, Model 410). 2.4 Treatment of ESCs and EBs with Gold Nanoparticles The gelatin coated tissue culture plates (24-well) with or without MEFs feeder layer were seeded with 250 ESCs per well containing 0.5 mL of the ESC medium. Likewise, 4 EBs/well were seeded in six-well culture plate containing 2.5 mL differentiation medium. Gold nanoparticles were sterilized by autoclaving. ESCs and EBs were treated with gold nanoparticles at concentrations ranging from 0.1 μg/mL to 100 μg/mL. The medium was changed every other day. The culture plates were observed daily using a light microscope to visualize the cell growth, number of colonies, colony size, and differentiation of the ESCs. In some cases the ESC colonies were stained with

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osmium prior to microscopic analysis. After trypsin treatment of culture plates, the total cell number was counted using a hemocytometer. Similarly, differentiation of EBs was monitored by light microscopy. 2.5 Cell Viability Assay Cell viability was determined by spectroscopic measurement of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide reduction (MTT; Sigma). This assay is based on the ability of a mitochondrial dehydrogenase enzyme from viable cells to cleave the tetrazolium rings of the pale yellow MTT to yield a dark blue insoluble formazan crystal, which is largely impermeable to healthy cell membranes. The insoluble formazan crystal is solubilized by a detergent. Cell viability is directly proportional to the level of the formazan product formed [37]. Briefly, the cells were washed with PBS and incubated in 1 mL of 0.5 mg/mL MTT solution for 2 h at 37 °C. The MTT solution was discarded, and formazan precipitate was dissolved in 500 μL of DMSO by agitating dishes for 5 min at 200 rpm on an orbital shaker. The absorbance at 570 nm was determined with a micro-plate reader (Bio-Rad, Hercules, CA). Each experiment was performed in triplicate. 2.6 Pluripotency of Embryonic Stem Cells The pluripotency of ESCs was tested by their ability to form EBs that differentiate into multilineage cells [38], produce teratomas in animals, and express ESC specific surface antigens and markers, such as Oct 4 [39]. 2.7 Analysis of Gold Nanoparticles Incorporated into ESCs Gold nanoparticles uptake was examined by TEM to confirm that the gold nanoparticles had entered the cells and were not just attached to the cell surface. For TEM analysis, cells were fixed in 3% (v/v)

glutaraldehyde (Sigma) dissolved in 0.1 M sodium phosphate buffer (pH 7.0) for 30 min at room temperature. They were then washed three times with 0.1 M phosphate buffer containing 8% sucrose, followed by post fixation for 45 min at room temperature with 2% (w/v) osmium tetroxide (EM grade) in sucrose-phosphate buffer. This was followed by washing with sucrose-phosphate buffer and sequential complete dehydration of the specimen in a series of increasing ethanol concentrations (60%-100%). The samples were then infiltrated and embedded in epoxy resin. Ultrathin sections (80 nm) were placed on TEM copper grids and analyzed. For the quantification of gold nanoparticles incorporated into the ESCs, the cells were harvested, washed three times with PBS and the gold was extracted using 1 N HCl and analyzed by atomic absorption spectrometry [40]. Standard curve of gold was also prepared in 1 N HCl and analyzed by atomic absorption spectrometry. 2.8 Extraction of RNA and RT-PCR Analysis Cells were detached from the cell culture plates and collected by centrifugation at 1,000 g for 3 min. The RNA was extracted from the cells using an RNeasy Kit (Qiagen Inc, Valencia, CA) following the manufacturer’s instructions as previously described [41]. Analysis of the RNA samples was performed using the one-step Reverse transcription polymerase chain reaction (RT-PCR) kit (Qiagen). PCR conditions used were as follows: reverse transcription, 50 °C, 30 min; Taq polymerase activation, 95 °C, 15 min; then thermal cycling, 94 °C, 30 s, 55 °C, 30 s, 72 °C, 30 s, for 35 cycles; followed by a single elongation step at 72 °C, 10 min. RT-PCR products were analyzed by 2% agarose gel electrophoresis. The forward and reverse primers used for mouse Oct 4 were GCAACTCAGAGGGAACCTCCT and TCTCCAACTTCACGGCATTG, respectively to yield an amplified product of 63 bp.

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2.9 Statistical Analysis The results are expressed as mean ± SEM (standard error of the mean). ANOVA (Analysis of variance) with repeated measures was used to analyze the effect of various concentrations of gold nanoparticles. Differences are considered significant of P < 0.05. Statistical analyses were carried out using SPSS for windows version 11.5 (SPSS Inc., Chicago, IL). All data are expressed as mean ± standard error. The results were statistically analyzed with SPSS for Windows, version 1 (SPSS-Inc, Chicago, IL, USA). A P value of less than 0.05 was considered significant.

(a)

3. Results 3.1 Embryonic Stem Cell Growth and Proliferation The general morphology of mouse and human ESCs in phase-contrast microscopy is shown in Fig. 1, where normal ESCs form dense, round or oval colonies. Under normal growth conditions, ESCs required splitting at 70%-80% confluency achieved within 2-4 days depending upon the amount of inoculum used. Once the ESC culture reached > 90% confluency, cells started to differentiate. ESC colonies differentiate even at lower than 80% confluency if grown for longer than 7 days. To study the effect of gold nanoparticles, we characterized the commercially available heterogeneous mixture of gold nanoparticles using TEM. The results, shown in Fig. 2, indicated a diverse particle size ranging from 1-100 nm of the gold nanoparticles in the analyzed mixture. mESCs cultured under the same conditions, but in the presence of the gold nanoparticles yielded more colonies (Fig. 3). There were 2-4 times more ESC colonies in the treated wells compared to the untreated wells. Furthermore, there were dramatic changes in the colony formation, size and morphology depending upon concentration of gold nanoparticles. At lower concentrations (< 5 μg/mL) of the gold nanoparticles, colonies were well-developed and somewhat larger

(b) Fig. 1 (a) Mouse ESC line D3 (10×) and (b) human ESC line H1 (4×) colonies cultured for 3 days and 4 days, respectively on the MEF layer in the respective stem cell media under normal culture conditions.

Fig. 2 TEM (transmission electron microscope) image of the commercially available heterogonous mixture of gold nanoparticles. Gold nanoparticles representing 1-100 nm were compared against the 500 nm bar (insert). Representative photomicrograph was from three independent experiments.

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5

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(a) (b) Fig. 3 Gold nanoparticles effected mouse ESC colony size and numbers. Mouse ESC line 7AC5 was cultured on MEF layer in the ESC medium. The six-well plates were seeded with 1,000 trypsinized ESCs/well. Light micrographs of 4 days old ESC cultures (a) untreated and (b) treated with 10 µg/mL of gold nanoparticles (10×). ESC colonies were relatively smaller but more compact and higher in number in gold nanoparticles treated wells as compared to the control wells. Representative photomicrographs were from three independent experiments.

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(a) (b) Fig. 4 Treatment of ESCs with gold nanoparticles. (a) Mouse ESC line 7AC5 and (b) line D3 were cultured with and without MEF layer, respectively in the ESC medium supplemented with LIF. The cell cultures were treated with various concentrations of the gold nanoparticles. 3 days old cell cultures were trypsinized and counted by hemocytometer. The average cell numbers in each treatment were plotted relative to the average cell numbers in the controls of three experiments. The asterisks indicate significant difference with the control at P < 0.05 (ANOVA with repeated measurement followed post-hoc pair-wise comparison).

than the control (approximately twice the size of control colonies) and grew for a longer period (> 10 days) without undergoing differentiation. At higher concentrations (> 10 μg/mL), the ESC colony size was significantly smaller as compared to the control (approximately 1/2 to 1/3 of the size of control colonies) (Fig. 4), and ESCs did not differentiate even after 15 days of culturing. A similar effect of gold nanoparticles on the growth and colony

formation was observed in the case of hESCs. At lower concentrations of gold nanoparticles hESC, colonies were larger but at higher concentrations the colonies were smaller, tighter and did not differentiate when compared to the control cell colonies. The fact that lower concentrations of gold nanoparticles enabled ESC colonies to grow larger than the control suggested that the larger colonies may have a higher number of cells.

Inhibition of Embryonic Stem Cell Differentiation by Gold Nanoparticles

We estimated the viable cell counts after treating the cells with trypan blue using hemocytometer and performing MTT assays. In both cases, the cell counts were higher in the gold nanoparticle treatments with up to 5 µg/mL of the medium (Fig. 5). The proliferation rate of ESCs treated with gold

249

nanoparticles was significantly increased and colonies grew larger with higher cell numbers at 1-5 µg/mL of gold nanoparticles but not at 50 µg/mL as compared with the control (Fig. 6). This concentration dependent two-phasic effect of gold nanoparticles on ESCs is interesting but requires further investigation.

Cell number in thousands

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Time (hrs) Fig. 5 Dual effect of gold nanoparticles on the rate of ESC proliferation. Mouse ESCs (D3) were cultured in the ESC medium supplemented with LIF in six-well plates. ESCs untreated and treated with gold nanoparticles were trypsinized at various intervals and counted by hemocytometer. Shown are ESC untreated (___▲___) and treated with 5 µg/mL and 50 µg/mL of gold nanoparticles (___ ___; and ___ ▄ ___, respectively). The average cell numbers in each treatment were plotted relative to average cell numbers in the controls of three experiments. The asterisks (all three lines) indicate significant difference with the control at P < 0.05 (ANOVA with repeated measurement followed post-hoc pair-wise comparison).

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Fig. 6 Cell viability of ESC treated with gold nanoparticles. ESCs (D3) were grown to 50% confluency in six-well plates and treated in triplicate with various doses of gold nanoparticles. After 12 h, the cells were washed with PBS and incubated in 1 mL of 0.5 mg/mL MTT solution for 2 h at 37 °C. The MTT solution was discarded, formazan precipitate was dissolved in 500 μL of DMSO, color intensity was determined using a micro-plate reader. The average cell numbers in each treatment were plotted relative to average cell numbers in the controls of three experiments. The asterisks indicate significant difference with the control at P < 0.05 (ANOVA with repeated measurement followed post-hoc pair-wise comparison).

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Inhibition of Embryonic Stem Cell Differentiation by Gold Nanoparticles

3.2 Embryonic Stem Cell Differentiation

treatment affected the self-renewal and differentiation

Since gold nanoparticles prolonged ESC self-renewal, it prompted us to investigate the influence of gold nanoparticles on the differentiation of ESCs and EBs. The results showed that gold nanoparticle treated (10 µg/mL to 50 µg/mL) ESC colonies remained small, tight, and undifferentiated for longer than 10 days after culturing, whereas the control ESC colonies differentiated after 2-5 days (Fig. 7). A similar effect of gold nanoparticles was observed on EBs treated with or without retinoic acid. The results shown in Fig. 8 indicate inhibition of both mouse and human EB differentiation when treated with gold nanoparticles. 3.3 Pluripotency and Expression of ESC-Specific Markers We

investigated

whether

gold

(a)

nanoparticle

properties of ESCs. Treated ESCs were passaged 5 times in the ESC medium containing 20 µg/mL gold nanoparticles and then subcultured in normal ESC medium. These cells behaved normally with respect to colony morphological characteristics and self-renewal for at least 10 passages. Treated ESCs were also tested for their ability to form EBs and differentiate into osteogenic, chondrogenic, adepogenic and neurogenic lineages, as well as, produce teratomas in mice. The results showed that ESCs exposed to the gold nanoparticles retained their ability to form EBs capable of differentiating into several cell types and produced teratomas when injected into the hind limbs of mice, suggesting that the pluripotency of ESCs was not affected. RT-PCR analysis of the ESC RNA showed that the cells treated with various concentrations of gold nanoparticles expressed Oct 4 in addition to SSEA

(b)

(c) (d) Fig. 7 Gold nanoparticles inhibited differentiation of ESC colonies. Light micrographs of ESC cultures untreated and treated with gold nanoparticles in a six-well culture plate. (a) Mouse ESC (7AC5) colonies in control wells were larger in size and differentiated (see arrow) after 4 days of incubation, and (b) treated with 50 µg of gold nanoparticles were relatively smaller, compact and did not differentiate even after 5 days of inoculation (10×). (c) Human ESC (H1) colony in a control well differentiated and (d) human ESC colony in a well, treated with 10 µg gold nanoparticles after 7 days of inoculation (4×). The control ESC colonies showed extensive differentiation while the gold nanoparticles treated cell colonies remained undifferentiated.

