High-Performance Liquid Chromatography (HPLC) as a Tool for Standardization of Complex Herbal Drugs.

Background: Herbal formulations have reached tremendous acceptability as therapeutic agents for several diseases mainly due to the indiscriminate use of modern medicine such as antibiotics, steroids and other synthetic drugs. The increasing popularity in plant-based drugs is leading to a fast growing market for plant-based drugs pharmaceuticals, nutraceuticals, functional foods, and even cosmeceuticals. Objective: The development of authentic analytical methods for complex herbal drugs, especially poly herbal formulations which can reliably profile the phytochemical composition, including quantitative analyses of marker/bioactive compounds and other major constituents, is a major challenge to scientists. Standardization is an important step for the establishment of a consistent biological activity, a consistent chemical profile, or simply a quality assurance program for production and manufacturing of herbal drugs. Methods/Results: HPLC as a tool has been widely used in the standardization of complex herbal drugs because of its ability to estimate the presence of active (chemical or biological) markers both qualitatively and quantitatively. Conclusions: An overview of various HPLC techniques that can be used for standardization of herbal drugs has been presented.

The genus spilanthes ethnopharmacology, phytochemistry, and pharmacological properties: a review.

Spilanthes spp. are popular, over-the-counter remedies; they are sold over the internet under various names and are widely used in traditional medicine in various cultures. This review will summarize the important reports on the ethnopharmacology, botany, phytochemistry, and pharmacological properties as described in the literature from recent years (1920 to 2013). Spilanthes spp. are used for more than 60 types of disorders. They are reported to contain a number of biologically active phytochemicals, although a large number of ethnopharmacological uses have been documented; only a few of these species have been investigated for their chemical and biological activities. The studies are carried out mainly on Spilanthes extracts and a few metabolites substantiate the uses of these plants in traditional medicine. Well-conducted pharmacological studies are still needed for several traditional indications, and the mechanisms of action by which the plant extracts and the active compounds exert their pharmacological effects remain to be studied. They are predominantly used as extracts in personal care products, traditional medicines, and the pharmaceutical and culinary areas. Suggestions are made regarding some of the possible mechanisms of action as to how the known compounds may exert their biological activity.

Validated reversed-phase column high-performance liquid chromatographic method for separation and quantification of polyphenolics and furocoumarins in herbal drugs.

A rapid column high-performance liquid chromatographic-photodiode array method has been developed for the separation and identification of secondary metabolites, especially different types of phenols and furocoumarins, in a 35 min chromatographic run. The method has been optimized and validated for selectivity, precision, recovery, and robustness with the aim of application for standardization of selected herbal drugs. Almost all of the tested compounds had linearity of >98%, with relative standard deviation <10% in terms of variation of retention time. Interday and intraday variability was <5%. The developed method has been successfully applied in identification and quantification of phenols and furocoumarins present in different plants, viz., Artemisia pallens (whole plant), Hibiscus rosa-sinensis DC (flower), Heracleum candicans DC (fruit), and Ficus carica Linn (bark). The results indicate that the method is rapid, accurate, and robust for the analysis of different types of phenols and furocoumarins and, hence, can be successfully used in the quality control and standardization of plant extracts and herbal drugs.

Novel mechanism of modulating natural antioxidants in functional foods: involvement of plant growth promoting Rhizobacteria NRRL B-30488.

