Discrepancies in the German Pharmacopoeia procedure for quality control of Quillaja saponin extracts.

This study focused on the evaluation of Quillaja saponin extracts with the additional quality designation DAB-which means the abbreviation of the German Pharmacopoeia (Deutsches Arzneibuch). This label suggests that Quillaja saponin extracts marked in this way are of pharmacopoeial quality and thus stand out from other Quillaja saponin extracts. The DAB ninth edition listed Quillaia saponin as a reagent. According to DAB, its quality must be checked by thin-layer chromatography (TLC), and three closely spaced zones in a defined retention factor (Rf) interval specify the saponin reagent. All the Quillaja saponin extracts obtained from different manufacturers and labeled as DAB quality complied with the TLC test. However, the analysis with high-performance liquid chromatography-quadrupole time-of-flight-mass spectrometry (HPLC-Q-ToF-MS) clearly showed additionally an intense peak pattern of Madhuca saponins in all measured samples. The TLC test for Mahua seed cake, which is the press residue from Madhuca longifolia, surprisingly showed the same three closely spaced zones in the defined Rf interval. The three zones could be identified as Mi-saponins from Madhuca after scraping and extracting them from the stationary phase of the TLC plate and subsequent measurement by HPLC-Q-ToF-MS. Therefore, the specification of the saponin reagent in DAB characterizes erroneously Madhuca saponins that are not listed as a saponin plant source for the saponin reagent.

Pentahydroxy flavonoid isolated from Madhuca indica ameliorated adjuvant-induced arthritis via modulation of inflammatory pathways.

Rheumatoid arthritis (RA) is an autoimmune disease associated with advanced joint dysfunction. Madhuca indica J. F. Gmel, from the family Sapotaceae, is an Indian medicinal plant reported to have an array of pharmacological properties. The aim of present investigation was to determine the anti-arthritic potential of an isolated phytoconstituent from methanolic leaf extract of Madhuca indica (MI-ALC) against FCA-induced experimental arthritis. Polyarthritis was induced in female rats (strain: Wistar) via an intradermal injection of FCA (0.1 mL) into the tail. Polyarthritis developed after 32 days of FCA administration. Then rats were treated orally with an isolated phytoconstituent from MI-ALC at doses of 5, 10, and 20 mg/kg. Findings suggested that High-Performance Thin-Layer Chromatography, Fourier-Transform Infrared Spectroscopy, and Liquid Chromatography-Mass Spectrometry spectral analyses of the phytoconstituent isolated from MI-ALC confirmed the structure as 3,5,7,3',4'-Pentahydroxy flavone (i.e., QTN). Treatment with QTN (10 and 20 mg/kg) showed significant (p < 0.05) inhibition of increased joint diameter, paw volume, paw withdrawal threshold, and latency. The elevated synovial oxidative stress (Superoxide dismutase, reduced glutathione, and malondialdehyde) and protein levels of Tumor necrosis factor-α (TNF-α) and Interleukin (ILs) were markedly (p < 0.05) reduced by QTN. It also effectively (p < 0.05) ameliorated cyclooxygenase-2 (COX-2), Nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-kß) and its inhibitor-α (Ikßα), and ATP-activated P2 purinergic receptors (P2X7) protein expressions as determined by western blot analysis. In conclusion, QTN ameliorates FCA-induced hyperalgesia through modulation of elevated inflammatory release (NF-kß, Ikßα, P2X7, and COX-2), oxido-nitrosative stress, and pro-inflammatory cytokines (ILs and TNF-α) in experimental rats.

Ameliorative activity of aqueous leaf extract from Madhuca longifolia against diclofenac-administered toxicity on rat stomach and intestine.