Inhibition of Embryonic Stem Cell Differentiation by Gold Nanoparticles

(a)

(b)

(c)

(d)

251

Fig. 8 Effect of gold nanoparticles on the differentiation of EBs. EBs prepared from mouse ESCs (D3) were induced by retinoic acid to differentiate and treated with 1 µg/mL to 10 µg/mL of gold nanoparticles. Light micrographs of the EBs (a) untreated and treated with (b) 1 µg, (c) 5 µg, and (d) 10 µg (10×). While untreated EBs differentiated readily, differentiation of treated EBs was noticeably inhibited by gold nanoparticles.

(ESC-specific cell surface antigen) genetic markers (data not shown) at levels comparable to the control ESCs. Fig. 9 depicts Oct 4 expression in ESCs treated with 20 µg/mL of gold nanoparticles.

These results as described in Fig. 11 demonstrated uptake of gold by the cells treated with gold nanoparticles in a concentration depended manner.

3.4 Uptake of Gold Nanoparticles by ESCs In order to determine whether the gold nanoparticles are actually incorporated into the cells and are not associated or externally bound to the cells, the treated cells were analyzed by TEM. The observations shown in Fig. 10 suggest the incorporation of gold nanoparticles in vivo. The uptake of gold nanoparticles by the cells was further investigated by extracting the treated cells and analyzing the cell extract for the amount of gold incorporated into the cell biomass using atomic absorption spectroscopy.

1

2

3

Fig. 9 Agarose gel electrophoresis of RT-PCR products of mRNA isolated from mouse ESCs (D3). Shown are the products of Oct 4 from ESCs treated with 5 µg/mL of gold nanoparticles, untreated ESCs and differentiated derivatives of ESCs (lanes 1-3, respectively).

Inhibition of Embryonic Stem Cell Differentiation by Gold Nanoparticles

252

(a) (b) Fig. 10 TEM (transmission electron microscopic) imaging of gold nanoparticles incorporated into ESCs. The cells were grown in six-well plates in the presence or absence of gold nanoparticles for 5 days. Shown are TEM of (a) untreated cells; and (b) cells treated with 20 μg/mL gold nanoparticles.

Gold recovered in the cells (ppb)

900 800 700 600 500 400 300 200 100 0 0

1

5

10

20

Gold nanoparticles in the medium (μg/mL) Fig. 11 Uptake of gold nanoparticles by embryonic stem cells. The cells treated with varying concentration of gold nanoparticles for 5 days were harvested and extracted using 1 N HCl. The extracts were analyzed by atomic absorption spectrometry.

4. Discussion It is quite evident that the commercial potential of nanotechnology offers vast new opportunities in fields such as: biomedicine, biotechnology, bioengineering, electronics, plastics, aerospace, and materials science [2, 5, 37, 39, 42-45]. The potential biomedical applications of nanoparticles are extensive.

For example, gold nanoparticles have been proposed for the treatment of ovarian cancer, multiple myeloma and arthritis [23, 46]. Studies using nanogold, with antiangiogenic and long-term delivery capabilities, showed no signs of retinal or optic nerve toxicity [47]. Studies have shown fullerene carbon nanomaterials to regrow hair in a mouse animal model and human skin [48]. It has been reported that gold nanoparticles

Inhibition of Embryonic Stem Cell Differentiation by Gold Nanoparticles

can be taken up by endocytosis, but show little cytotoxicity in human cells [25]. Additionally, gold nanoparticles taken up into vesicles or the nucleus showed no toxicity. However, along with the expanding range of uses in nanotechnology, there is growing concern that the exposure to nanomaterials could cause health hazards [42, 47] and our current understanding of the interactions of nanomaterials with living systems at cellular and molecular levels is severely limited. Studies in mice models have shown that PEG coated gold nanoparticles can accumulate in spleen, affecting the immune system [29]. Additionally, gold nanoparticles resulted in a significant increase in alanine transaminase and aspartate transminase, suggesting some liver damage. In another study, gold nanoparticles have been shown to be cytoxic in certain human epithelial cell lines in vitro and endothelial cells in vivo [26]. Clearly some studies implicate gold nanoparticles as toxic [25, 26, 29, 49-51] or nontoxic [24, 42], however, the understanding of the effects of gold nanoparticles on the processes of proliferation and development is lacking. We report here that the heterogeneous mixture of gold nanoparticles ranging from 1 nm to 100 nm in size (Fig. 2) influenced both the cell proliferation and differentiation processes. Results showed that at lower concentrations (< 10 μg/mL), gold nanoparticles promoted growth and the colonies were relatively larger in size, whereas the higher concentrations (> 10 μg/mL) inhibited ESC growth and colonies were smaller than the control. ESCs treated with gold nanoparticles remained undifferentiated long after the differentiation of the untreated cells. Despite both the positive and negative effects of gold nanoparticles on the colony formation (Fig. 3), cell proliferation (Fig. 4), and ability of ESC to self-renew (Fig. 5), their capacity to differentiate into various lineages was not affected. Preliminary results showed that the gold nanoparticles treated ESCs were capable of differentiating into osteogenic,

253

chondrogenic and myogenic lineages as well as producing teratomas in mouse model. Although untreated ESCs formed larger colonies, they differentiated earlier (within 3-4 days) as compared to the gold nanoparticle treated ESCs. Higher concentrations (> 20 µg/mL) of gold nanoparticles yielded smaller but more compact colonies, which remained undifferentiated even after 5 days of incubation (Fig. 7). These findings together, specifically the observations that gold nanoparticles prolonged undifferentiated growth of ESCs can potentially be helpful for maintaining ESCs for an extended period of time. The dual effect of gold nanoparticles on the growth of ESCs may be due to the reactive nature of these particles that could interact with multiple components at cellular and molecular levels. One of the hypotheses is that gold nanoparticles are involved in the inactivation of VEGF which promotes angiogenesis and thus interferes with signal transduction. Current studies have shown that gold nanoparticles act as anti-angiogenic agents by binding to heparin-binding growth factors like VEGF165 and bFGF [52]. Gold nanoparticles may also be cytotoxic or even embryotoxic particularly at higher concentrations. In that case, it would be important to investigate whether gold nanoparticles induce apoptosis or necrosis in these model systems. The gold nanoparticles used in this investigation had a heterogeneous mixture ranging from 1 nm to 100 nm. Therefore, the dual effect displayed by gold nanoparticles may also be due to the various sized particles. Future studies are required to be carried out using homogeneous size gold nanoparticles. It is significant to note that both lower and higher doses of gold nanoparticles had no effect on the pluripotency and stemness of ESCs, as judged by the expression of specific pluripotency markers including Oct 4 (Fig. 9), or on the potential to differentiate into various lineages despite the fact they caused an increase or decrease in cell proliferation. Oct 4 is a homeodomain

254

Inhibition of Embryonic Stem Cell Differentiation by Gold Nanoparticles

transcription factor of the POU (Pit-Oct-Unc) family. This protein is critically involved in the self-renewal of undifferentiated ESCs. The incorporation of gold nanoparticles into ESCs was clearly demonstrated by TEM analysis (Fig. 10). The uptake of the gold nanoparticles by ESCs was further confirmed by atomic absorption spectroscopy (Fig. 11). These results showed that incorporation of gold nanoparticles was increased with higher concentrations suggesting that the gold nanoparticles are actively transported across the cell membrane or taken up by endocytosis. The TEM images indicated the presence of gold nanoparticles in endosomes and organelles of ESCs, mostly in the form of aggregates. It is interesting to note that the gold nanoparticles accumulated in the cells inhibited cell proliferation but did not cause apoptosis or necrosis of the cells.

analysis, respectively.

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[5]

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5. Conclusions [7]

Production and use of nanomaterials are rapidly increasing. While toxicity of many of the nanomaterials is well established, biological effects of gold nanoparticles remain elusive. Our study has shown that gold nanoparticles adversely affected the growth and differentiation of ESCs. These results suggest that exposure to gold nanoparticles may pose health risks by influencing the development and differentiation processes in the embryo. Therefore, further studies on the molecular mechanism of gold nanoparticles action are warranted.

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Acknowledgments This research was supported by the OU-WB (Oakland University-William Beaumont) Interdisciplinary Research Program, Oakland University, Michigan Research Excellence Fund, and the SIBHI (Summer Institute of Bioengineering and Health Informatics), which was funded by the National Institutes of Health and the National Science Foundation. We are thankful for Ms. Loan Dong and Dr. Harvey Qu for TEM studies and statistical

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Journal of Pharmacy and Pharmacology 2 (2014) 257-268

DAVID PUBLISHING

Antioxidant and Antifungal Activities of the Essential Oils of Ocimum gratissimum from Yaoundé and Dschang (Cameroon) Jean Baptiste Hzounda Fokou1, Pierre Michel Jazet Dongmo 2, Fabrice Fekam Boyom1, Elizabeth Zeuko’o Menkem1, Issakou Bakargna-Via1, Ide Flavie Kenfack Tsague1, Marguerite Simo Kamdem1, Paul Henri Amvam Zollo3 and Chantal Menut4 1. Department of Biochemistry, University of YaoundéI, Yaoundé, Cameroon 2. Department of Biochemistry, University of Douala, Douala, Cameroon 3. Department of Food Science and Nutrition, National Advanced School of Agro-industrial Sciences, Ngaoundere, Cameroon 4. Institute of Biomolecules Max Mousseron, Faculty of Pharmacy, Montpellier 999019, France Abstract: Due to the harmful effect of free radical on physiological and pathological state of our body on one hand, and the increase of the fungal infection on the other hand, drug that can reduce free radical and inhibit fungal growth is needed. This work aims to evaluate the antioxidant and antifungal activities of the Ocimum gratissimum L. essential oils from center and west region of Cameroon. Essential oil was analyzed by GC (gas chromatography) and GC coupled with MS (mass spectrometry). The antioxidant activities of essential oils were studied by the Diphenylpicrylhydrazyl method and the β-carotene bleaching test. The antifungal activities were assessed using micro-dilution technique for yeasts and agar dilution method for Aspergillus. Thymol and γ-terpinene, eugenol and thymol were the major compounds for Yaoundéand Dschang, respectively. The scavenging capacity of sample from Dschang was higher than that of Yaoundé. Also, the β-carotene bleaching tests of the sample from Dschang were better than that from Yaoundé. The antifungal activity of the sample from Yaoundéwas higher than that from Dschang on yeasts and Aspergillus isolates, respectively. This work presents and compares the chemical composition, antioxidant and antifungal activity of Ocimum gratissimum essential oil from center and west region of Cameroon. Key words: Ocimum gratissimum L., antioxidant, antifungal, gas chromatography, gas chromatography coupled with mass spectrometry.

