Combining LC-MS/MS profiles with network pharmacology to predict molecular mechanisms of the hyperlipidemic activity of Lagenaria siceraria stand.

ETHNOPHARMACOLOGICAL RELEVANCE: Lagenaria siceraria Stand. (Family: Cucurbitaceae), popularly known as bottle gourd, is traditionally used in Ayurvedic medicine as a food plant, especially in hypertension and obesity. AIM OF THE STUDY: Investigations were undertaken to assign novel lead combinations from this common food plant to multi-molecular modes of actions in the complex disease networks of obesity and hypertension. LC-MS/MS based metabolite screening, in-vivo high fat diet induced hyperlipidemia animal study and network pharmacology explorations of the mechanism of action for lipid lowering effects including a neighbourhood community approach for molecular combinations were performed. MATERIAL AND METHODS: Major chemical constituents of the fruits of LS (LSFE) were analysed by HPLC-DAD-MS/MS-QTOF. Wistar albino rats (n = 36), divided into 6 groups (n = 6) received either no treatment or a high-fat diet along with LSFE or Atorvastatin. Lipid profiles and biochemical parameters were evaluated. In silico cross-validated network analyses using different databases and Cytospace were applied. RESULTS: Profiling of LSFE revealed 18 major constituents: phenolic acids like p-Coumaric acid and Ferulic acid, the monolignolconferyl alcohol, the flavonoid glycosides hesperidin and apigenin-7-glucoside. Hyperlipidemic animals treated with LSFE (200 mg/kg, 400 mg/kg, 600 mg/kg) showed a significant improvement of their lipid profiles after 30 days of treatment. Network pharmacology analyses for the major 18 compounds revealed enrichment of the insulin and the ErbB signalling pathway. Novel target node combinations (e.g. AKR1C1, AGXT) including their connection to different pathways were identified in silico. CONCLUSIONS: The combined in vivo and bioinformatics analyses propose that lead compounds of LSFE act in combination on relevant targets of hyperlipidemia. Perturbations of the IRS→Akt→Foxo1 cascade are predicted which suggest further clinical investigation towards development of safe natural alternative to manage hyperlipidemia.

A reversed-phase ultra-fast liquid chromatography-photodiode array detector (RP-UFLC-PDA) method for simultaneous estimation of ayapanin and umbelliferone in Ayapana triplinervis Vahl.

A rapid validated ultra-fast liquid chromatography-photodiode array detector (UFLC-PDA) method was developed to identify and quantify ayapanin (AY) and umbelliferone (UM) simultaneously in Ayapana triplinervis Vahl methanolic extract. The method was validated for linearity, limit of detection (LOD; 3:1σ/S), limit of quantification (LOQ; 10:1σ/S), precision, accuracy, specificity and robustness. The response was linear with a good correlation between concentration and mean peak area through a correlation coefficient of 0.9996, y = 7025.7x - 2269.8 and 0.9997, y = y = 16,262x - 946 with LOD of 6.256 ± 0.52 and 3.325 ± 0.36, and LOQ of 18.838 ± 0.18 and 8.870 ± 0.85 for AY (0.67% w/w) and UM (0.18% w/w), respectively. The relative standard deviation (%) of precision and recovery of AY and UM was <2.0%. The proposed method was simple, accurate, specific, precise and reproducible.

Therapeutic importance of Cucurbitaceae: A medicinally important family.

