Habb-e-Asgand, polyherbal Unani formulation, protects liver and antioxidative enzymes against paracetamol induced hepatotoxicity.

http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, 2014; 52(4): 506–515 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2013.863949

ORIGINAL ARTICLE

Habb-e-Asgand, polyherbal Unani formulation, protects liver and antioxidative enzymes against paracetamol induced hepatotoxicity Mehboob Ali1,2, Sagheer Ahmed Khan3, Peter S. Chang4, Rizwanul Haque5, Kanchan Bhatia1, and Saif Ahmad6 Department of Medical Elementology and Toxicology, Hamdard University, New Delhi, India, 2Research Institute-Perinatal Research, Nationwide Children’s Hospital, Columbus, OH, USA, 3Research & Development (R&D), Hamdard (Wakf) Laboratories, Ghaziabad, Uttar Pradesh, India, 4 Pharmacology and Experimental Therapeutics, Thomas Jefferson University, Philadelphia, PA, USA, 5Centre for Biological Sciences (Biotechnology), Central University of Bihar, Patna, Bihar, India, and 6Department of Biological Sciences, College of Science and Arts, King Abdul Aziz University, Rabegh, Saudi Arabia Abstract

Keywords

Context: Habb-e-Asgand, a polyherbal Homeopathy/Unani drug from Hamdard Wakf Laboratory, India, used in arthritis, gout and joint pain, is a mixture of many herbal medicinal plants. Scientific attempts to test and validate its efficacy are meager. Objective: To evaluate the hepatoprotective and antioxidative potential of Habb-e-Asgand against paracetamol toxicity. Materials and methods: Swiss albino male mice (n ¼ 5/group) were treated with Habb-e-Asgand (250 mg/kg, body weight (b.w.) in normal saline orally for 14 days followed by a single dose of paracetamol (400 mg/kg b.w./normal saline) intraperitoneally 24 h before euthanization. We estimated liver function (LFTs) using diagnostic kits, while antioxidant enzymes, cytochrome P450 (CYP) and lipid peroxidation (LPO) were measured using spectrophotometric methods. Results: Paracetamol alone induced LFTs enzymes significantly (p50.05 and p50.01, 0.001), serum glutamate pyruvate transaminase (SGPT, 70%), serum glutamate oxaloacetate transaminase (SGOT, 20%), alkaline phosphatase (ALP, 20%), total bilirubin (30%), CYP activity (50%) and LPO (45%), while it significantly inhibited the activity of antioxidant enzymes glutathione reductase (GR, 35%), glutathione peroxidase (GPx, 40%), glutathione S-tranferase (GST, 16%), catalase (CAT, 84%) and glutathione (GSH, 30%) contents. Habbe-Asgand alone and in combination of paracetamol significantly (p50.05, 0.01, 0.001) decreased LFT levels (20–25%), CYP activity (45%) and LPO level (25%), while it induced antioxidant enzyme activity (GR, 15%; GPx, 17%; GST, 20% and CAT, 60%). Discussion: Paracetamol metabolites may be mediating production of reactive oxidant species (ROS) and liver injury, which are attenuated by Habb-e-Asgand antioxidant constituents. Conclusion: Habb-e-Asgand may be used as a prophylaxis for ROS related liver injury.

Hepatoprotective, liver function test, oxidative stress

Introduction Liver is an essential organ playing a critical role in maintaining the body’s health (Papa et al., 2009). Its anatomical position facilitates hepatic control of the systemic supply of absorbed nutrients and metabolism of xenobiotics (Grattagliano et al., 2009; Mari et al., 2010) which are primarily detoxified by antioxidative enzymes and cytochrome P450s (CYPs) (Fasinu et al., 2012; Lupp et al., 2010). These enzymes also inactivate ROS in oxidative stress induced disorders (Hardwick & Cherrington, 2012; Mari et al., 2010) including liver injury, a secondary complication in many diseases such as diabetes, cancer, ischemia,

Alzheimer’s and Parkinson disease (Abu-Amara et al., Correspondence: Dr. Mehboob Ali, Research Institute at Children’s Hospital, Perinatal Research Department, Columbus, OH 43205, USA. Tel: 1 614 355 6748. Fax: 1 614 355 6799. E-mail: mehboob.ali@ nationwidechildrens.org; [email protected]

History Received 20 February 2013 Revised 17 October 2013 Accepted 2 November 2013 Published online 27 December 2013

