1 Introduction

Chronic liver diseases significantly contribute to morbidity and mortality in human populations. As a source of morbidity and mortality, they rank 15th and 11th, respectively. Globally in 2016, these diseases were responsible for 2.2 % of deaths and accounted for 1.5 % of disability-adjusted life years (Global Health Estimates, WHO, 2016). Treatment options for chronic liver diseases are limited in modern medicine. Therefore, there is a pressing need to explore alternative therapeutic sources. Plants have been utilized as medicine since time immemorial to treat various illnesses, including liver diseases.

Mallotus philippensis L. (MP) also known as “Kamala” in northern parts of India. This plant belongs to the family known as Euphorbiaceae. It is found in South China, Tibet, Nepal to North India and Southeast Asian countries (Gangwar et al., 2014b). It has been reported to exert antibacterial effects and generally used as folkloric medicine to treat meningitis and typhoid using a decoction of the stem (Cheenpracha et al., 2019; Kumar et al., 2020). The medicinal values of MP are revealed in several Indian and Chinese texts, such as the Sushruta Samhita, Charaka Samhita, Indian Materia Medica, and Indosyunic Medicine (Khare, 2008; Kunwar et al., 2021; Ye et al., 2021). Ayurveda and Yunani, two of the oldest medical traditions, also recommend its use to treat various disorders as an alexiteric, anthelmintic, appetiser, purgative, styptic (Khare, 2019; Quattrocchi, 2016). Commercially available drugs like Roghan Kameela and Zimad Jarb prescribed for dermatological conditions and Krikuthar Rasa prescribed for removing intestinal worms already contain MP as one of the active ingredients possessing therapeutic properties along with other active herbs. It has been documented that many people from different ethnic groups and geographical areas continue to use this plant traditionally as an ethnomedicine to treat various diseases. Various formulations made from this traditional medicinal plant have been used to treat a variety of health related issues, including diarrhoea, dysentery, bronchitis, jaundice, urinary complications, and parasitic infections (Barkatullah et al., 2015; Kunwar et al., 2021). It is frequently used to treat worm infestations and other gastrointestinal tract illnesses. Its hepatoprotective potential has been demonstrated in vitro on hepatoma cells using a derived compound Bergenin (Sriset et al., 2020), while its antioxidant activity of fruits and rottlerin (a derived polyphenolic compound) was analysed using in-vitro tests (Gangwar et al., 2014a; Lee et al., 2020). Despite folkloric claims of MP in jaundice, proper scientific validation is still lacking. Therefore, the present work was undertaken to provide a scientific basis for traditional use in hepatic disease.

2 Material and methods

2.3

2.3 Plant material

Dr Athar Ali Khan (Taxonomist) from the Department of Botany and Dr Rifat Afridi (Research Officer (Pharmacognosy) from the Regional Research Institute Unani Medicine, Aligarh Muslim University (A.M.U.) Aligarh identified the plant after it was obtained from a local market (5 kg of fresh fruit). A voucher specimen having reference number Rump-433 was placed in the herbarium of the Department of Botany at AMU.

The MP fruits were initially dried under shade and ground into a powder approximately of 3 kg. Subsequently, non-polar and polar organic solvents namely petroleum ether, benzene, chloroform, ethyl acetate, and methanol were used for extraction using a Soxhlet extractor (in the increasing order of polarity). Distillation was used to get rid of the solvent. Under reduced pressure, the petroleum ether and benzene extracts were concentrated to produce a greenish-sticky substance. An analysis using thin-layer chromatography (TLC) was conducted to determine the number of chemicals. Since the petroleum ether and benzene extracts behaved similarly during TLC examination, they were combined. Further silica gel column chromatography was performed on the fraction of the combined petroleum ether-benzene-and-ethyl acetate. Petroleum ether, petroleum ether-benzene (9:1, 8:2, 7:3, 1:1), benzene-ethyl acetate (9:1, 8:2, 7:3, 1:1), ethyl acetate, ethyl acetate–methanol (9:1, 8:2, 7:3, 6:4, 1:1), and finally, methanol was used to elute the column. Repeated column chromatography separated these fractions which upon recrystallisation produced four different compounds (A1 to A4; Isorotlerin, 5,7-Dihydroxy-8-methyl-6-prenylflavanone, 6″,6″-Dimethylepyrano (2″,3,“7,6)-5-hydroxyl-8-methylflavanone and 7,4′-Dihydroxy-3′′,3′′-dimethyl-(5,6-pyrano-2′′-one)-8-(3′′′,3′′′-dimethylallyl)-flavanone) (Supplementary file 1). A flavonoid compound in the form flavanone i.e., 7,4′-Dihydroxy-3′′,3′′-dimethyl-(5,6-pyrano-2′′-one)-8-(3′′′,3′′′-dimethyl allyl)-flavanone was isolated from ethyl acetate and methanol (1:1) fraction of MP, (Compound- A4, Fig. 1) (Rizvi et al., 2016). For additional analysis, a methanol extract in its raw form was also produced. Different solvents upon sequential extraction method afforded 11.70 % (Petroleum ether), 6.99 % (Benzene), 17.71 % (Chloroform), 39.88 % (Ethyl Acetate) and 79.13 % (Methanol) extracts, respectively.