Madhuca longifolia, a tropical tree used as medicine and food, is known to have a beneficial effect against stomach gastric toxicity. Madhuca longifolia is used in treating cough, skin disease and nerve disorders. Diclofenac, a non-steroidal anti-inflammatory drug (NSAID), with overdosage and prolonged use, is known to cause gastric toxicity. Silymarin (SLY), a polyphenolic antioxidant flavonoid, is a derivative of Silybum marianum extracted from milk thistle seeds and fruits, has been widely used in the treatment of gastric ulcer. SLY was used as the standard drug to compare the effects with the Madhuca longifolia aqueous leaf extract treatment. The aim of the current study is to understand the effect of Madhuca longifolia aq. leaf extract on rat stomach and intestine against diclofenac-administered toxicity. Rats (n = 30) were divided into Group I normal control, Group II treated with diclofenac, Group III treated with diclofenac and Madhuca longifolia leaf extract, Group IV treated with diclofenac and silymarin, and Group V was treated with Madhuca longifolia leaf extract alone. After the study duration, rats were euthanized and tissue samples were analyzed for antioxidant, cytokine, protein expression levels and histopathological changes. Diclofenac treated rats had significant (p < 0.05) changes in levels of antioxidants, cytokines, protein expression and pathological changes as compared to rats treated with Madhuca longifolia. This study demonstrated that Madhuca longifolia leaf extract had gastroprotective activity in rats treated with diclofenac.

Isolation, process optimisation and characterisation of the protein from the de-oiled cake flour of Madhuca latifolia.

This work reports the isolation of the protein from the flour of an underutilised agro waste, a de-oiled cake of Madhuca latifolia using the bis (2-ethylehexyl) sodium sulfosuccinate salt reverse micelle and the characterisation of the protein through various techniques. The experimental conditions for the extraction were optimised using Box-Behnken design. The highest yield of the protein was achieved when the extraction parameters, i.e. KCl concentration, KCl amount, and pH of the medium, were 0.5 M, 1.25 ml, and 9.02, respectively. The experimental yield (75.56%) obtained under the optimised conditions matched extremely well with the predicted yield (75.19%). The analysis of the biochemical composition envisaged the occurrence of 2S albumin, 7S globulin, and 11S globulin as the major components in the protein. The X-ray diffraction pattern supported the ß-sheets structure of the protein. The imaging of the protein through a scanning electron microscope revealed the shape and surface of the protein to be spherical and smooth, respectively. Thus, the protein isolate of the de-oiled cake flour of Madhuca latifolia could be utilised towards food product development and relevant fields.

Madhuca indica Inhibits Breast Cancer Cell Proliferation by Modulating COX-2 Expression.

BACKGROUND: Madhuca indica belongs to the family sapotaceae, commonly known as Mahua. It is primarily known for alcoholic beverage production and is reported to have anti-inflammatory, analgesic and antipyretic properties. Madhuca indica has also been reported to be effective in several diseases. OBJECTIVE: This study was undertaken to check the anticancer efficacy and chemopreventive effect of methanolic extract of Mahua flower (ME) on human breast cancer cell lines MCF-7 and MDA-MB-468. METHOD: The cytotoxic and anti-proliferative effects on MCF-7 and MDA-MB-468 cells were studied by MTT, hexosaminidase and colony formation assay. Expression of caspase 3/7 was assessed by flow cytometry and western blot analysis. Expression of COX-2 was evaluated by western blot analysis, luciferase assay and mRNA analysis. RESULTS: ME inhibited the proliferation of breast cancer cells by inducing apoptosis through up-regulating the expression of Caspase 3/7 (P < 0.0001). Our results showed a decrease in the expression of COX-2 mRNA and COX-2 protein in both MCF-7 and MDA-MB-468 cells with an increase in ME concentration. Furthermore synergistic effect of ME and chemotherapeutic drug paclitaxel was also studied in MCF-7 and MDA-MB- 468 cells which were found to be more effective (P < 0.0001) than treatment of either ME or paclitaxel alone. Results were analyzed by ANOVA and Pearson correlation analysis. CONCLUSION: All these experiments suggest that ME inhibits breast cancer cell proliferation and apoptosis by inhibiting the expression of COX-2 in MCF-7 and MDAMB- 468 cells. This work further highlighted that ME may enhance the potentiality of paclitaxel in breast cancer treatment.

Potential anti-proliferative activity of AgNPs synthesized using M. longifolia in 4T1 cell line through ROS generation and cell membrane damage.