The significance of plant growth-promoting rhizobacteria (PGPR) mediated increase in antioxidant potential in vegetables is yet unknown. The plant growth-promoting bacterium Bacillus lentimorbus NRRL B-30488 (B-30488) mediated induction of dietary antioxidant in vegetables ( Trigonella foenum-graecum, Lactuca sativa, Spinacia oleracea, and Daucus carota) and fruit ( Citrus sinensis) after minimal processing (fresh, boiled, and frozen) was tested by estimating the total phenol content, level of antioxidant enzymes, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) and superoxide scavenging activities along with integral radical scavenging capacity by photochemiluminescence assay and inhibition of lipid peroxidation. Minimal processing of vegetables showed that T. foenum-graecum had the highest phenol content in B-30488-treated plants followed by L. sativa, D. carota, and S. oleracea. Thermally treated vegetables T. foenum-graecum (26-114.5 GAE microg mg (-1)) had an exceptionally high total phenolic content, followed by D. carota (25.27-101.32 GAE microg mg (-1)), L. sativa (23.22-101.10 GAE microg mg (-1)), and S. oleracea (21.87-87.57 GAE microg mg (-1)). Among the vegetables and fruit used in this study for enzymatic estimation, induction of antioxidant enzymes, namely, polyphenol oxidase (PPO), ascorbate peroxidase (APX), catalase (CAT), and superoxidase dismutase (SOD), was observed in edible parts of T. foenum-graecum, L. sativa, S. oleracea, and D. carota, after inoculation with B-30488. The scavenging capacity of the vegetables treated with B-30488 against DPPH and superoxide anion radical activity was found to be significantly high as compared to nontreated control. Mild food processing had no adverse effect on radical scavenging capacity. Photochemiluminescence also ascertains the above findings. The ability of the plant extracts to protect against lipid peroxidation and its ability to prevent oxidation of reduced glutathione (GSH) was measured in rat liver homogenate, and the results suggested that the inoculated plant exhibited better activity in all of the screened plants. Significant increases in shoot length, root length, and dry weight, averaging 164, 132, and 135% in T. foenum-graecum, 174, 141, and 156% in L. sativa, 129, 141, and 59%, in S. oleracea, and 125, 146, and 42% in D. carota, respectively, over untreated controls, were attained in greenhouse trials. To the best of the authors' knowledge, this is the first report of PGPR-mediated induction of antioxidant enzyme activity (PPO, APX, CAT, and SOD) along with the antioxidant activity of the extracts in both in vitro (DPPH radical scavenging and superoxide scavenging) and ex vivo conditions using the rat liver tissue (percent inhibition of lipid peroxidation and prevention of oxidation of GSH) and phenolic content. The results demonstrate the PGPR-mediated induction of antioxidant level in vegetables and fruit controls oxidative damage even after minimal processing and thus is indicative of its potential as a viable substitute of synthetic antioxidants.

Effect of Desmodium gangeticum extract on blood glucose in rats and on insulin secretion in vitro.

Desmodium gangeticum is widely used in the indigenous system of medicine in India and is reported to contain flavone and isoflavonoid glycosides. It forms the ingredient of many Ayurvedic formulations used for diabetes. The present study was thus aimed at evaluating the insulin secretion and antidiabetic activity of Desmodium gangeticum. Treatment of diabetic rats with aerial parts of D. gangeticum extract (DG, 100 and 250 mg/kg body weight) for 3 weeks showed a significant reduction in blood glucose. D. gangeticum extract caused a significant increase in insulin secretion from MIN6 cells grown as monolayers and as pseudoislets, indicating that the antidiabetic activity may be as a result of increased insulin secretion. It also had a role on the lipid profile of the rats by causing reductions in cholesterol and triglycerides and increasing the HDL significantly (p < 0.05). This works supports the traditional use of D. gangeticum in the treatment of diabetes and this is likely to be due, at least in part, to its stimulation of insulin secretion by pancreatic islet cells.

Action of (2-->1)Fructo-oligopolysaccharide fraction of Chlorophytum borivilianum against Streptozotocin-Induced oxidative stress.