2010; Aliyazicioglu et al., 2012; Benabou & Waters, 2003; Singh et al., 2012). Because of the wide prevalence of these disorders, there has been increasing interest in finding agents with antioxidant potential while showing minimal side effects (Manna et al., 2010). In the last decade, complementary and alternative medicine (CAM) systems (e.g., acupuncture, ayurveda, traditional Chinese medicine, naturopathy and homeopathy) has received growing interest in treating various ailments because of the minimal side effects and virtuous safety profile. Thus, CAM has generated public attention (Figure 1) all over the world including developed countries like USA (Farooqui et al., 2012; Nahin et al., 2009; Nazik et al., 2012). Of importance, homeopathy (Unani Medicines) is the second most famous CAM system after acupuncture (Figure 1). Most Unani drugs are herbal or mineral or a combination of the two. The majority of these drugs are developed for specific indications but some are meant for general tonic and rejuvenating properties (George et al., 2008; Singh et al., 2007). In the absence of effective cures for liver disorders,

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Happ-e-Asgand protects liver

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Figure 1. Worldwide reliance in CAM systems including homeopathy. Among four major CAM systems, homeopathy is the most second accepted alternative method of treatment among adults. The highest percentage of homeopathy acceptance is in India (62%) followed by Brazil (58%), Saudi Arab (53%), UAE (49%), Chile (49%) and France (40%). Respondents are 18 years of age and above from ABC socio-economic group of urban areas. The percentage of acceptance is a clear indication of rise of interest in homeopathy as alternative method of treatment of various diseases.

Unani drugs have been studied extensively to develop innovative therapies for liver ailments and drug-induced liver toxicity (Ahmad et al., 2002; Dhuley, 2002; Kar et al., 2009; Nwaehujor & Udeh, 2011). Most Unani drugs are safe, but toxicity including liver injury has also been observed (Licata et al., 2013; Oberbaum et al., 2012). Moreover, there are very few scientific attempts attesting and validating the safety of Unani drugs (Leone et al., 2011; Rutten, 2011). Therefore, the present study evaluates the hepatoprotective and antioxidative potential of a Unani medicine, known as Habb-e-Asgand. Specifically, we evaluated the protective effect of Habb-e-Asgand against paracetamol-induced liver toxicity. At high doses, paracetamol is known to cause oxidative liver injury through generation of ROS (Colle et al., 2012; Craig et al., 2012).

Habb-e-Asgand is a popular polyherbal preparation prescribed for arthritis, gout and joint pain (CSIR, 1992). It is also known to possess aphrodisiac properties (CSIR, 1992). Habb-e-Asgand contains Withania somnifera (L.) Dunal. (Solanaceae) (English name: winter cherry, Hindi name: ashwagandha) as a main constituent (Table 1) which is known for its diverse medicinal uses (Elsakka et al., 1989). Briefly, Habb-e-Asgand therapy is provided in small, round and uniformly shaped pills made with the ingredients in the formulation composition given in Table 1. The blend included Ajwain Desi [Ptychotis ajowan DC (Apiaceae) seeds], Asgand Nagauri (W. somnifera), Chob Bidhara [Gmelina asiatica L. (Lamiaceae) wood], Pippl Kalan-Desi [Piper longum L. (Piperaceae) fruit, dried unripe], Pipla Mool (P. longum root) Moosli Siyah [Curculigo orchioides Gaertn. (Hypoxidaceae) stem], Satawar [Asparagus racemosus Willd.

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Table 1. Habb-e-Asgand constituents (batch size; 120 kg). S. no.

Plant common name

Plant botanical name

1 2 3 4 5 6 7 8 9

Ajwain Desi Asgand Nagauri Chob Bidhara Moosli Siyah Pippl Kalan (Desi) Pipla Mool Satawar Zanjabeel (Khushk) Qand Siyah Kohna

Ptychotis ajowan Withania somnifera Gmelina asiatica Curculigo orchiodes Piper longum Piper longum Asparagus racemosus Zingiber officinale Saccharum officinarum

(Asparagaceae) root), Zanjabeel-Khushk [Zingiber officinalis Roscoe (Zingiberaceae) rhizome) and Qand Siyah Kohna [Saccharum officinarum L. (Poaceae)] as the basic ingredients (CSIR, 1992). The high content of Qand Siyah Kohna is attributed to its use as a coating material in the preparation of pills.

