Bioactive solanidane steroidal alkaloids from Solanum lyratum.

Six previously unreported solanidane steroidal alkaloids, namely lyrasolanosides A-F, were isolated from Solanum lyratum. In addition, five known steroidal alkaloids were also identified. The structures of these compounds were determined through the use of NMR, HRESIMS,UV, IR and ECD analysis. To assess their bioactivities, the cytotoxic effects of the six previously unreported compounds were evaluated on A549 cells. The results revealed that lyrasolanoside B (2) exhibited the highest potency among them. Lyrasolanoside B (2) exhibited significant inhibition of cell migration, invasion, and adhesion dramatically. Mechanistically, it was found to suppress the activity of JAK2/STAT3 signaling pathway by downregulating the expression of phosphorylated JAK2/STAT3 in an exosome-dependent manner. In addition, lyrasolanoside B (2) was found to significantly upregulate the expression of E-cadherin and downregulate the expression of N-cadherin and vimentin. These findings indicate that lyrasolanoside B (2) inhibits the metastasis of A549 cells by suppressing exosome-mediated EMT. These findings suggest that lyrasolanoside B (2) may inhibit the metastasis of lung cancer by regulating A549-derived exosomes.

Pharmacological validation of Solanum mammosum L. as an anti-infective agent: Role of solamargine.

ETHNOPHARMACOLOGICAL RELEVANCE: Fungal and bacterial infections remain a major problem worldwide, requiring the development of effective therapeutic strategies. Solanum mammosum L. (Solanaceae) ("teta de vaca") is used in traditional medicine in Peru to treat fungal infections and respiratory disorders via topical application. However, the mechanism of action remains unknown, particularly in light of its chemical composition. MATERIALS AND METHODS: The antifungal activity of TDV was determined against Trichophyton mentagrophytes and Candida albicans using bioautography-TLC-HRMS to rapidly identify the active compounds. Then, the minimum inhibitory concentration (MIC) of the fruit crude extract and the active compound was determined to precisely evaluate the antifungal activity. Additionally, the effects of the most active compound on the formation of Pseudomonas aeruginosa biofilms and pyocyanin production were evaluated. Finally, a LC-HRMS profile and a molecular network of TDV extract were created to characterize the metabolites in the fruits' ethanolic extract. RESULTS: Bioautography-TLC-HRMS followed by isolation and confirmation of the structure of the active compound by 1D and 2D NMR allowed the identification solamargine as the main compound responsible for the anti-Trichophyton mentagrophytes (MIC = 64 µg mL-1) and anti-Candida albicans (MIC = 64 µg mL-1) activities. In addition, solamargine led to a significant reduction of about 20% of the Pseudomonas aeruginosa biofilm formation. This effect was observed at a very low concentration (1.6 µg mL-1) and remained fairly consistent regardless of the concentration. In addition, solamargine reduced pyocyanin production by about 20% at concentrations of 12.5 and 50 µg mL-1. Furthermore, the LC-HRMS profiling of TDV allowed us to annotate seven known compounds that were analyzed through a molecular network. CONCLUSIONS: Solamargine has been shown to be the most active compound against T. mentoagrophytes and C. albicans in vitro. In addition, our data show that this compound affects significantly P. aeruginosa pyocyanin production and biofilm formation in our conditions. Altogether, these results might explain the traditional use of S. mammosum fruits to treat a variety of fungal infections and respiratory disorders.

Solasodine, Isolated from Solanum sisymbriifolium Fruits, Has a Potent Anti-Tumor Activity Against Pancreatic Cancer.