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Fig. 1

Structure of 7,4′-Dihydroxy-3′′,3′′-dimethyl-(5,6-pyrano-2′′-one)-8-(3′′′,3′′′-dimethyl allyl)-flavanone isolated from M. philippensis.

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7,4′-Dihydroxy-3′′,3′′-dimethyl-(5,6-pyrano-2′′-one)-8-(3′′′,3′′′-dimethylallyl)-flavanone (compound A4).

Ethyl acetate–methanol (1:1) fraction yielded compound A4. Crystallization from the binary solvent system namely chloroform–methanol gave light yellowish solid (35 mg), melting point: 260–261 °C. Anal. Calc. for C₂₅H₂₆O₆; C, 71.07; H, 6.20; found: C, 71.09; H, 6.23. IR (KBr) ν cm⁻¹: 3411, 3326, 2916, 2848, 1681, 1623, 1586, 1443, 1365, 1298, 1107, 980, 801. Proton (¹H) NMR (400 MHz, CDCl₃, δ, ppm): 5.58 (dd, 1H, H-2, J = 13.6, 2.6 Hz), 2.83–2.88 (dd, 2H, H-3, J = 14.8, 2.6; J = 13.6, 17.2 Hz), 7.54 (dd, 1H, H-2′, J = 2.4, 10.0 Hz), 6.67 (d, 1H, H-3′, J = 10.0 Hz), 7.52 (d, 1H, H-5′, J = 10.8 Hz), 7.50 (dd, 1H, H-6′, J = 2.4, 10.8 Hz), 3.63 (brs, 2H, H- 1′′), 2.50 (brs, 3H, H-4′′), 1.98 (brs, 3H, H-5′′), 3.30–3.26 (d, 2H, H-1′′′, J = 12.6, 2.6 Hz), 5.54 (dd, 1H, H-2′′′, J = 2.6, 12.6 Hz), 1.57 (brs, 3H, H-4′′′), 1.59 (brs, 3H, H-5′′′). Carbon-13 (¹³C NMR) (100 MHz, CDCl₃, δ, ppm): 81.66 (C-2), 42.94 (C-3), 195.90 (C-4), 159.52 (C-5), 103.12 (C-6), 162.49 (C-7), 104.54 (C-8), 157.91 (C-9), 106.27 (C-10), 136.16 (C-1′), 115.61 (C-2′), 129.29 (C-3′), 157.52 (C-4′), 127.13 (C-5′), 126.50 (C-6′), 16.61 (C-1′′), 203.84 (C-2′′), 81.52 (C-3′′), 33.20 (C-4′′), 27.85 (C-5′′), 29.13 (C-1′′′), 130.16 (C-2′′′), 136.16 (C-3′′′), 27.60 (C-4′′′), 27.91 (C-5′′′). ESI-MS (m/z): 422.47 [M + H^+·] (Supplementary file 1).

2.3.1

2.3.1 Phytochemical analysis

Qualitative phytochemical analysis of ethyl acetate and methanol extracts of M. philippensis fruits was done to confirm the existence of reducing sugars, anthraquinones, terpenoids, alkaloids, flavonoids, saponins, tannins, phlorotannins, cardiac glycosides and steroids (Balamurugan, 2019) using double beam spectrophotometer.