Materials and methods Chemicals Bovine serum albumin (BSA), 1-chloro-2,4-dinitrobenzene (CDNB), ethylenediaminetetraacetic acid (EDTA) disodium salt and sulfosalicylic acid were obtained from Ameresco (Solon, OH). Dithio-bis-2-nitrobenzoic acid (DTNB), Folin reagent, glutathione (GSH) reduced, glutathione reductase (GR), sodium dithionite and nicotinamide adenine dinucleotide phosphate reduced (NADPH) were from Sigma Chemical Co. (St. Louis, MO). 2-Thiobarbituric acid (TBA) was from Hi-Media Lab (Mumbai, Maharashtra, India) and other routine chemicals were purchased from a local agent. Drug preparation, characterization and identification To prepare Habb-e-Asgand pills, 20.690 kg Gur Kohna (English name Jeggery) was dissolved in 25 L reverse osmosis water. The herbs were checked, weighed, converted into powder form and sieved through 80 no. mesh size sieve. This powder was used in Handa (a pot with upside opening) for the preparation of pills as given below to obtained the desired weight of pills as well as batch size (Table 1). Herbal powder (1 kg) was kneaded into Gur solution and sieved with 80 no. mesh size sieve. These small granules were then charged in Handa and the herbal powder was coated with the help of Gur solution. When the size of the pills becomes 20–30 g/100 pills, they were taken from the Handa and dried in air for 1 h. The small pills were again charged in Handa and the herbal powder was further coated with the help of Gur solution, when the size of the pills becomes 40–45 g/100 pills, these pills were taken out and dried in air and sieved. The pills of size 40–45 g/100 pills were again charged in Handa and the herbal powder was coated with the help of Gur solution. In this manner, a 92–93 g/100 pills size was ultimately achieved. The pills were dried in air for 2 h and sieved with 10.8 mm hole size sieve. The wet pills of 92–93 g/100 pills size were dried in fluid bed dryer (FBD) for 16 h at 85 . The dried pills were again sieved with 10.8 mm hole size sieve and finally the standard weight of dried pills was found to be 65 g/100 pills. Specification of Habb-e-Asgand mixture was evaluated by performing physicochemical analysis for appearance, color,

Plant part used Fruit Root Stem Root Fruit Root Root Rhizome Stem

Weight in kg 8.280 16.550 16.550 8.280 8.280 8.280 16.550 16.550 20.690

taste, smell, pH value, weight of 20 pills, average weight loss on drying at 105 C, ash value, etc. Physico-chemical analysis Water soluble extractive: 5 g of the air-dried drug mixture was macerated and coarsely powdered with 100 mL of water in a close flask for 24 h with frequent shaking through 6 h and allowed to stand for 18 h. The extract was filtered rapidly with precautions for the loss of solvent. A 25 mL aliquot of the filtrate was evaporated to dryness in a tarred flat bottom shallow dish at 105 C. The percentage of water extractive was calculated with reference to the air-dried drug (Anonymous, 2010). Total ash: 2–3 g of accurately weighted ground drug was incinerated in a tarred platinum or silica dish at a temperature not exceeding 450 C until free from carbon. The ash was cooled and weighed and the percentage of ash was calculated with reference to the air-dried drug (Anonymous, 2010). Test drug Habb-e-Asgand pills were crushed to a fine powder with a mortar and pestle. The powder was dissolved in a normal saline (0.90% w/v of NaCl) solution. Paracetamol was obtained from a local market and dissolved in normal saline. Animals Swiss albino male mice (30–35 g) were provided by the Central Animal Housing Facility of the University and the study was approved by the Institutional Animal Ethics Committee. Commercially available pellet diet and water were given ad libitum under standard laboratory conditions (temperature 25 2 C; photoperiod of 12 h). Dosage and experimental groups Four experimental groups (n ¼ 5 mice/group) were designated; control received normal saline (C), paracetamol treated (P), Habb-e-Asgand alone treated (HA) and Habb-e-Asgand and paracetamol treated (HA þ P). Briefly, Habb-e-Asgand was administered at the dose of 250 mg/kg b.w. per os for 14 days. Paracetamol was given as a single dose at 400 mg/kg b.w. intraperitoneally (IP) on the thirteenth day of drug treatment. The doses were selected based on the literature and on a pilot study. The pilot study showed no significant symptoms at doses below 250 mg/kg b.w. and showed the same symptoms for higher doses above 250 mg/kg b.w. Therefore, single dose investigations which showed


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significant differences from the control group are presented here. Further, in the absence of any known standard drug against paracetamol toxicity, we did not include it in present study. On completion of treatment at day 14, the animals were anaesthetized, blood was collected from the orbital sinus for LFTs, followed by euthanization by cervical dislocation and livers were removed to measure GSH, CYPs, LPO levels and the activities of GST, GPx, GR and CAT.

