Antiplasmodial and antitrypanosomal activity of plants from the Kingdom of Saudi Arabia

The antiplasmodial and antitrypanosomal activity of the methanol extracts of 42 plants collected from the Kingdom of Saudi Arabia and some fractions obtained thereof were evaluated. The antiplasmodial activity was tested in vitro against chloroquine-resistant strain (K1) and sensitive strain (FCR3), and the antitrypanosomal activity was tested in vitro against Trypanosoma brucei brucei GUTat 3.1 strain. For host cells, the cytotoxicity of the active extracts was also evaluated against the MRC5 human cell line. Only extracts of three samples demonstrated good antiplasmodial activity (IC₅₀ < 12.5 and > 1.56 μg/ml, score 2), the methanol extracts of Lycium shawii, Heliotropium zeylanicum and the petroleum ether-soluble fraction of the methanol extract of Caralluma tuberculata, while extracts of the remaining 42 plants were inactive (IC₅₀ > 12.5 μg/ml, score 1). As for the antitrypanosomal activity, the methanol extract of Solanum schimperianum demonstrated the highest activity (IC₅₀ 0.061 μg/ml), followed by the petroleum ether-soluble fraction of the methanol extract of C. tuberculata (IC₅₀ 0.5 μg/ml). The chloroform-soluble fraction of the methanol extract of C. tuberculata was moderately active (IC₅₀ 3.5 μg/ml), with low cytotoxicity (IC₅₀ 62.6 μg/ml) and moderate selectivity index (SI 17.9). The methanolic extracts of 34 plants showed good activity with score 2 (IC₅₀ < 12.5 and > 1.56 μg/ml), while the extracts of seven plants were inactive (IC₅₀ > 12.5 μg/ml, score 1).     

Antihyperglycaemic and aldose reductase inhibitory potential of Acacia catechu hard wood and Tectona grandis leaves

The ethanolic extract of Acacia catechu hard wood (Ac) and the ethanolic as well as aqueous extracts of Tectona grandis leaves (Tg) showed marked antihyperglycaemic activity in both normoglycaemic and streptozotocin-induced diabetic rats at 250 mg/kg dose levels. The ethanolic and aqueous extracts of Ac and Tg also showed marked inhibition on AR from eye lens of normal rats with IC 50 values of 9.30 and 9.08 and 3.05, 4.51 μg/ml, respectively. The ethanolic as well as aqueous extracts of both Ac and Tg also showed marked inhibition on AR from eye lens of STZ-induced diabetic rats with IC₅₀ values of 4.70 and 4.91 and 4.71 and 4.83 μg/ml. These results suggest that Acacia catechu hard wood and Tectona grandis leaves could be a new approach in the development of therapeutic or preventive agents for diabetic late-stage complications.     

Inhibitory effects of compounds from Anemarrhenae Rhizoma on α-glucosidase and aldose reductase and its contents by drying conditions

An aqueous extract of Anemarrhena asphodeloides Bunge (Anemarrhenae Rhizoma) showed inhibitory effects on α-glucosidase and aldose reductase (AR). We obtained two active compounds, mangiferin (α-glucosidase: IC₅₀ = 96.1 μg/ml, AR:IC₅₀ = 0.13 μg/ml) and mangiferin 7-O-β-D-glucopyranoside (α-glucosidase: IC₅₀ = 158 μg/ml, AR:IC₅₀ = 65.7 μg/ml ), from Anemarrhenae Rhizoma. In addition, the amount of mangiferin in Anemarrhenae Rhizoma changed with the method of preparation (drying conditions), namely, low-temperature, long-term drying conditions and harvesting in August were most effective.     

Brazilian marine sponge <Emphasis Type="Italic">Polymastia janeirensis</Emphasis> induces apoptotic cell death in human U138MG glioma cell line,but not in a normal cell culture

