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Cytotoxicity and Suppressive Effect of Leaves of Mimusops laurifolia on Carbon Tetrachloride-induced Liver Injury in Rats and its Bioactive Constituents
Abstract
Since the genus Mimusops is one of the important genera in the Indian traditional medicine, and is represented in Egypt with species; Mimusops laurifolia (Forssk.) Friis., thus the plant is selected for our investigation to reveal its biological activities and phytochemically analyze its bioactive fractions. The ethanolic extract of its leaves (LEE) and its different fractions: n-hexane (HF), chloroform (CF), ethyl acetate (EAF) and n-butanol (BF) were evaluated for in vivo hepatoprotective activity against CC14 induced hepatic cell damage in rats and for in vitro cytotoxicity against human liver cancer cell line (HEPG2); consequently the bioactive constituents were defined whereby EAF evidenced statistically significant hepatoprotection. Moreover, HF and α-amyrin (major compound isolated from HF) showed promising cytotoxicity against HEPG2. Structures of isolated compounds were established on the basis of physicochemical properties and spectral analysis. The bioactive fractions were examined for the isolation of 14 compounds for the first time from Mimusops laurifolia (Forssk.) Friis. From the lipophilic fractions: Lupeol acetate, α-amyrin, chondrillasterol, oleanolic acid, chondr111 asterol-3-O-β-D-galactoside, meamsitrin, myricetin and quercetin were isolated. While, from EAF: mearnsitrin, myricitrin, myricetin- 3 - O -β-D -g alactoside, quercetin- 3 - O - β-D-g lucoside, rutin and myncetin-3-0-β- D-glucuronide were isolated. Leaves of Mimusops laurifolia (Forssk.) Friis can be considered as a natural medicinal plant with a potential anticancer and hepatoprotection due to its bioactive ingredients in both HF and EAF, respectively.
The increase in cases of various cancers, the number of cancer patients, and the serious side effects of current chemical treatments have encouraged researchers to discover novel and more effective drugs from plant sources. In this review, PubMed and Scopus were searched for English-language peer-reviewed articles published since 1994, using the keywords: Medicinal plants, Egypt, and Cancer. The inclusion criteria for this systematic review were English peer-reviewed original research articles. A total of 286 studies were included. Studies have shown that the active compounds in certain plants can have anticancer activities by various mechanisms, such as cell cycle arrest, apoptosis induction, and antioxidant effects. Additionally, it is evident that medicinal plants can act as inhibitors of cancer cells. Plants can also act as immune checkpoint inhibitors, which inhibit proteins such as PD-L1 on tumor cells, PD-1 and CTLA-4 on T cells, and help to regulate immune responses. This review also discusses the efficacy of nanoparticles of these plants against cancer cells. The findings indicate the high potential of investigating the bioactive anticancer components of Egyptian medicinal plants for advancing novel treatments that are more effective in combating cancer. The extracts and active compounds of the medicinal plants detailed in this review could provide the foundation for further clinical trials to be conducted, to develop new anticancer drugs.
Mimusops laurifolia (Forssk.) Friis is an endangered medicinal plant and natural propagation of this plant through seeds is found to be very difficult due to its hard seed coat. To conquer this problem, an efficient in vitro seed germination as well as subsequent in vitro regeneration technique has been developed in conserving this endangered plant species. Seed germination was achieved on half strength of MS (HMS), full strength MS (FMS) and MS medium supplemented with different concentration and combination of Plant Growth Regulators (PGR) like BA, Kn and GA. The highest seed germination rate (18%) was obtained from water soaking de-coated seeds inoculated on full strength of MS (FMS), on the other hand, 12% germination of seeds was recorded on HMS medium, but both the conditions required more than 90 days to germinate. A similar rate of germination was also found on MS with PGRs, but it required 55-60 days. Best responses towards seed germination with elongated shoots (2.8 cm and 3.0 cm) were obtained when seeds were inoculated on MS with 3.0 mg/l BA, 0.5 mg/l Kn and 0.2 mg/l GA. After a long period of period of 8 months following germination, seedlings produced elongated tap roots only. Rooted seedlings were successfully transferred to soil for their further growth and development. Apart from this, for in vitro regeneration of shoots, explants of nodal segments and shoot tips from the natural plant were cultured on woody plant medium (WPM) supplemented with various combination and concentration of BAP, Kn and TDZ. In these cases, there was no positive response recorded towards the initiation of shoots from nodal segment and shoot tips. Plant Tissue Cult. & Biotech. 33(2): 143-153, 2023 (December)
In the last few decades there has been an exponential growth in the field of herbal medicine. Herbal medicines have been the basis of treatment and cure for various diseases and physiological conditions in traditional methods of practice such as Ayurveda, Unani and Siddha. Medicinal components from plants play an important role in conventional as well as western medicine. They were the sole source of active principles capable of curing man's ailments. Thus natural products have been a major source of drugs for centuries. Mimusops elengi, commonly called 'Bakul' is a medicinally important plant of family sapotaceae. All parts of the plant including stem, bark, leaves, fruit, root and seeds in all stages of their maturity have medicinal properties. Taking into consideration the medicinal importance of the plant, the present review has made to explore the phytochemical and pharmacological potential of Mimusops elengi.