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(Sigma, St. Louis, MO), 0.1 mM NADPH and 10% PMS in a total volume of 2.0 mL. The enzyme activity was quantitated at 25 C by measuring the rate of consumption or the disappearance of NADPH absorbance at 340 nm. The enzyme activity was calculated as nmole NADPH oxidized/ min/mg protein using a molar extinction coefficient of 6.22 103 M1 cm1. GSH measurement


Preparation of post mitochondrial supernatant The liver was homogenized in chilled phosphate buffer (0.1 M, pH 7.4) containing KCl (1.17%), using a Potter Elvehjem homogenizer. The homogenate was centrifuged at 10 500 g for 30 min at 4 C to obtain the post mitochondrial supernatant (PMS), which was used for biochemical analyses (Haque et al., 2003). Liver function tests The liver function tests (LFTs) included serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), ALP and total bilirubin and were measured in serum using a diagnostic kit according to the method of Reitman and Frankel (1957) for SGPT and SGOT, King and King (1954) for ALP, Varley (1980) for bilirubin as described by Pawa and Ali (2006) and Najmi et al. (2010). Levels of SGOT and SGPT were expressed as IU/L and the total bilirubin and ALP were expressed as mg % and U/L, respectively. Antioxidant enzymes measurement Catalase (CAT) activity was assayed by the method of Claiborne (1985) with modification. The reaction suspension consisted of 0.1 M phosphate buffer (pH 7.4), 0.019 M hydrogen peroxide and 10% PMS in a total volume of 3 mL. CAT activity was expressed as nmol H2O2 consumed/ min/mg protein. The method of Habig et al. (1974) with some modifications was used to estimate the glutathione S-transferase (GST) activity. In a final volume of 2 mL, the reaction mixture consisted of 0.1 M phosphate buffer, 1 mM reduced glutathione, 1 mM CDNB and PMS (10%). The GST activity determined was expressed as nmol CDNB conjugate formed/min/mg protein using a molar extinction coefficient of 9.6 103 M1 cm1. Glutathione peroxidase (GPx) activity was assayed according to the method of Mohandas et al. (1984) with modifications. The assay mixture consisted of 1.44 mL phosphate buffer (0.05 M, pH 7.0), 0.1 mL EDTA (1 mM), 0.1 mL sodium azide (1 mM), 0.05 mL GR (1 IU/mL), 0.1 mL GSH (1 mM), 0.1 mL NADPH (0.2 mM), 0.01 mL H2O2 (0.25 mM) and 0.1 mL of PMS (10%) in a total volume of 2 mL. Oxidation of NADPH was recorded spectrophotometrically at 340 nm at room temperature. The enzyme activity was calculated as nmol NADPH oxidized/min/mg of protein, using a molar extinction coefficient of 6.22 103 M1 cm1. GR activity was assayed by the method of Carlberg and Mannervik (1975) as modified by Mohandas et al. (1984). The reaction system consisted of 0.1 M phosphate buffer (pH 7.4), 0.5 mM EDTA, 1 mM oxidized glutathione, GSSG

GSH was determined in PMS according to, the method of Jollow et al. (1974). Sulfosalicylic acid (4.0%) in the ratio of 1:1 was used to precipitate PMS. The samples were kept at 4 C for 1 h followed by centrifugation at 1200 g for 15 min at 4 C. The assay mixture contained supernatant, 0.1 M phosphate buffer and DTNB (stock ¼ 100 mM in 0.1 M phosphate buffer) in a total volume of 3 mL. The optical density was read at 412 nm of reaction product on a spectrophotometer and GSH contents were expressed as nmol GSH/g tissue. CYP measurement The method of Omura and Sato (1964) was used to determine the microsomal CYP content using carbon monoxide (CO) difference spectra of sodium dithionite reduced samples and a molar extinction coefficient of 91 mM/cm. The assay mixture consists of 0.25 mL microsome suspension, 1.5 mL phosphate buffer and a pinch of sodium dithionite. The optical density of mixture was measured at 450 nm and 490 nm using split-beam spectrophotometer and results expressed as nmole CYP content/mg protein. Lipid peroxidation estimation Lipid peroxidation was measured using the method of Mihara and Uchiyama (1978). The rate of lipid peroxidation (LPO) was calculated as nmoles of thiobarbituric acid reactive substance (TBARS) formed/h/g of tissue using a molar extinction coefficient of 1.56 105 M1 cm1. Protein measurement Protein was estimated by the method of Lowry et al. (1951) using Folin’s reagent. BSA was used as a protein standard. Statistical analysis Means standard error (SE) were calculated for each experimental group, and data were analyzed for statistical significance between control and experimental groups using Student’s t-test or a one-way analysis of variance (ANOVA). The statistical significance was reached at p ¼50.05.