Summary  Marine sponges have been prominently featured in the area of cancer research. Here, we examined the anti-proliferative effects of crude extracts (aqueous and organic) of the Brazilian marine sponge Polymastia janeirensis in the U138MG human glioma cell line. Moreover, we examined the effects of extracts on selective cytotoxicity in the glioma cells in comparison with a normal cell culture. Exposure of glioma cells to treatments (24 h) resulted in cell number decrease at all doses tested, with both aqueous and organic extracts (IC₅₀ <20 and <30 μg/ml, respectively). Parallel to this result, sponge extracts reduced glioma cell viability (IC₅₀ <15 μg/ml for both extracts). However, higher doses (50 and 100 μg/ml) induced a stronger cytotoxic effect when compared to the lower dose tested (10 μg/ml), inhibiting more than 80% of cellular growth and viability. Propidium iodide uptake and flow cytometry analysis further showed that sponge extracts caused necrosis in the glioma cell line at higher doses, while a high percentage of apoptotic glioma cells were observed at 10 μg/ml. Moreover, apoptosis was prevented by the pan-caspase inhibitor Z-VAD, suggesting that marine sponge extracts, at lower doses, induce caspase-dependent apoptosis in U138MG glioma cells. Surprisingly the extracts herein tested were more effective than temozolomide, a potent inductor of apoptosis used for the treatment of malignant gliomas. Furthermore, our results suggested a selectivity cytotoxic effect on glioma cell line in comparison with a normal cell culture, since the effect on viability found in glioma cells was not observed in astrocyte cultures with the lower dose (10 μg/ml). Thus, this marine sponge may be considered a good candidate for development of new cancer medicines with antitumor activity against gliomas.     

In vitro anti-plasmodial and in vivo anti-malarial activity of some plants traditionally used for the treatment of malaria by the Meru community in Kenya

Extracts of seven medicinal plant species used for treatment of malaria in traditional/cultural health systems of the Ameru people in Kenya were tested in vitro and in vivo against Plasmodium falciparum (D6 and W2 strains) and P. berghei, respectively. Of the plants tested, 28.57% were highly active (IC₅₀ <10 μg/ml) and 42.86% moderately active (IC₅₀ 10–50 μg/ml), while 28.57% had weak activity of 50–125 μg/ml in vitro. The water and methanol extracts of Boscia salicifolia Oliv. and Artemisia afra Jacq. (ex-Willd.) were the most active against both the chloroquine (CQ)-sensitive (D6) and the CQ-resistant (W2) P. falciparum strains. Artemisia afra and Rhus natalensis Bernh. (ex-Krauss) exhibited the highest parasite clearance and chemo-suppression (>70%) in vivo (in mice). The plants with high in vitro anti-plasmodial (low IC₅₀ values) and high anti-malarial activity (high chemo-suppression) in vivo are potential sources of novel anti-malarial drugs.     

Potent antimalarial activity of newly synthesized substituted chalcone analogs in vitro

Abstract   Several new chalcone analogues were synthesized and evaluated as inhibitors of malaria parasite. Inhibitory activity was determined in vitro against a chloroquine-sensitive Plasmodium falciparum strain of parasites. The compound 3-(4-methoxyphenyl)-1-(4-pyrrol-1-yl-phenyl)prop-2-en-1-one was found to be the most active with 50% inhibition concentration (IC₅₀) of 1.61 μg/ml. This inhibitory concentration is comparable to a prototype phytochemical chalcone, licochalcone A, with an IC₅₀ of 1.43 μg/ml. The present study suggests that small, lipophilic nitrogen heterocyclic ring A together with small hydrophobic functionality at ring B can enhance antimalarial activity. These results suggest that chalcones are a class of compounds that provides an option of developing inexpensive, synthetic therapeutic antimalarial agents in the future. Graphical Abstract  Claisen-Schmidt condensation method was employed to synthesize various substituted chalcones. Among all, 3-(4-Methoxyphenyl)-1-(4-pyrrol-1-yl-phenyl)prop-2-en-1-one was found to be most effective with IC₅₀ value of 1.6 μg/ml in vitro against chloroquine sensitive strain (3D7) of Plasmodium falciparum.      

Antibacterial, antioxidant and cytotoxic activities of extracts of Conyza canadensis (L.) Cronquist growing in Tunisia

Antibacterial antioxidant and cytotoxic activities of petroleum ether, ethyl acetate and methanol extracts of Conyza Canadensis (L.) Cronquist were investigated. Antibacterial activity was evaluated using the agar diffusion and microwell dilution assays against four strains of bacteria. Antioxidant activity was measured by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) method and the cytotoxic activity was tested against Hep-2 cells (laryngeal carcinoma cell line) using methylene blue assays. Among tested extracts, the methanolic extract exhibited important antibacterial activity. It also showed good antioxidant activity with 50% inhibition concentration (IC₅₀) of 120 μg/ml. Cytotoxicity of extracts was time depend, increasing with exposure time and concentration. At 72 h of incubation, the ethyl acetate and petroleum ether extracts demonstrated effective cytotoxic activity against Hep-2 cells with IC₅₀ values of 45 and 50 μg/ml, respectively.     