To overcome the problem of breast cancer, silver nanoparticles (AgNPs) synthesized using Indian medicinal plant Madhuca longifolia could be explored as an alternative anticancer medicine. Synthesized AgNPs were studied their characteristics and their anti-proliferative property was investigated in breast cancer cell line (4T1). Based on zeta sizer analysis, the size of the AgNPs was 103.5 nm and potential -9.57 eV. Fe-SEM results showed particle size of 69.4-99.4 nm while TEM images indicated the particle size of 18-24 nm. In dose-dependent study, AgNPs showed 93% of anti-proliferative activity at 50 µg/ml whereas the methanolic extract of M. longifolia showed 80% activity only at 10-fold increased concentration (500 µg/ml). AgNPs exhibited higher level of cytotoxicity in breast cancer cell line than extract through cell wall degradation and ROS generation. Such effective AgNPs could be investigated further through in vivo models with a view to develop anticancer drug.

Antibacterial potential of silver nanoparticles synthesized using Madhuca longifolia flower extract as a green resource.

The green and one-step synthesis of silver nanoparticles (AgNPs) has been proposed as simple and ecofriendly. In the present study, a flower extract of Madhuca longifolia was used for the reduction of silver nitrate into AgNPs, with phytochemicals from the flower extract as a reducing and stabilizing agents. The synthesized AgNPs were spherical and oval shaped and about 30-50 nm sizes. The appearance of a brown color in the reaction mixture is a primary indication of AgNPs formation, and it was confirmed by observing UV-visible spectroscopy peak at 436 nm. The Energy Dispersive X-ray spectra and X-ray diffraction analysis results together confirm that the synthesized nanoparticles contain silver and silver chloride nanoparticles. The Zeta potential analysis indicates presence of negative charges on synthesized AgNPs. The FT-IR study represents involvement of functional groups in AgNPs synthesis. Synthesized AgNPs shows potential antibacterial activity against Gram-positive and Gram-negative pathogens. M. longifolia flower is a good source for AgNPs synthesis and synthesized AgNPs are applicable as antibacterial agent in therapeutics.

Aqueous leaves extract of Madhuca longifolia attenuate diclofenac-induced hepatotoxicity: Impact on oxidative stress, inflammation, and cytokines.

Diclofenac is a Non-Steroidal Anti-inflammatory drug which is used as an analgesic. It is known to cause heptotoxicity on over dose and long term usage. Madhuca longifolia is an evergreen tree found widely in India that is known to have several ethnomedical uses. The aim of our study is to evaluate the beneficial effect of the aqueous leaf extract of M. longifolia against diclofenac-induced toxicity. Rats were dived into five groups of six rats each. Group-I was normal control. Group-II was administered with diclofenac (50 mg/kg. b.w./day, i.p) on 4th and 5th day. Group-III rats were treated with aqueous leaf extract of M. longifolia (500 mg/kg b.w./day, oral) for 5 consecutive days and diclofenac (50 mg/kg. b.w./day, i.p) was given on 4th and 5th day. Silymarin (25 mg/kg. b.w./day, oral) was used as standard drug which was given to the rats of group-IV along with diclofenac on 4th and 5th day. Aqueous leaf extract of M. longifolia (500 mg/kg b.w./day, oral) alone was administered in group-V. After the study period, the rats were evaluated for liver enzyme markers, antioxidant parameters, histopathological changes, and cytokines levels. The hepatic proinflammatory mediator cytokines like TNF-α, IL-6, and IL-1ß were evaluated through ELISA. The protein expression of Caspase-3, COX-2, and NF-κB were analysed through Western blotting techniques. Aqueous leaves extract of M. longifolia was able to normalize the changes caused by diclofenac. Current study indicatesthe protective effect of the aqueous leaves extract of M. longifolia against diclofenac-induced toxicity.

Diclofenac-induced renal toxicity in female Wistar albino rats is protected by the pre-treatment of aqueous leaves extract of Madhuca longifolia through suppression of inflammation, oxidative stress and cytokine formation.

CONTEXT: Kidney has a vital role in renal clearance, maintenance of blood pressure, elimination of toxic products and formation of prostaglandins. Certain medications are known to cause renal injury on its frequent usage and high dosage. Diclofenac is a non-steroidal anti-inflammatory drug which is used in the treatment of pain and arthritis. Madhuca longifolia is a deciduous tree which is known to the have anti-microbial, anti-ulcer, hepatoprotective, anti-diabetic, anti-inflammatory and analgesic activity. The aim of the present study is to evaluate the beneficial effect of aqueous leaf extract of Madhuca longifolia against DFC-induced renal toxicity in female Wistar albino rats. METHODS: Thirty female Wistar albino rats were divided into five groups and the drugs were administrated specifically on each group. After the treatment period, the rats were sacrificed to evaluate the significant changes in renal enzyme markers, antioxidant activities in kidney tissue homogenate and plasma, renal histopathology and protein expression levels. The cytokines like TNF-α, IL-6 and IL-1ß were measured through ELISA techniques and the levels of Caspase-3, COX-2 and NF-κB were measured through western blotting techniques. DiscussionMadhuca longifolia was observed to show a better result in normalizing the toxicity caused by diclofenac. CONCLUSION: The significant result of the aqueous leaf extract ofMadhuca longifolia was due to its ability in restoring renal function by restoring antioxidants and preventing cellular damages.

