In vitro analysis of synergistic effect of honey against chemically induced hepatic insult in Balb/c mice.

Prashant Kumar Gupta¹*, Chandradeo Narayan², Kamal Shah³, Arvind Kumar⁴, Brij Mohan Singh⁵

¹Shri NPA Govt Ayurveda College, Raipur, Chhattisgarh India.

²Post graduate Institute of Medical Science, Chandigarh, India.

³Institute of Pharmaceutical Research, GLA University, Mathura, India.

⁴School of Biotechnology, Banaras Hindu University, Varanasi, India.

⁵Faculty of Ayurveda, Banaras Hindu University, Varanasi, India.

*Corresponding Author E-mail: prashantgupta27@gmail.com, shahkamal.123@gmail.com, k_arvindk@rediffmail.com, hodkb2006@yahoo.co.on

ABSTRACT:

Human being knows honey since thousands of years for its nutritional and medicinal values. Traditional medicine systems like Ayurveda have elaborated honey as boon for health and patient care. Synergistic effect of honey was research but to a limited extend. Honey is advised with Picrorhiza kurroa for hepatic disorders or hepato-protection in Ayurveda. Here we have examined in vitro synergistic effect of honey when given with Picrorhiza kurroa in acetaminophen-induced hepatotoxicity in Balb/c mice model. We obtained Serum and liver lysate to check levels of hepatic markers, Alkaline phosphatase and Glutathione. The level of alkaline phosphatase raised while glutathione level is reduced during hepatic offence. We observed a compensation of above markers when P. Kurrua, honey and its combination were used in acetaminophen induced hepatic toxicity. We observed a better mice weight gain in combination group (Group IV) compared to control group. This study can pave a way for future research on honey as a better adjuvant for hepatotoxic drugs and other herb-drug interactions researches.

KEYWORDS: Ayurveda, Yogavahi, Synergism, hepato-protection, honey.

Table 1- Studies on Yogavahi or synergistic property of honey

Study	Study model	Preparation	Results	Reference
In vitro study	15 bacterial strains (7 Pseudomonas and 8 Klebsiella species) isolated from bile, blood, pus, ascitic fluid etc.	Honey+ Gentamicin, Amikacin and Ceftazidime	Synergistic action seen in the case of Pseudomonas species, not with Klebsiella species.	Karayil S, et al., 1998
In vitro study	Aspergillus niger (ATCC 16404)	Five variety of honey+ Starch	Synergistic effect, MIC % five varieties of honey with starch ranged between 6% and 19.5%.	Boukraa L, et al., 2008)
In vivo study	Acetaminophen induced hepatotoxicity Balb/c mice model	Honey+ Picrorhiza kurroa	Enhanced hepatoprotective, hepatoregenerative ability	Gupta P, et al., 2016
Clinical study	30 subjects of pediatric age.	Honey + Mandur bhasma (herbo-mineral preparation)	Raised Hb gm% observed in honey+ mandur bhasma in comparison to mandur bhasma alone.	Gupta P, et al., 2015
In vitro study	2 clinical pathogenic bacterial isolates- S. aureus, Strept. Pyogenes and 2 reference strains- S. aureus (ATCC 14775), Strept. Pyogens (ATCC 19615)	Sidr and sommor honey+ Ofloxacin, Piperacillin, Amoxicillin+ clavulanic acid, Sulphamethoxazole + Trimethoprim,	Showed synergistic effect against tested bacterial strains, highest synergistic effect was observed against Strept. Pyogenes clinical isolate	Masoud E, et al., 2015

In Ayurveda, Honey (Madhu) is described significantly for its therapeutic value especially for Yogawahi property (Enhances/ complement potency of lead compound while not disturbing/reducing own potency) (Indu commentary on A.S. Su. 43/93). This Yogavahi property is equated with the synergistic effect of drug. Studies on Yogavahi or synergistic property of honey are summarised in table 1.