1. Introduction The metabolism of oxygen continuously produces small quantity of reactive oxygen derivatives. The physiological role of these molecules can have a function in cell signalization, phagocytosis and cell cycle [1]. The production of free radicals species grow up and induce the stress state called oxidative stress [1, 2]. Due to its capacity to damage almost all the type of molecules in the organism, the oxygen derivative species are implicated in a large number of pathologies Corresponding author: Pierre Michel Jazet Dongmo, Ph.D., associate professor, research field: biochemistry. E-mail: [email protected].

as well as chronic or acute diseases [3]. On the other hand, these reactive oxygen derivatives are produced during fungal infection [3]. The incidence of these fungal infections has dramatically increased with the number of immunocompromized patients. The genus frequently involved is Aspergillus, Candida and Cryptococcus [4, 5]. They cause 90% of death on the infected persons [6]. The genus Aspergillus is responsible for 20% of total fungal infections and is involved in 66%-99% of death on the pulmonary Aspergillosis, sinuses and brain infections [7]. The genus Candida is responsible for 64.7% of fungal infections with about 26.4% of death cases [6]. The

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Antioxidant and Antifungal Activities of the Essential Oils of Ocimum gratissimum from Yaoundé and Dschang (Cameroon)

genus Cryptococcus accounts for about 90% in HIV positive and represents 11.7% of death among these individuals [8]. The reasons for this upsurge of infection are the aggressive therapeutic agents, transplantations, immuno-depressions [9], alcoholic cirrhosis [10], increase of the drug costs [11] and the development of fungi resistance to available drugs [12]. As the available antioxidant molecules, the antifungal drugs possess side effects. Thus, searching for alternative antioxidant and antifungal compounds from natural source has been a major concern in recent years. Plant extracts are used as natural source of antioxidant and antifungal molecules due to their potent pharmacological activities, low side effect and economic viability [13-15]. Ocimum gratissimum belongs to the group of plants known as spices [16]. The plant is found throughout the tropics and subtropics, and its greatest variability occurs in tropical Africa and India [17]. In Cameroon, some studies have shown the antioxidant activity of Ocimum gratissimum L. essential oils from different sites in the east region [16]. The essential oils of this plant also exhibit toxic potentials when taken in certain quantity [18]. Its antibacterial activity was showed by Matasyoh et al. [19] on Kenyan’s species. The biological activities of essential oils are due to its chemical composition [13, 14, 16] which depends on the site of growth of the plant [16, 17, 20]. The aim of this study is to characterize the chemical composition of the essential oils of Ocimum gratissimum L. from Yaoundé and Dschang (center and west region of Cameroon, respectively), and to compare their antioxidant and antifungal activities.

2. Materials and Methods 2.1 Plant Material The leaves were collected from Nkolondom II for the Yaoundéspecies and Bafou for Dschang species, on August 2009. The identification of the plant was done in the Cameroon National Herbarium and

voucher specimen was deposited under identification number 5817/SRF/Cam. 2.2 Fungal Strains The fungal material was made of three mould isolates of Aspergillus genus (Aspergillus flavus, Aspergillus fumigatus and Aspergillus niger) and three yeasts (Candida albicans ATCC24433, Candida parapsilosis

ATCC22019

and

Cryptococcus

neoformans IP95026) obtained from “Centre Pasteur du Cameroun”. These fungal strains and isolates were maintained on Sabouraud Dextrose Agar. 2.3 Extraction of Essential Oils The plant samples were hydro-distilled for 5 h using a Clevenger-type apparatus. Essential oils obtained were dried over anhydrous sodium sulphate and stored at 4 °C until use for further experiments. The extraction yields were calculated in percentage (w/w) relatively to the starting plant material. 2.4 Chemical Analysis of the Essential Oils The essential oils were analysed by GC (gas chromatography) and GC coupled with MS (mass spectrometry). 2.4.1 Gas Chromatography The oil was analysed on a Varian CP-3380 GC with flame ionisation detector fitted with a fused silica capillary column (30 m × 0.25 mm coated with DB5, film thickness 0.25 μm); temperature program 50 -200 °C at 5 °C/min, injector temperature 200 °C, detector temperature 200 °C, carrier gas N2 at 1 mL/min. The linear retention indices of the components were determined relatively to the retention times of a series of n-alkanes and the percentage compositions were obtained from electronic integration measurements without taking into account relative response factors. 2.4.2 Gas Chromatography/Mass Spectrometry GC/MS analyses were performed using a Hewlett-Packard apparatus equipped with an HP1 fused silica column (30 m × 0.25 mm, film thickness

Antioxidant and Antifungal Activities of the Essential Oils of Ocimum gratissimum from Yaoundé and Dschang (Cameroon)

0.25 μm) and interfaced with a quadrupole detector (GC-quadrupole MS system, model 5970). Column temperature was programmed from 70-200 °C at 10 °C/min; injector temperature was 200 °C. Helium was used as carrier gas at a flow rate of 0.6 mL/min, the MS was operated at 70 eV. 2.4.3 Identification of Components The identification of the constituents was assigned on the basis of comparison of their retention indices and their mass spectra with those given in Refs. [21-23] with the data bank NBS75K and with the stored laboratory mass spectral library [16, 20]. 2.5 Determination of the Antioxidant Activities 2.5.1 DPPH Test The antiradical activity was determined using DPPH (2, 2-diphenyl-1-picrylhydrazyl), which was dissolved in ethanol to give 100 μm solution. The accurate CDPPH (DPPH concentration) was determined by spectrophotometric method following Eq (1): A517 = 9832 × CDPPH (1) where, 9,832 is the molecular extinction coefficient of DPPH determined independently in ethanol. 2.0 mL of the ethanolic solution of DPPH was added 100 μL of a methanolic solution of a reference molecule BHT (butylated hydroxyl toluene) at different concentrations. The oil was tested using the same method. The control (without antioxidant) was represented by the DPPH ethanolic solution containing 100 μL of methanol. Decrease in absorption was measured at 517 nm after 2 h, at room temperature. The decrease in absorption induced by the test compound was calculated by subtracting that of the control. The concentration required for SC50 (50% scavenging concentration) was determined graphically. All the spectrophotometric measurements were performed using a SAFAS UV-mc2 Spectrophotometer, equipped with a multi-cell/multi-kinetic measurement system and with a thermostated cell-case [13, 14]. 2.5.2 β-carotene Bleaching Test Antioxidant activity was evaluated using a

259

β-carotene/linoleate model system. A solution of β-carotene from Fluka (7235-40-7) was prepared by dissolving 2.0 mg of β-carotene in 10 mL chloroform. 1.0 mL of this solution was pipetted into a round-bottomed flask which contained 20 μL purified linoleic acid from Avocado (60-33-3) and 200 mg Tween 40 emulsifier from Aldrich (9005-66-7). After chloroform was removed under vacuum using a rotary evaporator at 40 °C, 50 mL aerated distilled water was added to the flask with vigorous shaking. The antioxidant activity was evaluated by measuring, at 470 nm, the kinetics of discoloration of β-carotene in the absence (control) and presence of the antioxidant solution (10 μL methanolic solutions containing different concentrations of essential oil or BHT for comparative purposes) at 50 °C [13, 24]. A blank was prepared under the above conditions but without β-carotene. All the kinetics were obtained according to Eq (2): A = A0e-kt + C (2) where, A0 is the absorbance at time zero, C is the absorbance at infinite time and k is the degradation rate constant of β-carotene, from which Ip (inhibition percentages) were calculated using Eq (3): Ip = 100 (k0 - k)/k0 (3) where, k0 and k are the degradation rate constants of β-carotene in the absence and presence of inhibitor. The plot of the inhibition percentage as a function of the inhibitor concentration enabled the determination of the IC50 of the sample. All the spectrophotometric measurements were performed using the same apparatus as in scavenging assay. 2.6 Antifungal Activities 2.6.1 Antifungal Susceptibility Testing Agar dilution and agar disc diffusion methods were respectively used to assess the sensitivity of moulds and yeasts to essential oil. For moulds, the experiments were designed as previously described [25]. For yeasts, the disc diffusion method was used. Briefly, 10 mL of

260

Antioxidant and Antifungal Activities of the Essential Oils of Ocimum gratissimum from Yaoundé and Dschang (Cameroon)

Sabouraud Dextrose Agar were aseptically poured on 54 mm Petri dishes and allow solidifying. After solidification, 2 mL of 2.5 × 105 CFU/mL inoculums from each yeasts were inoculated on Petri dishes, allowed to stand for 10-15 minutes after what, 15 µL of essential oil (1 mg/mL to 0.125 mg/mL) were place aseptically on sterilized piece of filter papers and the prepared filter paper was placed at the center of the prepared Petri dishes. The Petri dishes were sealed using paraffin paper and incubated at 37 °C for 48 h. The test was done in triplicate. Nystatin (1.33 mg/mL) was used as positive control and treated as essential oils. The negative controls contain filter paper with no antifungal substance. The susceptibility of microorganisms was determined by measuring the diameter of the inhibition zone after incubation. 2.6.2 Determination of MIC (Minimal Inhibitory Concentration) and MFC (Minimal Fungicidal Concentration) Agar dilution and broth micro-dilution methods were respectively used to assess MIC and MFC of moulds and yeasts of essential oil using the method previously described [25].

Rather, yield was similar to that obtained by Pessoa et al. [26] (0.2%). This variation in yield can be explained by the geographic difference, period of harvest, postharvest treatment and extraction conditions [16, 20].

2.7 Statistical Analysis

The identifiable constituents of our essential oils on the qualitative point were generally the same for the

Results are presented as means ± SD (standard deviation) of the three measurements. The analyses of variance followed by LSD (least significant difference) post hoc determination were performed using the statistical software Statgraphics 5.0 for Windows. Statistical significance was set at P < 0. 05.

two species. Nevertheless, there was a slight difference,

3. Results and Discussion 3.1 Yields of Essentials Oil’s Extraction The yield of 0.19% with yellowish color was obtained with the species of Yaoundé and 0.61% for that of Dschang. The yield of Ocimum gratissimum-Yaoundé was lower than that obtained by Tchoumbougnang [20] who had a percentage yield of 0.47% and 0.42% from the samples of Yaoundé and Bazou, respectively.

3.2 Chemical Composition After GC and GC/MS analyses, the chemical composition of our essential oils is shown in Table 1. From Table 1, the essential oil from Yaoundéwas rich in monoterpenes (91.6%) on which monoterpene hydrocarbon and oxygenated monoterpenes were 46.1% and 45.5%, respectively. Sesquiterpenes form only 6.1% of the essential oil. The major compounds were thymol (40.7%), γ-terpinene (24.5%) and p-cymene (5.9%). As in the Yaoundé species, the essential oil from Dschang was rich in monoterpene (41.2%) on which monoterpene hydrocarbon was 18.6% and oxygenated monoterpene was 22.6%, with thymol as the major component; on the other hand, the oil was characterized by a high content of eugenol (46.2%). Sesquiterpenes form only 10.6% of the whole essential oil, of which sesquiterpene hydrocarbons represent 9.1% and oxygenated sesquiterpenes only 1.5%.

with the presence of oxygenated sesquiterpene in the essential oil from Dschang (0.2%), while there was none in the Yaoundé species. The presence of some chemical compounds is dependent on the geographical situation of the plant. These results were similar to those previously obtained by Ndoye [16] for the essential oils from six localities of east Cameroon. This author obtained a variation in the presence of oxygenated sesquiterpene (0%-2.5%) among the six species. Matasyoh et al. [19] obtained 0% oxygenated sesquiterpene on the Kenyan’s species. Based on the majority of compounds, the essential oil from Yaoundé showed similar results with that previously obtained by Ndoye [16], Tchoumbougnang [20] and Nguefack [27] for plants collected in East,

Antioxidant and Antifungal Activities of the Essential Oils of Ocimum gratissimum from Yaoundé and Dschang (Cameroon) Table 1 Chemical composition of essential oils. KI

