Asoca Bark Research Articles

Antiproliferative Effect of Methanolic Extract of Saraca asoca bark and its Possible Targets of Action

Background: The use of phytochemicals as anticancer drugs has gained attention in scientific and industrial approaches. In this context, the present study was undertaken to determine the antiproliferative effect of methanolic extract of Saraca asoca bark in the C127I cell line and its possible targets of action by in silico analysis. Method: Methanolic extract of S. asoca bark was assessed for its cytotoxicity in the C127I cell line by 3-(4,5-dimethyl thazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay at concentrations of 320, 160, 80, 40 20 and 10 µg/mL and the half maximal inhibitory concentration (IC50) was calculated using Graph Pad Prism 5.0. The cells were seeded in 6 well plates at a concentration were treated for 24 hours with extract of S. asoca bark at IC50 concentration. The cells were trypsinised and subjected to Acridine orange - Ethidium bromide staining (AOEB) staining for morphological evaluation of apoptosis. Fourier transform infrared (FTIR) spectroscopic analysis was performed to identify the chemical nature of the extract. In silico analysis was done to assess the affinity of various phytochemicals in the extract towards Caspase and BCl2 proteins. Results: Dose-dependent reduction in cell viability was noticed when the cells were subjected to different concentrations of the extract and the IC50 value of S. asoca was found to be 16.55 µg/mL. AO/EB staining detected proliferating cells with green fluorescence in the control cells whereas the cells with S.asoca extract showed a dose-dependent shift from orange to red fluorescence indicating apoptosis in treated cells. Ellagic acid present in the extract was found to have a maximum affinity towards Bcl2 and Caspase proteins. Conclusions: From the study, it could be concluded that the methanolic extract of Saraca asoca was found to possess an antiproliferative effect.

Therapeutic exploration of polyherbal formulation against letrozole induced PCOS rats: A mechanistic approach

ObjectiveThis study aimed to develop an effective alternative medicine with multi potential herbs against polycystic ovarian syndrome (PCOS) in rats induced by letrozole treatment. Materials and methodPolyherbal syrup was prepared with a combination of S. asoca bark, G. sylvestre leaves, P. daemia aerial parts, C. zeylanium stem bark, C. bonduc seeds, and W. somnifera roots ethanolic extract. In vitro cell viability study, adenosine monophosphate-activated protein kinase (AMPK), and glucose transporter 4 (GLUT4) gene expression assay were carried out on the Chinese Hamster Ovarian (CHO) cell line. For the PCOS induction letrozole (1 mg/kg p. o.) was given for 21 consecutive days. The PCOS induction was confirmed by measuring estrus irregularity, insulin resistance by oral glucose tolerance test (OGTT), and hyperandrogenism by measuring serum total testosterone level 21 days after completion of letrozole treatment. After induction of PCOS, metformin (155 mg/kg p. o.), and polyherbal syrup (100 mg/kg, 200 mg/kg, and 400 mg/kg p. o.) were administered for further 28 days. The treatment efficacy was measured by measuring serum lipid profile, fasting insulin level, sex hormones level, ovarian steroidogenic enzymes, ovarian tissue insulin receptor, AMPK, and GLUT4 protein expression levels, and histomorphological studies. The post-treatment effect was confirmed by reproductive performance studies. ResultsLetrozole-induced PCOS rats showed significant estrus irregularity, abnormal sex hormones levels, and hyperandrogenism indicated by showing increased free androgenic index and decreased sex hormones binding globulin (SHBG) level. The insulin resistance in PCOS rats was indicated by increased fasting glucose levels with impaired glucose clearance in the OGT test. Homeostasis Model Assessment Index of Insulin Resistance (HOMA-IR) increased level, also decreases INSR, GLUT4, and AMPK mRNA expression in ovarian cells confirming the insulin resistance in PCOS rats. Ovarian histology in PCOS rats also showed many follicular cysts, atretic follicles, and the absence of corpus luteum. The administration of polyherbal syrup, in a dose-dependent manner, effectively restored these alterations. The treatment of polyherbal formulation 400 mg/kg possesses highly significant efficacy over the treatment of metformin in PCOS rats. It mainly acts by reducing peripheral and ovarian hyperandrogenism and improves insulin sensitivity via activating the insulin receptor and AMP-activated kinase-mediated transcription and translation of GLUT4 from the cytoplasm to the ovarian membrane improves glucose uptake and promotes the follicular development and ovulation. The higher fertility rate, delivery index, and survival of delivered pups confirm the broader and superior efficacy of PCOS. These beneficial actions are mainly attributable to the formulation's inclusion of the key secondary metabolites flavonoids and phytosterols. In conclusion, the prepared polyherbal syrup was found to be the safest and most effective alternative medicine for both endocrinal and metabolic complications of PCOS women.