Mimusops laurifolia is a native species restricted to the Red Sea mountains and Gulf of Aden. Its leaves contain saponins with wide range of biological activities. The presented research aimed to prepare saponins-rich extract from n-butanol fraction of M. laurifolia leaves and screen it for promising antimicrobial activities. Minimum inhibitory concentration (MIC) of the prepared saponins against Candida albicans, and their potential anti-pathogenic and antivirulence effects were determined. Different concentrations of the saponins-rich extract were investigated for their antimicrobial potential, particularly against C. albicans, using the agar well diffusion method. To assess the potential antivirulence and anti-pathogenic effects, we carried out molecular docking of the bioactive saponins against four key enzymes in C. albicans, which are involved in virulence and/or pathogenicity. Different concentrations of the investigated mixture showed notable antifungal activity against C. albicans with an MIC value of 6.4 μg ml-1. Docking analysis of the investigated saponins showed their affinity toward the docked enzymes, particularly saponin 1 with secreted aspartic proteinase 3 and saponin 6 with secreted aspartic proteinase 5. Thereafter, the stability of these two protein-ligand interactions was investigated using molecular dynamics (MD) simulation. The molecular interactions between saponins and the enzymes' active sites were analyzed and discussed.
Ethnopharmacological Relevance
Egyptian plants are a rich source of natural molecules, representing considerable biodiversity due to climate variations between the northern and southern and eastern and western regions of the country. Sinai is considered a precious nature reserves preserving flora, fauna, marine organisms, and historical habitats with ancient origins. Here, traditional medicinal approaches have been used for hundreds of years. Healthy lifestyles, low levels of stress and microbial infections, and a dependence on flora and herbal medicine might in combination explain why the burden of cancer is lower in some regions than in others.
Aim of the study
The primary aim of this review is to document the plants and natural products that are used as foods and medicines in Egypt, in general, and in Sinai, in particular, with a focus on those with demonstrated anticancer activities. The documented traditional uses of these plants are described, together with their chemical and pharmacological activities and the reported outcomes of clinical trials against cancer.
Materials and Methods
A literature search was performed to identify texts describing the medicinal plants that are cultivated and widely grown in Egypt, including information found in textbooks, published articles, the plant list website (http://www.theplantlist.org/), the medicinal plant names services website (http://mpns.kew.org/mpns-portal/), and web databases (PubMed, Science Direct, and Google Scholar).
Results
and Discussion: We collected data for most of the plants cultivated or grown in Egypt that have been previously investigated for anticancer effects and reported their identified bioactive elements. Several plant species, belonging to different families and associated with 67 bioactive compounds, were investigated as potential anticancer agents (in vitro studies). The most potent cytotoxic activities were identified for the families Asteraceae, Lamiaceae, Chenopodiaceae, Apocynaceae, Asclepiadaceae, Euphorbiaceae, Gramineae, and Liliaceae. The anticancer activities of some species, such as Punica granatum L., Nerium oleander L., Olea europea L., Matricaria chamomilla L., Cassia acutifolia L., Nigella sativa L., Capsicum frutescens L., Withania somnifera L., and Zingiber officinale Roscoe, have been examined in clinical trials. Among the various Egyptian plant habitats, we found that most of these plants are grown in the North Sinai, New-Delta, and Giza Governorates.
Conclusion
In this review, we highlight the role played by Egyptian flora in current medicinal therapies and the possibility that these plants may be examined in further studies for the development of anticancer drugs. These bioactive plant extracts form the basis for the isolation of phytochemicals with demonstrated anticancer activities. Some active components derived from these plants have been applied to preclinical and clinical settings, including resveratrol, quercetin, isoquercetin, and rutin.