Compounds

Monoterpenes Monoterpene hydrocarbons 928 α-thujene 938 α-pinene 954 camphene 970 sabinene 977 β-pinene 991 myrcene 1,002 α-phellandrene 1,010 Δ-3-Carene 1,014 α-terpinene 1,018 p-cymene 1,027 limonene 1,039 (E)-β-ocimene 1,057 γ-terpinene 1,081 p-cymenene 1,085 terpinolene Oxygenated monoterpenes 1,074 sabinene hydrate 1,093 linalool 1,107 thujone 1,127 limonene oxide 1,135 t-pinocarveol 1,159 isoborneol 1,163 borneol 1,175 terpinen-4-ol 1,197 α-terpineol 1,230 neral 1,280 thymol methyl-ether 1,288 thymol Sesquiterpenes Sesquiterpene hydrocarbons 1,388 β-elemene 1,397 isocaryophyllene 1,398 longifolene 1,434 β-caryophyllene 1,450 α-humulene 1,453 (E)-β-farnesene 1,456 (Z)-β-farnesene 1,468 α-humulene 1,480 germacrene D 1,494 α-selimene 1,497 α-curcumene 1,500 (E,E)-α-farnesene 1,511 β-selimene 1,530 Δ-cadinene 1,535 epi-α-selinene Oxygenated sesquiterpenes 1,593 humulene oxide 1,597 caryophyllene oxide 1,633 t-cadinol Aromatic compound 1,354 eugenol

O. gratissimum (Dschang) 41.2 18.6 0.6 0.2 0.1 0.1 1 tr tr 0.7 5.9 1.7 0.1 7.6 0.6 tr 22.6 0.7 tr 0.2 tr 0.9 0.2 tr 20.6 10.6 9.1 0.7 0.1 3.0 0.4 tr 0.3 0.1 0.1 tr 3.0 0.8 0.4 0.2 1.5 0.1 1.3 0.1 46.2 46.2

tr = percentage lower than 0.1%; - = not present; KI = Kovats index.

Percentages O. gratissimum (Yaoundé) 91.6 46.1 3.8 1.0 0.1 0.7 3.8 0.3 0.2 3.0 5.9 1.6 0.2 24.5 0.8 0.2 45.5 0.2 3.0 0.2 tr 0.5 0.2 1.3 tr tr 40.70 6.1 5.9 0.3 0.1 2.7 0.9 0.2 0.2 0.5 0.4 0.6 0.2 0.2 0.6 0.6

261

262

Antioxidant and Antifungal Activities of the Essential Oils of Ocimum gratissimum from Yaoundé and Dschang (Cameroon)

Littoral and Center region of Cameroon, respectively. These essential oils from the Center and East Regions of Cameroon can be classified as thymol/γ-terpinene chemotype. Meanwhile, that of Dschang can be classified as eugenol/thymol chemotype and its chemical composition does not correspond to anyone in literature. 3.3 Antioxidant Activity 3.3.1 DPPH Test 3.3.1.1 BHT Activity Fig. 1 represents the dose response curve of DPPH radical scavenging activity of BHT. This graph enabled us to determine the scavenging concentration of BHT. An SC50 value of 0.07 ± 0.01 mg/mL was obtained. 3.3.1.2 Essentials Oils Activity The curves of Fig. 2 show the scavenging capacity of the different essential oils. From the curve, it shows that scavenging capacity of the DPPH radical increases as concentration increases. The SC50 values were extrapolated and are shown in

Table 2. From these SC50 values, the effective concentration, that is, the concentration of essential oil necessary to capture 50% of DPPH radical (EC50) and the antiradical capacity was calculated and shown in Table 2. It was found that O. gratissimum from Dschang (5.88 ± 0.99) × 10-4 and BHT (1.22 ± 0.00) × 10-3 had the highest antiradical activities than O. gratissimum of Yaoundé(3.37 ±0.19) × 10-5. There was a statistically significant difference (P < 0.05) among the activities of the essential oils, but there was no statistical significance difference (P < 0.05) among the activities of O. gratissimum-Dschang and BHT. Our essentials oils are rich in phenolic compounds (41.3% for essential oil from Yaoundéand 66.8% for essential oil from Dschang). Both oils showed antiradical activity, but the relative high activity of essential oil of Dschang can be attributed to its high phenolic content. This activity could be explained by the capacity of phenolic compounds to stabilize DPPH by hydrogen donation and formed DPPH-H as previously reported by Brand-Williams et al. [28].

100

Percentage of inhibition (%)

90 80 70 60 50 40

BHT

30 20 10 0 0

0.02

0.03

0.05

0.1

concentration (µg/mL) Fig. 1 DPPH scavenging activities of BHT. BHT = butylated hydroxytoluen.

0.2

0.3

Antioxidant and Antifungal Activities of the Essential Oils of Ocimum gratissimum from Yaoundé and Dschang (Cameroon)

263

Percentatge of inhibition (%)

100 90 80 70 60 50 40

OGd

30

OGy

20 10 0 0

0.2

0.3

0.5

1

2

3

concentration (mg/mL) Fig. 2 DPPH scavenging activities of essentials oils. OGd = Ocimum gratissimum from Dschang, OGy = Ocimum gratissimum from Yaoundé. Table 2 SC50, EC50 and AC values of the essential oils and BHT. SC50 ±SD (%) CE50 ±SD (%) AC ±SD (%) O. gratissimum-YDE (mg/mL) 2.39 ±0.13a (2.97 ±0.16) ×104c (3.37 ±0.19) ×10-5e b 3d O. gratissimum-DSC (mg/mL) 0.15 ±0.00 (1.70 ±0.40) ×10 (5.88 ±0.99) ×10-4f BHT (mg/mL) 0.07 ±0.01b (8.23 ±0.00) ×102d (1.22 ±0.00) ×10-3f O.gratissimum-YDE = Ocimum gratissimum from Yaoundé; O. gratissimum-DSC = Ocimum gratissimum from Dschang; SC50 = scavenging concentration 50; EC50 = efficient concentration 50; AC = anti oxydant capacity; SD = standard deviation; a, b, c, d, e and f = statisticaly significantl difference.

3.3.2 Β-carotene Bleaching Test 3.3.2.1 BHT Activity The antioxidant activity of BHT (used as the reference antioxidant) was analyzed and the results are plotted as shown in Fig. 3. The inhibitory concentration of the BHT to the oxidation of linoleic acid was determined. An IC50 value of 0.10 ±0.00 was obtained. 3.3.2.2 Essential Oils Activity The antioxidant activity of the respective essential oils was analyzed and the results are plotted as shown in Fig. 4. From the curves of Fig. 4, the inhibitory concentration of the essential oils was determined. For O. gratissimum-Dschang, the IC50 value of (0.30 ±0.03) × 10-3 mg/mL was obtained, while an IC50 value of (3.28 ± 0.10) × 10-3 mg/mL was obtained from O. gratissimum-Yaoundé. The values obtained showed

that the sample of Dschang was a better antioxidant than that of Yaoundéwith a statistical difference (P < 0.05). With respect to the reference, there was no statistical difference (P < 0.05) between O. gratissimum-Dschang and BHT. The activities observed can be explained by the higher content of phenolic compounds. The antioxidant activity can be attributed to the phenol content of the essential oil [13, 14, 16], but other compounds in the essential oil can also increase these activities as previously reported [29, 30]. Our essentials oils are rich in other compounds such asγ-terpinene, α-terpinene and terpinolene for both Yaoundé and Dschang samples. Kim et al. [30] reported that the essential oil from tea tree had higher antioxidant than BHT. This oil was rich in α-terpinene, γ-terpinene and terpinolene. Therefore, we can suggest that the antioxidant activities of our essential oil could

264

Antioxidant and Antifungal Activities of the Essential Oils of Ocimum gratissimum from Yaoundé and Dschang (Cameroon)

100 Percentage of Inhibition (%)

90 80 70 60 50 40

BHT

30 20 10 0 0

0.13

0.2

0.33

0.66

1.3

Concentration (µg/mL) Fig. 3 Antioxidant activity of BHT. BHT = butylated hydroxytoluen.

100 Percentage of inhibition (%)

90 80 70 60 50 EOOG DSC

40

EOOG YDE

30 20 10 0 0

0.33

3.33

8.4

16.4

33.33

Concentration(mg/mL) Fig. 4 Antioxidant activities of essentials oils. EOOG DSC = Ocimum gratissimum from Dschang, EOOG YDE = Ocimum gratissimum from Yaoundé.

be due to the synergistic action between terpinene (in his various configurations), terpinolene and phenol compounds. 3.4 Antifungal Activity The disc diffusion method is actively used to

evaluate the biological activities of natural substances and plant extracts. But in the case of the solutions with a weak activity, high quantity of extracts are needed and the countenance of disc is very poor [3], in addition the essential oils contain high amount of hydrophobic compound and the interaction between disc surface and

Antioxidant and Antifungal Activities of the Essential Oils of Ocimum gratissimum from Yaoundé and Dschang (Cameroon)

these molecules is very weak. Therefore, this method was used in this study just to find any active concentration as previously used by Mohammedi [31]. 3.4.1 Results of MIC and MFC on Moulds The results from Table 3 show the inhibitory effect of essentials oils on three moulds tested. All these essential oils revealed statistically the same activity (P < 0.05) and all the Aspergillus were resistant to the Amphotericine B (Fungizone) and Griseofulvin. In fact, the MIC is higher than 2 mg/mL. The MIC of Aspergillus flavus obtained in this study was smaller than that obtained by Tchoumbougnang [32]. In fact, this author, using Ocimum gratissimum harvested in Yaoundéand dried before essential oil extraction, obtained the MIC of 1.25 µL/mL on Aspergillus flavus FPR023. Using microathmosphere technique [33] has enable us to obtained the MIC of 80 × 10-3 mg/disc on Aspergillus niger. The MFC/MIC ratio revealed that the essential

265

oils tested have fungicidal activity on tested strains. 3.4.2 Results of MIC and MFC on Yeasts Table 4 gives the results. In general, these two essential oils revealed good anti-yeast activity as compared to the reference drug found in Cameroon markets: Nystatin. From Table 4, we observed that the essential oil from Yaoundé revealed the high activity on the tested yeasts strains with 0.32 mg/mL ≤ MIC ≤ 2.57 mg/mL. The most sensitive strain was Candida albicans with MIC of 0.32 mg/mL and 2.50 mg/mL, respectively. The most resistant was Cryptoccocus neoformans with MIC of 2.57 mg/mL and 4.68 mg/mL for essential oils from Yaoundé and Dschang, respectively. Instead, the activities observed here on another Candida albicans strain were weaker than that obtained by Lemos et al. [34]. In fact, these authors, using the same method, obtain MIC of 0.25 mg/mL.