In vitro antioxidant activity and total phenolic content of ethanolic leaf extract of Stevia rebaudiana Bert.

The aim of this study was to assess the in vitro potential of ethanolic leaf extract of Stevia rebaudiana as a natural antioxidant. The DPPH activity of the extract (20, 40, 50, 100 and 200 μg/ml) was increased in a dose dependent manner, which was found in the range of 36.93–68.76% as compared to ascorbic acid 64.26–82.58%. The IC₅₀ values of ethanolic extract and ascorbic acid in DPPH radical scavenging assay were obtained to be 93.46 and 26.75 μg/ml, respectively. The ethanolic extract was also found to scavenge the superoxide generated by EDTA/NBT system. Measurement of total phenolic content of the ethanolic extract of S. rebaudiana was achieved using Folin–Ciocalteau reagent containing 61.50 mg/g of phenolic content, which was found significantly higher when compared to reference standard gallic acid. The ethanolic extract also inhibited the hydroxyl radical, nitric oxide, superoxide anions with IC₅₀ values of 93.46, 132.05 and 81.08 μg/ml, respectively. However, the IC₅₀ values for the standard ascorbic acid were noted to be 26.75, 66.01 and 71.41 μg/ml respectively. The results obtained in this study clearly indicate that S. rebaudiana has a significant potential to use as a natural antioxidant agent.     

Antimalarial activity of Gomphostemma crinitum leaf extracts

Abstract  Antimalarial efficacy of Gomphostemma crinitum leaf extracts were studied in vitro against the chloroquine-sensitive MRC-02 strain of Plasmodium falciparum. The CHCl₃ extract (IC₅₀ 37.28 μg/mL) was found to be better than the aqueous extract (IC₅₀ 111.4 μg/mL). The major compound present in CHCl₃ extract was coded as GC-7 (12,17-diacetoxy, 15-hydroxy, 2-oxo, 3, 13 E (14)-diene clerodane). Seasonal variation of GC-7 was studied and correlated to the antimalarial efficacy of the crude extracts of leaves collected in different months. GC-7 was found to be the only regular compound present in G. crinitum leaves throughout the year. A good correlation between antimalarial efficacy (IC₅₀ 9.3 μg/mL) and concentration of GC-7 in crude extracts was observed. The mode of action of GC-7 was found to be different from that of chloroquine. Graphical Abstract  Antimalarial activity of Gomphostemma crinitum leaf extracts B.N Acharya, Deepika Saraswat and M.P Kaushik^* Discovery Centre, Process Technology Development Division Defence R & D Establishment, Jhansi Road, Gwalior-474002 (MP) INDIA ^#Entomology division, Defence Research and Development Establishment,Gwalior-474002, India Antimalarial activity of chloroform extract of G. crinitum leaves and an isolated compound GC-7 (12,17-diacetoxy,15-hydroxy,2-oxo,3,13 E (14)-diene clerodane) against a chloroquine susceptible strain of Plasmodium falciparum is described.     

<Emphasis Type="Italic">In-vitro</Emphasis> evaluation of antioxidant potential of <Emphasis Type="Italic">rauwolfia vomitoria</Emphasis> root extract and its inhibitory effect on lipid peroxidation as indication of aphrodisiac properties

Rauwolfia vomitoria (RV) Afzel (Apocynaceae) is a medicinal plant used in traditional medicinal practice for the treatment of hypertension. This research is devoted to phytochemical constituents, in particular, some specific alkaloids present in the RV root extract. The phytochemical evaluation revealed the presence of alkaloids, tannins, saponins, and flavonoids in this extract. The antioxidant activity of the RV root extract was also evaluated in a series of in vitro assays involving free radicals. The extract exhibited significant hydrogen peroxide scavenging effect relative to ascorbic acid (p < 0.05, IC₅₀ = 98 μg/ml), nitric oxide scavenging effect (50.37 ± 0.4% after 150 min), and metal chelating activity (89.08 ± 2.62%). In addition, it exhibited significant ferric reducing power relative to ascorbic acid (p < 0.05). The total content of phenolic substances was 233.3 ± 2.9 mg/g. The extract was also studied for its inhibitory capacity on lipid peroxidation as a possible mechanism of its aphrodisiac effect, by measuring thiobarbituric acid reactive substances in various male-cow tissues incubated in a 5% solution of the RV root extract, distilled water, and antioxidant vitamins C and E upon keeping the samples frozen for 35 days. Tissues incubated in the test solution had lower levels of malondialdehyde (MDA) compared to those in the samples incubated in distilled water. Results obtained from this study indicate that the RV root extract can be a potential source of natural antioxidants.     