ANTI-ULCER ACTIVITY OF HYDRO-ALCOHOLIC EXTRACT OF SARACA ASOCA STEM BARK IN RATS

In the present study effort has been made to establish the antiulcer activity of Saraca asoca (Roxb.), De Wild (family-Caesalpinaceae) stem bark hydroalcoholic-extract using ethanol induced ulceration model in albino rats, because no systematic study has been reported for its antiulcer activity. The effect of hydro-alcoholic extract (60:40) of S. asoca bark was investigated by carrying out an acute toxicity study in albino rats to evaluate the antiulcerogenic activity by using ethanol induced gastric ulcer model compared with omeprazole as standard drug. The preliminary photochemical screening of this medicinal plant identified the presence of important secondary metabolites like flavonoids and tannins. Acute toxicity study did not manifest any toxicological signs in rats. Oral administration of absolute ethanol (99.5 %, 5 mL kg -1, p.o.) produced significant ulcers index (41.00 ± 1.75) in the control group. Standard drug omeprazole (40 mL kg -1, p.o.) produced ulcer index (12.17 ± 0.74). Ulcer index (24.17 ± 1.40) is significantly (p<0.001) protected by hydro-alcoholic extract of S. asoca (stem bark) at a dose of 200 mL kg -1, p.o. The present finding suggests that hydro-alcoholic extract of S. asoca (stem bark) promotes ulcer protection activity.

Evaluation of Anti-quorum Sensing Potential of Saraca asoca (Family Caesalpiniaceae) against Chromobacterium violaceum and Pseudomonas aeruginosa PA01

Aim: The present study was performed to evaluate the anti-quorum sensing (QS) potential of traditional medicinal herb Saracaasoca (family Caesalpiniaceae) stem bark extract against Chromobacterium violaceum and Pseudomonas aeruginosa PA01.
 Study Design: First, the test sample (bark extract) was screened for anti-QS activity. Then systematic in-vitro and biochemical tests were performed to evaluate the effect of the test sample on the QS mediated virulence factors.
 Place and Duration of Study: All the experimental works were performed in Lab 311, pharmacology division, CSIR-NBRI Lucknow from June 2019 to October 2019.
 Methodology: The samples of Saraca asoca stem bark were washed, dried and extracted using 70% methanol. The minimum inhibitory concentration (MIC) of the prepared Sarca asoca bark extract was determined using the Alamar blue assay, and the anti-QS activity was screened using standard agar overlay method against CV 12472 at subinhibitory concentrations 100, 200 and 300 µg (< MIC value). SAE effect on biofilms formation was assessed by growing biofilms on glass slides in a static culture of PA01. Anti-virulence effect of SAE on the production of QS-regulated virulence factors such as Pyocyanin, proteases, elastases, rhamnolipid and alginate in Pseudomonas aeruginosa was determined using the supernatant of a 24 hours old broth culture of PA01 supplemented with SAE. Using the agar plate technique, the swimming and swarming motility assays were conducted on 0.3% and 0.5% agar plates respectively. One-way ANOVA was used to analyze the data, presented as mean ± SD (standard deviation) of three independent experiments.
 Results: Preliminary screening results showed significant QS inhibition against CV 12472 in an agar overlay disk diffusion assay in a concentration-dependent manner. Data from the biofilm assay showed loose, distorted, irregular PA01 biofilm formation at 200 µg (48%) and 300 µg (65%). SAE caused a significant drop in virulence factor production, with maximum reduction in pyocyanin (58%), proteases (67%), elastases (52%), rhamnolipid (53%), and alginate (44%) observed at 300 µg concentration. At SAE sub-lethal concentrations (200 and 300 µg), both the swimming and swarming motility of PA01 were significantly inhibited.
 Conclusions: The present study demonstrates the broad-spectrum anti-QS potential of SAE, reported for the first time, suggesting that SAE could be considered as an alternative herbal source to develop antimicrobial agents which can be either solitary or synergistically with conventional antimicrobial drugs.