3 Results

3.4

3.4 Results of liver function tests

The ability of the MP fruit's methanol and ethyl acetate extracts to prevent the rise of serum transaminases was evidence of their strong hepatoprotective efficacy. The methanol and ethyl acetate extracts had a greater impact at 500 mg/kg (p < 0.001), although the liver-specific enzyme ALT did not significantly decrease at 300 mg/kg. Additionally, the active ingredient A4 considerably (p < 0.05) reduced ALT. When compared to the corresponding control groups, no discernible difference in the bilirubin levels of the test groups or the negative control groups was observed. (Fig. 2: a-d). Hepatoprotection was calculated for all test groups as a function of AST, ALT, and ALP as indicated in Table 3.

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Fig. 2

(a-d): Effect of M. philippensis fractions on liver function tests.

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Table 3 Percentage of hepatoprotection offered by M. phillipensis fractions.

Groups	AST (%)	ALT (%)	ALP (%)
POSITIVE CONTROL (SILYMARIN)	82.6	81.5	74.7
MPM (300 mg/kg)	24.0	19.6	28.7
MPM (500 mg/kg)	45.9	32.1	65.0
MEA (300 mg/kg)	30.3	29.2	54.5
MEA (500 mg/kg)	61.1	55.4	63.2
A4 (50 mg/kg)	29.3	26.9	58.9

MPM- Methanol fraction of M. phillipensis, MEA-Ethyl acetate fraction of M. phillipensis, A 4 Active compound (Effects in decreasing order MEA 500 > MPM 500 > MEA 300 > A4 > MPM 300).

3.5

3.5 Results of antioxidants

The ethyl acetate fraction produced significant changes in all variables (Catalase, GSH and MDA) at both doses. When compared to the CCl₄ group, the catalase levels were elevated (p < 0.01) by the MEA fraction at 500 mg and the A4 compound. Similarly, the MDA levels of the MEA 500 mg group were raised (p < 0.01). In contrast, the A4 compound group was raised to 3.5 ± 0.30 μmoL/mg, which was also higher (p < 0.05) than the negative control group. On the other hand, MDA levels were significantly lowered in the MEA 500 and the A4 compound group, implying a substantial decrease in hepatocyte membrane damage due to fatty acid oxidation. At 300 mg/kg, methanol extract produced a significant (p < 0.01) effect for GSH only i.e.0.3.8 ± 0.23 μmoL/mg (Fig. 3: a-c).

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Fig. 3

(a-c): Effect of M. philippensis fractions on antioxidants. The negative control group was compared with the normal control group, and all other groups were compared with the Negative control group; *p < 0.05, **p < 0.01 and ***p < 0.001 were considered significant. MPM- Methanol fraction of M. phillipensis, MEA-Ethyl acetate fraction of M. phillipensis, A 4 Active compound.

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3.6

3.6 Results of serum cytokines

As shown in Fig. 4 (a-c), MPM 500 mg/kg and MEA 300 mg/kg produced a significant (p < 0.05) change in TNF α only. At the same time, the MEA fraction at 500 mg/kg decreased all the cytokines TNF α (p < 0.05), IL-6 (p < 0.05) and IL-1 (p < 0.05). The active compound produced a positive effect on TNF alpha (p < 0.05) and Interleukin-1 (p < 0.05).

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Fig. 4

(a-c): Effect of M.philippensis fractions on serum cytokines. The negative control group was compared with the normal control group, and all other groups were compared with the Negative control group;* p < 0.05, **p < 0.01 and ***p < 0.001 were considered significant. MPM- Methanol fraction of M. phillipensis, MEA-Ethyl acetate fraction of M. phillipensis, A 4 Active compound.

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3.7

3.7 Results of histopathological analysis

Histopathological examination of liver tissue sections was done on different control and treated groups. CCl₄-induced changes like sinusoidal integrity, hepatocytic degeneration, centrilobular changes, and inflammatory infiltrates were considered parameters for microscopic examination; these changes were reduced by pre-treatment with silymarin, fractions and isolated compound of M. phillipensis (Fig. 5: A-H). Table 4 shows the histopathological scoring of liver sections (Kamisan et al., 2014).