In vitro aneugenic effects of the fungicide thiabendazole evaluated in human lymphocytes by the micronucleus assay

Thiabendazole is a benzimidazole-derived compound widely employed in agriculture as anthelmintic and fungicide. It is also used as a post-harvest fungicide for imported citrus fruits during transport and storage, and thus, it was found at high concentration in fruits and vegetables. Several studies have analyzed the potential genotoxic effect of thiabendazole on different prokaryotic and eukaryotic systems, but in many cases, results were contradictory. In the present study, the genotoxic potential of thiabendazole have been evaluated, by micronucleus assay in freshly isolated human peripheral lymphocytes. The cells were incubated with 0.5, 5 and 50 μg/ml concentrations of the tested substance for 48 h at 37°C. Mitomycin C at final concentration of 0.01 μg/ml culture was used as a positive control. The results indicated that the thiabendazole significantly (P < 0.05) increased the micronucleus frequency compared with the negative control in all treatment concentrations, indicating a potential aneugenic hazard of thiabendazole in cultured human peripheral lymphocytes. The cytokinesis-block proliferation index value, however, was not decreased significantly compared with the negative control. Significant (P < 0.05) differences in the micronuclei frequency were also found between the lower dose (0.5 μg/ml) and the other two analyzed doses of thiabendazole. In contrast, no differences were found between 5 and 50 μg/ml of thiabendazole and between DMSO and negative control. Finally, control cultures treated with the known mutagen MMC showed a very consistent increase in MN with respect to the negative controls.     

Cytotoxicity of fumonisin B1: implication of lipid peroxidation and inhibition of protein and DNA syntheses

The effects of fumonisin B₁ (FB₁) from Fusarium moniliforme on lipid peroxidation and protein and DNA syntheses were studied in monkey kidney cells (Vero cells). FB₁ was found to be a potent inducer of malondialdehyde (MDA), one of the secondary products formed during lipid peroxidation. At 0.14 μM (0.1 μg/ml), FB₁ induced 0.496 ± 0.1 nmoles of MDA/mg protein, compared to the control level 0.134 ± 0.01 nmoles of MDA/mg protein (P < 0.005). No inhibition of protein or DNA synthesis was observed at this concentration of FB₁. Inhibition of protein and DNA syntheses was observed at FB₁ concentrations >14 μM (10 μg/ml) with an IC₅₀ of 33 μM for both protein synthesis and DNA synthesis. These results indicate that lipid peroxidation is a very sensitive cellular response to the mycotoxin fumonisin B₁ observed at concentrations lower than that required to inhibit cellular synthesis of macromolecules, protein and DNA. This oxidative damage induced by FB₁ concentrations encountered in naturally contaminated foodstuffs and feed might lead to mutagenicity and genotoxicity. Received: 18 August 1997 / Accepted: 17 November 1997     

Neolignans from North American Magnolia Species with Cyclooxygenase 2 Inhibitory Activity

Objectives:  Based upon reported ethnomedicinal use by Native Americans, extracts and pure isolates from leaves and seeds of Magnolia grandiflora, M. virginiana, M. acuminata and M. macrophylla, all native to the Southeastern United States, were investigated for their anti-inflammatory potential against cyclooxygenase 2 (COX-2). Material and methods:  The extracts and pure compounds from Magnolia species were tested for their production of prostaglandin E₂ (PGE₂) using a mouse macrophage (RAW 264.7) assay where cells were stimulated by lipopolysaccharide. Results:  Leaf extracts were moderately active (44–58% inhibition at 50 μg/ml) whereas seed extracts showed significant activity of 54–88% inhibition, respectively. In the seed extract of M. grandiflora, honokiol, magnolol and 4’-O-methylhonokiol strongly inhibited COX-2 (IC₅₀: 1.2–2.0 μg/ml), 3-O-methylmagnolol was moderately active while a new compound was inactive towards COX-2. The neolignans were not cytotoxic to macrophages (RAW 264.7) and kidney fibroblast (VERO) cells in vitro. Conclusions:  The results indicate that the reported ethnomedicinal use of the investigated Magnolia species is in agreement with anti-inflammatory activity of their respective compounds. Received 8 December 2007; accepted 16 January 2009     