Results No mortality and significant body weight changes were observed in the animals. Drug preparation and characterization Habb-e-Asgand pills were brown colored and spherical (solid) with characteristic taste and smell of Asgand (a multimedicinal properties plant). The pills were stored in a cool

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and dark place in tightly closed containers, protected from moisture, light and temperature. Physico-chemical analysis showed slightly basic pH and the ash value ranging 5.17–5.22% (Table 2). Liver function tests Effect on SGOT, SGPT and ALP

A single dose of paracetamol caused an increase in the levels of SGPT (p50.001), SGOT (p50.05) and ALP (p50.05) activities indicating hepatotoxicity. Habb-e-Asgand treatment alone lowered the levels of these markers compared to the control (Figure 2). Significant protection was observed for SGOT (p50.001), SGPT (p50.001) and ALP (p50.001) in the animals treated with Habb-e-Asgand and paracetamol compared to the paracetamol only treated group. Effect on total bilirubin

Table 2. Habb-e-Asgandh physico-chemical analysis.


Test type Appearance Color Taste Smell pH – value(1% solution) Water soluble extract Weight of 20 pills Average weight Diameter Loss on drying at 105 C Total ash value

Result Spherical pills Brown Slightly mucilaginous and acrid Odorless 4.68 (limit 4.60–6.80) 23.32% (limit 20.50–5.70%) 13.00 g (limit 12.740–13.260 g) 650 mg (2%) 10.58 mm (limit 10.20–10.80 mm) 4.18% (NMT 5%) 7.53% (NMT 10%)

Figure 2. Blood serum enzyme levels of control, Habb-e-Asgand (HA), paracetamol (P) and Habb-e-Asgand þ (HA þ P) exposed Swiss albino mice. The values are expressed as means SE (n ¼ 5). Enzymes level was measured as U/L for alkaline phosphatase (ALP) and IU/L for SGOT and SGPT which are expressed here as percent change with respect to the control group. The p values observed were *p50.05 and ***p50.001 when compared with control group values. ###p50.001 when values compared with the paracetamol-treated group.

Paracetamol caused a significant increase in serum total bilirubin (p50.01) levels indicating hepatotoxicity. Treatment with Habb-e-Asgand alone caused a significant (p50.01) decrease in total bilirubin compared to control (Figure 3). The combination of Habb-e-Asgand and paracetamol prevented the rise in total bilirubin levels (p50.001) compared to paracetamol alone. Biochemical investigations Effect on antioxidant enzymes

Figure 3. Total bilirubin values in serum of control and Habb-e-Asgand (HA), paracetamol (P) and Habb-e-Asgand þ paracetamol (HA þ P) exposed Swiss albino mice. The values are expressed as means SE (n ¼ 5). The values obtained as mg% which are expressed here as percent change with respect to the control group. The p values observed were **p50.01 and *** p50.001 when compared with control group values. ### p50.001 when values compared with the paracetamol-treated group.