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Fig. 5

Histopathology of rat liver. (10X, H & E stain), Fig. 5 A: Photomicrograph of liver from Normal Control Group (Propylene glycol) showing normal liver microstructure central vein (A) with parallel hepatic cords (B) with normal foci of the portal triad (C). Fig. 5 B: Photomicrograph of rat liver from Negative Control Group (CCI₄) showing spotty necrosis with periportal fibrosis (A) with diffuse mild acute parenchymal inflammation (B) with diffuse ballooning degeneration(C). Fig. 5 C: Photomicrograph of rat liver from Positive Control Group (Silymarin) showing mild sinusoidal dilatation with mild portal fibrosis (A) and mild lymphocytic infiltration (B). Fig. 5 D: Photomicrograph of rat liver from Test Group (CCI₄ + MP Methanol 300 mg/kg) showing focal hepatocytic (A) necrosis with dilated central vein and mild lymphocytic infiltration (B) in the parenchyma. Fig. 5 E: Photomicrograph of rat liver from Test Group (CCl₄ + MP Methanol 500 mg/kg) showing a decrease in necrosis of hepatocytes with irregular and thickened hepatocytes with mitosis and moderate interface hepatitis (A). Fig. 5 F: Photomicrograph of rat liver from Test Group (CCI₄ + MP Ethyl acetate 300 mg/kg) showing mild periportal lymphocytic infiltrate (A) with sinusoidal congestion (B). Fig. 5 G: Photomicrograph of rat liver from Test Group (CCl₄ + MP Ethyl acetate 500 mg/kg) showing mildly dilated central vein (A) with mild periportal fibrosis (B). Fig. 5 H: Photomicrograph of rat liver from Test Group (CC1₄ + A-450 mg/kg) showing irregular and thickened hepatocyte plate (A) with bi-nucleated forms and mild fibrosis (B) and focal chronic inflammation.

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Table 4 Histopathological scoring of liver sections.

	Groups	Dose	Inflammation	Necrosis	Degeneration	Fibrosis	Congestion
1	Normal Control	1 ml/Kg	–	–	–	–	–
2	Negative control	1 ml/Kg	+++	+++	++	+	+
3	Positive Control	50 mg/Kg	+	+	–	+	++
4	Methanol Extract	300 mg/Kg	++	++	–	–	+
5	Methanol Extract	500 mg/Kg	+	+	–	+	+
6	Ethyl acetate extract	300 mg/Kg	++	+	–	+	++
7	Ethyl acetate extract	500 mg/Kg	+	–	–	++	+
8	A4	50 mg/Kg	+	+	+	+	+

The severity of hepatic injury was evaluated based on the following scoring pattern: - Absent, + Mild effect, ++ Moderate effect, +++ Severe effect.

4 Discussion

Traditional medicinal plants have been used as a source of medicine for all forms of diseases since time immemorial. Plants have shown remarkable activity in treating ailments which has a definite rationale in modern pharmacology and therapeutics (Sen and Samanta, 2015; Colalto, 2018; Alhassan et al., 2019; Benchoula et al., 2019). It has been discovered that a number of plant products guard against liver damage. A milk thistle bioflavonoid called silymarin was found to be useful in preventing liver damage brought on by CCl₄ (Gillessen and Schmidt, 2020), as was colchicine, the toxin from autumn-blooming flowering plant crocus i.e., Colchicum autumnale L. (Awad et al., 2020). Both have antioxidant properties. Since the liver has great potential to regenerate, therefore, the aim should be to come up with a molecule that could reduce the injurious effect or preserve the architecture and the physiological function of the liver disturbed by the hepatotoxin (Dezső et al., 2017).

A substantial community from many ethnic groups and geographical areas continues to use M. phillipensis in the traditional way as an ethnomedicine to cure medical ailments. Different formulations made from this tree have been used for the treatment of worm infestations and other gastrointestinal tract ailments, as well as bronchitis, jaundice, urinary complications (Barkatullah et al., 2015; Kunwar et al., 2021).