ETHNOPHARMACOLOGICAL RELEVANCE: Medicinal plants of Cucurbitaceae family consist of several edible fruits and vegetables consumed worldwide since ancient times. The plants of this family have played an essential role in the ethnopharmacological as well as traditional medicinal system globally and their evidence is well established in several traditional literatures. Various plant parts have been used to treat several human ailments viz. Pandu (anemia), Pliharoga (splenomegaly), Sopha (inflammation), Gulma (tumor growth), Adhmana (indigestion. acidity), Garavisa (poisoning) etc. AIM OF THE REVIEW: This review article aims to systematically document and bridge scientific evidences with the ethnopharmacological, ethnoveterinary and folklore claims along with the therapeutic efficacy with mechanism of action found in different literature, books, and scientific articles belonging to the Cucurbitaceae family. MATERIALS AND METHODS: To construct the manuscript a comprehensive literature review was done based on the information collected from Ayurvedic Pharmacopoeia of India; books, research articles and databases such as ScienceDirect, Wiley Online Library, SciFinder, Scopus, Springer, Google Scholar, Web of Science, ACS Publications and PubMed. RESULTS: The plants of Cucurbitaceae family are rich in phytochemicals like terpenoids, glycosides, alkaloids, saponins, tannins, steroids, etc., responsible for the therapeutic effect. Various parts of these plants such as leaves, stems, flowers, fruits, seeds, roots etc. exhibit a plethora of pharmacological activity viz. hypolipidemic, antihyperglycemic, anticancer, antimicrobial, analgesic, anti-inflammatory, anti-stress and immunomodulatory activities. Also, in-vitro and in-vivo reports suggest strong inhibitory potential against α-glucosidase, α-amylase, lipase, carbonic anhydrase enzyme along with antioxidant, anti-inflammatory, antidiabetic, anti-tumor, antifungal, etc. Furthermore many reports suggest these plants are beneficial for nutritional, economical and ethnoveterinary uses. CONCLUSIONS: The current review enlightens the therapeutic potential of the gourd family, comprising of the geographical origins, morphology, phytochemistry, ethnopharmacology, ethnoveterinary, nutritional importance, therapeutic benefits, safety, efficacy and related aspects. The phytochemical and pharmacological potential indicated will popularize this family as a potential source of novel therapeutic agents and functional foods. This study will help to validate the therapeutic claims of several ethnomedicinal uses of this plant family. Furthermore the Cucurbitaceae family needs to be evaluated based on the combine approaches of chemoprofiling and bioexploration to develop the concept of food as medicine for the development of new generation therapeutics leading to the human wellness.

Lagenaria siceraria and it's bioactive constituents in carbonic anhydrase inhibition: A bioactivity guided LC-MS/MS approach.

INTRODUCTION: Lagenaria siceraria, is a popular food plant among Indians, contains a large number of phenolic compounds with several medicinal benefits, mentioned in Indian System of Medicine (ISM). OBJECTIVES: To investigate the carbonic anhydrase inhibitory potential and inhibitory mechanism of the most potent fraction of L. siceraria fruits. MATERIALS AND METHODS: The extract and fraction of dried fruit of L. siceraria screened for their in vitro carbonic anhydrase II (bCA II) inhibitory activity. The active fraction was purified by using flash chromatography. The bioactive compounds were identified and quantified through liquid chromatography quadrupole time-of-flight tandem mass spectrometry (LC-QTOF-MS/MS) and reverse-phase high-performance liquid chromatography (RP-HPLC). Finally, the underlying carbonic anhydrase inhibitory mechanism of the compounds was explained by enzyme kinetics and molecular docking study. RESULTS: The LC-QTOF-MS based identification of the most active fraction revealed the presence of phenolic compounds. The results of the enzyme inhibition assay revealed that coniferyl alcohol, ferulic acid and p-Coumaric acid inhibited bCA II activity [half maximal inhibitory concentration (IC50 ) value range of 80 to 250 µM) in a dose dependent manner. The kinetics study of enzyme inhibition revealed that p-Coumaric acid binds to the enzyme competitively whereas the non-competitive type of inhibition was observed for ferulic acid and coniferyl alcohol. The molecular docking study explored the interaction mechanism of phenolic compounds at the active site of bCA II. CONCLUSION: The present research led us to conclude that, the phenolic compounds from L. siceraria serve as major contributors for carbonic anhydrase inhibition, which could play a useful role in the management of oedema, hypertension, obesity and related metabolic disorders.

LC-QTOF-MS-based metabolite profiling and evaluation of α-glucosidase inhibitory kinetics of Coccinia grandis fruit.

Coccinia grandis is an important food crop of the Cucurbitaceae family, widely used for culinary purposes in India. It is reported to possess hypoglycemic, hypolipidemic and antioxidant activities. The current study was aimed to explore the inhibition kinetics as well as major constituents of the active fraction of C. grandis against α-glucosidase. The kinetic study was performed through spectrophotometric assay, with p-nitrophenyl-α-d-glucopyranoside as a substrate with varying concentrations. An in vitro antioxidant study was performed by DPPH assay. In addition, UPLC-QTOF-MS analysis was carried out for metabolite profiling of the bioactive fraction of C. grandis. The results showed that the difference between the α-glucosidase inhibitory activity of the ethyl acetate fraction of C. grandis (EFCG) (IC50 2.43 ± 0.27 mg/ml), and standard inhibitor, acarbose (2.08 ± 0.19 mg/ml), was not statistically significant at a P-value of 0.05. The enzyme kinetics confirmed the inhibition mode in a mixed manner. The EFCG also showed the highest antioxidant activity (101.74 ± 1.95 µg/ml) among all of the fractions. A significant correlation between antioxidant and α-glucosidase inhibitory activity of EFCG was observed. The LC-QTOF-MS study of the EFCG putatively identified 35 metabolites, which may be responsible for its antioxidant and α-glucosidase inhibitory properties. Thus, C. grandis fruits can serve as a functional food to address diabetes-related disorders associated with α-glucosidase.

