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

Figures & data

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.

Table 1. Habb-e-Asgand constituents (batch size; 120 kg).

S. no.	Plant common name	Plant botanical name	Plant part used	Weight in kg
1	Ajwain Desi	Ptychotis ajowan	Fruit	8.280
2	Asgand Nagauri	Withania somnifera	Root	16.550
3	Chob Bidhara	Gmelina asiatica	Stem	16.550
4	Moosli Siyah	Curculigo orchiodes	Root	8.280
5	Pippl Kalan (Desi)	Piper longum	Fruit	8.280
6	Pipla Mool	Piper longum	Root	8.280
7	Satawar	Asparagus racemosus	Root	16.550
8	Zanjabeel (Khushk)	Zingiber officinale	Rhizome	16.550
9	Qand Siyah Kohna	Saccharum officinarum	Stem	20.690

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

Test type	Result
Appearance	Spherical pills
Color	Brown
Taste	Slightly mucilaginous and acrid
Smell	Odorless
pH – value(1% solution)	4.68 (limit 4.60–6.80)
Water soluble extract	23.32% (limit 20.50–5.70%)
Weight of 20 pills	13.00 g (limit 12.740–13.260 g)
Average weight	650 mg (±2%)
Diameter	10.58 mm (limit 10.20–10.80 mm)
Loss on drying at 105 °C	4.18% (NMT 5%)
Total ash value	7.53% (NMT 10%)

Figure 2. Blood serum enzyme levels of control, Habb-e-Asgand (HA), paracetamol (P) and Habb-e-Asgand + (HA + P) exposed Swiss albino mice. The values are expressed as means ± SE (n = 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 *p < 0.05 and ***p < 0.001 when compared with control group values. ^###p < 0.001 when values compared with the paracetamol-treated group.

Figure 3. Total bilirubin values in serum of control and Habb-e-Asgand (HA), paracetamol (P) and Habb-e-Asgand + paracetamol (HA + P) exposed Swiss albino mice. The values are expressed as means ± SE (n = 5). The values obtained as mg% which are expressed here as percent change with respect to the control group. The p values observed were **p < 0.01 and *** p < 0.001 when compared with control group values. ^###p < 0.001 when values compared with the paracetamol-treated group.

Table 3. Habb-e-Asgand effect on liver enzymatic antioxidants against paracetamol drug toxicity.

Organs →	Liver
Treatment → parameters ↓	Control	HA	P	HA + P
GR (nmole NADPH oxidized/min/mg protein)	20.86 ± 0.5	23.97 ± 1.3	13.80 ± 0.6***	21.97 ± 0.4^###
GPx (nmole NADPH oxidized/min/mg protein)	264.90 ± 9.5	309.23 ± 9.4*	162.44 ± 10.0***	236.78 ± 10.9^##
GST (nmole CDNB oxidized/min/mg protein)	396.40 ± 1.2	432.23 ± 2.0***	336.57 ± 1.4***	484.13 ± 3.4***^###
CAT (µmoles H2O2/min/mg protein)	141.82 ± 0.8	234.06 ± 0.7***	23.61 ± 0.5***	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 + 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 µM/min/mg protein for catalase activity. Values are means ± SE of five mice.

*p < 0.05 and ***p < 0.001 indicate values significantly different from controls.

^##p < 0.01 and ^###p < 0.001 indicate values significantly different from the paracetamol-treated group.

Figure 4. GSH values in liver of control and Habb-e-Asgand (HA), paracetamol (P) and Habb-e-Asgand + paracetamol (HA + P) exposed Swiss albino mice. The values are expressed as means ± SE (n = 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 **p < 0.01 and ***p < 0.001 when compared with control group values and ^###p < 0.001 when compared with the paracetamol-treated group.

Figure 5. Cytochrome P450 values in liver of control and Habb-e-Asgand (HA), paracetamol (P) and Habb-e-Asgand + paracetamol (HA + P) exposed Swiss albino mice. The values are expressed as means ± SE (n = 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 ***p < 0.001 when compared with control group values and ^###p < 0.001 when compared with the paracetamol-treated group.

Figure 6. LPO values in liver of control and Habb-e-Asgand (HA), paracetamol (P) and Habb-e-Asgand + paracetamol (HA + P) exposed Swiss albino mice. The values are expressed as means ± SE (n = 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 **p < 0.01 and ***p < 0.001 when compared with control group values and ^###p < 0.001 when compared with the paracetamol-treated group.

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.