Hepatoprotective Activity of an Herbal Composition,MAP,a Standardized Blend Comprising Myristica fragrans,Astragalus membranaceus,and Poria cocos

Abstract

Historically, botanicals have been reported to possess good antioxidative activities as demonstrated by their free radical scavenging property rendering their usage in liver protection. In this study, we describe the potential use of MAP, a standardized blend comprising three extracts from Myristica fragrans, Astragalus membranaceus, and Poria cocos, in ameliorating chemically induced acute liver toxicities. Acetaminophen (APAP) and carbon tetrachloride (CCl₄)-induced acute liver toxicity models in mice were utilized. Hepatic functional tests from serum collected at T24, histopathology analysis, and merit of blending three standardized extracts were evaluated. MAP administered at doses of 150–400 mg/kg showed statistically significant and dose-correlated inhibitions of serum alanine aminotransferase (ALT) ranging from 30.8% (P ≤ .05) to 88.1% (P = .0001) in the APAP and 66.9% (P = .002) to 83.7% (P = .0002) in the CCl₄ models, respectively. Moreover, MAP resulted in up to 75.7%, 60.9%, and 33.3% reductions in serum aspartate aminotransferase (AST), bile acid, and total bilirubin, respectively. Mice treated with oral doses of composition of MAP at 300 mg/kg showed statistically significant reduction in hepatocyte necrosis when compared with vehicle control. Unexpected synergistic protection of liver damage was also observed. Therefore, the composition, MAP, could be potentially utilized as an effective hepatic detoxifying agent for the protection of liver damage.

Introduction

Incidences of liver disease have continued to rise globally; for example, at least 30 million people (or 1 in 10 Americans) have some form of liver disease.¹ Liver disease is a result of habitual repeated alcohol consumption, exposure to some xenobiotics, drug usage, and drug interactions, which are some of the major causes of morbidity and mortality where variable symptoms manifested ranging from asymptomatic elevation of the liver enzymes to sudden hepatic failure. While damage of oxidative stress affects the whole body as a system, the impact becomes more detrimental when it involves vital organs such as the liver where primary detoxification takes place to remove and metabolize harmful toxins.² The most frequently observed pathological conditions of the liver such as fatty liver, hepatitis, fibrosis, and cirrhosis in conjunction with cellular lipids, proteins, and DNA oxidation have been demonstrated in multiple experimental animal models.³ Among these models, the acetaminophen and carbon tetrachloride (CCl₄)-induced hepatotoxicity models have been most frequently used in assessing hepatoprotective agents.

Among chemicals known to cause hepatotoxicity, acetaminophen (n-acetyl-p-aminophenol or acetaminophen [APAP], also known as tylenol, paracetamol) and CCl₄ are generally utilized to develop animal models that mimic the human type of liver toxicity with similar mechanisms of action. Acetaminophen is a safe and effective analgesic and antipyretic drug at therapeutic dosage. However, APAP overdose can cause severe liver toxicity characterized by depletion of glutathione (GSH), protein adduct formation,^4,5 generation of highly active free radicals, mitochondrial damage, and nuclear DNA fragmentation⁶ that leads to cell death and hence necrosis. While some species such as rat are relatively resistant to APAP toxicity, the mouse is the preferred animal for the model development as several studies have demonstrated dose-dependent response to either oral or intraperitoneal APAP challenge.^7,8 Similarly, CCl₄, a halogenated alkane with restricted usage as industrial chemical/solvent, is a well-known hepatotoxin that is widely used to induce acute toxic liver injury in a large range of laboratory animals. CCl₄ toxicity is initiated by cytochrome P450 s primarily of (CYP) 2E1⁹ to yield reactive metabolic products, trichloromethyl free radicals (CCl₃ ⁻), which can initiate lipid peroxidation and ultimately results in the overproduction of reactive oxygen species (ROS) and hepatocyte injuries.^10,11 These radicals can also react with oxygen to form the trichloromethyl peroxy radical, CCl₃OO−, a highly reactive species that could initiate the chain reaction of lipid peroxidation leading to cell death. Therefore, it could be inferred that regardless of the chemical agents used to induce the hepatotoxicity, both the acetaminophen and CCl₄ models share the critical step in oxidative stress induced by ROS generated by excess intermediate metabolites leading to protein oxidation, lipid peroxidation, and DNA damage.