Chemically characterised extract of Saraca asoca improves the sexual function in male Wistar rats.

In this study, methanolic extract of Saraca asoca bark was evaluated for its aphrodisiac potential using male and female Wistar albino rats. Male rats were dosed daily for 54days at a dose of 100mg/kg p.o. Sexual activity of male rats was assessed after 14, 28, 42 and 54days of the study. Male rats were placed in a glass chamber lit with a dim red light (10W) followed by the introduction of sexually receptive female rats in a ratio of 1:1. Improvement in sexual behaviour of male rats was characterised by an increase in both mount frequency and intromission frequency and decrease or reduction in mount latency and intromission latency compared to normal control. After completion of the study, the effect of the S.asoca extract on sperm count, sperm motility and sperm morphology was also assessed. The extract of S.asoca bark was found to be safe as it did not affect these sperm parameters. From this study, it was found that methanolic extract of S.asoca bark plays a role in enhancing sexual behaviour and potential without causing reproductive toxicity.

Evaluation of antidepressant activity of methanolic extract of Saraca asoca bark in a chronic unpredictable mild stress model.

It is well established that chronic exposure to stressful events plays an important role in the etiology of depression. Saraca asoca (Roxb.), De. wild, or Saraca indica, belonging to family Fabaceae, is endogenous to India. The flowers, seeds, bark, and leaves of the plant have been used widely in Ayurveda medicine. The bark extract of S. asoca has shown chemoprotection, myeloprotection, and antioxidant potential. Owing to the above-mentioned properties of the plant, the present study sought to evaluate the effect of a methanolic extract of S. asoca bark in rats exposed to chronic unpredictable mild stress (CUMS) daily for 8 weeks using a forced-swim test, an open-field test, and a sucrose-preference test. The effect of the extract on endogenous antioxidant levels in the brain was also assessed using catalase activity, superoxide dismutase activity, reduced glutathione levels, and malondialdehyde levels in the brain. Male Sprague-Dawley rats received 100 mg/kg (oral) of the extract daily 1 h before daily stress exposure for 8 weeks. The extract showed a significant reduction in the immobility time in the forced-swim test, increased the total number of line crossing, rearing, and grooming in the open-field test, and increased the sucrose consumption as well as the levels of endogenous antioxidants significantly in comparison with the CUMS control group. Therefore, S. asoca might be a useful agent for the treatment or alleviation of symptoms associated with depression possibly by reducing CUMS-induced oxidative stress and reactive oxygen species in the brain.

Tyrosinase Inhibitory Effects of Saraca Asoca Bark, Leaf and Seed

Tyrosinase Inhibitory Effects of Saraca Asoca Bark, Leaf and Seed

Extraction, quantification and antioxidant activities of flavonoids, polyphenols and pinitol from wild and cultivated Saraca asoca bark using RP-HPLC-PDA-RI method