BACKGROUND: Increasing evidences have revealed that solasodine, isolated from Solanum sisymbriifolium fruits, has multiple functions such as anti-oxidant, anti-tumor and anti-infection. However, its role in pancreatic cancer has not been well studied. METHODS: To explore the role of solasodine in pancreatic cancer, human pancreatic cell lines including SW1990 and PANC1 were treated with different concentrations of solasodine for 48 h, and cell viability was evaluated by MTT assay, cell invasion and migration were evaluated by Transwell assay. The effect of solasodine on the apoptosis of SW1990 and PANC1 cells was detected by flow cytometry. To further explore the antitumor effect of solasodine in vivo, an SW1990 tumor-bearing mouse model was constructed. The effects of solasodine on cytokines in the serum of SW1990 tumor-bearing mice were also evaluated by ELISA assay. RESULTS: Specifically, in vitro, solasodine could significantly inhibit the proliferation of pancreatic cancer cell lines SW1990 and PANC1 cells. Flow cytometric analysis indicated that solasodine could induce apoptosis of SW1990 and PANC1 cells. Western blot assay indicated that solasodine could significantly inhibit the activation of Cox-2/Akt/GSK3ß signal pathway. Meanwhile, the release of Cytochrome c from mitochondria to cytoplasm which can raise the caspases cascade (C-caspase 3 and C-caspase 9) was significantly enhanced by solasodine. In vivo, the results showed that solasodine had potent anti-tumor activities with a lower cytotoxicity. In addition, the serum TNF-α, IL-2 and IFN-γ levels in SW1990 tumor-bearing mice after the treatment of solasodine was significantly increased. CONCLUSION: Taken together, our results suggested that the solasodine could prevent the progression of pancreatic cancer by inhibiting proliferation and promoting apoptosis, as well as stimulating immunity, suggesting that solasodine might be a potential therapeutic strategy for pancreatic cancer.

Effect of ethanolic extract of Solanum virginianum Linn. on neuropathic pain using chronic constriction injury rat model and molecular docking studies.

The present research work was designed to examine the neuroprotective effect of ethanolic extract of Solanum virginianum Linn. (SV) in chronic construction injury (CCI) of sciatic nerve-induced neuropathic pain in rats. The extract was initially standardized by high-performance thin-layer chromatography using solasodine as a biomarker and was then subjected to assess the degree of mechanical allodynia, thermal allodynia, mechanical hyperalgesia, thermal hyperalgesia and biochemical evaluations. Administration of SV (100 and 200 mg/kg; p.o.) and pregabalin (10 mg/kg; p.o.) as a reference standard significantly debilitated hyperalgesia and allodynia and notably restored the altered antioxidant level and pro-inflammatory cytokine (IL-1ß and TNF-α) expression in a dose-dependent manner. Further, to appraise the mechanistic approach of solasodine, docking simulation studies were done on the 3D structure of the voltage-gated N-type calcium channel (Cav 2.2), R-type calcium channel (Cav 2.3) and sodium channel (Nav 1.7), and the results revealed that solasodine properly positioned into Phe 19, Leu 32, Met 51 and Met 71 (FLMM pocket) of Cav 2.2 and Cav 2.3 and being a competitor of Ca2+/N-lobe it may inactivate these calcium channels but did not bind into the desired binding pocket of Nav 1.7. Thus, the study confirmed the role of solasodine as a major biomarker for the observed neuroprotective nature of Solanum virginianum.

The Steroidal Glycoalkaloids from Solanaceae: Toxic Effect, Antitumour Activity and Mechanism of Action.

Steroidal glycoalkaloids present in Solanaceae are toxic compounds biosynthesised for the protection of the plants. However, many health benefits of these compounds have been reported so far. One of their promising targets might be cancer, as demonstrated in a large number of studies. However, the main mechanism of action seems to be unclear. It could include the induction of apoptosis or trigger a necrosis with a subsequent inflammatory response. The relatively high systemic toxicity of steroidal compounds is another effect that must be taken into account in anticancer research. The main aim of this work was to summarise the recent progress in the investigation of the mechanisms of their antitumour action and to discuss their potential.

Molluscicidal activity of Solanum elaeagnifolium seeds against Galba truncatula intermediate host of Fasciola hepatica: Identification of ß-solamarine.