Elucidation of protective efficacy of Pentahydroxy flavone isolated from Madhuca indica against arsenite-induced cardiomyopathy: Role of Nrf-2, PPAR-γ, c-fos and c-jun.

BACKGROUND: Madhuca indica J. F. Gmel. (Sapotaceae) is widely used ethnobotanically as anti-diabetic, antipyretic, hepatoprotective, anti-inflammatory and analgesic. It was shown to possess potent anti-apoptotic property. THE AIM OF THE STUDY: To evaluate the possible mechanism of action of isolated phytoconstituent from Madhuca indica Leaves methanolic extract (MI-ALC) on arsenic-induced cardiotoxicity in rats. MATERIALS AND METHODS: The 3,5,7,3',4'-Pentahydroxy flavone (QTN) was isolated and characterized by using HPTLC, 1H NMR, and LC-MS from MI-ALC. QTN (5, 10 and 20mg/kg, p.o.) was administered in arsenic intoxicated rats (5mL/kg, p.o.) for 28days and evaluated for various behavioral, biochemical, molecular and ultra-histological changes. RESULTS: Treatment with QTN (10 and 20mg/kg, p.o.) significantly inhibited (p<0.05) arsenic-induced electrocardiographic, hemodynamic and left ventricular function alterations. Elevated levels of cardiac markers (LDH, CK-MB, AST, ALT, and ALP), altered lipid metabolism (total cholesterol, triglyceride, LDL, HDL, and VLDL) was significantly restored (p<0.05) by QTN. It also significantly inhibited (p<0.05) altered cardiac oxido-nitrosative stress, Na-K-ATPase level and mitochondrial enzymes (I-IV) activity after arsenite administration. QTN significantly increased (p<0.05) myocardial Nrf-2, PPAR-γ and significantly decreased (p<0.05) myocardial c-fos and c-jun mRNA expressions. Flow cytometric analysis showed that treatment with QTN (10 and 20mg/kg) significantly inhibited (p<0.05) arsenite-induce ROS and apoptosis. It also reduced ultra-histological aberrations induced by sodium arsenite. CONCLUSION: Administration of 3,5,7,3',4'- Pentahydroxy flavone (i.e. Quercetin (QTN)) isolated from MI-ALC showed significant protection against arsenic-induced oxido-nitrosative stress and myocardial injury via modulation of Nrf2, PPAR-γ, and apoptosis.

Isolation of a New Homomonoterpene from Madhuca pasquieri and Effect of Isolated Compounds on NO Production.

A new homomonoterpene, 1,3,3-trimethyl-7-oxabicyclo[3.1.1]hexa-9-en-10-oic acid, named madhusic acid A (1), together with ten known compounds (2-11) were isolated from the methanolic extract of the dried leaves of Madhuca pasquieri (Dubard) H. J. Lam. The structure of the new compound was elucidated on the basis of 1D, 2D NMR (COSY, HMQC, and HMBC) and mass spectral analyses. We examined the effects of the isolated compounds against LPS-induced NO production in macrophage RAW264.7 cells and compound 2 showed effective activity with an IC50 value of 14.5 µM.

Anti-inflammatory Activity of Pyrrolizidine Alkaloids from the Leaves of Madhuca pasquieri (Dubard).

A novel pyrrolizidine alkaloids, madhumidine A (1), and two known alkaloids, lindelofidine benzoic acid ester (2) and minalobine B (3) were isolated from the leaves of Madhuca pasquieri (Dubard) H. J. LAM. The chemical structures of these alkaloids were established mainly by NMR techniques and mass spectrometry. Their anti-inflammatory activity was evaluated against lipopolysaccharide-induced nitric oxide production in macrophage RAW264.7 cell. In addition, the cytotoxic activity of all isolated compounds was tested against a panel of cancer cell lines.