Saraca asoca (Roxb.) De Wilde Syn. Saraca indica Linn belonging to the family Leguminosae, is one of the ancient medicinal plant of India and its bark is used in Ayurvedic preparations. In the present work, the barks were collected from mature trees of different geographical locations. Besides, one of the best chemotype is cultivated under different fertilizer treatments and the barks from 4year old plants were taken up for analytical studies. The barks were extracted with methanol as well as aqueous-methanol solvent systems, and chemical compositions were compared using RP-HPLC-PDA-RI analysis. Different classes of compounds viz. pinitol, flavonols and polyphenols were detected through the analytical protocol developed by our laboratory. It was found that the barks collected from Uttar Pradesh (BLA, BLK), Sirsi, Karnataka (BSS) and Empress Garden, Pune (BEG) gave higher yield of the extracts and were more enriched with the above classes of compounds. For getting barks in a short period of time, the BSS plant saplings were experimentally cultivated under different fertilizer treatments. It was found that the plants treated with a combination of fertilizers such as poultry waste+biofertlizer, vermicompost+biofertlizer and farm yard manure+biofertlizer+diammonium phosphate yielded reasonably higher amounts of extracts, which were chemically close with the parent bark extracts. Among these treatments, the vermicompost+biofertlizer gave pinitol (729mg), flavonoids (2872mg) and polyphenols (1150mg) in 100g bark extracts. The antioxidant activities were evaluated by using 1,1-diphenyl-2-picrylhydrazyl radical-scavenging assay and interestingly it was noticed that the extracts contained improved percentage of flavonoids and pinitol showing better activity.

Biotransformation of lignan glycoside to its aglycone by Woodfordia fruticosa flowers: quantification of compounds using a validated HPTLC method

Context: Saraca asoca Linn. (Caesalpiniaceae) is an important traditional remedy for gynaecological disorders and it contains lyoniside, an aryl tetralin lignan glycoside. The aglycone of lyoniside, lyoniresinol possesses structural similarity to enterolignan precursors which are established phytoestrogens. Objectives: This work illustrates biotransformation of lyoniside to lyoniresinol using Woodfordia fruticosa Kurz. (Lythraceae) flowers and simultaneous quantification of lyoniside and lyoniresinol using a validated HPTLC method. Materials and methods: The aqueous extract prepared from S. asoca bark was fermented using W. fruticosa flowers. The substrate and fermented product both were simultaneously analyzed using solvent system:toluene:ethyl acetate:formic acid (4:3:0.4) at 254 nm. The method was validated for specificity, accuracy, precision, linearity, sensitivity and robustness as per ICH guidelines. Results: The substrate showed the presence of lyoniside, however, it decreased as the fermentation proceeded. On 3rd day, lyoniresinol starts appearing in the medium. In 8 days duration most of the lyoniside converted to lyoniresinol. The developed method was specific for lyoniside and lyoniresinol. Lyoniside and lyoniresinol showed linearity in the range of 250–3000 and 500–2500 ng. The method was accurate as resulted in 99.84% and 99.83% recovery, respectively, for lyoniside and lyoniresinol. Conclusion: Aryl tetralin lignan glycoside, lyoniside was successfully transformed into lyoniresinol using W. fruticosa flowers and their contents were simultaneously analyzed using developed validated HPTLC method.

Fermentation process of traditional Asokarista using Wickerhamomyces anomalus and its optimization using three-factor, three-level Box–Behnken design

ABSTRACTThe aim of the study was to determine the effect of selected variables on fermentation of decoction of Saraca asoca bark to develop a suitable method for preparing its polyherbal formulation. S. asoca bark has been fermented using Wickerhamomyces anomalus (nonsaccharomyces yeast) and the method was optimized using response surface methodology (RSM). The independent variables, substrate volume (ml), temperature (°C), and time (hours), were optimized using three-factor, three-level Box–Behnken design to obtain the good content of secoisolariciresinol, lyoniresinol, and 5-methoxy isolariciresinol. Secoisolariciresinol and 5-methoxy isolariciresinol were first isolated from S. asoca bark. The method yielded a quadratic polynomial equation to predict the effect of independent variables on selected responses (<0.0001). All three variables significantly (p-value < 0.0001–0.0068) affected the selected responses. The experimental values agreed closely with the predicted values and the analysis of variance signified a good model fit. The optimum conditions were 67.03 ml substrate volume, 30.95°C, and 64.86 h resulting in 23.24, 21.20, 25.06 μg of secoisolariciresinol, lyoniresinol, and 5-methoxy isolariciresinol content. This is the first report of biotechnological production of Asokarista using W. anomalus in 48 h rather than 35 days as mentioned in traditional method. Also, secoisolariciresinol has been found in fermented S. asoca bark for the first time. It has been ascertained that W. anomalus causes deglycosylation in traditional ayurvedic formulations during fermentation. Present study proved the possibility of using pure strain, Wi. anomalus, for formulating traditional formulation.