The oral administration of dried Pircorhiza kurrua rhizome and roots has been claimed as a cure for human viral hepatitis, Hepatic insult and Damage, Asthma, Allergy, Mushroom’s Poisoning^, Hypoxia, Vitiligo, Antidiabetic, Antioxidant, Heart ailment and abdominal pain.(Jadhao M et al., 2009)

Alkaline phosphatase (ALP) manifest as a critical biomarker of numerous diseases and a major tool enzyme of experiments. The majority of sustained elevated ALP levels are associated with disorders of the liver or bone, or both. Since production is increased in response to cholestasis, serum ALP activity provides a sensitive indicator of obstructive and space-occupying lesions of the liver.

Reduced glutathione, a tripeptide, serves as a vital antioxidant in animal, plant, fungi and bacteria by providing free thiol. In healthy human cells, majority (90-95%) of glutathione exist in reduced form and GSH/GSSG ratio is critical indicator for cell health. Oxidative stress deranges GSH/GSSG ratio by decrease in GSH and increase in GSSG (oxidized form of Glutathione). Antioxidant property of GSH is a prominent area of research and projects it as a possible therapeutic agent for several diseases.

Hence a study was planned to evaluate the synergistic effect of honey when administered with known hepato-protective like Picrorhiza kurrua against an acetaminophen induced hepatic insult.

MATERIAL AND METHODS:

A design was made to evaluate the synergistic effect (Yogavahi) property of honey by experimental study in mice by inducing the hepatotoxicity with Paracetamol (Acetaminophen) and therapeutic effect of honey as well as the synergistic effect with P. Kurrua were assessed by getting data of SGPT, SGOT, Alkaline Phosphatase enzymes and glutathione reductase and cytotoxicity assay in control, Group A (Acetaminaphen), GroupB (Acetaminophen and honey), Group-C (Acetaminophen and P. Kurrua) and the Group D (Acetaminophen + P. Kurrua + Honey). Due to huge data collection, a part of study was published earlier and referenced here (Gupta et al. 2016), the data related with levels of Alkaline phosphatase and Glutathione reductase in Serum and liver lysate are compiled in present study.

Animal care:

Screening, acclimatisation of mice was carried out from the Animal House, School of biotechnology B.H.U. Balb/c male mice, 5-6 weeks old, healthy, young age and having weight between (25-35 gm) were selected for the study. Standard mice chow and water were available ad libidum. Required treatment was given according to group distribution and mice were accordingly weighted and sacrificed on 2, 4, 6 and 8th day of experiment by cervical dislocation.

Group distribution and Dose calculation

Mice were divided randomly, and each group containing 5 mice.

Group I: Acetaminophen

Group II: Acetaminophen + Honey

Group III: Acetaminophen + P. Kurrua

Group IV: Acetaminophene + Honey + P. Kurrua

Group V: Control (Double distilled water)

A dispersible 1000mg/tab acetaminophen (paracetamol)(Apex Laboratory, India) was taken to produce hepatotoxicity in above four mice groups in a sub-lethal dose of acetaminophen i.e. 625 mg/kg body wt. orally. Air-dried rhizome of P. Kurrua Royle extract was taken from Ayurvedic Pharmacy, Faculty of Ayurveda, Institute of Medical Sciences, Banaras Hindu University. Extraction (Aqueous) of P. Kurrua dried root and rhizome was concentrated in vacuum and dried in Department of Rasa Shastra, Institute of Medical Sciences, Banaras Hindu University and stored in a dark and dry place. P. Kurrua was given in the dose 200mg/kg P.o. in aqueous form. Honey was given in diluted to 1:1 (in D.D.W.) with dose 0.25 ml per oral.

Chemicals and Reagents:

Enzyme assay kit for alkaline phosphatase (Cat No. - K 753-100) was purchased from Hysel, new delhi and stored under advised temperature till further use.

Sample collection and tissue homogenate preparation:

Two mice from each group was sacrificed randomly at a set time interval by cervical dislocation, Blood samples were collected in centrifuge tubes/2 ml syringe via cardiac puncture and the serum was separated. The abdomen was then cut open and liver samples were removed. In order to prevent RBC contamination, samples were cut into small slices, rinsed thoroughly in ice-cold 0.9 % NaCl, and blotted with blotting paper, weighted and homogenised in ice-cold sodium pyrophosphate buffer (ph 8.3) and preserved in recommended temperature (4⁰ C) till further use. Serum samples were frozen immediately in a deep freezer at – 20⁰C and enzyme assays were performed on the same day or next day.