Table 3 Different parameters of the anti Aspergillus activities of essential oils and reference molecules. IC50±SD(mg/mL)×10-3 MIC ±SD(mg/mL)×10-3 MFC MFC/MIC Strains OGd OGy OGd OGy AmB Gris A.Fl 0.27b±0.00 0.15b±0.02 0.34ce±0.01 0.28de±0.06 >2 >2 0.47a 0.43a 1.43 1.76 a a A.Fu 0.27b±0.01 0.17b±0.01 0.34ce ±0.01 0.31ce±0.01 >2 >2 0.45 0.40 1.41 1.47 A.Ng 0.37bc±0.01 0.28b±0.02 0.42ce±0.01 0.33ce±0.01 >2 >2 0.45a 0.40a 1.14 1.40 IC50 = inhibitory concentration 50; MIC = minimal inhibitrory concentration; A.Fl = Aspergillus flavus; A.Fu = Aspergillus fumigatus; A.Ng = Aspergillus niger; OGd = Ocimumgratissimum harvested in Dschang; OGy = Ocimumgratissimum harvested in Yaoundé; AmB = Amphotericine B (fungizone); Gris = griseofulvine; a, b, c, d and e = no significant differences. Table 4 Anti-yeast parameters. Candida albicans Candida parapsilosis ATCC24433 ATCC22019 OGy OGd N OGy OGd N MAC ND ND ND ND ND ND IC50 ND ND ND ND ND ND Micro dilution MIC 0.32 2.50 25.90 0.64 2.50 ≤3.24 MFC ND ND ND ND ND ND MAC 0.08 1.12 8.00 0.25 0.24 8.00 IC50 2.80 33.60 152.00 0.83 0.24 12.00 Absorbance (625nm) MIC ≥80 ≥80 584.00 11.97 5.06 72.00 MFC ND ND ND ND ND ND MAC ND ND ND ND ND ND IC ND ND ND ND ND ND Subculture and 50 colony count MIC ND ND ND ND ND ND MFC 0.32 10 25.9 5.15 10 25.90 MAC = minimal antifungal concentration; IC50 = inhibitory concentration 50; MIC = minimal minimal fungicidal contration. Methods

Parameters (mg/mL)

Cryptoccocusneoformans IP95026 OGy OGd N ND ND ND ND ND ND 2.57 4.68 25.90 ND ND ND 1.12 2.99 8 10.39 8.55 12 ≥80 ≥150 72 ND ND ND ND ND ND ND ND ND ND ND ND 2.57 4.68 51.00 inhibitory concentration; MFC =

266

Antioxidant and Antifungal Activities of the Essential Oils of Ocimum gratissimum from Yaoundé and Dschang (Cameroon)

Table 5 MFC/MIC ratios. Parameters OGy OGd Nystatin

Candida albicans 1 4 1

MFC/MIC Candida parapsilosis 8 4 >4

Cryptoccocus neoformans 1 1 1.96

OGY = Ocimum gratissimum from Yaoundé; OGD = Ocimumgratissimum from Dschang.

The change of coloration in this method is due to the presence of acetic acid. These compounds are produced during the fungal growth by hydrolysis of glucose contained in the medium [35]. This acetic acid is a weak acid and to change the coloration of the medium, the presence of high amount is needed. Therefore, it is possible that we consider that the inhibition of yeasts is complete when there are a small number of yeast cells. Using these three methods, the anti-yeast parameters obtained here are high than those revealed by the Nystatin as previously shown by Nakamura et al. [36]. In fact, these authors showed that Ocimum gratissimum L. from Brazil is more active on yeast than Nystatin. The variation observed in the result of spectrophotometric method can be explained by the effect of the oils on yeast. In fact, this method is based on the evaluation of the turbidity of the solution but the subculture revealed that the yeasts observed in the solution are not viable. These observations confirm the previous description of the effect of the eugenol rich essential oils of Ocimum gratissimum L. on some Candida species [37]. Using electronic transmission microscopy on yeasts previously treated with sub-MIC concentration, these authors observed that the deleterious effect of the essential oil on the cell wall of the yeast may be the main reason for the decrease in the rate of yeast growing, because the integrity of the cell wall is necessary for cell division. In the aim to assess the fungicidal degree of essential oil, we calculate the MFC/MIC ratio and the results are in Table 5 Table 5 shows that the essential oils and Nystatin are fungicidal for Cryptococcus neoformans with MFC/MIC lower than 4, and fungistatic for Candida parapsilopsis with MFC/MIC ratio greater than 4. The

essential oil from Yaoundéand Nystatin are fungicidal on Candida albicans, instead, the essential oil from Dschang is fungistatic on this same strain. Many authors have shown that phenolic compounds have an important antifungal activity [27, 31]. The antifungal properties of essential oils were good. But the essential oil from Ocimum gratissimum harvested in Yaoundé revealed statistically the same antifungal activities than that harvested in Dschang on moulds (P < 0.05). Except on Candida albicans, these two essential oils revealed the same activities on yeasts. According to their chemical composition, essential oils from Yaoundé contain less phenolic compound than that of Dschang. This observation reveals that the inhibitory effects on yeast observed are not only due to the presence of the phenolic compounds. These activities can also be caused by the hydrocarbon monoterpene as previously shown. Our essential oils contained monoterpen hydrocarbons in the range of 46.1% in the essential oil from Ocimum gratissimum L. harvested in Yaoundéand 18.6% for the one harvested in Dschang. Bouzouita et al. [37], starting with essential oil containing α-pinene (59.1%), obtained the MIC of 7.5 µg/mL.

4. Conclusions The results of this study show that the essential oils of Ocimum gratissimum from Yaoundé and Dschang belongs to two different chemotypes and has antioxidant as well as antifungal activities. In general, Ocimum gratissimum from Dschang has a better antioxidant activity compared to that of Yaoundébut the contrary was observed with regard to the antifungal activities. The best antioxidant potential and the considerable antifungal activity exhibited by Ocimum

Antioxidant and Antifungal Activities of the Essential Oils of Ocimum gratissimum from Yaoundé and Dschang (Cameroon)

gratissimum show that this essential oil is a source of compounds which could be used for the prevention of oxidative stress related diseases. The antioxidant properties of this extract probably explain partly, the use of these plants in traditional medicine for the treatment of infectious diseases and inflammations. However, if this essential oil is to be used for preservation or medicinal purposes, issues of safety and toxicity need to be addressed.

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2849-2854. [31] Z. Mohammedi, Study of microbial and antioxidant power of essential oils and flavonoids of some plants from the region of Tlemcen, Ph.D. Thesis, University Abou Bakar Belkkaïde Tlemcen, 2006. [32] F. Tchoumbougnang, Contribution to the determination of contains, chemical characteristics and antifungal activities of essential oils of some aromatic condiments and medicinal plants in Cameroon, Doctoral Thesis, University of YaoundéI, 1997. [33] J.F. Terezinda, S.F. Rafael, Y. Lidiane, R.P.S.J. osé, K.I. Noemia, M.B. Anali, Antifungual activity of essential oil isolated from Ocimum gratissimum L. (Eugenol chemotype) against phytopathogénic fungi, Brazilian Archive of Biotechnological Technics 49 (6) (2006) 867-871. [34] J.A. Lemos, X.S. Passos, O.F. L. Fernades, J.R. Paula, P.H. Ferri, L.K.H. Souza, et al., Antifungal activity from Ocimum gratissimum L. towards Cryptoccocus neoformans, Memorial Institute Oswaldo Cruz 100 (1) (2005) 55-58. [35] L. Bousmaha, L. El moualdi, O. El Yachioui, Isolated strains of Candida guilliermondii brine carrot producing an extracellular beta-fructofuranosidase, Pharmaceutical Society Bulletin Bordeaux 146 (2007) 51-62. [36] C.V. Nakamura, K. Ishida, L.C. Faccin, B.P.D. Filho, D.A.G. Cortez, S. Rozental, et al., In vitro activity of essential oil from Ocimum gratissimum L. against four Candida species, Research in Microbiology 155 (2004) 579-586. [37] N. Bouzouita, F. Fachouri, B.M. Halina, M.M. Chaabouni, Chemical composition and antioxidant activity, antimicrobial and insecticidal activity of essential oil of Funiperus phaeniceae, Journal of Social Chemistry 10 (2008) 119-125.

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Central Sensitization of HPA Axis in Modulation of Neuropathic Pain in Diabetic Rats Vandana Sharma, Rohit Goyal, Shaila Khah and Babita Thakur Department of Pharmacology, School of Pharmaceutical Sciences, Himachal Pradesh 173212, India Abstract: The present study was designed to investigate the possible role of HPA (hypothalamic-pituitary-adrenal) axis in neuropathic pain in streptozotocin induced diabetic rats. Wistar albino rats of either sex weighing 180-220 g (n = 6) were employed. Diabetes was induced by administering STZ (streptozotocin) (45 mg/kg, i.p.) once. Neuropathy was induced by the ligation of sciatic nerve in diabetic animals. A glucocorticoid receptor antagonist: ketoconazole 175 mg/kg, p.o. and glucocorticoid receptor agonist: hydrocortisone 1 mg/kg, i.p. were given. Assessment of neuropathic pain was achieved using hot plate and hot immersion tests. Tissue biochemical: lipid peroxidation, NO and glutathione were estimated on 28th day spectrophotometrically. Plasma cortisol level was also estimated using HPLC (high performance liquid chromatography). Sciatic nerve ligation to diabetic animals caused significant increase in nociceptive responses as hyperalgesia and allodynia, tissue lipid peroxidation and NO products, and decrease in GSH (glutathione) level, in comparison to SC (saline control). Ketoconazole administration produced decreased pain responses as well as decrease in oxidative stress and inflammatory mediators. Administration of hydrocortisone resulted in suppression of HPA axis. The activation of HPA axis in diabetic animals resulted in significant pain response. This may be due to increased corticosterone level which in turn desensitizes HPA axis and decreased nociceptive response in diabetic animals observed. Key words: Diabetes, neuropathy, HPA axis, sciatic nerve ligation.

1. Introduction Diabetes mellitus is a syndrome which is characterized by hyperglycemia and impaired insulin signaling. It occurs mainly as altered state of carbohydrate, fat and protein metabolism [1, 2]. The macrovascular diseases like cardiomyopathy, nephropathy, retinopathy and neuropathy ultimately result to disruption of cell and organ [3, 4]. DN (diabetic neuropathy) affects somatic and autonomic divisions of peripheral nervous system and causes nerve damage throughout the body [5]. DN can be classified as peripheral (pain or loss of feeling in toes, feet, legs, hands and arms), autonomic (changes in digestion, glandular and sexual responses, and cardiac activity), and proximal or focal (pain in thighs, hips, and weakness in muscle). The prevalence of pain is 35% in patients with type 1 Corresponding author: Rohit Goyal, Ph.D., associate professor, research field: neuropharmacology. E-mail: [email protected].

and type 2 diabetes and greater in females with diabetes than in males (38%) [6]. Neuropathic pain is an unpleasant biological response which occurs due to progressive distal neurodegeneration and damage of nerves from spinal cord and CNS (central nervous system) [7, 8]. It is characterized by spontaneous pain, allodynia (pain response to normally innocuous stimuli), and hyperalgesia (aggravated pain evoked by noxious stimuli). Nociceptive information is relayed through multiple parallel pathways to higher brain structures which initiate appraisal of pain [9]. Neuropathic pain is mediated through sympatho-adrenal and HPA (hypothalamic-pituitary-adrenal) axis [10]. The etiology of HPA axis involves an enhanced central drive of hormone production and release or resistance to circulating glucocorticoids as hyper- or hypofunction, respectively [9]. Activation of HPA axis causes in increased secretion of CRH (corticotropin-releasing hormone) into

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Central Sensitization of HPA Axis in Modulation of Neuropathic Pain in Diabetic Rats

hypophyseal-portal circulation. The CRH released stimulates synthesis and breakdown of POMC (proopiomelanocortin) into ACTH (adrenocorticotrophic hormone) in corticotrophs. Thus, ACTH acts to secrete corticosterone, the major glucocorticoid in rats (cortisol in humans) at the adrenal cortex. Increased cortisol level negatively regulates glucocorticoid pathway in hippocampus, hypothalamus, and anterior pituitary [11]. Physical sensations, such as touch and pain, are relayed through peripheral sensory neurons to the central nervous system and are transduced with higher threshold by specialized receptor complexes, called nocciceptors [10, 12]. Thus, the spinal reflex arc is completed by activation of motor neurons located in the ventral horn. Nociceptive information also ascends from the spinal cord dorsal horn via brainstem and thalamic nuclei to activate neurons within the somatosensory cortex to result in the sensory perception of pain [13]. The parallel activation of limbic (emotional) structures mediates the sympathoadrenal and neuroendocrine component of the pain response [9]. The pain transmission pathways sensitize neuronal function at multiple levels which may lead to the occurrence of maladaptive cascades for chronic pain [14]. Thus, the possible role of HPA axis is hypothesized in central sensitization of neurons in diabetic neuropathy.