Natural antioxidants from natural sources have been extensively studied through the years. In the past, some botanicals containing phenolic compounds have been reported to be associated with antioxidative actions in biological systems, acting as scavengers of free radicals rendering their usage in liver protection. We postulated that combining such plant materials with historical efficacy and safety data would give a beneficial boost in their indication for overall liver health. To test this hypothesis, we screened a series of plant extracts collected through legacy mining, which led to the discovery of a composition designated as MAP, which comprised standardized extracts from three medicinal plants: Myristica fragrans (M), Astragalus membranaceus (A), and Poria cocos (P).

M. fragrans seeds, belonging to the Myristicaceae family, possess variety of pharmacological properties, including antidiarrhea, antiomitting, soothing stomach pain, analgesic, hypnotic, neuroprotective, and appetite stimulating.^12,13 The aromatic oil from the seeds is a key principal active ingredient of this herb medicine.^14,15 The main chemical constituents of M. fragrans are myristicin, myristic acid, elemicin, safrole, eugenol, palmitic, oleic, lauric, and other acids.^14,16 Myristicin, one of the major constituents of essential oil, was reported with potent hepatoprotective activity in the lipopolysaccharide (LPS)/D-galactosamine-induced liver injury model. Myristicin also possesses potent antifungal, antioxidant, and anti-inflammatory properties.¹⁷

A. membranaceus root is one of the most popular Chinese herbs from the Fabaceae family (legumes), with common name as Radix Astragali, Astragalus root, or huang qi. Huang qi is one of the 50 fundamental herbs used in traditional Chinese medicine (TCM) and was included in many TCM preparations with a wide range of biological functions.^18,19 It has been recently studied as a treatment for cardiovascular disorders, hepatitis, kidney disease, and diabetics.²⁰ Their liver protective property has been confirmed by modern study in CC1₄-induced animal models.²¹ The major components in the extract of Radix Astragali are reported as flavonoids, saponins, and polysaccharides. Among these compounds, polysaccharides (Astragalus polysaccharide [APS]) were considered as the principal hepatoprotective components of A. membranaceus root. APS consistently ameliorated the liver fibrosis induced with CCl₄ in the mouse model.²² Astragalus polysaccharides were also reported with antiviral, anti-inflammation, antitumor, antiatherosclerosis, hematopoiesis, neuroprotection, and antidiabetic properties in different studies.²³ Immunomodulatory effect of the Radix Astragali has been attributed to its polysaccharides especially for radiation and chemotherapy patients in cancer treatments.^24
–26

P. cocos wolf, a fungus in the family Polyporaceae, is a medicinal mushroom growing on the roots of Chinese red pine trees or other conifer trees, with common names as fuling in China, and matsuhodo in Japan, and also known as hoelen, poria, tuckahoe, or China root. Traditionally, it has been used for treating nausea, vomiting, diarrhea, loss of appetite, and stomach ulcer, as well as insomnia and amnesia.^27,28 The mechanism of anti-inflammation of P. cocos ethanol extracts is demonstrated as through inhibition of iNOS, COX-2, IL-1β, and TNF-α through inactivation of the NF-κB signaling pathway in LPS-stimulated RAW 264.7 macrophages.²⁹ The major constituents of fuling are polysaccharides (Pachyman) in the form of β-glucan. Variable biological functions have been reported for P. cocos polysaccharides, such as antioxidant, antihyperglycemic, soothing the stomach pain, anti-inflammation, anticancer, and immunological modulation.³⁰ Triterpenoids were also identified as active components in fuling, which are actively researched, mainly on anticancer, anti-inflammatory efficacies, and potential immunological activity as well.^27,31

Recently, we have demonstrated the liver protection activity of MAP using ethanol-induced acute hepatotoxicity in an animal model of binge drinking.³² In this model, excessive increases of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were significantly inhibited by orally delivered MAP at 46.3% and 43.6%, respectively. MAP replenished the depleted superoxide dismutase (SOD) by more than 60% while causing significant stimulation of GSH productions. MAP showed statistically significant reduction in ballooning degeneration, vascular steatosis, cytoplasmic or nuclear condensation, and shrinkage, as well as inflammations, when compared with a vehicle-treated alcohol-induced liver toxicity model. Mice treated with MAP also showed statistically significant reduction in alcoholic steatohepatitis scoring when compared with vehicle control.