Table 3. Habb-e-Asgand effect on liver enzymatic antioxidants against paracetamol drug toxicity. Organs ! Treatment ! parameters # GR (nmole NADPH oxidized/min/mg protein) GPx (nmole NADPH oxidized/min/mg protein) GST (nmole CDNB oxidized/min/mg protein) CAT (mmoles H2O2/min/mg protein)

Liver Control

HA

P

HA þ P

20.86 0.5 264.90 9.5 396.40 1.2 141.82 0.8

23.97 1.3 309.23 9.4* 432.23 2.0*** 234.06 0.7***

13.80 0.6*** 162.44 10.0*** 336.57 1.4*** 23.61 0.5***

21.97 0.4### 236.78 10.9## 484.13 3.4***### 156.14 0.9***###

Paracetamol (P) was administered in Swiss albino mice at a dose of 400 mg/kg body weight (b.w.) intraperitoneally and euthanized at 24 h post-treatment. Habb-e-Asgand (HA) was given at a dose of 250-mg/kg b.w. per os for 14 days. The Habb-e-Asgand and paracetamol-treated group (HA þ P) received HA for 14 days and a single dose of P on 13th day. Unit of expression: nM/min/mg protein for glutathione reductase (GR), nM/min/mg protein for glutathione peroxidase (GPx), nM/min/mg protein for glutathione-S-transferase and mM/min/mg protein for catalase activity. Values are means SE of five mice. *p50.05 and ***p50.001 indicate values significantly different from controls. ## p50.01 and ###p50.001 indicate values significantly different from the paracetamol-treated group.


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A single dose of paracetamol caused a significant decrease in GR (p50.001), GPx (p50.001), CAT (p50.001) and GST (p50.001) activities compared to control (Table 3). The decrease in activities might reflect responses to overwhelming production of ROS and oxidative damage. Habb-e-Asgand alone significantly increased the activities of GPx (p50.05), GST (p50.001) and CAT (p50.001), compared to control but no differences were observed in GR activity. The Habb-e-Asgand and paracetamol-treated groups demonstrated a significant increase in GR (p50.001), GPx (p50.01) GST (p50.001) and CAT (p50.001) activity compared to paracetamol alone and the control group.


Effect on GSH content Paracetamol treatment alone caused a reduction in GSH content (p50.001) compared to control (Figure 4) at 24 h. Habb-e-Asgand treatment alone (p50.001) increased GSH content compared to the control group. Furthermore, Habb-e-Asgand attenuated the decrease in GSH levels caused by paracetamol (p50.001).

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decreased in LPO levels (p50.001) compared to paracetamol alone.

Discussion Liver protection has been shown by numerous studies investigating single plant or plant constituent (Achliya et al., 2004; Ahmad et al., 2002). In this regard, Sho-saiko-to (SST), or Xiao-chai-Hu-Tang (Chinese name), is widely used for treating chronic liver diseases in Japan (Tajiri et al., 1991). The present study further characterized the hepatoprotective effects of Habb-e-Asgand. Habb-e-Asgand is a Unani polyherbal preparation manufactured by Hamdard Wakf Laboratories, India. This preparation was evaluated for both hepatoprotective and oxidative/antioxidative potential against paracetamol induced toxicity (Figure 7).

Effect on CYP content Treatment of animals with paracetamol alone resulted in a significant increase in CYP content (p50.001) compared to control (Figure 5). Habb-e-Asgand treatment alone for 14 days demonstrated a significant decrease in the CYP content (p50.001) compared to control while Habbe-Asgand in combination with paracetamol prevented the increase in CYP content (p50.001) compared to paracetamol alone.

Paracetamol significantly increased LPO levels (p50.001) compared to control. Habb-e-Asgand alone caused no significant change in LPO levels (Figure 6). Habb-e-Asgand in combination with paracetamol caused a significant

Figure 5. Cytochrome P450 values in liver of control and Habb-eAsgand (HA), paracetamol (P) and Habb-e-Asgand þ paracetamol (HA þ P) exposed Swiss albino mice. The values are expressed as means SE (n ¼ 5). Values obtained as nmole of CYP /mg protein which are expressed here as percent change with respect to the control group. The significance levels observed is ***p50.001 when compared with control group values and ###p50.001 when compared with the paracetamol-treated group.

Figure 4. GSH values in liver of control and Habb-e-Asgand (HA), paracetamol (P) and Habb-e-Asgand þ paracetamol (HA þ P) exposed Swiss albino mice. The values are expressed as means SE (n ¼ 5). GSH values obtained as nmole GSH/g tissue which are expressed here as percent change with respect to the control group. The significance levels observed is **p50.01 and ***p50.001 when compared with control group values and ###p50.001 when compared with the paracetamoltreated group.

Figure 6. LPO values in liver of control and Habb-e-Asgand (HA), paracetamol (P) and Habb-e-Asgand þ paracetamol (HA þ P) exposed Swiss albino mice. The values are expressed as means SE (n ¼ 5). LPO values obtained as nmole of TBARS formed/mg tissue which are expressed here as percent change with respect to the control group. The significance levels observed is **p50.01 and ***p50.001 when compared with control group values and ###p50.001 when compared with the paracetamol-treated group.