CONTEXT: The persistence of fascioliasis in many developing countries urges the search for simple, cheap, and effective substances. In this view, plants provide interesting molluscicidal activities thanks to the secondary metabolites they produce. The genus Solanum is known for its potent effect on vector snails. OBJECTIVE: The molluscicidal activity of Solanum elaeagnifolium Cav. (Solanaceae) seeds against Galba truncatula Müll. (Lymnaeidae), intermediate host of Fasciola hepatica L. (Fasciolidae), was evaluated. MATERIALS AND METHODS: Solanum elaeagnifolium seeds were powdered and successively extracted using n-hexane, methylene chloride, acetone, and methanol, for 20 h each. After filtration, solvents were evaporated. An acid-base treatment was conducted on seed methanolic extract to isolate total alkaloids and ß-solamarine. Total saponins fraction was obtained after successive macerations and evaporations. The molluscicidal activity was evaluated by subjecting snails, in groups of 10, for 48 h to 500 mL of extracts, fractions, and pure product aqueous solutions, each containing amounts, ranging from 1 to 50 mg of plant material in 5 mg increments. RESULTS: The methanolic extract of seeds, ß-solamarine isolated for the first time from this plant and total saponins fraction showed very potent activities on snails, giving respective median lethal concentrations (LC50) of 1.18, 0.49, and 0.94 mg/L. Total alkaloids fraction obtained from the methanolic extract was less active giving an LC50 value of 14.67 mg/L. DISCUSSION AND CONCLUSION: This study emphasizes that glycoalkaloids and saponins of Solanum elaeagnifolium are potent molluscicidal agents. Seed methanolic extract, ß-solamarine, and total saponins fraction may be used as molluscicides.

Natural product solasodine-3-O-ß-D-glucopyranoside inhibits the virulence factors of Candida albicans.

Candida albicans undergoes yeast-to-hyphal transition that has been recognized as a virulence factor as well as the key point for the development of mature biofilm. In this study, we found that a natural product, solasodine-3-O-ß-D-glucopyranoside (SG), a steroidal alkaloid glycoside, isolated from Solanum. nigrum L., could attenuate the virulence of C. albicans by inhibiting the adhesion and morphological transition. Moreover, SG dramatically inhibited the biofilm formation and displayed killing activity against the mature biofilm. In vivo study using Caenorhabditis elegans showed that SG prolonged the survival time of C. albicans infected worms. The mechanism investigation revealed that SG could inhibit the expression of adhesions and hyphae-specific genes by regulating Ras-cAMP-PKA signaling pathway. The inhibitory effects on yeast-to-hyphal conversion and biofilm formation caused by SG could be rescued by addition of exogenerous cAMP, suggesting that the retarded cAMP synthesis is responsible for these actions. Taken together, our work uncovers the underlying mechanism of SG-dependent inhibition of the yeast-to-hyphal switch and biofilm formation and provides a potential application in treating relevant fungal infections.

Steroidal glycoalkaloid profiling and structures of glycoalkaloids in wild tomato fruit.

Steroidal glycoalkaloids (SGAs) constitute one of the main groups of secondary metabolites in tomato fruit. However, the detailed composition of SGAs other than α-tomatine, dehydrotomatine and esculeoside A, remains unclear. Comparative SGA profiling was performed in eight tomato accessions, including wild tomato species by HPLC-Fourier transform ion cyclotron resonance mass spectrometry (HPLC-FTICR/MS). On the basis of molecular formulae obtained from accurate m/z and fragmentation patterns by multistage MS/ MS (MS(n)), 123 glycoalkaloids in total were screened. Detailed MS(n) analysis showed that the observed structural diversity was derived from various chemical modifications, such as glycosylation, acetylation, hydroxylation and isomerization. Total SGA content in each tomato accession was in the range of 121-1986 nmol/gfr.wt. Furthermore, the compositional variety of SGA structures was distinctive in some tomato accessions. While most tomato accessions were basically categorized as α-tomatine-rich or esculeoside A-rich group, other specific SGAs also accumulated at high levels in wild tomato. Here, five such SGAs were isolated and their structures were determined by NMR spectroscopic analysis, indicating three of them were presumably synthesized during α-tomatine metabolism.

Purification, antitumor activity in vitro of steroidal glycoalkaloids from black nightshade (Solanum nigrum L.).

Six steroidal glycoalkaloids (1-6) were isolated and purified from Solanum nigrum L. (SNL) by acid extraction and alkaline precipitation, various chromatographic techniques, and their structures were elucidated by spectroscopic data. Antitumor activity, structure-activity and its molecular mechanism were investigated by methyl thiazolyl tetrazolium (MTT) method, flow cytometry, colorimetric assay and an immunocytochemical method. Experimental results showed that compounds 1 (solasonine), 2 (ß1-solasonine), 3 (solamargine) and 6 (solanigroside P) have cytotoxicity to MGC-803 cells. Compounds with three sugar units and a-l-rhamnopyranose at C-2 or a hydroxyl group on the steroidal backbone may be potential candidates for the treatment of gastric cancer. The mechanism of action may be related to the decrease of mutation p53, the increase of the ratio of Bax to Bcl-2 and the activation of caspase-3 to induce apoptosis.