In this study, we implemented the most frequently used animal models with practical clinical implications such as APAP and CCl₄-induced hepatotoxicity to assess the effect of the composition, MAP, in protecting the liver from such damages. In addition, the merit of combining M. fragrans, A. membranaceus, and P. cocos was also evaluated.

Materials and Methods

The composition

The composition, MAP, comprises M. fragrans seed extract (M), A. membranaceus root extracts (A), and P. cocos whole fruit body extract (P). M. fragrans extract was produced as 70% ethanol extract of the seeds at 7:1 extraction ratio with no less than 2% myristicin. A. membranaceus root extract was standardized as 4:1 water extract with no less than 20% polysaccharides. P. cocos extract (P) was produced by ethanol extraction, followed by water extraction of the fungus fruiting body at a final extraction ratio of 16:1 with no less than 15% triterpenoids and 6% polysaccharides.

Animals and housing

Purpose-bred female CD-1 mice, weighing 18–24 g, were purchased from a USDA-approved laboratory animal vendor (Charles River Laboratories, Inc.) and acclimated upon arrival for a week. Individual cages were identified with a cage card indicating project number, test article, dose level, group, and animal number. Harlan T7087 soft cob beddings were used and changed at least twice/week. Animals were provided with fresh water and rodent chow diet # T2018 (Harlan Teklad) ad libitum and were housed in a temperature-controlled room (22.2°C) on a 12-h light–12-h dark cycle.

Model inductions

A balanced therapeutic schedule was generated and optimized as follows to address prophylaxis and intervention: for the APAP-induced hepatotoxicity model, APAP (Lot# MKBQ8028 V; Sigma) at a dose of 400 mg/kg dissolved in warm saline (Lot#132908; G-Biosciences, Lot# 720729 from Quality Biological) (heated to 60°C and cooled down to ambient temperature) was orally administered to overnight-fasted CD-1 mice to induce toxicity.^7,8 For the CCl₄-induced hepatotoxicity model, CCl₄ (Lot#SHBD5351 V; Sigma) at a dose of 25 μL/kg dissolved in corn oil was administered intraperitoneally to overnight-fasted CD-1 mice to induce toxicity.^11,22 For both models, materials were administered at −48,−24, and −2 h before APAP or CCl₄ administrations and +6 h after induction. In total, the mice received three doses before the chemical induction and a dose after the chemical induction; 10% Tween 20 (Lot# 0134C141; Amresco) for MAP and 1% MC (Lot#SLBK4357 V) for Silymarin were used as carrier vehicles. Control mice without APAP or CCl₄ received carrier vehicle only.

Hepatic function test

Serum was isolated from blood drawn at T24 using a serum separator tube after a 30-min room temperature clot and spun at 704 g 10 min for ALT, AST, total bilirubin, conjugated and unconjugated bilirubin, bile acid, total protein, albumin, globulin, and alkaline phosphatase (ALP) monitoring in an automated colorimetric assay using Beckman Coulter AU2700 at Phoenix Laboratories.

Histopathology

Liver tissues from normal control (n = 12), CCl₄+vehicle (n = 12), and CCl₄+MAP (300 mg/kg, n = 12)-treated groups were fixed in 10% buffered formaldehyde and embedded in paraffin wax for histological examination. The highest dosage of the composition used in the liver function test, that is, 300 mg/kg, was selected for a maximum impact evaluation. All microsectioned (5 μm) slides were stained with hematoxylin/eosin, and the entire stained field was assessed for any cellular and structural changes under multiple magnification and subjected to histopathological scoring using the modified Nonalcoholic Steatohepatitis Clinical Research Network³³ for ballooning degeneration (severity score 0–4), microvascular steatosis (severity score 0–2), cytoplasmic condensation (severity score 0–4), hepatocyte vacuolation (severity score 0–4), and necrosis (severity score 0–4).