Among several mechanisms, oxidative stress i.e., the generation of free radicals, is one important explanation of liver injury (Li et al., 2015). CCl₄ is often used for screening hepatoprotective drugs, it is a potent hepatotoxin that results in centrilobular liver inflammation and necrosis (Dong et al., 2016; El Rabey et al., 2021). The stages of liver damage include fat accumulation, CCl₃-OO* radical generation, lipid peroxidation, membrane damage, loss of Ca²⁺ sequestration, apoptosis, fibrosis, dehalogenation, covalent binding of the resulting radicals, protein synthesis inhibition (especially of apolipoprotein synthesis), assembly, packaging, and release of VLDL and HDL (Lackner and Tiniakos, 2019; Weber et al., 2003, Fareed et al., 2023). In our study, the administration of CCl₄ enhanced the serum markers of hepatotoxicity in the negative control group (Fig. 2). Additionally, it depleted tissue GSH. It increased lipid peroxidation (Fig. 3). Significant hepatoprotective activity was seen in the M. phillipensis methanol and ethyl acetate fractions, as evidenced by their capacity to restrain the increase in serum transaminases. The active compound A4 also significantly decreased ALT, although its effect was less as compared to the ethyl acetate extract. The reason might be due to the presence of some important bioactive molecules more active than the A4 in it. It also produced a significant antioxidant effect and reduced lipid peroxidation. Our results indicate that the two extracts and the active compound possess antioxidant activity, decreased lipid peroxidation, and protect the liver cells exposed to CCl₄ from oxidative stress, possibly through the membrane stabilizing effect of extracts and compound. A4 compound has shown lesser activity as compared to the high dose of ethyl acetate extract. It may be due to the fact that apart from the flavonoid (including A4) ethyl acetate fraction also contains other phytochemicals like steroids, alkaloids and saponins.

Besides free radical injury, inflammation is an important cause of hepatitis. The liver injury induced by either infection or free radicals is mediated by inflammation as well as other innate immune cells (Tacke and Zimmermann, 2014, Rani et al., 2024), during which pro-inflammatory cytokines like TNF-α are produced by liver cells (Brenner et al., 2013; Niederreiter and Tilg, 2018). Proliferation, inflammation, and the control of immunological responses that result in apoptosis and necrosis are only a few of the physiological and pathological processes that the pro-inflammatory cytokine TNF- α is involved in (Chu, 2013). According to a study, alcoholic liver disease is initiated and promoted in part by TNF-α. and has been hypothesised to cause acute liver failure (Chastre et al., 2012). TNF-α is typically overexpressed in fatty liver disease and alcohol-related liver disease; cytokines that contribute to liver protection include IL-6 and IL-10 (Tiegs and Horst, 2022). TNF- α is another possible therapeutic target in individuals with the condition, as evidenced by its significant role in animals with steatohepatitis (Sehrawat et al., 2020).

In our research, we observed reduced concentrations of proinflammatory cytokines supporting the protective role of MP in CCl₄ induced hepatotoxicity (Fig. 4). In our previous studies, we also have reported that MP attenuates proinflammatory cytokines (Rizvi, 2016).

The pro-inflammatory cytokines like IL-6 and IL-1 modulates the inflammatory process in the hepatocytes through JAK- STAT3 activation (Scheller et al., 2011) which regulates the gene transcription of anti-apoptosis and proliferative regulatory proteins (Tiegs and Horst, 2022). The levels of IL-6 decrease in the presence of IL-10 (Braun et al., 2013). Apart from IL to 6, IL-10 an anti-inflammatory cytokine produced by Kupffer cells which targets immune cells also activates STAT3 signalling pathway (Wang et al., 2011). Although we did not measure the levels of IL-10, it seems that decrease in IL-6 could possibly be due to the presence of IL-10.

The histological investigation, which revealed repair of architecture and decreased inflammation and necrosis of hepatocytes, supports, and confirms the findings of the biochemical tests. This could be attributed to flavonoids, terpenoids, alkaloids and steroids in the two fractions of M. phillipensis.

5 Conclusion

We conclude that the ethyl acetate and methanol fractions of MP has good hepatoprotective activity. It was also found that a flavanone (A4; 7,4′-Dihydroxy-3′′,3′′-dimethyl-(5,6-pyrano-2′′-one)-8-(3′′′,3′′′-dimethylallyl)-flavanone) has potential hepatoprotective activity. The hepatoprotective activity might be due to the compound's antioxidant properties and cytokine-lowering effect. Further research is essential to ascertain the exact mechanism of action of the compound.

CRediT authorship contribution statement

Waseem Rizvi: Conceptualization, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Writing – original draft. Syed Shariq Naeem: Investigation. Ompal Singh: Data curation, Investigation. Shagufta Moin: Data curation, Formal analysis. Kafil Akhtar: Formal analysis, Investigation. Syed Najmul Hejaz Azmi: . Zubair Ahmed: Funding acquisition, Methodology, Visualization, Writing – review & editing. Monowarul Mobin Siddique: Formal analysis. Zainul Amiruddin Zakaria: Data curation, Writing – review & editing. Qamar Uddin Ahmed: Formal analysis, Methodology, Resources, Writing – review & editing.