In vitro leishmanicidal and cytotoxic activities of the glycoalkaloids from Solanum lycocarpum (Solanaceae) fruits.

Leishmaniasis is an infection caused by a protozoan parasite of the genus Leishmania and is the second most prevalent parasitic protozoal disease after malaria in the world. We report the in vitro leishmanicidal activity on promastigote forms of Leishmania amazonensis and cytotoxicity, using LLCMK2 cells, of the glycoalkaloids from the fruits of Solanum lycocarpum, determined by colorimetric methods. The alkaloidic extract was obtained by acid-base extraction; solamargine and solasonine were isolated by silica-gel chromatography, followed by reversed-phase HPLC final purification. The alkaloidic extract, solamargine, solasonine, as well as the equimolar mixture of the glycoalkaloids solamargine and solasonine displayed leishmanicidal activity against promastigote forms of L. amazonensis, whereas the aglycone solasodine was inactive. After 24 and 72 h of incubation, most of the samples showed lower cytotoxicities (IC50 6.5 to 124 µM) as compared to leishmanicidal activity (IC50 1.1 to 23.6 µM). The equimolar mixture solamargine/solasonine was the most active with an IC50 value of 1.1 µM, after 72 h. Likewise, solamargine was the most active after 24 h with an IC50 value of 14.4 µM, both in comparison with the positive control amphotericin B.

Immunomodulatory effect of the alkaloidic extract of Solanum lycocarpum fruits in mice infected with Schistosoma mansoni.

Schistosomiasis is a chronic disease caused by trematode flatworms of the genus Schistosoma; it accounts for more than 280,000 deaths annually. In this work we investigated the effect of the alkaloidic extract obtained by acid-base extraction of the dried fruits of Solanum lycocarpum on schistosomiasis. We used this extract at concentrations of 10, 20, and 40 mg/kg to treat mice infected with Schistosoma mansoni in different phases of the parasite cycle, and we compared its effect with that of the positive control praziquantel (60 mg/kg). We evaluated the results on the basis of the number of macrophages, eggs, and granulomas; we also assessed nitric oxide (NO) and interferon-gamma (IFN-γ) production. Animals treated with a daily dose of 10 or 20 mg/kg alkaloidic extract between the 37th and 41st day of infection showed increased number of macrophages, elevated NO and IFN-γ concentrations, and reduced number of eggs and granulomas in the liver. The alkaloidic extract of S. lycocarpum fruits displayed an immunomodulatory effect on mice infected with S. mansoni, so its potential to treat schistosomiasis deserves further studies.

Complete (1) H and (13) C NMR spectral assignments for the glycoalkaloid dehydrocommersonine.

The normal levels and types of glycoalkaloids found in commercial varieties of potato (Solanum tuberosum) appear to present no hazard to human health. However when wild Solanum species are used in breeding endeavors, new and untested glycoalkaloids may be introduced. Recent studies of domestic crosses with a wild Solanum oplocense accession indicated that the levels of a non-indigenous glycoalkaloid appeared associated with reduced defoliation by the Colorado potato beetle. The non-indigenous glycoalkaloid was isolated from foliage of the wild S. oplocense accession and unambiguously characterized by high-resolution electrospray ionization mass spectrometry and NM analysis as the glycoalkaloid dehydrocommersonine.

Solanum incanum and S. heteracanthum as sources of biologically active steroid glycosides: confirmation of their synonymy.

A new spirostanol saponin (1), along with four known saponins, dioscin (2), protodioscin (3), methyl-protodioscin (4), and indioside D (5), and one known steroid glycoalkaloid solamargine (6) were isolated from the two synonymous species, Solanum incanum and S. heteracanthum. The structure of the new saponin was established as (23S,25R)-spirost-5-en-3ß,23-diol 3-O-{ß-D-xylopyranosyl-(1→2)-O-α-L-rhamnopyranosyl-(1→4)-[O-α-L-rhamnopyranosyl-(1→2)]-ß-D-glucopyranoside}, by using a combination of 1D and 2D NMR techniques including (1)H, (13)C, COSY, TOCSY, NOESY, HSQC and HMBC experiments and by mass spectrometry. The compounds 1, 3, 4 and 5 were evaluated for cytotoxicity against five cancer cell lines and for antioxidant and cytoprotective activity.