Determination of cucurbitacin E in some selected herbs of ayurvedic importance through RP-HPLC.

BACKGROUND: The consumption of the fruits of cucurbitaceae plants is widely popular among Indians due to their various nutritional and medicinal purposes. Some of these plants are well reported in Ayurveda due to their potential therapeutic importance. In particular, the plants of this family are well-characterized by the presence of its bitter principle, Cucurbitacin E which differs within the species due to its genetic variations. OBJECTIVES: The objective of the study was to develop a validated RP-HPLC method for standardization in some widely consumed cucurbits with cucurbitacin E as a marker compound. MATERIALS AND METHODS: The RP-HPLC method was developed with a reverse phase C18 column, using acetonitrile and water (1% glacial acetic acid) as mobile phase (70:30 v/v). The flow rate and λmax were optimized at 1 mL/min and 230 nm respectively. The HPLC method was validated in terms of accuracy, specificity, sensitivity, and repeatability as per ICH guideline. RESULTS: The calibration curve was found linear in the concentration range of 1-100 µg/mL. The % RSD of precision and recovery was found to be <2%, which confirms high repeatability of the method. The results indicated that the content of cucurbitacin E was highest (0.0663% w/w) in Cucurbita pepo whereas Lagenaria siceraria contains the lowest (0.0356% w/w). CONCLUSION: The study was able to explore the variation of cucurbitacin E content in some selected food plants of Cucurbitaceae family. The applicability of the method can be established in nutraceutical industry for the effective quality control of cucurbits for safe human consumption.

RP-HPLC analysis of methanol extract of Viscum articulatum.

BACKGROUND: Viscum articulatum Burm. (Family: Loranthaceae) is commonly known as mistletoe. In ayurveda, the plant parts are used in "Kapha", "Vata", diseases of the blood, ulcer, and epilepsy. The plant parts are also used in urinary tract infection and wound infection. The plant contains five triterpenoids such as α-amyrin, lupeol, betulin, betulinic acid and oleanolic acid, exhibiting several pharmacological activities including antimicrobial, anti-HIV, antitumor, antiviral activity. OBJECTIVE: To ensure the content of uniformity of oleanolic acid, a RP-HPLC method has been developed for estimation of oleanolic acid in V. articulatum aerial part. MATERIAL AND METHODS: The RP-HPLC method was carried out in reverse phase C18 column, using methanol and water as mobile phase in the ratio of 95:5 (v/v), at the flow rate of 1 mL/min. The pH of aqueous phase was adjusted 3.2 with 1% (v/v) glacial acetic acid. The λmax was set at 210 nm. RESULTS: The retention time of oleanolic acid was found at 21.5 ± 0.05 min. The linearity of the response was found to be 10-800 µg/mL. The coefficient of determinants of oleanolic acid was found to be (r2) 0.995 and equation Y = 19462X + 16,172. The LOD and LOQ were found to be for oleanolic acid (1.96% w/w) 0.197 ± 0.63 and 0.623 ± 0.87 µg/mL, respectively. The developed method was accurate, specific, precise and reproducible. CONCLUSION: This RP-HPLC may be useful for quantitative estimation of the chemical constituents present in the plant extract as well as the quality assessment of the herbal product.

RP-HPLC and HPTLC Methods for Analysis of Selected Herbs Used as Complexion Promoters in Ayurveda and Unani Systems of Medicine.