Statistical analysis

Data were analyzed using Sigma plot (version 11.0). The results are represented as mean ± standard deviation. Statistical significance between groups was calculated by means of single-factor analysis of variance and by a t-test. P-values less or equal to .05 were considered as significant. When normality test failed, for nonparametric analysis, data were subjected to Mann–Whitney sum ranks for t-test and Kruskal–Wallis one-way analysis of variance on ranks for analysis of variance (ANOVA). The merit of combining extracts from M. fragrans, A. membranaceus, and P. cocos was determined using Colby's equation.³⁴ In this method, a formulation of two or more materials together will presume to have unexpected synergy if the observed value of a certain endpoint measurement is greater or equal to the hypothetically calculated values. Colby's equation: Expected = (A + B − C) where A = (x + y + z); B = (xyz)/10,000; and C = [(xy)+(xz)+(yz)]/100; x = percent change mean ± SD of M, y = percent change mean ± SD of A, and z = percent change mean ± SD of P.

Results

ALT inhibition by individual plant extracts

Each plant extract was tested individually in CCl₄ and APAP-induced hepatotoxicity models for its liver protection activity using serum ALT as a measure of efficacy at a dose of 500 and 400 mg/kg, respectively. Percent reductions of 37.6%, 34.1%, and 38.1% in the CCl₄ model and 94.4%, 50.9%, and 41.0% in the APAP model were observed for mice treated with Myristica, Astragalus, and Poria, respectively, when compared with vehicle-treated diseased mice (Fig. 1). Among these reductions, while only Myristica extract showed statistically significant reductions in serum ALT in the APAP model, Astragalus and Poria extracts showed statistical significance in the CCl₄ model.

FIG. 1.

Hepatoprotective activity of plant extracts in APAP and CCl₄-induced models. APAP at a dose of 400 mg/kg dissolved in warm saline (heated to 60°C and cooled down to ambient temperature) was orally administered to overnight-fasted CD-1 mice to induce toxicity. Similarly, CCl₄ at a dose of 25 μL/kg dissolved in corn oil was injected intraperitoneally to overnight-fasted CD-1 mice to induce toxicity. Materials (M = Myristica, A = Astragalus, P = Poria) were administered at −48, −24, and −2 h before CCl₄ injection and +6 h after induction. Control mice without APAP/CCl₄ received carrier vehicle (10% Tween 20) only. Serum ALT was determined at T24. * P ≤ .05. ALT, alanine aminotransferase; APAP, acetaminophen; CCl₄, carbon tetrachloride.

Composition efficacy confirmation

Following individual efficacy data, a study was designed to identify the most effective combination of these three plant extracts. While multiple ratios were tested at multiple doses, here we reported the most optimally effective ratio for the composition, MAP, 1 M:4A:1.25P. Mice treated with the composition at the dose of 400 mg/kg showed reductions in serum ALT of 80.8% and 92.7% in the CCl₄ and APAP models, respectively, when compared with vehicle-treated diseased mice (Fig. 2). Both reductions were statistically significant when compared with the vehicle control.

FIG. 2.

Hepatoprotective activity of the composition, MAP, in APAP and CCl₄-induced models. Test compounds were administered at −48, −24, and −2 h before CCl₄ injection and +6 h after induction. Control mice without APAP/CCl₄ received carrier vehicle (10% Tween 20) only. Serum ALT was determined at T24. *P ≤ .01; ^† P ≤ .001.