Synergistic effect of (+)-pinitol from Saraca asoca with β-lactam antibiotics and studies on the in silico possible mechanism

Saraca asoca bark has been used in the Ayurvedic system of medicine for female urino-genital disorders. We have recently reported the isolation and characterization of several compounds as markers to develop HPLC profiling of its methanol and aqueous methanol extracts. Now, a HPLC-PDA inactive compound, (+)-pinitol has been isolated and characterized from the bark of this medicinally important tree. Pinitol is a well known bioactive compound for a variety of biological activities, including hypoglycemic and anti-inflammatory activities. A process for the isolation of relatively good concentration of (+)-pinitol from S. asoca bark has been developed and its in vitro anti TNF-α and anti-inflammatory activities against carragenan-induced edema confirmed. While conducting experiments on the possible agonistic activity, it was found that (+)-pinitol showed up to eight fold reduction in the doses of β-lactam antibiotics. The mechanism of its agonistic activity was studied by docking experiments which showed that different conformations of (+)-pinitol and antibiotics were actually in the same binding site with no significant change in the binding energy. These docking simulations, thus predict the possible binding mode of studied compounds and probable reason behind the synergistic effect of (+)-pinitol along with β-lactam antibiotics.

Optimization of microwave-assisted extraction conditions for preparing lignan-rich extract from Saraca asoca bark using Box–Behnken design

Context: Lyoniside is the major constituent of Saraca asoca Linn. (Caesalpiniaceae) bark. There is an immediate need to develop an efficient method to isolate its chemical constituents, since it is a therapeutically important plant.Objective: A rapid extraction method for lyoniside based on microwave-assisted extraction of S. asoca bark was developed and optimized using response surface methodology (RSM).Materials and methods: Lyoniside was analyzed and quantified by high-performance liquid chromatography coupled with ultraviolet detection (HPLC-UV). The extraction solvent ratio (%), material solvent ratio (g/ml) and extraction time (min) were optimized using Box–Behnken design (BBD) to obtain the highest extraction efficiency. The optimal conditions were the use of 1:30 material solvent ratio with 70:30 mixture of methanol:water for 10 min duration.Results: The optimized microwave-assisted extraction yielded 9.4 mg/g of lyoniside content in comparison to reflux extraction under identical conditions which yielded 4.2 mg/g of lyoniside content. Under optimum conditions, the experimental values agreed closely with the predicted values. The analysis of variance (ANOVA) indicated a high goodness-of-fit model and the success of the RSM method for optimizing lyoniside extraction from the bark of S. asoca.Discussion: All the three variables significantly affected the lyoniside content. Increased polarity of solvent medium enhances the lyoniside yield.Conclusion: The present study shows the applicability of microwave-assisted extraction in extraction of lyoniside from S. asoca bark.

Effect of Saraca asoca (Asoka) on estradiol-induced keratinizing metaplasia in rat uterus.