BACKGROUND: In Ayurvedic and Unani texts, many herbs and formulations are mentioned as complexion promoters having skin brightening and whitening effects that downregulate melanin synthesis. However, with the assistance of chromatographic techniques, further validation of scientific standardization is required. OBJECTIVE: To validate individual herbs and formulations in the ancient literature, some scientific aspects are to be focused on, including standardization of herbs or herbal products consisting of the active compounds. METHODS: Out of many herbs having complexion promoting properties, three herbs (Myristica fragrans, Hemidesmus indicus, and Inula racemosa) were selected for the study. In the present study, validated reverse phase (RP)-HPLC and high-performance TLC (HPTLC) methods were developed for standardization of the herbs. RESULTS: It was observed that the quercetin present in M. fragrans was 0.62% (w/w) with a retention time (Rt) of 6.23 min, the ferulic acid present in H. indicus was 1.39% (w/w) with an Rt of 8.083 min, and the chlorogenic acid present in I. racemosa was 1.03% (w/w) with an Rt of 3.19 min. The HPTLC method showed 1.23% (w/w) of quercetin present in M. fragrans with a retardation factor (Rf) of 0.48, ferulic acid in H. indicus was 1.52% (w/w) with an Rf of 0.44, and chlorogenic acid was 1.09% (w/w) in I. racemosa with an Rf of 0.52. CONCLUSIONS: This specific and precise validated method can be useful for the quality evaluation and quantitative determination of the constituents in complexion-promoting herbs from Ayurveda and Unani systems of medicine. HIGHLIGHTS: The exploration of complexion promoters from Ayurveda and Unani; quality evaluation of complexion promoters herbs from Ayurveda and Unani; HPTLC and RP-HPLC analysis of herbal extracts used and their validation.

UPLC-QTOF-MS analysis of a carbonic anhydrase-inhibiting extract and fractions of Luffa acutangula (L.) Roxb (ridge gourd).

INTRODUCTION: Luffa acutangula (L.) Roxb, commonly known as ridge gourd (cucurbitaceae), is a common vegetable cultivated in India. It is also a well-used medicinal plant in Indian traditional medicine. OBJECTIVES: To analyse the phenolics content of the most potent carbonic anhydrase-inhibiting fraction from an extract of L. acutangula. MATERIALS AND METHODS: An aqueous ethanol extract of dried fruits of L. acutangula was successively fractionated into petroleum ether, dichloromethane and ethyl acetate. The extract and subsequent fractions were assessed for carbonic anhydrase-inhibitory activity and the enzyme inhibition kinetics were determined for the most active fraction. Total phenolic and flavonoid content of the extract and subsequent fractions were determined spectrophotometrically. Ultra-performance liquid chromatography-quadrupole/time-of-flight mass spectrometry (UPLC-QTOF-MS) analysis was used to tentatively identify the major phenolics in the most active fraction. RESULTS: The concentration of total phenolics and total flavonoids in the extract and each fraction thereof correlated with the level of carbonic anhydrase inhibition activity. The ethyl acetate fraction of the aqueous ethanol extract of L. acutangula had the highest carbonic anhydrase inhibition activity. The enzyme kinetics analysis indicated a mixed mode of inhibition. UPLC-QTOF-MS analysis of the ethyl acetate fraction indicated a number of phenolic acids, hydroxycoumarins, flavones, flavanones, and flavonoids. CONCLUSION: The correlation of total phenolic content with carbonic anhydrase inhibition suggested further research that might confirm that phenolic compounds of L. acutangula offer potential therapeutic benefits against carbonic anhydrase-related disorders.

Study of pancreatic lipase inhibition kinetics and LC-QTOF-MS-based identification of bioactive constituents of Momordica charantia fruits.

The different parts of Momordica charantia have been reported to have several therapeutic applications against hyperglycemia and hypercholesterolemia associated with pancreatic lipase (PL). Inhibition of this enzyme prevents the absorption of dietary triglyceride in the intestine, and thus exerts an anti-obesity effect. This study aimed to investigate the bioactive constituents of the fruits of M. charantia (MCF) extract and fractions against pancreatic PL followed by study of their inhibition kinetics. The PL inhibitory assay was performed spectrophotometrically by measuring the change in absorbance of the products at 405 nm, using p-nitrophenylcaprylate as substrate. The results indicated that the ethyl acetate fraction of MCF (EFMC) offered significant, dose-dependent inhibition against PL, compared with the positive control, Orlistat. The enzyme kinetics study revealed the inhibition to be a mixed type in nature. Additionally, the total phenol and flavonoid content of the fractions was estimated. A positive correlation between phenolic content of EFMC and its PL inhibitory activity was established statistically, which implied that higher inhibition potential was contributed by the phenolic compounds. The identification of the bioactive constituents was further confirmed by LC-QTOF-MS study. This finding suggested that phenolic compounds of MCF can serve as functional food components to address obesity-related disorders linked with PL.