Dose–response effect of MAP

Furthermore, the optimum dosage of the composition of MAP that incurs significant liver protection was evaluated in both APAP and CCl₄-induced models. Mice were gavaged orally with the composition, MAP, at doses of 200, 300, and 400 mg/kg suspended in 10% Tween 20. As seen in Figure 3, in the APAP group, dose-correlated inhibitions in serum ALT were observed for the composition; 25.8% (P = .49), 62.9% (P = .01), and 88.1% (P = .0001) inhibitions were observed for mice treated with doses of 200, 300, and 400 mg/kg MAP, respectively. Similarly, in the CCl₄ group, dose-correlated inhibitions in serum ALT were observed for the composition; 66.9% (P = .002), 80.0% (P = .0002), and 83.7% (P = .0002) inhibitions were observed for mice treated with doses of 200, 300, and 400 mg/kg MAP, respectively. At least in the CCl₄ model, the composition, MAP, has provided statistically significant protection in liver damage at a dosage level as low as 200 mg/kg as determined by the serum ALT level when compared with vehicle-treated diseased mice.

FIG. 3.

Dose-correlated hepatoprotective activity of the composition, MAP (1:4:1.25), in APAP and CCl₄-induced models. Test materials were administered at −48, −24, and −2 h before CCl₄ injection and +6 h after induction. Control mice without APAP/CCl₄ received carrier vehicle (10% Tween 20) only. Serum ALT was determined at T24.

Unexpected synergy

The efficacy of individual plant extracts was tested, including Myristica, Astragalus, and Poria, at a dosage equivalent to each plant extract ratio in the composition of MAP (such as 32, 128, and 40 mg/kg of Myristica, Astragalus, and Poria extracts, respectively) as they appear in 1 M:4A:1.25P at an oral dose of 200 mg/kg. While mice treated with Myristica showed a 40.7% decrease in serum ALT, both Astragalus and Poria extracts exacerbated the liver damage at these specific dosages. In contrast, the composition resulted in 66.9% reduction in serum ALT when compared with the vehicle-treated diseased model. Colby's equation was utilized to evaluate the benefit of combining extracts from M. fragrans, A. membranaceus, and P. cocos in the CCl₄ model. As shown in Table 1, the observed values were greater than the expected hypothetical values, indicating the existence of synergy in formulating three extracts at a specific ratio in MAP. Hence, the merit of blending Myristica, Astragalus, and Poria was confirmed by their synergistic protection of liver damage caused by CCl₄ induction.

Table 1.

Unexpectedly Enhanced Liver Protection Activity of MAP in the CCl ₄-Induced Hepatotoxicity Model

Composition	Extract	Dose (mg/kg)	n	Percent change of CCl₄ +vehicle Serum ALT @T24
MAP	Myristica fragrans	32	10	40.7 ± 20.8 (x)
	Astragalus membranaceus	128	10	−18.1 ± 11.2 (y)
	Poria cocos	40	10	−13.5 ± 8.5 (z)
	Expected^a	—	—	20.5 ± 4.4 (A + B − C)
	Observed^b	200	10	66.9 ± 20.3

Data are expressed as mean ± SD.

Colby's equation (A + B − C): A = (x + y + z); B = (xyz)/10,000; and C = [(xy) + (xz) + (yz)]/100.

Calculated value according to Colby's method.

Data observed when mice were orally administered with MAP at 200 mg/kg according to previously described treatment schedule.

ALT, alanine aminotransferase; CCl₄, carbon tetrachloride.

Moderation of hepatic functional panel

Liver panels such as AST, ALT, total bilirubin, conjugated and unconjugated bilirubin, bile acid, total protein, albumin, globulin, and ALP have been used as a standard screen method for liver health. CCl₄-induced liver toxicity models were utilized to compare the liver protection activity of the composition of MAP (given at 150, 200, and 300 mg/kg) against Silymarin (200 mg/kg) using liver panel data as a measure of efficacy. Besides serum ALT, liver panels such as total protein, total bilirubin, direct and indirect bilirubin, albumin, globulin, AST, bile acid, ALP, and γ-glutamyl transferase were monitored.