An investigation of the hepatoprotective and antioxidant activities of the chloroform, ethyl acetate and n-butanol fractions of the leaves of Albizia lebbeck L. was performed. The first two fractions expressed the best results regarding the suppression of the increased levels of plasma amino-transferases and alkaline phosphatase in liver-damaged mice (after intoxication with CCl4) compared with silymarin and the significantly increased GSH content of alloxan-induced diabetic rats compared with Vitamin E (tests and reference drugs were orally administered). The bioactive fractions of Albizia lebbeck L. were subjected to chromatographic analysis to investigate their phenolic contents using a HPLC-PDA-ESI-MS/MS technique in the negative ion mode. The results constitute the first report of the presence of seven compounds in the genus Albizia, three of which were identified as 3-O-caffeoylquinic acid, cafeic acid, myricetin; four other flavonoids (in mg 100 g⁻¹ dry powder ± SD), myricetin 3-O-rhamnoside (0.129 ± 0.0052), quercetin 3-O-dideoxypentoside (0.011 ± 0.001), kaempferol 3-O-glucoside (0.015 ± 0.002), quercetin 3-O-dihexoside (0.138 ± 0.002); quercetin 3-O-rutinoside at a level of 0.135 ± 0.004; and the aglycones quercetin, luteolin and kaempferol. Method validation was performed, providing an analytical technique that can be used to detect trace amounts of the identified compounds in Albizia extracts with rapid sample preparation.
The phytochemical analysis of Vaccinium corymbosum var bluecrop leaves and callus biomass revealed ursolic acid, oleanolic acid, alpha-amyrin and beta-amyrin in both plant materials. Beta-sitosterol was determined only in callus biomass. The structure of isolated compounds was elucidated by TLC co-chromatography with standards and with spectroscopic methods (1H NMR, 13C NMR, EI-MS).
The hepatoprotective activity of flavonol glycosides rich fraction (F-2), prepared from 70% alcohol extract of the aerial parts of V. calcarata Desf., was evaluated in a rat model with a liver injury induced by daily oral administration of CCl4 (100 mg/kg, b.w) for four weeks. Treatment of the animals with F-2 using a dose of (25 mg/kg, b.w) during the induction of hepatic damage by CCl4 significantly reduced the indices of liver injuries. The hepatoprotective effects of F-2 significantly reduced the elevated levels of the following serum enzymes: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH). The antioxidant activity of F-2 markedly ameliorated the antioxidant parameters including glutathione (GSH) content, glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), plasma catalase (CAT) and packed erythrocytes glucose-6-phosphate dehydrogenase (G6PDH) to be comparable with normal control levels. In addition, it normalized liver malondialdehyde (MDA) levels and creatinine concentration. Chromatographic purification of F-2 resulted in the isolation of two flavonol glycosides that rarely occur in the plant kingdom, identified as quercetin-3, 5-di-O-beta-D-diglucoside (5) and kaempferol-3, 5-di-O-beta-D-diglucoside (4) in addition to the three known compounds identified as quercetin-3-O-alpha-L-rhamnosyl- (1-->6)-beta-D-glucoside [rutin, 3], quercetin-3-O-beta-D-glucoside [isoquercitrin, 2] and kaempferol-3-O-beta-D-glucoside [astragalin, 1]. These compounds were identified based on interpretation of their physical, chemical, and spectral data. Moreover, the spectrophotometric estimation of the flavonoids content revealed that the aerial parts of the plant contain an appreciable amount of flavonoids (0.89%) calculated as rutin. The data obtained from this study revealed that the flavonol glycosides of F-2 protect the rat liver from hepatic damage induced by CCl4 through inhibition of lipid peroxidation caused by CCl4 reactive free radicals.
Two flavonol glycosides have been isolated and characterised from leaves of Syzygium samarangense. One is the rare mearnsitrin (1) while the second, 2′-C-methyl-5′-O-galloylmyricetin-3-O-a-l-rhamnopyranoside (2), is new. Detailed spectral data are provided for both.
Two novel triterpenoid saponins, mimusopin ( 3-O-β-D-glucopyranosyl-2β, 3β, 6β, 23-tetrahydroxyolean-12-en-28-oic acid 28-O-α-L-rhamnopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)[a-L-rhamnopyranosyl-(1→ 3)]-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside)(1) and mimusopsin 3-O-[β-D-glucopyranosyl-(1→3)β-D-gluco-pyranosyl]-2β, 3β, 6β, 23-tetrahydroxyolean-12-en-28-oic acid 28-O-α-L-rhamnopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside (2) were isolated from the seeds of Mimusops elengi. Their structures were elucidated by a combination of 2D-NMR (COSY, HOHAHA, HETCOR, HMBC and NOESY), FAB-MS/MS and strategic chemical degradation. In addition, molecular mechanics and dynamics studies showed that the lack of a 13C glycosylation shift at the C-4 of the inner rhamnose in 1 could be correlated with distortion in the corresponding torsion angles.
The absolute configuration at C-24 of C-24 epimeric 24-ethyl-5α-cholesta-7,E-22-dien-3β-ols I–IV previously isolated from
tea seed oil, shea fat, and gourd and sponge cucumber seed oils, respectively, was studied by proton nuclear magnetic resonance
spectroscopy. The results showed that the sterols I and II are identical with spinasterol (24S/α-ethyl group), whereas the
sterols III and IV are identified as its 24R/β-epimer, chondrillasterol. This study has thus for the first time properly documented
the presence in tracheophytes of a 24β-ethylsterol in which Δ25(27)-bond is reduced.