2. Materials and Methods 2.1 Animals Wistar albino rats weighing 180-220 g of either sex were procured from Animal house, Shoolini University, Solan, HP. They were maintained at temperature 22-24 °C, relative humidity 55% ± 5% and light and dark cycles (12/12 h), and provided with standard pellet diet and water ad libitum. The animals were acclimatized to laboratory conditions for 7 days before commencement of the experiment. The experiment was conducted in accordance with the guidelines of CPCSEA (Committee for the Purpose of Control and

Supervision of Experiments on Animals). The experimental protocol was duly approved from IAEC (Institutional Animal Ethics Committee) (No. IAEC/SU-PHARM/12/025). 2.2 Induction of Neuropathy Induction of neuropathy to the animals was performed using SNL (sciatic nerve ligation) model. Thirty six Wistar albino rats were divided into six groups, each comprising of six animals (n = 6) viz. NC (normal control), SC (sham control), DNC (diabetic neuropathic control), Keto (Ketoconazole 175 mg/kg), Hydro (hydrocortisone 1 mg/kg), and Keto + Hydro. At the outset, diabetes was induced by administering streptozotocin (45 mg/kg, i.p., once) and serum glucose level was estimated using biochemical kit using glucose oxidase-peroxide method. STZ treated animals were provided with 10% glucose solution in water ad libitum. The animals having blood glucose level > 300 mg/dL were considered to be diabetic. The animals were anesthetized with pentobarbital sodium (50 mg/kg, i.p.). The left sciatic nerve was exposed below the division of the semitendinosis branch. Four loose ligatures using 4.0 chromic gut were made around the nerve within a 1.0-1.5 mm gap to each other. The muscle and skin layers were sutured and the animals were allowed to recover [15]. A glucocorticoid receptor antagonist: KTZ (Ketoconazole) 175 mg/kg, p.o.

and

a

glucocorticoid

receptor

agonist:

hydrocortisone 1 mg/kg, i.p. were given daily. The sham operated animals were treated with surgical procedures. 2.3 Pharmacological Assessments The periodic checkup for nocciceptive response was assessed using Eddy’s hot plate and hot immersion tests. The animals were sacrificed and blood was collected for plasma corticosterone estimation. Sciatic nerve was excised out and homogenized using buffer solution for biochemical estimations.

Central Sensitization of HPA Axis in Modulation of Neuropathic Pain in Diabetic Rats

2.4 Estimation of Plasma Corticosterone Using HPLC Plasma corticosterone level was estimated using HPLC/photodiode array system (Agilent, USA). Briefly, 500 μL of plasma containing known quantity of dexamethasone was extracted with 5 mL of dichloromethane. The dichloromethane extract was evaporated to dryness and dissolved in 100 μL mobile phase. The mobile phase used was methanol: water (70:30) at the flow rate of 1.0 mL/min. 20 μL of sample was injected into HPLC system for quantification and detected at 250 nm using PDA (photo-diode array) detector. Agilent Eclipse XDB-C18, 15 μm, 4.6 × 150 mm column (made in USA) was used. Dexamethasone was used as an internal standard. The chromatogram was recorded and analyzed with Empower software [16]. 2.5 Estimation of Lipid Peroxidation Products The lipid peroxidation products as MDA (malondialdehyde) in sciatic nerve tissue were assessed by the method of Ohkawa et al. [17]. Briefly, 500 µL aliquot was added to equal volume of Tris buffer and sample was incubated for 2 h at 37 °C. 1 mL of 10% TCA (trichloroacetic acid) was added into the test tubes and centrifuged at 5,000 rpm for 10 min. 1 mL of supernatant was pipetted out and 1 mL of 0.67% of TBA (thiobarbituric acid) solution was added. The tubes were kept in boiling water for 15 min, cooled immediately under running tap water and 1.0 ml of water in each test tube was added. Absorbance of the sample was recorded at 532 nm. The amount of lipid peroxidation products was expressed as nM of MDA/mg of tissue. 2.6 Estimation of Nitrate/Nitrite Level Nitric oxide expression in the form of stable nitrate, /nitrite products was assessed by the method of Green [18]. Briefly, 500 µL sample (prepared in phoshphate buffer 0.1 M, pH 7.4) was mixed with 500 µL Griess reagent (1% of sulfanilamide solution in 2.5% ortho-phosphoric acid and 0.1% of NEDA in distilled water). Samples were incubated at room temperature

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for 10 min and absorbance was recorded at 540 nm. The concentration of NO products was determined from standard curve prepared using 10-100 µM solution of sodium nitrite and expressed as µM/mg of tissue. 2.7 Estimation of Reduced Glutathione GSH Level Reduced glutathione level was estimated by the method of Ellman [19]. 1 mL of tissue homogenate in phosphate buffer (0.1 M, pH7.4) was mixed with 1mL of 20% (w/v) TCA reagent containing 1 mM EDTA, and kept for 10 min at room temperature. Proteins were precipitated and filtrate was removed carefully after centrifugation at 2,000 rpm for 10 min. 0.2 mL filtrate was taken and 1.8 mL of Ellman’s reagent (0.1 nM of DTNB in 0.3 M phosphate buffer with 1% sodium citrate). The pale yellow color was developed and optical density was measured at 412 nm. The level of reduced glutathione was expressed as µM/mg of tissue. 2.8 Statistical Analysis The findings from present study were expressed as mean ± SD (standard deviation), analyzed by one way and two-way ANOVA (analysis of variance) followed by Bonferroni’s multiple comparison test. P value < 0.05 was considered to be statistically significant. A Graph pad Prism Instate software was used as statistical tool.

3. Results 3.1 Effect of Pharmacological Interventions on Pain Perception by Hot Plate Test Sciatic nerve ligation to diabetic rats of DNC group showed significant (P < 0.05) increase in pain perception in the form of hyperalgesia, in comparison to SC on 3rd day onward. Administration of ketoconazole 175 mg/kg to diabetic neuropathic rats of Keto group produced significant (P < 0.05) increase in latency of pain, as compared to DNC group on 7th, 14th, 21st and 28th day significantly. Treatment with hydrocortisone (1 mg/kg) showed decreased pain

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perception and administration of ketoconazole + hydrocortisone to diabetic neuropathic rats also showed a significant decrease in pain perception on 7th, 14th, 21st and 28th day. The effect produced from the treatment of Keto + Hydro was insignificant to that produced from Keto group on 7th, 14th, 21st and 28th day (Fig. 1). 3.2 Effect of Pharmacological Interventions on Pain Perception by Hot Immersion Test SNL to diabetic rats exhibited a significant (P < 0.05) decrease in latency time of pain response in the form of allodynia in DNC group, as compared to SC on 3rd day onward. Administration of ketoconazole (175 mg/kg) antagonized the pain response in Keto group, as compared to DNC group on 7th, 14th, 21st and 28th day. Treatment with hydrocortisone (1 mg/kg) to the rats of Hydro group showed a significant (P < 0.05) increase in latency time of pain, and that of Keto + Hydro group showed similar effect on 7th, 14th, 21st and 28th day. The effect produced from Keto + Hydro was insignificant to that produced from Keto group on 7th, 14th, 21st and 28th day (Fig. 2).

cortisol level, as compared to SC. Administration of KTZ (175 mg/kg) to Keto group produced a significant (P < 0.05) decrease in cortisol level, as compared to DNC group. The animals treated with hydrocortisone (1 mg/kg) of hydro group, and KTZ + hydrocortisone of KTZ + Hydro group showed significant (P < 0.05) increase in cortisol level, in comparison to DNC group (Table 1). 3.4 Effect of Pharmacological Interventions on Lipid Peroxidation Reaction Induction of diabetes and SNL produced significant (P < 0.05) increase in tissue MDA level, as compared to SC. Animals from the Keto group showed significant (P < 0.05) decrease in MDA, as compared to DNC group. Administration of hydrocortisone (1 mg/kg) to Hydro group showed significant (P < 0.05) decrease in MDA and the administration of KTZ + hydrocortisone to diabetic neuropathic animals also showed significant decrease in MDA level, as compared to DNC. The level of MDA observed in Keto + Hydro group was insignificant (P > 0.05) to that of Keto group (Fig. 3).

3.3 Effect of Pharmacological Interventions on Plasma Cortisol Estimated Using HPLC

3.5 Effect of Pharmacological Interventions on Reduced Glutathione Level

Induction of neuropathy to diabetic animals produced significant (P < 0.05) increase in plasma

Sciatic nerve ligation to diabetic animals produced

Fig. 1 Effect of pharmacological interventions on pain perception by hot plate test; results are expressed as mean ± SD analyzed by two way ANOVA followed by Bonferroni’s multiple comparison test; a: P < 0.05 vs. NC; b: P < 0.05 vs. DNC; c: P > 0.05 vs. KTZ.

Central Sensitization of HPA Axis in Modulation of Neuropathic Pain in Diabetic Rats

273

Fig. 2 Effect of pharmacological interventions on pain perception by hot immersion test; results are expressed as mean ±SD analyzed by two way ANOVA followed by Bonferroni’s multiple comparison test; a: P < 0.05 vs. NC; b: P < 0.05 vs DNC; c: P > 0.05 vs. KTZ.

significant (P < 0.05) decrease in tissue GSH level, as compared to SC. Treatment with KTZ (175 mg/kg) in Keto group showed significant increase in GSH level, as compared to DNC group. Administration of hydrocortisone (1 mg/kg) to hydro group showed significant increase (P < 0.05) in GSH level and that of KTZ followed by hydrocortisone to Keto + Hydro group also showed significant increase in GSH level, in comparison to DNC group. The effect produced by the administration of Keto + Hydro in restoring GSH level was insignificant (P > 0.05) to that of KTZ group (Fig. 4).

in Keto + Hydro group was not significant (p > 0.05) to that of KTZ group (Fig. 5). Table 1 Effect of pharmacological interventions on plasma cortisol estimated using HPLC. S. No.

Group

1

Standard

Plasma cortisol level (Area: mAU) 6,270,041

2

NC

6,125

3

DNC

176,739

4

KTZ

46,973

5

Hydro

82,055

6

KTZ + Hydro

60,634

3.6 Effect of Pharmacological Interventions on Nitrite/Nitrate Level Treatment with STZ and SNL to rats produced significant (P < 0.05) increase in tissue NO level in animals of DNC group, as compared to SC. Administration of KTZ (175 mg/kg) to Keto group showed significant (P < 0.05) decrease in NO, as compared to DNC group. Administration of hydrocortisone (1 mg/kg) to hydro group showed significant (P < 0.05) decrease in NO level and the administration of KTZ and hydrocortisone to Keto + Hydro group also showed significant decrease in NO level, in comparison to DNC. The level of NO observed

Group Fig. 3 Effect of pharmacological interventions on lipid peroxidation reaction; results are expressed as mean ± SD analyzed by one way ANOVA followed by Bonferroni’s multiple comparison test: a: P < 0.05 vs. SC; b: P < 0.05 vs. DNC; c: P > 0.05 vs. KTZ.

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Central Sensitization of HPA Axis in Modulation of Neuropathic Pain in Diabetic Rats

Group Fig. 4 Effect of pharmacological interventions on reduced glutathione level; results are expressed as mean ± SD analyzed by one way ANOVA followed by Bonferroni’s multiple comparison test; a: P < 0.05 vs. SC; b: P < 0.05 vs DNC; c: P > 0.05 vs. KTZ.