Superoxide anion radical scavenging activity of Cassia siamea and Cassia javanica

This study was designed to explore the antioxidant potential of hexane, chloroform, ethyl acetate, methanol, and water extract of bark and leaves of Cassia siamea and Cassia javanica by superoxide anion radical scavenging assay. The different extracts showed significant inhibition of superoxide radicals in a dose-dependant manner. Among all the bark extracts of C. siamea, the methanol extract showed the maximum inhibition of 60.5% at 800 μg/ml concentration and water extract also showed strong antioxidant potential of 51.3% at 1000 μg/ml concentration. The water extract of bark of C. javanica showed strong antioxidant potential of 55% at 1000 μg/ml concentration. The various leaf extracts of C. siamea showed moderate antioxidant potential of 25–50% at 1000 μg/ml, whereas methanol leaf extract of C. javanica showed strong antioxidant potential of 50.4% at 300 μg/ml concentration. This preliminary study indicates the antioxidant activity of the bark and leaves of C. siamea and C. javanica.     

Anti-oxidative and anti-inflammatory activities of some isolated constituents from the stem bark of Allanblackia monticola Staner L.C (Guttiferae)

Stem bark of Allanblackia monticola has been used in association with others plant in the Cameroonian folk medicine for the treatment of various diseases such amoebic dysentery, diarrhoea, lung infections, and skin diseases. The methylene chloride fraction, its isolated compounds like α-mangostin, lupeol and acid betulinic were screened for antioxidant activity using free radical scavenging method. These isolated compounds were further tested for anti-inflammatory properties using carrageenan-induced model. Methylene chloride fraction, showed concentration-dependent radical scavenging activity, by inhibiting 1,1-diphenyl-1-picryl-hydrazyl radical (DPPH) with an IC₅₀ value of 14.60 μg/ml. α-Mangostin and betulinic acid (500 μg/ml), showed weak radical scavenging activity with a maximum inhibition reaching 38.07 μg/ml and 26.38 μg/ml, respectively. Betulinic acid, lupeol and α-mangostin (5 mg/kg and 9.37 mg/kg) showed anti-inflammatory activity with a maximum inhibition of 57.89%, 57.14% and 38.70%, respectively. Methylene chloride fraction of Allanblackia monticola and some derivatives, have antioxidant and anti-inflammatory activities. Received 7 July 2008; accepted 7 October 2008     

Effect of hexane fraction of leaves of <Emphasis Type="Italic">Cinnamomum tamala</Emphasis> Linn on macrophage functions

The leaves of Cinnamomum tamala Linn (Lauraceae), component of Indian spices are associated with hypoglycemic property in Ayurveda; however, no report is available towards its immunomodulation property, which has been explored here. The dried powder of CT leaves was extracted with hexane and solvent free extract (CTH) was given orally to rats for 10 days, in various doses. Its effect was studied on peritoneal macrophage functions, and was compared with ascorbic acid (1,000 mg/kg, immune-stimulant) and cyclophosphamide (10 mg/kg, immune-suppressant). CTH significantly suppressed phagocytosis activity (EC₅₀ 2,355 ± 52.45 mg/kg), reduced production of superoxide (EC₅₀ 275.91 ± 10.21 μg/ml) and cellular NADPH (EC₅₀ 384.959 ± 4.85 μg/ml) content in concentration dependent manner. It also inhibited LPS induced production of nitric oxide (EC₅₀ 143.75 ± 3.40 μg/ml) and iNOS protein expression (EC₅₀ 183.132 μg/ml). Thus, it could be suggested that non-polar hexane fraction of leaves of C. tamala possesses immunosuppressive property, which is mediated through modulation of innate immunity.     