Two new steroidal glycosides [1 and 2] have been isolated from Mimusops elengi and characterized as the 3-O-beta-D-glucopyranoside and the 3-O-beta-D-galactopyranos ide of (24R)-stigmast-7,22(E)-dien-3 alpha-ol, respectively.
Chlorinated hydrocarbons differ in their capacity to produce liver damage. The comparative effects of carbon tetrachloride (CCl4), chloroform, 1,1,1trichloroethane and 1,1,2-trichloroethane were studied in rats. CCl4 produced the highest increase in hepatic triglycerides and also produced elevated levels at the lowest dose (0.03 ml/kg). Chloroform was intermediate in producing elevated liver triglyceride levels; with 1,1,2-trichloroethane, enhanced triglyceride levels were demonstrated only at near-lethal dosages. No enhanced hepatic triglyceride level was demonstrated with 1,1,1-trichloroethane. Significant dose-related decreases in glucose 6-phosphatase activity were demonstrated with doses of 0.3 ml/kg of CCl4 or greater. With the other chlorinated hydrocarbons, no decrease in hepatic glucose 6-phosphatase activity was detected. When the hydrocarbons were added directly to liver homogenates, only those incubations containing CCl4 exhibited increased lipid peroxidation (enhanced thiobarbituric acid reactants). In vivo, evidence of lipid peroxidation (diene conjugates) in the liver was obtained 15 min after CCl4 treatment; this effect reached its peak at 30 min. Enhanced diene conjugates were detected with doses of 0.3 ml/kg of CCl4 or greater. However, with the other three hydrocarbons, no increase in diene conjugates was detected. Thus, this study shows that while the temporal relationships of CCl4-induced hepatotoxicity are compatible with the lipid peroxidation hypothesis, the dosage relationship is weaker. Also the lack of qualitative similarity in the results obtained with chloroform is disturbing.
A method for the investigation of the acute toxicity of an unknown chemical substance, with an estimation on the LD50, is described. Using this, it is possible to obtain with 13 experimental animals adequate information on the acute toxicity and on the LD50. This method has no limitations and applies to drugs, agricultural and industrial chemicals. It can be used for every route of administration.
Dried leaves of Licania densiflora yielded five myricetin glycosides: myricetin 3'-methylether-3-O-glucoside (1), myricetin 3'-methylether-3-O-galactoside (2), myricetin 4'-methylether-3-O-rhamnoside (3), myricetin 3',5'-dimethylether-3-O-glucoside (4), and myricetin 3',5'-dimethylether-3-O-rhamnoside (5). Their structures were elucidated by spectroscopic methods including mono- and bi-dimensional NMR techniques.
The present study was designed to investigate the effect of Mimusops elengi (Sapotaceae) against experimental gastric ulcers. The 50% alcoholic extract of Mimusops elengi (Ext E) and its different fractions namely ethyl acetate (Ext E1), n-butanol (Ext E2), methanol (Ext E3) and aqueous (Ext E4) were studied (p.o.) against ethanol-induced gastric damage. Ext E1 was also studied in ethanol-induced, pylorus-ligated and water-immersion plus stress-induced gastric ulcer models. Ranitidine HCl (80 mg kg(-1)) was used as a reference standard. In ethanol-induced gastric ulcer model, pantoprazole (20 mg kg(-1)) was also used as a reference standard. Ext E1 tested in mice up to the dose of 5000 mg kg(-1) (p.o.) did not produce any sign of toxicity. Ext E at the doses of 50, 100, 300 and 500 mg kg(-1) and its different fractions (100 mg kg(-1)) showed reduction in gastric ulceration (P < 0.05). Ext E1 at the doses of 10, 50 and 100 mg kg(-1) showed dose-dependent inhibition of gastric lesions against ethanol-induced gastric damage. In 19 h pylorus-ligated animals, Ext E1 at 50 and 100 mg kg(-1) doses showed significant reduction in ulcer index (P < 0.05). Significant reduction was also observed in total acidity, volume of gastric acid secretion, total acid output and pepsin activity (P < 0.05) when compared with the control group. Besides, Ext E1 showed increase in the mucosal glycoproteins that was evident from significant rise in total carbohydrates to protein ratio (TC:PR ratio) (P < 0.05), which is an indication of mucin activity. Ext E1 also showed protection against water-immersion plus stress-induced gastric lesions that was evident from dose-dependent decrease in ulcer index (P < 0.05), score for intensity (P < 0.05) and total lesion area (P < 0.05) when compared with the control group. It can be concluded from our study that Ext E1 possesses anti-ulcer activity against experimental gastric ulcers. The mechanism of anti-ulcer activity can be attributed to decrease in gastric acid secretory activity along with strengthening of mucosal defensive mechanisms.