Estrogen-mediated uterus endometrium instability is considered as one of the etiological factors in dysfunctional uterine bleeding (DUB) and uterine cancer. Saraca asoca (Family: Fabaceae) and its fermented preparation, Asokarishta, are extensively used as uterine tonic to treat gynecological disorders in Ayurveda. The present study evaluated the effect of S. asoca (Asoka) on estrogen-induced endometrial thickening of rat uterus. Endometrial thickening was induced by intraperitoneal injection of estradiol (20 μg/kg b.wt) to 8-day-old immature rats for alternate 5 days. Methanolic extract (200 mg/kg b. wt) from S. asoca bark was given orally along with estradiol. Uterus endometrial thickening was analyzed histopathologically and serum estrogen level by radioimmunoassay (RIA). Cyclooxygenase (COX-2) expression in rat uterus was also estimated by Western blot. Anti-inflammatory activity of the extract was analyzed by formalin- and carrageenan-elicited paw edema models in mouse. Uterus endometrium proliferation and keratinized metaplasia with seven to eight stratified epithelial layers on day 16 was observed in rats administered with estradiol. Treatment with S. asoca reduced the thickening to two to four layers and the serum estrogen level diminished significantly to 82.9±12.87 pg/mL compared to rats administered with estrogen alone (111.2±10.68 pg/mL). A reduction of formalin- and carrageenan-induced paw edema in mouse by S. asoca extract was observed. Lower level of lipopolysaccharides (LPS)-induced COX-2 enzyme in rat uterus by the extract further confirms its anti-inflammatory activity. Present study reveals the antiproliferative and antikeratinizing effects of S. asoca in uterus endometrium possibly through its anti-estrogenic and anti-inflammatory properties.

Anti-inflammatory flavanol glycosides from Saraca asoca bark

Saraca asoca (Roxb.) de Wilde, a common tree of India, is popularly used in the Ayurvedic and modern herbal systems of medicine for genito-urinary problems of women. Considering the reported antimicrobial or anti-inflammatory effect of S. asoca bark against such infections, we studied the anti-inflammatory activity-guided isolation of active compounds from methanol extract. The methanol extract of bark has yielded 10 compounds out of which 3′-deoxyepicatechin-3-O-β-d-glucopyranoside (6) and 3′-deoxycatechin-3-O-α-l-rhamnopyranoside (8) have been found to be in vitro and in vivo active. 3′,5-Dimethoxy epicatechin (3), 3′-deoxyepicatechin-3-O-β-d-glucopyranoside (6), 3′-deoxycatechin-3-O-α-l-rhamnopyranoside (8) and epigallocatechin (9) are being reported for the first time from S. asoca.

Molluscicidal activity of Saraca asoca and Thuja orientalis against the fresh water snail Lymnaea acuminata

The molluscicidal activity of bark powder of Saraca asoca, leaf powder of Thuja orientalis against the snail Lymnaea acuminata was studied. The molluscicidal activity of all the plant products was found to be both time and concentration dependent. The 96 h LC 50 of T. orientalis leaf powder against L. acuminata was 250.5 mg/l. Ethanol extracts were more toxic than other organic extracts. The ethanol extract of T. orientalis leaf (24 h LC 50: 32.74 mg/l) was more effective than that of S. asoca bark (24 h LC 50: 82.38 mg/l). The 24 h LC 50 of column purified fraction of T. orientalis leaf and S. asoca bark powder was 29.25 and 64.89 mg/l, respectively. Saponin and thujone were identified as active molluscicide components in the bark of S. asoca and leaf of T. orientalis, respectively. The product of S. asoca and T. orientalis may be used as potent molluscicides.

The action of Saraca asoca Roxb. de Wilde bark on the PGH2 synthetase enzyme complex of the sheep vesicular gland.

Extracts of S. asoca bark and pure compounds isolated from the bark were tested for properties that might inhibit the conversion of arachidonic acid by the PGH2 synthetase. They were assayed spectrophotometrically with adrenaline as cofactor. Methanol- and ethyl acetate extracts inhibited the conversion. The observed inhibition was confirmed in an oxygraphic assay. Two procyanidin dimers from the ethyl acetate extract showed enzyme catalyzed oxidation in our assay. The ether extract of the bark was also found to contain yet unknown substances which were capable of being oxidised by the PGH2 synthetase. The combined action of the components of the bark may explain the mode of action of the drug Asoka Aristha, the main ingredient of which is the bark of S. asoca. The drug is traditionally used in Sri Lanka to treat menorrhagia.