Inhibition of Hep G2 hepatic cancer cell growth and CCl4 induced liver cytotoxicity in Swiss albino mice by Mahua extract.

Mahua flower extract may provide protective effects against hepatotoxicity. The effect of Mahua flower extract (ME) was investigated on Hep G2 cell line and carbon tetrachloride (CCl4)-induced liver damages in Swiss albino mice. To investigate its cytotoxic effect in liver cancer, Hep G2 cells were treated with different doses of ME, and cell proliferation as well as colony formation assays demonstrated dose-dependent cytotoxicity of ME towards Hep G2 cells in tissue culture. Further gene expression studies showed significant down-regulation of AKT1/2/3, p-AKT, and COX-2 proteins including up-regulation of active caspase-3 in ME treated Hep G2 cells. In in vivo experiments, the mice were pretreated with ME for 15 days. On the 16th day CCl4 was injected intraperitoneally and after 24 h all mice were sacrificed. The antioxidant enzyme activities were measured in liver homogenates. CCl4-induced hepatotoxicity was evidenced by significant increase in lipid peroxidation and decrease in activities of antioxidant enzymes such as GST, GSH, SOD, CAT, and GPx. Histological studies showed CCl4-induced centrilobular necrosis and formation of fatty vacuoles in cirrhotic mice liver. Treatment with ME at a dose of 2 mg and 4 mg/kg exhibited the potential to prevent significant liver toxicity. The expression of active caspase-3 protein was down-regulated in ME treated groups compared to CCl4 exposed animals. This study demonstrated ME mediated antioxidant activity and hepatoprotective effects; therefore it could be used in the future for treating hepatic disorders including liver cancer, especially in combination with chemotherapeutics.

Negative effects of a nonhost proteinase inhibitor of ~19.8 kDa from Madhuca indica seeds on developmental physiology of Helicoverpa armigera (Hübner).

An affinity purified trypsin inhibitor from the seed flour extracts of Madhuca indica (MiTI) on denaturing polyacrylamide gel electrophoresis showed that MiTI consisted of a single polypeptide chain with molecular mass of ~19.8 kDa. MiTI inhibited the total proteolytic and trypsin-like activities of the midgut proteinases of Helicoverpa armigera larvae by 87.51% and 76.12%, respectively, at concentration of 5 µg/mL with an IC50 of 1.75 µg/mL against trypsin like midgut proteinases. The enzyme kinetic studies demonstrated that MiTI is a competitive inhibitor with a K i value of 4.1 × 10(-10) M for Helicoverpa trypsin like midgut proteinases. In vivo experiments with different concentrations of MiTI in artificial diet (0.5, 1.0, and 1.5% w/w) showed an effective downfall in the larval body weight and an increase in larval mortality. The concentration of MiTI in the artificial diet to cause 50% mortality (LD50) of larvae was 1.5% w/w and that to cause reduction in mass of larvae by 50% (ED50) was 1.0% w/w. Nutritional indices observations suggest the toxic and adverse effects of MiTI on the growth and development of H. armigera larvae. The results suggest a strong bioinsecticidal potential of affinity purified MiTI which can be exploited in insect pest management of crop plants.

Composition and thermal analysis of binary mixtures of mee fat and palm stearin.

Seed fat of Madhuca longifolia known as mee fat (MF) has been considered as a potential plant fat for producing fat mixture to simulate the properties of lard. A study was carried out to evaluate the effect of addition of palm stearin (PS) on the solidification behavior of MF to formulate a mixture to become similar in solidification characteristics of lard. Three fat mixtures were prepared by blending MF with palm stearin PS in different ratios: MF:PS (99.5:0.5), MF:PS (99:1), MF:PS (98:2) (w/w), and identified by the mass ratio of MF to PS. The fat mixtures were compared with lard in terms of their fatty acid and triacylglycerol compositions, differential scanning calorimetric (DSC) thermal profiles and solid fat content (SFC) characteristics. Results showed that there were considerable differences between lard and MF:PS fat mixtures with regard to fatty acid and triacylglycerol compositions. The increasing proportion of PS in MF:PS fat mixtures caused a general increase in SFC at different temperatures with respect to the SFC profile of native MF. Of the three binary mixtures, MF:PS (99:1) was found to show the least difference to lard in terms of SFC values throughout the temperature range.