Patchouli alcohol: in vitro direct anti-influenza virus sesquiterpene in <Emphasis Type="Italic">Pogostemon cablin</Emphasis> Benth.

During the screening of anti-influenza virus substances from traditional herbal medicines, the methanol extract from the leaves of Pogostemon cablin Benth. showed potent in vitro antiviral activity (99.8% inhibition at a concentration of 10 μg/mL) against influenza virus A/PR/8/34 (H1N1). The anti-influenza virus principle was isolated from the hexane-soluble fraction, through solvent fractionation, repeated silica gel column chromatography, and reversed-phase HPLC. The major active principle was a volatile substance that was identified as a sesquiterpene, patchouli alcohol (1), on the basis of its spectral analyses. When anti-influenza virus activity against A/PR/8/34 was evaluated by the plaque forming assay, patchouli alcohol reduced the number of plaques by 75% at 2 μg/mL and 89% at 10 μg/mL. Patchouli alcohol showed dose-dependent anti-influenza virus activity, and its IC₅₀ value was estimated to be 2.635 μM. Although 11 different sesquiterpenes were tested for antiviral activity against influenza virus A/PR/8/34, no or negligible activity was observed except for patchouli alcohol. Patchouli alcohol did not show anti-influenza virus activity against A/Guizhou/54/89 (H3N2), but showed weak activity against B/Ibaraki/2/85 (IC₅₀ = 40.82 μM). Patchouli alcohol did not show inhibitory activity against influenza virus neuraminidase.     

Anti-oxidative and inhibitory activities on nitric oxide (NO) and prostaglandin E<Subscript>2</Subscript> (COX-2) production of flavonoids from seeds of <Emphasis Type="Italic">Prunus tomentosa</Emphasis> Thunberg

Chemical investigation of the 80% Me₂CO extract from the seeds of Prunus tomentosa led to the isolation and identification of six flavonoids: kaempferol (1), kaempferol 3-O-α-L-rhamnopyranoside (2; afzelin), kaempferol 3-O-β-D-(6-acetyl)-glucopyranosyl(1→4)-α-L-rhamnopyranoside (3; multiflorin A), kaempferol 3-O-β-D-glucopyranosyl(1→4)-α-L-rhamnopyranoside (4; multiflorin B), quercetin 3-O-α-L-rhamnopyranoside (5; quercitrin), and quercetin 3-O-β-D-glucopyranosyl (1→4)-α-L-rhamnopyranoside (6; multinoside A). Anti-oxidative and inhibitory activities on nitric oxide (NO) and prostaglandin E₂ production in interferon-γ (INF-γ) and lipopolysaccharide (LPS)-activated RAW 264.7 cells in vitro (COX-2) of the isolated compounds were evaluated. Compounds 1, 5, and 6 exhibited potent anti-oxidative activity in the DPPH radical scavenging assay with IC₅₀ values of 57.2, 59.4, and 54.3 μg/mL respectively. The positive control, ascorbic acid, had an IC₅₀ of 55.5 μg/mL. Compounds 1, 5, and 6 also reduced COX-2 levels in a dose dependent manner with IC₅₀ values of 10.2, 8.7, and 9.6 μg/mL respectively, with the positive control, indomethacin, having an IC₅₀ of 5.1 μg/mL. All six compounds inhibited NO production in a dose dependent manner with IC₅₀ values of 35.1, 42.8, 40.0, 44.8, 43.7, and 43.9 μg/mL respectively, while the positive control, L-NMMA, had an IC₅₀ of 42.1 μg/mL.     

Cytotoxic constituents of<Emphasis Type="Italic">diadema setosum</Emphasis>

5,8-Epidioxycholest-6-en-3-ol (1), cholesterol (2), glycerol 1-palmitate (3) and glycerol 1,3-dioleate-2-stearate (4) were isolated from the methanol extract of the sea urchinDiadema setosum, which was collected from the Halong sea, Vietnam. Chemical structures were established based on extensive 1D, 2D-NMR, FAB-MS, EI-MS spectroscopic data and GC-MS analysis. The NMR spectral data of compound 1 were reassigned by using HMQC and HMBC. Compound1 was found to have strong cytotoxic effect against various cancer cell lines, such as KB (IC₅₀, 2.0 μg/mL), FL (IC₅₀, 3.93 μg/mL), and Hep-2 (IC₅₀, 2.4 μg/mL) byin vitro assay.