Group Fig. 5 Effect of pharmacological interventions on nitrite/nitrate level; results are expressed as mean ± SD analyzed by one way ANOVA followed by Bonferroni’s multiple comparison test; a: P < 0.05 vs. SC; b: P < 0.05 vs. DNC; c: P > 0.05 vs. KTZ.

3.7 Effect of Pharmacological Interventions on Serum Glucose Level Induction with STZ (45 mg/kg) produced a significant (P < 0.05) increase in blood glucose level to the animals of DNC group, as compared to SC. However, level of blood glucose level observed in Keto, Hydro and Keto + Hydro was insignificant to that of DNC group (Fig. 6).

Group Fig. 6 Effect of pharmacological interventions on serum glucose level; results are expressed as mean ± SD analyzed by one way ANOVA followed by Bonferroni’s multiple comparison test; a: P < 0.05 vs. NC; b: P > 0.05 vs. DNC.

4. Discussion The present study demonstrated the central sensitization of HPA axis in modulation of neuropathic pain in diabetic rats. Neuropathic pain is characterized by increased nocciceptive response due to injuries to spinal cord or peripheral nerve [7]. The nerve damage was accompanied by “hyperalgesia” and/or “allodynia” response [15]. Streptozotocin at the dose of 45 mg/kg is reported to cause marked hyperglycemia due to direct destruction of beta cells of islets of langerhans. In present study, a significant increase in glucose level was observed, hence, the animals were considered to be diabetic. The sciatic nerve ligation model was designed to assess neuropathic pain in animals. Ligation of sciatic nerve causes edema and decrease in perineural blood supply which eventually results in peripheral axonal damage. Sciatic nerve ligation in diabetic animals is reported to potentiate neuropathy by central mechanisms in early pathological stages as abnormal gait, posture, guarding behaviors, sudden licking of hind paw and pain [20]. Neuropathic pain occurs due to sensitization of peripheral and central nerve responses [21]. A neuropathic pain was induced on 3rd day by ligation of sciatic nerve which evidently resulted to hyperalgesia and allodynia, as compared to normal untreated rats in

Central Sensitization of HPA Axis in Modulation of Neuropathic Pain in Diabetic Rats

present study. There are reports indicating direct effects of hyperglycemia on spinal cord which modify sensory mechanisms, contributing to behavior indices of neuropathic pain [22]. It is also attributed to increased production of oxidative and nitrosative stresses and release of pro-inflammatory mediators: tumor necrosis factor alpha (TNF-α), interleukins-2 (IL-2) and interferon gamma (IFN-γ), leading to progression of neuropathy in pre-diabetic rats. In present study, on 28th day tissue biochemical estimation was made, which showed significant increase in lipid peroxidation, NO and decrease in GSH levels. However, drug treatment in all the groups produced significant decrease in oxidative stress, free radical production and restoration of defensive antioxidant system. The above finding is supported by the fact that the peripheral nerve injury increases TNF-α and IL-1β immunoreactivity in dorsal root ganglia of both injured and uninjured ipsilateral adjacent afferents. The increased cytokine level is associated with reduced mechanical and thermal withdrawal thresholds in rats [23]. In diabetic patients, hyperglycemia and ketoacidosis is poorly controlled which results hyperactivation of hypothalamic-pituitary-adrenal

axis

and

elevated

circulating cortisol [24]. The conditions of both type 1 and 2 diabetes have been characterized with elevated circulating cortisol along with increased urinary free cortisol [11]. Treatment with ketoconazole (a specific

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The neurochemical and morphological changes within sensory neuron and dorsal horn manifest together as central sensitization [26]. The etiology of HPA axis involve an enhanced central drive of hormone production and release or resistance to the circulating glucocorticoids resulting hyper- or hypofunction of HPA axis. In the face of chronic activation, appropriate negative feedback within the axis can be disrupted to such an extent that could lead to some maladaptive changes [9]. Treatment with hydrocortisone (1 mg/kg) suppressed hyperactivation of HPA axis through negative feedback mechanism and produced decrease in plasma corticosterone level. These changes are associated with significant decrease in hyperalgesia and allodynia on 7th, 14th, 21st and 28th day, as compared to diabetic neuropathic control rats in present study. Administration of hydrocortisone subsequent to ketoconazole caused desensitization of HPA axis due to hyper cortisol state and thereby improved anti-nocciceptive response. This effect was insignificant to the hydrocortisone alone to experimentally neuropathic rats. Thus, the findings observed suggest that HPA axis modulation may contribute for the anti-nociceptive effect of ketoconazole and hydrocortisone in neuropathic pain.

5. Conclusions In conclusion, the findings from present study may postulate that the activation of HPA axis in

glucocorticoid receptor antagonist) caused hypofunction of HPA axis which was expressed as

hyperglycemic state resulted in increased cortisol and

decreased plasma corticosterone level. It was evident in

cortisol desensitizes HPA axis and thereby resulted decreased nociceptive response.

the form of decreased pain responses on 7th, 14th, 21st and 28th day in both the tests, as compared to diabetic neuropathic control rats. Spinal nerve ligation effectively simulates spinal root damage owing to a lumbar disk herniation. Pain is accompanied by fast autonomic and delayed neuroendocrine responses which are mediated by sympathoadrenal and HPA (hypothalamic-pituitary-adrenal) axis respectively [25].

significant neuropathic pain. The hypersecretion of

Acknowledgments The authors are thankful to the management of Shoolini University for providing research facilities.

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Study of Immunomodulating Activity of Rectal Suppositories with an Extract of Licorice Root Larisa Vasilevna Yakovleva1, Tatiana Grigoryevna Yarnykh2, Elena Yuryevna Koshevaya1, Olga Anatolievna Rukhmakova2 and Galina Nikolaevna Melnik2 1. Central Scientific Research Laboratory, National University of Pharmacy, Kharkov 61002, Ukraine 2. Department of Drugs Technology, National University of Pharmacy, Kharkov 61168, Ukraine Abstract: The article is devoted to the study of the immunomodulating activity of rectal suppositories with an extract of licorice root and essential oils of chamomile and tea tree. Taking into account that medicine is intended and prescribed for children, all the experiments were performed on immature nonlinear one-month rats with weight of 60.0-80.0 g. As a result, it was found out that rectal insertion of suppositories with an extract of licorice root causes an increase in the phagocytic function of neutrophils and increases the quantity of antibodies in the spleen and titers of hemagglutinins and hemolysins in serum, that indicating on the activation of nonspecific and humoral immunity of immature animals. Moreover, the prospects of possible application of suppositories with an extract of licorice root are showed for prevention and treatment of various immunodependent diseases, viral in particular. Key words: Rectal suppositories, extract of licorice root, immunomodulating activity, study.

1. Introduction The rate of disease incidence of children and adolescents has been significantly increased in recent years, which is caused by the current intensive loads inadequate to adaptive capacity of the growing organism. In modern conditions, the child is under the influence of various factors (unfavorable environment and bad ecological situation, uncontrolled use of antibiotics) that fully affect metabolic processes and regulatory systems which leads to immune status change. This causes the strong intention of pediatricians to use medicines with immunomodulating action for prophylaxis and children treatment [1-3]. For effective treatment of immune system diseases and their prophylaxis, it is necessary to apply only safe and effective medicines. However, among all modern immunotropic medicines synthetic ones Corresponding author: Olga Anatolievna Rukhmakova, associate professor, research field: drugs technology. E-mail: [email protected].

dominate, which are either immunostimulating or immunosuppressive agents, and as a result of active intervention into the immune system, they cause its depletion. At the same time, there is a number of herbs that have exactly immunomodulating properties, which increase the body’s resistance in the most natural and physiological way [4]. In terms of this aspect, licorice represents special scientific interest because its root contains glycyrrhizin acid, flavonoids, pectin, sugar, starch and other biologically active substances. Its anti-inflammatory, antispasmodic, wound-healing effects have been investigated as well [5-7]. Glycyrrhizin acid is not only capable to inhibit reproduction of human immunodeficiency virus, DNA and RNA viruses, but it also has antiviral, anti-inflammatory, immunomodulating activity. Over the years, the most prominent and outstanding scholars were involved in the technology development and standardization of the licorice root extract [5]. Several medicines with glycyrrhizin acid were

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Study of Immunomodulating Activity of Rectal Suppositories with an Extract of Licorice Root

developed [6, 7]. Traditionally, medicines containing licorice root are used in pediatrics as expectorants prescribed for the treatment of bronchitis and pneumonia. But rectal medicines with a licorice root extract in the form of suppositories do not exist at Ukrainian pharmaceutical market. Taking into account both rapid growth of different immunodependent diseases, especially of viral nature, and the safeness of such medicinal form as suppositories in pediatrics, nowadays it is very important to develop rectal suppositories with an extract of licorice root for various immunodependent children diseases treatment.

2. Materials and Methods All experiments have been conducted in correspondence to the rules of the ―European convention for the protection of vertebrate animals used for experimental and scientific purposes‖ [8]. The objects of the research were rectal suppositories with an extract of licorice root and essential oils of chamomile and tea tree developed at the Department of Drugs Technology of National University of Pharmacy. The aim of this study was to investigate the immunomodulating activity of suppositories with an extract of licorice root on rats with normal immune status. Considering that medicine is intended for children, studies were performed on immature nonlinear one-month rats with weight of 60.0-80.0 g. According to methodological recommendations [9], the age of 1 month rat corresponds to a person’s age of 4-7 years and is optimal for the study of medicine’s immunological properties used in pediatrics [10]. During the experiment, the animals were kept in a vivarium at t = 18-24 °C, 50%-60% humidity, natural light the ―day-night‖ in plastic cages on a balanced diet in accordance with the applicable rules. The immunomodulating activity of suppositories with an extract of licorice roots was discovered in

animals with normal immune status. To determine the nature of immunomodulating activity of suppositories, their influence on humoral, cellular and non-specific immunity of rats was studied. As a relevant medicine by its pharmacological action Echinacea tincture was used because it revealed distinct immunotropic properties according to the scientific literature and our own research. Its dose had been previously installed in the experimental studies [11]. The following groups of animals were used per eight species in each group: (1) group of immunized animals; (2) group of animals that were administered with rectal suppositories with an extract of licorice root for 2 weeks at a dose of 30 mg/kg (by the contents of the active substance); (3) group of animals that were administered with rectal suppositories with an extract of licorice root for 2 weeks at a dose of 50 mg/kg (by the contents of the active substance); (4) group of animals that were administered with Echinacea tincture for 2 weeks at a dose 1 mL/kg. The impact on the phagocytic activity of neutrophils was detected in the following way: 1% suspension of unpainted latex with a particle size of 1.5-2 µm microns was added to the heparin venous blood, obtained from the tail of the rat, and was incubated in a thermostat at 37 °C for 40 min. After the incubation period smears on glass slides were prepared, they were stained and examined under the microscope in conditions of immersion. Expression of the phagocytic activity of neutrophils was assessed by parameters: phagocytic index—the percentage of phagocytic cells on 100 cells, phagocytic number—the average number of particles of latex which were absorbed by neutrophils, phagocytic integral index—the average number of particles of latex for 100 neutrophils [12, 13]. To determine the number of autoimmune complexes and hemagglutinin, rats were immunized by single intraperitoneal injection of 3% suspension of sheep erythrocytes in a dose of 1.0 mL/100 g animals’ body weight. On the fifth day after immunization, the