As seen in Table 2 below, clear dose-correlated inhibitions in many of the major liver toxicity biomarkers were observed for mice treated with MAP. Staying with similar methods of composition efficacy analysis described above, the composition, MAP, resulted in statistically significant, 30.8–71.1%, inhibitions in ALT and 41.7–75.7% inhibitions in AST when compared with vehicle-treated injured mice. Statistically significant increase in serum albumin and total protein was observed for all dosages. Complementing the above biomarkers, even at the lowest dosage used (150 mg/kg), statistically significant 50.3% reduction in bile acid was detected. Administered at 300 mg/kg, MAP resulted in 60.9% and 33.3% reductions in serum bile acid and total bilirubin, respectively. For comparison purpose, at least in this model, Milk thistle showed only marginal liver protection in all the parameters measured.

Table 2.

Hepatic Function Test Results for Composition of MAP

				MAP (mg/kg)
Analyte	Control	CCl₄ (25 μL/kg)	Milk thistle (200 mg/kg)	150	200	300
ALT (U/L)	17.1 ± 3.1	11,238.4 ± 4980.6	10,894.9 ± 2000.4	7781.5 ± 2236.4^*	5072.9 ± 1772.9^†	3244.6 ± 1440.6^‡
AST (U/L)	58.3 ± 12.0	6981.9 ± 3386.0	6726.3 ± 1698.9	4067.4 ± 1564.8^*	2427.1 ± 1565.9^‡	1700.0 ± 996.9^‡
Bile acid (μM)	1.0 ± 0.0	48.8 ± 28.3	23.9 ± 7.2^*	24.3 ± 15.1^*	21.4 ± 34.1^*	19.1 ± 36.6^*
GGT (U/L)	0.2 ± 0.4	1.3 ± 0.7	1.1 ± 0.3	1.3 ± 1.2	1.3 ± 0.8	0.8 ± 0.4
ALP (U/L)	79.7 ± 18.2	119.0 ± 26.9	131.6 ± 34.4	99.2 ± 21.4	76.7 ± 16.0^‡	71.4 ± 21.6^‡
T. bilirubin (mg/dL)	0.1 ± 0.0	0.3 ± 0.1	0.4 ± 0.1	0.3 ± 0.1^*	0.3 ± 0.1	0.2 ± 0.1^‡
D. bilirubin (mg/dL)	0.0 ± 0.0	0.1 ± 0.0	0.1 ± 0.0	0.1 ± 0.1	0.0 ± 0.0^†	0.0 ± 0.0^*
I. bilirubin (mg/dL)	0.1 ± 0.0	0.3 ± 0.1	0.3 ± 0.1	0.2 ± 0.0	0.2 ± 0.1	0.2 ± 0.1^*
T. protein (g/dL)	4.4 ± 0.2	4.8 ± 0.2	4.7 ± 0.2	4.6 ± 0.1^*	4.6 ± 0.3^*	4.5 ± 0.2^†
Albumin (g/dL)	2.3 ± 0.1	2.7 ± 0.2	2.7 ± 0.1	2.6 ± 0.1^*	2.6 ± 0.2^*	2.5 ± 0.1^‡
Globulin (g/dL)	2.1 ± 0.1	2.1 ± 0.1	2.0 ± 0.2	2.0 ± 0.1	2.0 ± 0.1	2.1 ± 0.1

Test compounds were administered at −48, −24, and −2 h before CCl₄ injection and +6 h after induction. Control mice without CCl₄ received carrier vehicle (10% Tween 20) only. Serum ALT was determined at T24. Data are expressed as mean ± SD.

P ≤ .05; ^† P ≤ .001; ^‡ P ≤ .0001.

ALP, alkaline phosphatase; AST, aspartate aminotransferase.

Histopathology findings

Pathological interpretation is based on observed significance either on necrosis alone or sum of all parameters together. If statistical significance is attained for hepatocellular necrosis for the test compound-treated group compared with vehicle control, the outcome is considered positive improvement regardless of the other parameters considered. As seen in Table 3 and Figure 4, mice treated with oral doses of composition of MAP at 300 mg/kg showed statistically significant reduction in necrosis when compared with vehicle control. Substantiating the clinical chemistry data, the histopathology findings indicated improved liver protection for mice treated with the composition.