Yield and nutritional content of Pleurotus sajor caju on wheat straw supplemented with raw and detoxified mahua cake.

The effect of supplementation of wheat straw (WS) with raw/detoxified mahua cake (MC) on yield and nutritional quality of Pleurotus sajor caju was studied. Raw cake significantly enhanced the yield compared to control and could be tolerated up to a 10% addition. Detoxification further improved the mushroom yield giving a maximum of 1024.7 g kg(-1) from WS supplemented with 20% saponin free detoxified mahua cake. Chemical analysis of fruit bodies revealed that they are rich in proteins (27.4-34.8%), soluble sugars (28.6-32.2%) and minerals. Glucose, trehalose and glutamic acid, alanine were the major sugars and amino acids detected by HPLC analysis, respectively. HPLC studies further confirmed the absence of saponins (characteristic toxins present in MC) in both fruit bodies and spent. Degradation of complex molecules in spent was monitored via FTIR. The study proved beneficial for effective management of agricultural wastes along with production of nutrient rich and saponin free fruit bodies/spent.

Novel application of Mahua (Madhuca sp.) flowers for augmented protease production from Aeromonas sp. S1.

The present study explored the utilization of Mahua (Madhuca sp.) flowers, a major non-timber forest product (NTFP) of India, as a low-cost, natural substrate for protease production under submerged fermentation. Bacterial strain Aeromonas sp. Si1, previously reported by us, was used as the protease producer. Using Mahua flower extract (MFE) as the medium additive, the protease production could successfully be enhanced by 5.6-fold (564.5 UmL-1) after 24 h of fermentation under optimized conditions compared with initial production of 99.9 UmL' in the absence of MFE. The cultural parameters for optimum production of protease were determined to be: incubation time-24 h; pH-7.0; MFE concentration-5% (v/v); inoculum size-0.3% (v/v) and agitation rate-200 rpm. The results obtained demonstrate the potential of cheaper and abundantly available Mahua flowers for induction of proteases, and thus offer a new approach for value addition to this biomass through industrial enzyme production.

Neuropharmcological potential of methanolic extract and a triterpene isolated from Madhuca longifolia L leaves in mice.

The methanolic extract of M. longifolia (MLME) and a compound a triterpene, derivative of madhucic acid (dMA) isolated from the leaves of M. longifolia, were investigated for their possible neuropharmacological activities in mice using phenobarbitone induced sleeping time, spontaneous motor activity, marble burying test and Eddy's hot plate method. LD50 for MLME and dMA were 100 and 10 mg/kg of body weight, respectively. Both MLME and dMA (10 mg/kg and 2 mg/kg oral route respectively) exhibited significant increase in phenobarbitone induced sleeping time, greater reduction in spontaneous motor activity and marble burying activity, confirming their sedative nature. Both MLME and dMA also exhibited considerable antinociceptive activity in experimental animals. The results suggest that both MLME and dMA have CNS depressant activity in mice.

Biosynthesis of anisotropic gold nanoparticles using Maduca longifolia extract and their potential in infrared absorption.

Metal nanoparticles, in general, and gold nanoparticles, in particular, are very attractive because of their size- and shape-dependent properties. Biosynthesis of anisotropic gold nanoparticles using aqueous extract of Madhuca longifolia and their potential as IR blockers has been demonstrated. The tyrosine residue was identified as the active functional group for gold ion reduction. These gold nanoparticles were characterized by of UV-Vis spectrophotometer, FTIR, TEM and HrTEM. The presence of proteins was identified by FTIR, SDS-PAGE, UV-Vis and fluorescence spectroscopy. The micrograph revealed the formation of anisotropic gold nanoaprticles. The biologically synthesized gold nanotriangles can be easily coated in the glass windows which are highly efficient in absorbing IR radiations.

Arbutin derivatives from the seeds of Madhuca latifolia.

A new arbutin derivative, madhuglucoside (1), along with three known arbutin derivatives were isolated from the seeds of Madhuca latifolia in addition to seven other known constituents. Their structures were established on the basis of spectral analysis. Compounds 1a, 2a and 3a were obtained in a pure state after acetylation of the mother fraction and characterized as their acetyl derivatives.