Study of Immunomodulating Activity of Rectal Suppositories with an Extract of Licorice Root

number of antibodies in spleen and titers of hemagglutinin in serum were determined [14, 15]. Determination of autoimmune complexes quantity in the spleen was performed by the method of local hemolysis in gel. This method is based on the ability of lymphoid cells of experimental animals immunized by alien erythrocytes to secrete antibodies that cause lysis of red blood cells in the presence of complement. By the number of macroscopically visible zones of hemolysis around the antibody cells, the number of antibody producers on lymphoid organs was counted [16]. Titers of hemagglutinins and hemolysins were detected in the serum of immunized animals by serial dilutions in polystyrene plates. Agglutination test is based on the ability of antibodies (agglutinins), contained in the serum of immunized animals, to glue sheep red blood cells (used as antigen) in isotonic solution of sodium chloride. The cellularity of organs of immunogenesis (spleen, thymus) was determined. Lymphoid organs were removed on the date of determination of the number of autoimmune complexes. Spleen and thymus were weighed and weight increase was measured. After the disintegration of lymphoid organs using homogenizer, the number of cells was counted in the Goryaev camera and their concentration in the suspension was determined by conventional method. The increase in body weight and mass ratios of spleen and thymus served as a criteria of rat’s physiological state. The functionality of the cellular immunity of animals was determined by the slow type of hypersensitivity reaction by Kitamura’s method [17]. The reaction is aimed to identify the ability of investigational medicinal products to affect the sensitized effectors of mediators by T-lymphocytes that cause tissue infiltration of cellular elements. Introduction of antigen into an animal paw leads to the development of local edema. The following groups of animals were used per

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eight species in each group: (1) group of non-immunized animals that were not subjected to any impact; (2) group of immunized animals; (3) group of animals that were administered with rectal suppositories with an extract of licorice root for 2 weeks at a dose of 30 mg/kg (by the contents of the active substance); (4) group of animals that were administered with rectal suppositories with an extract of licorice root for 2 weeks at a dose of 50 mg/kg (by the contents of the active substance); (5) group of animals that were administered with rectal suppositories ―Viburkol‖ (Germany) for 2 weeks at a dose of 100 mg/kg. Rats were immunized by 3% suspension of sheep erythrocytes in the dose of 0.2 mL/20 g. The final dose of suspension of sheep erythrocytes was administered under aponeurotic plate of one of the hind limbs (research paw) of rats on the 5th day. The same amount of saline was administered in the contralateral paw (control paw). After 24 h, the animals were taken out of the experiment by shift of cervical vertebrae under ether anesthesia. Feet hind limbs of animals were cut at the level of shin joint and weighed on torsion scales. Severity of local reactions was assessed by the ratio of the mass of the paw in the experimental and control group of animals, the response index was calculated using the formula: RI = (Мres. paw – Мcont. paw)/Мcont. paw × 100% (1) where: Мres. paw—mass of research paw; Мcont. paw—mass of control paw. The experimental data were processed by methods of variation statistics using a standard statistical software package ―Statistic, v. 6.0‖. For statistical conclusions when comparing samples of relative variables were used single-factor ANOVA (variance analysis) and Newman-Keyls criteria or nonparametric methods: Kruskal-Wallis test and Wilcoxon Mann-Whitney criteria. Differences between control and experimental groups were considered statistically significant at P < 0.05.

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Study of Immunomodulating Activity of Rectal Suppositories with an Extract of Licorice Root

3. Results and Discussion

differences from the group of intact control. Thus, the

According to the data of experiment, the 2-week administration of suppositories with an extract of licorice root has led to an increase in the phagocytic function of neutrophils of immature rats. Under the influence of the medicine in the dose of 30 mg/kg, a significant increase of the integral phagocytic index was observed by increasing the phagocytic number (number of latex particles absorbed by a neutrophil). Increasing the dose of the medicine to 50 mg/kg caused an increase in both the phagocyte number and integral phagocytic index (Table 1). The identified differences are reliable with respect to the intact control group and demonstrate the activation of phagocytic activity of neutrophils, namely absorptive function. Under the influence of the comparison medicine, Echinacea tincture, it is also observed an increase in the phagocytic activity of neutrophils, but due to the large disparity between the group data, the founded effect has no significant

2-week administration of suppositories with an extract of licorice root activates the rat’s phagocytic function of neutrophils. The highest efficiency of the medicine was detected at the dose of 50 mg/kg. Table 2 shows the results of determining the number of immune complexes in the spleen, and hemagglutinins and hemolysins in serum of rats. The 2-week administration of the medicine promoted the increase of antibodies genesis—the number of immune complexes in the spleen and titers of hemagglutinins and hemolysins in serum were increased. Moreover, with the dose increasing the enhancement effect was observed, indicating a dose-dependent nature of the medicine. It should be noted that for the ability to increase the number of immune complexes suppositories with an extract of licorice root significantly dominates comparison medicine, Echinacea tincture, the introduction of which also caused a significant increase in the number of immune complexes, hemagglutinins and hemolysins.

Table 1 Effect of suppositories with an extract of licorice root on the phagocytic activity of neutrophils of rats compared with Echinacea tincture Ме (Q25; Q75). Groups of animals

na

Intact control

8

Suppositories with an extract of licorice root, 30 mg/kg Suppositories with an extract of licorice root, 50 mg/kg Echinacea tincture, 1 mL/kg

8 8 8

Phagocytic index 55.0 (51.5; 62) 55.5 (52.0; 69.5) 65.0 (59.0; 70.0) 62.0 (55.5; 63)

Phagocytic number 6.3 (5.9; 7.1) 7.4 (6.7; 8.7) 8.2* (7.2; 10.0) 7.7 (5.8; 8.1)

Integral phagocytic index 3.5 (3.1; 3.97) 4.7* (4.1; 5.1) 5.5* (5.0; 6.3) 4.2 (3.4; 5.4)

na: number of animals in each group; *: reliable differences on the values of intact control, Р < 0.05. Table 2 Effect of suppositories with an extract of licorice root on humoral immune response of rats compared with Echinacea tincture. Groups of animals

na

Number of immune complexes in the spleen (mean  St. er) 1,940.0  365.9

Hemagglutinins titers, Log 2 Ме (Q25; Q75) 7.0 (6.5; 7.0)

Hemolysinins titles, Log 2 Ме (Q25; Q75) 3.5 (3.0; 5.0)

Immunized control 8 Suppositories with an extract of 8 20.0 (19.5; 24)* 7.0 (5.5; 12.0)* 4,360.0  861.2* licorice root, 30 mg/kg Suppositories with an extract of 8 24.0 (22.5; 24)* 6.0 (4.5; 10.5)* 5,960.0  448.5*/** licorice root, 50 mg/kg Echinacea tincture, 1 mL/kg 8 20.5 (19.0; 24)* 7.0 (5.5; 12.0)* 4,115.6  535.5* na: number of animals in each group; *: reliable differences on the values of immunized control, Р < 0.05; **: reliable differences on the values of comparison medicine, Р < 0.05.

Study of Immunomodulating Activity of Rectal Suppositories with an Extract of Licorice Root

Research of spleen cellularity (Table 3) showed that under the influence of medicines, the number of splenocytes increases, which may indicate the activation of recycling immune cells and immune genesis. These data are consistent with the determination of the number of immune complexes in the spleen and confirm the above conclusion about activation of antibodies genesis under the influence of suppositories with an extract of licorice root. The studied medicines affected the thymus cellularity not significantly. Important integral indicator characterizing the physiological state of animals and assessing the impact of the medicine on the general trophic processes of the body of rats, especially immature, is an indicator of growth in body weight. As seen from the data presented in Table 4, 2-week administration of suppositories with an extract of licorice root to immature rats did not have any negative impact on the physiological state of animals: Growth in body weight in the experimental group was not significantly different from the data in the control intact group.

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While determining the nature of the influence of suppositories with an extract of licorice root on cellular immunity, it has been discovered that the 2-week medicine administration does not affect the development of slow type hypersensitivity reactions of immature animals. Index of reactions in the groups of animals that was administered with suppositories with an extract of licorice root as in the group of immunized animals was significantly higher than values in control intact group that testifies to the normal immune response of rats to entering thymus-dependent antigen. Under the influence of the comparison medicine suppositories ―Viburkol‖ a clear trend towards the inhibition of slow type hypersensitivity reactions is observed (Table 5), due to anti-inflammatory properties of the medicine. Therefore, analyzing the data obtained, we can conclude that the introduction of suppositories with an extract of licorice root to one-month rats with normal immune status causes an increase in the phagocytic function of neutrophils, increases antibody cells in the spleen, titers of hemagglutinins and hemolysinins in

Table 3 Effect of suppositories with an extract of licorice root on spleen and thymus cellularity of rats compared with Echinacea tincture Ме (Q25; Q75). Groups of animals

na

Amount of splenocytes (109/L) 4,395 (4,165; 5,960)

Indicators Amount of thymocytes (109/L) 2,860 (1,785; 3,490)

Immunized control 8 Suppositories with an extract of 8 5,805 (4,050; 6,150) 2,790 (2,300; 3,370) licorice root, 30 mg/kg р1 Suppositories with an extract of 6,665 (6,070; 7,830) */ 8 2,990 (2,612.5; 3,365) licorice root, 50 mg/kg Echinacea tincture, 1 mL/kg 8 6,710 (5,280; 8,780)* 2,425 (2,010; 2,772.5) a n: number of animals in each group; *, reliable differences on the values of immunized control, Р < 0.05; р1: reliable differences on the values of suppositories with an extract of licorice root in the dose of 30 mg/kg at P = 0.059. Table 4

Effect of suppositories with an extract of licorice root on the growth in body weight of rats Ме (Q25; Q75).

Groups of animals

Dose (mg (mL)/kg)

Intact control Suppositories with an extract of licorice root Echinacea tincture a

n: number of animals in each group.

30 mg/kg 50 mg/kg 1 mL/kg

na 8 8 8 8

(g) 35 (30; 37.5) 35 (22,5; 37.5) 37.5 (25; 42.5) 35 (25; 37.5)

Growth in body weight (%) 40 (33; 47) 42 (26; 48) 42 (27; 59) 40 (28; 47)

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Study of Immunomodulating Activity of Rectal Suppositories with an Extract of Licorice Root

Table 5 Effect of suppositories with an extract of licorice root on cellular immunity of rats compared with suppositories “Viburkol”, М (min/max) (n = 10). Groups of animals Intact control Immunized control Suppositories with an extract of licorice root Suppositories ―Viburkol‖

Dose (mg/kg) 30 50 100

Index of slow type hypersensitivity reactions 1.6 (0.9; 2.6) 3.5 (2.9; 5.0)* 3.9 (2.8; 4.9)* 4.7 (3.7; 6.1)* 2.8 (2.5; 3.1)**

*: reliable differences on the intact control, Р < 0.05; **: reliable differences on the intact control at P = 0.057.

serum, indicating the activation of humoral and nonspecific immunity of immature animals. Increasing the number of splenocytes confirms the above conclusion about the activation of the humoral immune response of one-month rats under the influence of studied medicine. The highest efficiency of suppositories with an extract of licorice root was detected in the dose of 50 mg/kg, which significantly dominates the efficiency of comparison medicine Echinacea tincture. The results of the study of the impact of investigated medicine on the development of the slow type hypersensitivity reactions in rats indicate a lack of immunostimulating or suppressive effects on cellular immunity of rats. These data contribute to the prospects of a possible application of suppositories with an extract of licorice root for the prevention and treatment of various immunodependent diseases, particularly viral.

4. Conclusions (1) A study of immunomodulating activity of rectal suppositories with an extract of licorice root and essential oils of chamomile and tea tree is conducted. (2) It is established that rectal insertion of an extract of licorice root causes an increase in the phagocytic function of neutrophils, increases antibodies in the spleen and titers of hemagglutinins and hemolysins in serum, indicating the activation of nonspecific and humoral immunity of immature animals. (3) The prospects of possible application of suppositories with an extract of licorice root are presented for the prevention and treatment of various

immunodependent diseases, particularly viral.

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