Tyrosinase inhibitory mechanism of betulinic acid from Dillenia indica.

The fruit of Dillenia indica L. is extensively used as a food additive. Betulinic acid (BA) is the most prominent secondary metabolite present in D. indica. This study screened the bioassay guided isolation of BA from D. indica and explored its tyrosinase inhibitory mechanism. Half maximal inhibitory concentration (IC50) of BA were calculated as 13.93µM and 25.66µM for diphenolase and monophenolase. Enzyme kinetic analysis revealed that BA inhibited tyrosinase activity non-competitively. Further, conformational analysis of tyrosinase with BA was measured by fluorescence and circular dichroism spectroscopy. These results implied that diminish rigidity of enzyme might disturb the catalytic conformation of tyrosinase. Moreover, In-silico analysis confirmed probable binding polar and non-polar region on the active site of tyrosinase. Based on these findings, we suggest that BA from D. indica may be useful in preventing enzymatic browning reactions in food products.

Development of Ayurveda - Tradition to trend.

ETHNOPHARMACOLOGICAL RELEVANCE: Ayurveda entails a scientific tradition of harmonious living and its origin can be traced from ancient knowledge in Rigveda and Atharvaveda. Ayurveda is a traditional healthcare system of Indian medicine since ancient times. Several Ayurvedic medicines have been exploiting for treatment and management of various diseases in human beings. The several drugs have been developed and practiced from Ayurveda since ancient time to modern practice as 'tradition to trend'. The potential of Ayurvedic medicine needs to be explored further with modern scientific validation approaches for better therapeutic leads. AIM OF THE STUDY: The present study was aimed to explore the various aspects of Ayurveda and inspired drug discovery approaches for its promotion and development. MATERIALS AND METHODS: We have reviewed all the literature related to the history and application of Ayurvedic herbs. Various aspects for the quality control, standardization, chemo-profiling, and metabolite fingerprinting for quality evaluation of Ayurvedic drugs. The development of Ayurvedic drugs is gaining momentum with the perspectives of safety, efficacy and quality for promotion and management of human health. Scientific documentation, process validation and several others significant parameters are key points, which can ensure the quality, safety and effectiveness of Ayurvedic drugs. RESULTS: The present review highlights on the major goal of Ayurveda and their significant role in healthcare system. Ayurveda deals with several classical formulations including arka, asavas, aristas, churna, taila, vati, gutika, bhasma etc. There are several lead molecules that have been developed from the Ayurvedic herbs, which have various significant therapeutic activities. Chemo-profiling of Ayurvedic drug is essential in order to assess the quality of products. It deals with bioactive compound quantification, spurious and allied drug determination, chromatographic fingerprinting, standardization, stability and quality consistency of Ayurvedic products. CONCLUSIONS: Scientific validation and the documentation of Ayurvedic drugs are very essential for its quality evaluation and global acceptance. Therapeutic efficacy of Ayurvedic herbs may be enhanced with high quality, which can be achieved by identity, purity, safety, drug content, physical and biological properties. Ayurvedic medicines need be explored with the modern scientific approaches for its validation. Therefore, an attempt has been made in the present review to highlight the crucial aspects that need to be considered for the promotion and development of Ayurvedic medicine.

RP-HPLC simultaneous estimation of betulinic acid and ursolic acid in Carissa spinarum.

Carissa spinarum is a well-known medicinal plant which has been reported for its anthelmintic, antipyretic, antiviral, antimicrobial and antitumour activities. In this study, a reverse-phase high-performance liquid chromatographic method was developed for the simultaneous estimation of betulinic acid (BA) and ursolic acid (UA) in the methanol extract of C. spinarum root. The method was further validated for linearity, limit of detection (LOD = 3.3σ/S), limit of quantification (LOQ = 10σ/S), precision, accuracy and ruggedness. The linear response was obtained using the equation, y = 511.5x+17603 (r(2) = 0.9920) and y = 2886x+6821 (r(2) = 0.9935) for BA and UA, respectively. The LOD and LOQ were found to be 0.268 ± 0.520 µg mL(-1), 0.878 ± 0.183 µg mL(-1) for BA (0.58% w/w) and 3.140 ± 0.36 µg mL(-1), 8.820 ± 0.85 µg mL(-1) for UA (1.09% w/w), respectively. The %RSD of precision and recovery of BA and UA was < 2.0%. The proposed method was simple, accurate, specific, precise and reproducible.