Effect of Habb-e-Asgand on LPO


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Habb-e-Asgand physico-chemical investigations confirmed the moderate size, smooth texture and a characteristic color, taste and smell of W. somnifera (Table 2). Most drugs are designed with consideration to parameters such as size, color, taste, smell and pH to attract physicians and patient interest. The drugs with small to moderate size, smooth texture and pleasant smell are easily accepted by the patients and are easy to swallow (Allen, 2008; Allen et al., 2011). In this regard, our drug of interest possesses these characters and the slightly acidic pH makes it suitable for solubility in acidic stomach environment. Paracetamol-induced hepatotoxicity has been associated with elevated serum levels of enzymes such as alanine transaminase (ALT), aspartate transaminase (AST), ALP and bilirubin in various previous investigations (Jaeschke et al., 2012; Rajesh et al., 2009; Zimmerman & Maddrey, 1995). In parallel, an increase in these same parameters was observed in our study with paracetamol treatment. Habb-e-Asgand alone caused a decrease in the levels of LFTs and it reversed the effect of paracetamol when given in combination. Furthermore, several other studies on polyherbal formulations and their components have also reported similar trends (Rajesh & Latha, 2004; Rajesh et al., 2009; Rajeswary et al., 2011; Tasaduq et al., 2003). The mixed function oxygenase (MFO) pathways based on CYP activities result in production of toxic metabolites that cause increase in LPO through ROS generation and thus causing disruptions in the redox cycle. Habb-e-Asgand alone and in combination with paracetamol caused a reduction in LPO and CYP while paracetamol alone increased these levels. These findings are consistent with previous findings that polyherbal preparations such as Siotone and Eumil (Bhattacharya et al., 2000, 2002), Maharasnadhi quathar,

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Weldhehi choornaya (Thabrew et al., 2001) and W. somniferal, A. racemosus and species of Piper normalize elevated LPO and CYP due to ROS (Dhuley, 1998; Kamat et al., 2000; Lei et al., 2003). GSH is an important antioxidant, protecting cells against ROS (Hellou et al., 2012; Main et al., 2012). Many studies have revealed that low levels of serum and hepatic GSH increase susceptibility to toxic substances (Hellou et al., 2012; Lauterburg & Velez, 1988; Main et al., 2012). Moreover, paracetamol in high doses reduces GSH content, which increases the likelihood of tissue injury due to ROS and/or paracetamol metabolites (Jaeschke et al., 2003; Lauterburg & Velez, 1988). Depletion of GSH results in the reduction of GPx activity, which makes the cells more vulnerable to oxidative stress because GPx neutralizes hydrogen peroxide (Gaetani et al., 1989). Habb-e-Asgand treatment significantly induced while paracetamol reduced the levels of GSH and activity of GPx. Habb-e-Asgand in combination with paracetamol prevented these changes. Therefore, Habb-e-Asgand showed the potential to prevent depletion of GSH and suppression of GPx activity and could act as a therapeutic agent in oxidative stress associated diseases. In support of our findings a large number of herbal plants including the Aswagandha (W. somnifera) and A. racemosus, the constituents of Habb-e-Asgand are reported to induce GSH production and increase GPx activity in hepatotoxicity and other diseases (Anilakumar et al., 2009; Dhuley, 1998; Palanisamy & Manian, 2012). Moreover, polyherbal preparations such as Dihar, Maharasnadhi quathar, Weldehi choornaya mentioned above and the Rhinax have also been reported as potent enhancers of GSH content and GPx activity (Dhuley, 2002; Patel et al., 2009; Thabrew et al., 2001).

Figure 7. Schematic presentation of possible mechanism of Habb-e-Asgand liver protection against paracetamol (acetaminophen) induced toxicity. Up arrows are to show increase while down arrows to show decrease. It is proposed that paracetamol is converted into a reactive metabolite through CYP and thus suppresses the antioxidant system and results in over production of ROS and thus liver toxicity. Habb-e-Asgand by decreasing cytochrome P450 and increasing the antioxidant levels is protecting liver against paracetamol reactive metabolites such as NAPQI induced overwhelming production of ROS-mediated toxicity.