Structure elucidation and NMR assignments of new spirosolane alkaloids from Solanum campaniforme.

From the leaves of Solanum campaniforme, two new spirosolane alkaloids ß-acetoxyl-(25S)-22ßN-spirosol-4-en-3-one (1) and ß-hydroxyl-(25S)-22ßN-spirosol-4-en-3-one (4) were isolated along with two other known alkaloids of the same class (25S)-22ßN-spirosol-1,4-dien-3-one (2) and (25S)-22ßN-spirosol-4-en-3-one (3), which are reported for the first time as natural products. The structures of all alkaloids were established after an extensive analysis by 1D and 2D NMR spectroscopy (COSY, HSQC, HMBC and NOESY) as well as HRESIMS.

Evaluation of the schistosomicidal activity of the steroidal alkaloids from Solanum lycocarpum fruits.

Solanum lycocarpum (Solanaceae), a Brazilian medicinal plant known as "wolf fruit," contains about 1.5% of glycoalkaloids in its dried fruits, consisting mainly of solamargine and solasonine. The present work reports the obtainment of the alkaloidic extract of the S. lycocarpum fruit by acid-base extraction and the isolation of the major alkaloid heterosides by chromatographic means, as well as the evaluation of their in vitro schistosomicidal activities. The in vitro schistosomicidal activities of the alkaloidic extract of S. lycocarpum fruits and its isolated steroidal alkaloids were undertaken against adult worms of Schistosoma mansoni. The alkaloidic extract (20, 32, and 50 µg mL(-1)), solasonine (50 µM), solamargine (32 and 50 µM), and equimolar mixture of glycoalkaloids (20, 32, and 50 µM) lead to the separation of all couple worms and extensive disruption on their teguments, such as sloughing, as well as their deaths within 24 h of incubation. In addition, the alkaloidic extract (10 and 15 µg mL(-1)), solasonine (50 µM), solamargine (10, 15, and 20 µM), and equimolar mixtures of glycoalkaloids (10 and 15 µM) reduced the development of eggs produced by the adult worms. Solamargine, containing the sugar chain moiety chacotriose, was more active than the solasonine, which contains solatriose sugar chain moiety. A synergistic effect was also observed for a mixture of solamargine and solasonine. Therefore, the alkaloidic extract of S. lycocarpum, and its major components, solamargine and solasonine, showed promising schistosomicidal activity.

Induction of apoptosis in human hepatoma SMMC-7721 cells by solamargine from Solanum nigrum L.

ETHNOPHARMACOLOGICAL RELEVANCE: Solanum nigrum L. (SNL), a traditional Chinese medicinal herb endowed with diuretic, antipyretic and hepatoprotective effects, has been used as a major ingredient of folk prescriptions for anticancer treatment in China. AIM OF THE STUDY: The purpose of this study was to evaluate the inhibitory effect of solamargine (SM), a major steroidal alkaloid glycoside purified from SNL, on human hepatoma SMMC-7721 cells and investigate the possible mechanism of SM. MATERIALS AND METHODS: The MTT assay was used to evaluate the IC(50) on tumor cell lines. The effect on morphology was observed with a light or fluorescence microscopy. The rate of apoptosis and the cell cycle were measured using flow cytometry (FCM). The expression of caspase-3 protein was measured by colorimetric assay. RESULTS: SM significantly inhibited the growth of SMMC-7721 and HepG2 cells and induced cell apoptosis. Cell cycle analysis revealed that SM caused cell cycle arrest at the G2/M phase. Moreover, SM could up-regulate the expression of caspase-3. CONCLUSIONS: This study indicated that SM exerted potential anticancer activity on SMMC-7721 cells in vitro through the activation of caspase-3 and the regulation of the cell cycle progression to induce apoptosis and inhibit hepatoma cells proliferation.

[Extraction, purification technology and antineoplastic effects of solamargine].