FIG. 4.

H&E staining of liver tissues from mice treated with MAP at 300 mg/kg in the CCl₄-induced hepatotoxicity model. (A) Normal control+vehicle = diffuse centrilobular vacuolization. CV, central vein (100 × ). (B) CCl₄ (25 μL/kg)+vehicle = there are occasional areas of severe necrosis with disruption of normal architecture and tissue loss with replacement by hemorrhage (100 × ). (C) CCl₄ (25 μL/kg)+MAP (300 mg/kg). Higher magnification of coalescing areas of centrilobular of necrosis (N) and hydropic degeneration (arrow). CV, central vein (100 × ). (D) CCl₄ (25 μL/kg)+vehicle = there are occasional areas of severe necrosis with disruption of normal architecture and tissue loss with replacement by hemorrhage (100 × ). (E) CCl₄ (25 μLkg)+MAP (300 mg/kg). Higher magnification of coalescing areas of centrilobular of necrosis (N) and hydropic degeneration (arrow), CV, central vein (100 × ). Color images available online at www.liebertpub.com/jmf

Table 3.

Histopathological Scoring

Histopathology changes	Normal control+vehicle	CCl₄ (25 μL/kg)+vehicle	CCl₄ (25 μL/kg)+MAP (300 mg/kg)
Ballooning	0.04 ± 0.13	1.46 ± 0.63	1.71 ± 0.77
Steatosis	2.17 ± 0.37	1.04 ± 0.78	0.63 ± 0.30
Condensation	0.04 ± 0.14	2.13 ± 0.74	1.17 ± 0.37^*
Vacuolation	2.08 ± 0.28	0.54 ± 0.38	0.67 ± 0.42
Necrosis	0.00 ± 0.00	2.63 ± 0.94	1.29 ± 0.48^**

Histopathological scoring using modified Nonalcoholic Steatohepatitis Clinical Research Network³² for ballooning degeneration (severity score 0–4), microvascular steatosis (severity score 0–2), cytoplasmic condensation (severity score 0–4), hepatocyte vacuolation (severity score 0–4), and necrosis (severity score 0–4).

P ≤ .001; ^** P ≤ .0001.

Discussion

Historically, many plant extracts have been traditionally used for treatments of human ailments over centuries. Among these plants, extracts from M. fragrans, A. membranaceus, and P. cocos have been utilized as anti-inflammatory, antioxidant, and hepatoprotection agents.^17,21,22,31

In the current study, legacy mining approach was used to search Medline, pharmacopeia of TCM, Ayurveda medicine, and Unigen's Phytologix collection database for plants that have potentials for liver protection and liver-related indications. As such, we selected and tested 54 plant extracts on APAP and CCl₄-induced liver toxicity models that led to the discovery of three extracts from M. fragrans (M), A. membranaceus (A), and P. cocos (P). Given the analyses of the above results and data, it is reasonable to infer that a composition comprising these three plant materials possesses significant antioxidation, anti-inflammation, and immune regulation activities and hence protects the liver from oxidative stress-caused damages. To the best of our knowledge, these three plant extracts have never been reported to combine together before at specific ratios to yield the MAP composition on the basis of literature search. In our study, while all three extracts produced very similar ranges of liver protection efficacy in the CCl₄ model, M. fragrans showed a marked reduction in serum ALT in the APAP model and hence liver protection to a magnitude of 94.4% inhibition when compared with vehicle-treated mice. This finding led us to set a criterion for the composition to possess balanced reductions in serum ALT in both models to be considered as a lead. Previously, a potent hepatoprotective effect with significant inhibition on both serum ALT and AST levels in an LPS plus d-galactosamine model¹⁷ and hepatoprotective and antioxidant activity in another study against isoproterenol-induced hepatotoxicity and oxidative stress³⁵ by M. fragrans extracts have been reported. Similarly, the liver protection activity of APS also has been demonstrated in CCl₄-induced liver damage.^22,36 In their study, APS significantly inhibited the activities of serum AST, ALT, and LDH levels in serum and reduced the content of malondialdehyde in liver tissues and enhanced SOD and GSH levels.