DOI: 10.3109/13880209.2013.863949

Paracetamol significantly reduced GST activity compared to control. Habb-e-Asgand given alone and with paracetamol significantly increased GST activity. Many single herbal plants extract such as Adhatoda vesica Nees (Acanthacea), Aloe vera L. (Aloaceae), and Curcuma longa L. (Zingiberaceae), W. somnifera, A. racemosus, some of them constituents of Habb-eAsgand have been reported to induce the activity of GST (Palanisamy & Manian, 2012; Piper et al., 1998; Singh et al., 2000a,b; Yarru et al., 2009). Here, induction of GST activity by Habb-e-Asgand might favor the elimination of N-acetyl-pbenzo-quinone imine (NAPQI), a toxic metabolite of paracetamol. Further, we found that paracetamol caused significant decreases in the activities of CAT and GR, other GSH conjugating enzymes. Habb-e-Asgand alone and along with paracetamol significantly increased their activities. These findings are in line with other polyherbal preparations and their ingredients such as W. somnifera (Chaurasia et al., 2000; Das et al., 2010; Dhuley, 2002). One possible mechanism of paracetamol toxicity involves the generation of the toxic metabolite, NAPQI by CYP. NAPQI induces the production of ROS, which attack surrounding tissue and disturb the redox homeostasis, subsequently increasing LPO levels (Ferner et al., 2011; Jaeschke et al., 2012). NAPQI is converted into a nontoxic metabolite by conjugation with glutathione and ultimately forms mercapturic acid and related conjugates that are excreted in urine. Thus, in the present investigation paracetamol induced LPO and CYP while reducing glutathione and its conjugating enzymes which are associated with liver damage as evident by high levels of LFTs. Habb-e-Asgand alone and in combination prevented the paracetamol induced hepatotoxicity through its antioxidative and hepatoprotective potential. We proposed here that the active ingredients of the major the herbs constituting Habb-e-Asgand might be contributing for this effect. We support this notion from previous findings done to screen the hepatoprotective and anti-stress effect of other polyherbal preparations such as Rhinax (Dhuley, 2002), EuMil (Bhattacharya et al., 2002; Muruganandam et al., 2002), Maharasnadhi quathar and Weldehi choornaya (Thabrew et al., 2001) and Jigrine (Najmi et al., 2010). Moreover, polyherbal preparations are known to be used as safe natural remedies against various liver ailments and other infectious diseases (Patel et al., 2009; Vidyashankar et al., 2011). W. somnifera, A. racemosus, P. longum and other species of Piper, Z. officinalis and P. ajowan, the main constituents of Habb-e-Asgand, are also present in such polyherbal preparations. Concomitantly, these constituents and their active ingredients have been reported to possess antioxidative, antibacterial, antitumor, hepatoprotective and free radical scavenging properties (Choudhary et al., 2010; Mandal et al., 2000; Singh et al., 2004; Sunila & Kuttan, 2004; Takahashi et al., 2011; Palanisamy & Manian, 2012). In this context, glycowithanolides and withaferrins active principles of W. somnifera, piperine an active principle of P. longum, polysaccharides from black pepper and about 40 antioxidative compounds from Zingiber officinalis were reported to possess antioxidative and hepatoprotective activity against free radical mediated stress in diseases including liver ailments (Christina et al., 2006; Choudhary et al., 2010; Koul & Kapil, 1993; Nakatani, 2000; Singh et al., 2004).

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We also propose further work to investigate how the metabolites of paracetamol interact with the active ingredients of Habb-e-Asgand, driving its antioxidative and hepatoprotective activity. In addition, Habb-e-Asgand as a mixture of several antioxidative and anticancer herbs offers a promising option for testing against cancer in search of an effective anticancer regime (Shibaguchi et al., 2012).

Conclusion Habb-e-Asgand is hepatoprotective homeopathy polyherbal drug possessing antioxidative potential and could be used against free radical induced diseases including liver disorders. Although it is out of the scope of the present study but worthy to mention here that Habb-e-Asgand is highly attractive as an anticancer drug because this drug is a combination of several anticancerous activity possessing herbs and their active ingredients. To further substantiate this prediction, combination therapy tests are prevalent in cancer investigations.

Acknowledgements The author highly appreciate the help of Dr. Lynette Rogers, Associate Professor, Research Institute-Perinatal Research, Nationwide Children’s Hospital, Columbus, Ohio for editing the English language, format and style of this manuscript.

Declaration of interest The authors report no conflicts of interest.

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