Enzyme assay:

The serum samples were subjected to assay for hepatic marker enzymes such as Alkaline phosphatase (ALP). Values were expressed as IU/dl ALP activity was measured using the method of Bower, G.N. and McComb. All six test tubes were taken and marked as blank, I, II, III, IV and V. Buffered substrate was taken in all test tubes except blank and brought it to 25⁰ to 37⁰ C then tissue lysate 0.02 ml was added in all test tubes, mixed it and after 1 minute initial absorbance was taken at wavelength 405nm at every 1 minute and alkaline phosphatase activity was calculated by multiplying change in absorbance per minute to the factor (2713).

(IU/Lt) = Δ A/min x factor (2713)

Method for Glutathione estimation:

The assay involves mainly using glutathione reductase and Ellman’s reagent (DTNB). Glutathione reductase reduces GSSG to GSH. DTNB (5-5’- dithiobis [2-nitrobenzoic acid]) reacts with GSH to form yellow color chromophore, 5- thionitrobenzoic acid (TNB) with absorbance maxima at 415 nm and GS-TNB. GS-TNB is further reduced to GSH and TNB by glutathione reductase, thus this enzymatic recycling of GSH enhances the sensitivity of the assay.

All six test tubes were taken and marked as blank, I, II, III, IV and V. 0.2 ml of sample were taken in all test tubes except blank then 2 ml of substrate was added in all test tubes, mixed well and NADPH 1 ml was also added in all test tubes ,mixed and start times simultaneously then absorbance was taken at 1^st minute , 2^nd minute, 3 rd minute and 4^th minute at wavelength 340nm, temperature 37⁰ C and glutathione activity was calculated by multiplying change in absorbance per minute to 4983 (Factor).

RESULTS:

We estimated Alkaline phosphatase and Glutathione which showed that sub acute dose of paracetamol resulted in significant increased Alkaline phosphatase decreased Glutathione level. Honey, P. Kurrua and combination of both decreased the raised activity of Alkaline phosphatase while at the same time it increased the reduced activity of glutathione reductase.

Serum Alkaline Phosphatase activity:

Group I (acetaminophen feed) showed increased serum activity of ALP by 1.46, 2.36, 3.94 and 7.81 fold while in Group II (Acetaminophen + honey) it ranged increased serum ALP activity by 1.24, 1.87, 3.09 and 5.24 fold in comparison to control group on day 2 , 4, 6, 8th day respectively. Group III (Acetaminophen + P. Kurrua) expressed increased serum ALP activity by 1.16, 1.89, 2.06, and 3.94 fold while in group IV (Acetaminophen + P. Kurrua + honey) increased activity of serum ALP by 1.17, 1.29, 1.74, and 2.05 fold in comparison to control group on day 2, 4, 6, 8th day respectively. Results related with serum alkaline phosphatase are summarised in table 3 and figure 1.

Liver lysate Alkaline Phasphatase activity:

Group I (acetaminophen feed )showed increased liver lysate activity of ALP by 1.27, 2.06, 2.56 and 5.5 fold while Group II (Acetaminophen + honey) showed increased liver lysate ALP activity by 1.09, 1.56, 1.88 and 4.11 fold in comparison to control group on on day 2 , 4, 6, 8th day respectively. Group III (Acetaminophen + P. Kurrua) expressed increased ALP activity by 1.1, 1.45, 1.70, and 3.85 fold while in group IV (Acetaminophen + P. Kurrua + honey) increased activity of liver lysate ALP by 1.06, 1.09, 1.52, and 2.17 fold in comparison to control group on day 2, 4, 6, 8th day respectively. Results related with liver lysate alkaline phosphatase are summarised in table 3 and figure 2

Serum Glutathione Level:

Group I (acetaminophen feed )showed reduced serum level of glutathione by 0.70, 0.37, 0.22 and 0.07 fold while in Group II (Acetaminophen + honey) it ranged decreased serum glutathione activity by 0.85, 0.54, 0.33 and 0.14 fold in comparison to control group on day 2 , 4, 6, 8th day respectively. Group III (Acetaminophen + P. Kurrua) expressed reduced serum glutathione activity by 0.94, 0.69, 0.25, and 0.15 fold while in group IV (Acetaminophen +P. Kurrua+ honey) decreased activity of serum glutathione was observed by 0.95, 0.73, 0.65, and 0.37 fold in comparison to control group on day 2, 4, 6, 8th day respectively. Results related with serum glutathione are summarised in table 3 and figure 3

Liver lysate Glutathione Level:

Group I (acetaminophen feed )showed reduced liver lysate level of glutathione by 0.52, 0.37, 0.35 and 0.30 fold while in Group II (Acetaminophen + honey) it ranged decreased liver lysate glutathione activity by 0.83, 0.60, 0.51 and 0.34 fold in comparison to control group on day 2 , 4, 6, 8th day respectively. Group III (Acetaminophen + P. Kurrua) expressed reduced liver lysate glutathione activity by 0.81, 0.72, 0.68, and 0.35 fold while in group IV (Acetaminophen +P. Kurrua + honey) decreasSSed activity of liver lysate glutathione was observed by 0.94, 0.83, 0.81, and 0.47 fold in comparison to control group on day 2, 4, 6, 8th day respectively. Results related with liver lysate glutathione are summarised in table 3 and figure 4

Table 2- Experiment observations of all group and all activities (Serum and Liver lysate )

Group A		Sr. Alkaline Phasphatase (U/ml)	Liver lysate Alk. Phosphotase (U/ml)	Sr. Glutathione (U/ml)	Liver Lysate Glutathione (U/ml)
	Day 0 Control DDW	31.99 ± 1.56	52.55± 1.32	142.52 ± 1.66	279.01 ± 1.75
	Day 2^nd Acetaminophen	46.99 ± 1.12	66.98± 1.62	100.53 ± 1.01	145.50 ± 1.95
	Day 4^th Acetaminophen	75.58 ± 1.54	108.52± 1.33	53.94 ± 1.09	103.77 ± 1.22
	Day 6^th Acetaminophen	126.18 ± 1.62	134.72± 1.54	32.05 ± 1.08	97.66 ± 1.65
	Day 8^th Acetaminophen	250.03 ± 1.92	289.53± 1.93	11.04 ± 1.02	85.75 ± 1.68
Group B	Day 0 Control DDW	30.62 ± 1.72	52.16± 1.62	143.36 ± 1.67	288.00 ± 1.78
	Day 2^nd Acetaminophen + Honey	38.14 ± 1.97	57.21± 1.32	121.70 ± 1.62	239.68 ± 1.96
	Day 4^th Acetaminophen + Honey	57.24 ± 1.26	81.49± 1.33	77.61 ± 1.11	173.40 ± 1.54
	Day 6^th Acetaminophen + Honey	94.83 ± 1.53	98.183± 1.53	47.94 ± 1.18	148.29 ± 1.29
	Day 8^th Acetaminophen + Honey	160.50 ± 1.62	214.76± 1.79	19.38 ± 1.47	99.11 ± 1.11
Group C	Day 0 Control DDW	31.66 ± 1.12	52.40± 1.67	141.11 ± 1.64	288.6 ± 1.12
	Day 2^nd Acetaminophen + P. Kurrua	37.00 ± 1.36	57.87± 1.53	133.68 ± 1.66	234.00 ± 1.40
	Day 4^th Acetaminophen + P. Kurrua	59.84 ± 1.73	76.31± 1.11	97.90 ± 1.36	210.12 ± 1.18
	Day 6^th Acetaminophen + P. Kurrua	65.27 ± 1.54	89.42± 1.69	35.37 ± 1.54	196.96 ± 1.46
	Day 8^th Acetaminophen + P. Kurrua	125.012 ± 1.66	201.87± 1.16	22.22 ± 1.66	101.31 ± 1.16
Group D	Day 0 Control DDW	30.62± 1.92	51.98± 1.77	143.36 ± 1.53	287.4 ± 1.72
	Day 2^nd Acetaminophen + Honey + P. Kurrua	36.12 ± 1.73	55.54± 1.64	136.3 ± 1.03	242.88
	Day 4^th Acetaminophen + Honey + P. Kurrua	39.79± 1.50	57.054± 1.12	105.88 ± 1.54	236.00 ± 1.32
	Day 6^th Acetaminophen + Honey + P. Kurrua	53.51± 1.32	79.11± 1.79	94.42 ± 1.81	217.12 ± 1.38
	Day 8^th Acetaminophen + Honey + P. Kurrua	62.914± 1.32	113.91± 1.27	53.26 ± 1.64	172.40 ± 2.54