In the present study, other than Myristica in the APAP model, each component of the composition showed modest individual performances in modulating toxicities induced by the disclosed chemicals, reinforcing the idea of combining these plant extracts for a better outcome in both models. This hypothesis needs to be confirmed in a way that the composition of MAP should demonstrate a boosted and balanced protection of liver damage elicited by both APAP and CCl4. Moving forward, documenting individual liver protection data for lead plants, a search for unexpected or enhanced outcome using unpredicted blending for these plant materials was carried out using multiple ratios, leading to an excelled and well-balanced liver protection activity when these plant extracts were formulated at 1 M:4A:1.25P ratio. When the combination of these three plant extracts was tested, clearly interesting, yet an unexpected, synergy was observed from the MAP composition, which exceeded the predicted effect based on simply summing the effect observed for each individual extract at the given ratio. In fact, when administered alone, Astragalus and Poria extracts did not show liver protection activity at the magnitude equivalent to the one noted for the composition in both models separately. This comparison holds true for Myristica extract in the CCl₄ model. Furthermore, following this consistent liver protection activity of composition, MAP, in both APAP and CCl₄ models, additional comprehensive efficacy confirmatory study was carried out using the CCl₄-induced hepatotoxicity model. Data from this study documented for liver function test, including AST, ALT, bile acid, total protein, total bilirubin, conjugated bilirubin, albumin, and total protein, demonstrated that the composition has indeed liver protection activity when compared with the vehicle-treated control animals with liver injury. Despite the fact that there were differences in degrees of serum ALT inhibition caused by the composition between models, taking inhibitions in the CCl₄ model into consideration, it can be inferred that the composition, MAP, could incur its liver protection activity at a dose level as low as 150 mg/kg. Moreover, these findings were substantiated by the histopathological observations signifying a liver protection capability of the MAP composition.

For years, as an individual plant, extracts from M. fragrans, Astragalus membranous, and P. cocos have been used separately in many traditional medicines and contemporary pharmaceutical compounding for multiple indications. This wide array of usage of these plants for varieties of disorders, well before the existence of modern pharmacotoxicology data, suggests their relative tolerance in humans. Possible mechanisms of action of actives from these plants have also been reported at multiple occasions. For instance, induction of glutathione S-transferase,³⁷ inhibition of NO,³⁸ and TNF-α production¹⁷ by M. fragrans; free radical scavenging,²² inhibition of TNF-α production and NOS induction,³⁹ and increase in tissue SOD and GSH-px⁴⁰ by A. membranous; and inhibition of iNOS and TNF-α production²⁹ by P. cocos have been documented. Given these facts, it is possible to imply that their standardized blend, MAP, would possibly possess triple mechanism actions such as induction of endogenous phase II antioxidant enzymes, free radical scavenging, and anti-inflammatory activities to counteract the sudden or habitual insult to the liver. Hence, the enhanced liver protection activities observed in the composition, MAP, could be, in part, due to these aggregated contributions of Myristica, Astragalus, and Poria.

Summary

To sum up, based on analyses of data from the hepatic function test, synergistic activity, and histopathological findings, we strongly believe that combining these extracts from traditionally well-known folk medicinal plants, M. fragrans, A. membranaceus, and P. cocos, into the ratio of 1 M:4A:1.25P provided a significantly enhanced liver protection activity. Therefore, the composition of MAP could potentially be considered as a mitigating agent for alcohol and/or chemical-induced hepatotoxicity.

Footnotes

Acknowledgments

The authors would like to express their best gratitude to Dr. Wenwen Ma, Dr. Min Chu, Mrs. Lidia, Brownell, Breanna Moore, Sabrina Cleveland, and Unigen team for their incalculable support for the completion of this research. The authors would like to extend their utmost gratitude to Mr. Bill Lee, the owner of Econet/Unigen, Inc., who supported the project described in this article.

Author Disclosure Statement

All authors are current Unigen employees and they have competing financial interests.

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