OBJECTIVE: To extract and purify of solamargine from Solanum nigrum, and to research its antineoplastic effects. METHOD: S. nigrum was extracted refluently with 80% alcohol, solamargine was purified with silica gel column chromatography and recrystallization, and then conducted its structure identification and purity checks. Screened the effect on human tumor cell groth inhibition in vitro by MTT assay, and researched on the features in mice with H22 liver cancer or Ehrlich ascites tumor of solamargine. RESULT: The concent of solamargine reached 97.9%. Solamargine had significantly inhibition on 6 tumor cells in vitro, and it had significantly inhibition on mice with H22 liver cancer or ehrlich ascites tumor in the 2.4 mg x kg(-1) dose of i.v. CONCLUSION: Solamargine have the antineoplastic effect.

Anti-inflammatory activity of an alkaloid from Solanum trilobatum on acute and chronic inflammation models.

The solasodine isolated from Solanum trilobatum has been examined for possible anti-inflammatory activity in acute and chronic inflammatory animal models. Solasodine (5, 30 and 75 mg kg⁻¹) exerted statistically significant and dose-dependent anti-inflammatory activity in carrageenan-induced rat paw oedema, which was similar to that of indomethacin (10 mg kg⁻¹), a known anti-inflammatory agent. At the dose of 75 mg kg⁻¹, solasodine inhibited the arachidonic acid-elicited rat paw oedema 1 h after arachidonic acid challenge. Topical application of solasodine significantly inhibited the ear inflammation induced by multiple applications of tetradecanoyl-phorbol 13-acetate. It also suppressed the volume of exudates, total leucocytes and amount of neutrophil migration into the rat pleural cavity. Administration of solasodine at a dose of 75 mg kg⁻¹ significantly inhibited the adjuvant-induced rat paw oedema. These results suggest that solasodine exerts anti-inflammatory activity, at least partly through the inhibition of cyclooxygenase and 5-lipoxygenase pathways.

Anticonvulsant activity of solasodine isolated from Solanum sisymbriifolium fruits in rodents.

CONTEXT: Solanum sisymbriifolium Lam. (Solanaceae), commonly known as sticky nightshade, is traditionally used for central nervous system (CNS) disorders. Although solasodine has been isolated from this plant, little is known about its anticonvulsant and CNS depressant actions. OBJECTIVE: We investigated anticonvulsant and CNS depressant effects of solasodine isolated from S. sisymbriifolium using several experimental models. MATERIALS AND METHODS: Swiss albino mice (n=6) were employed for pentylenetetrazole (PTZ) and picrotoxin (PCT)-induced convulsions and thiopental-induced sleep time. Different groups of Wistar albino rats (n=6) were subjected to maximal electroshock (MES) test. Solasodine, a steroidal glycoalkaloid, was isolated from dried fruits of S. sisymbriifolium and identified by GC-MS. RESULTS: The results showed that intraperitoneal (i.p.) injection of solasodine (25 mg/kg) significantly delayed (p < 0.01) latency of hind limb tonic extensor (HLTE) phase in the PCT-induced convulsions. In the MES model, solasodine significantly reduced (p < 0.001) duration of HLTE at 25, 50, and 100 mg/kg, i.p. in a dose-dependent manner. Interestingly, solasodine did not produce any significant reduction in PTZ-induced convulsions. Prior treatment of solasodine (25, 50, and 100 mg/kg, i.p.) significantly potentiated thiopental-provoked sleep in a dose-dependent manner (p < 0.001). DISCUSSION AND CONCLUSION: Our study, for the first time, shows potent anticonvulsant and CNS depressant activities of solasodine. It is likely that solasodine, in part, is responsible for the anticonvulsant and sedative properties of S. sisymbriifolium. The future study should focus on the exact mechanism of action of solasodine.

Antinociceptive activity of steroid alkaloids isolated from Solanum trilobatum Linn.

Solasodine (1) was isolated for the first time from the roots of Solanum trilobatum Linn., a member of the Solanaceae, and assessed for its presumed antinociceptive activity using several experimental murine models, viz. the writhing, formalin, and hot plate tests. When used at doses of 2, 4, and 8 mg/kg, this steroidal alkaloid caused a significant and dose-dependent decrease in the nociception induced by an intraperitoneal injection of acetic acid (p < 0.001). It also led to a significant reduction of the painful sensation caused by formalin in both phases of the formalin test (p < 0.001). Furthermore, the alkaloid produced a significant increase in the reaction time in the hot plate test (p < 0.001). These results suggest that solasodine elicited antinociceptive activity through both central and peripheral mechanisms.