DISCUSSION:

In traditional medicine therapeutics, Honey is popular for its various medicinal properties such as cardio-tonic, better for eye sights, hypo-lipidemic, appetizer etc. since ancient period but recently it has been found to possesses Antibacterial activity (Eteraf-Oskouei T, et al., 2013; Mandal, et al., 2011), hypoglycemic (Bobis et al., 2018; Erejuwa et al., 2012), antioxidant (Erejuwa et al., 2010; Ahmad et al., 2018), wound healing (Febriyenti et al., 2019; Oryan et al., 2016) and gastric ulcer protective, (Fazalda et al., 2018) properties.

The present study is aimed to evaluate the synergistic role of honey and picrorrhiza in combination against the paracetamol (Acetaminophen) toxicity in mice model.

Experimental data based on activities of Alkaline phosphatase (serum/liver) and glutathione level (serum/liver) suggest the protective role of honey against paracetamol (Acetaminophen) adverse effects to Heart and liver (Hazai et al., 2002). Sulfahydryl compounds are known as the most important endogenous antioxidants. NAPQEI binds with the GSH resulting in conversion of GSH to GSSG. Decreased GSH levels in cell resulting in increased cell damage. In such conditions, other proteins sulfhydryl groups present in the cells provide an alternative protection (Genet et al., 2000).

Antioxidant activity and hepatoprotective potential of honey has been described in a rat models against CCl₄. (Khadr et al., 2002). The CCl₄ administration causes increase in SGOT and SGPT at significant level in rat (Khadr et al., 2002). This agree with our observations in respect to alkaline phosphatase which increased in mice after administration of paracetamol (Acetaminophen) after 2 days, 4 days, 6 days and 8 days. We further observed that enzyme activity of alkaline phosphatase increased in serum maximum approximately 7 fold at day 8 in respect to day 2nd after paracetamol treatment. Honey when given with CCl₄along with black seeds and silymarin (Khatr et al.) showed improvement in antioxidant status. Our data also suggest that honey, picrorrhiza and combination of both honey and picrorrhiza when administered along with paracetamol (Acetaminophen) resulted decrease in the activity of ALP at significant level on day 2nd, 4th, 6th, 8th. We can calculate that toxicity of liver and heart might be due to the elevated enzyme activity of ALP as well as decreased Glutathione reductase. In the current study, we estimated Alkaline phosphatase and Glutathione which showed that toxic dose of paracetamol resulted in significant increase in the activity of alkaline phosphatase tremendous decrease of the Glutathione level. Honey, picrorrhiza and combination of both decreased the activity of alkaline phosphatase while at the same time it increased the activity of glutathione reductase. At conclusion, honey and picrorhiza could be given along with paracetamol (Acetaminophen) to avoid liver and heart cells Damages.

ACKNOWLEDGEMENT:

We acknowledge Dr Kishor Sonawane, and Dr Rashmi Sahu Raipur, India for contributing in table design and data mining. We acknowledge Dr Nagendra Chauhan, senior scientific officer, DTL, Raipur, India for his support for manuscript structuring.

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Received on 01.07.2020 Modified on 29.07.2020

Research J. Pharm.and Tech 2021; 14(12):6309-6314.

DOI: 10.52711/0974-360X.2021.01091