A fructo-oligosaccharide was isolated from Chlorophytum borivilianum and identified as O-beta-D-fructofuranosyl-(2-->1)-(beta-D-fructofuranosyl) (n)-(2-->1)-alpha-D-glucopyranoside (n = 5 - 30) using high-pressure anion exchange chromatography, MALDI-MS, NMR, GC, HPTLC and chemical analysis. The extract and the fructo-oligosaccharide were found to have significant antidiabetic activity with the blood sugar levels being 118.32 +/- 3.56 and 110.21 +/- 4.22, respectively, as compared to the control value of 231.25 +/- 3.03 along with moderate antioxidant activity in streptozotocin-induced diabetic animals.

Antimicrobial, wound healing and antioxidant activity of Plagiochasma appendiculatum Lehm. et Lind.

Plagiochasma appendiculatum (Aytoniaceae) of the order Marchantiales is widely used in the form of paste ethnomedicinally by Gaddi tribe in Kangra valley for treating skin diseases. In this context, antimicrobical potential of Plagiochasma appendiculatum against a wide range of microorganisms was studied. To validate the ethnotherapeutic claims of the plant in skin diseases, wound healing activity was studied, besides antioxidant activity to understand the mechanism of wound healing activity. The plant (alchoholic and aqueous extract) showed significant antibacterial and antifungal activity against almost all the organisms: Micrococcus luteus, Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, Streptococcus pneumoniae, Enterobacter aerogenes, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella typhimurium, and eight fungi Candida albicans and Cryptococcus albidus-dimorphic fungi, Trichophyton rubrum-dermatophyte fungi, Aspergillus niger, Aspergillus flavus, Aspergillus spinulosus, Aspergillus terreus and Aspergillus nidulans-systemic fungi, with especially good activity against the dermatophyte (Trichophyton rubrum) and some infectious bacteria (Escherichia coli, Proteus mirabilis and Salmonella typhimurium) with an MIC of 2.5 microg/disc. The results show that Plagiochasma appendiculatum extract has potent wound healing capacity as evident from the wound contraction and increased tensile strength. The results also indicated that Plagiochasma appendiculatum extract possesses potent antioxidant activity by inhibiting lipid peroxidation and increase in the superoxide dismutase (SOD) and Catalase activity.

Free radical scavenging potential of Chlorophytum tuberosum Baker.

Chlorophytum tuberosum Baker commonly referred as 'Musli' has been widely used as a potent 'Rasayana' drug in 'Ayurveda' as a rejuvenator and tonic. Antioxidant potential of Chlorophytum tuberosum has been investigated for their ability to scavenge 1,1,diphenyl picryl hydrazyl (DPPH), nitric oxide radical along with their capacity to reduce lipid peroxidation in rat liver homogenate, chelation of ferrous ion, radical scavenging potential using chemiluminescence and their total antioxidant capacity. Sugar, starch, protein, and Vitamin C content were estimated spectrophotometrically along with the percentages of the individual amino acids by HPLC and individual sugars by using HPTLC as standardization tool. The extract has been found to possess antioxidant activity in all the models tested as evident by IC50 values being 225.31, 888.44, 809.22 and 422.97 microg/ml for scavenging of DPPH, nitric oxide, lipid peroxidation and ferry bi-pyridyl complex, respectively, along with a integral anitoxidant activity of 2.986 nmol ascorbic acid/g equivalents in photochemiluminescence assay.

Antioxidant activity of Desmodium gangeticum and its phenolics in arthritic rats.

Total alcoholic extract of Desmodium gangeticum, which exhibited significant anti-inflammatory activity, was evaluated for the possible mode of action by studying its antioxidant potential in adjuvant-induced arthritic rats. Activity guided fractionation and isolation were carried out. The phenolics fraction showed maximum potency. Solid phase extraction followed by preparative HPLC of the active phenolic fraction yielded for the first time two potent antioxidant compounds, caffeic acid and chlorogenic acid, from this plant. The biological antioxidant defense system, involving superoxide dismutase, glutathione and catalase, showed a significant increase with their levels close to the normal control with a decrease in the lipid peroxide content upon administration of D. gangeticum extract (100 mg kg(-1)) and its phenolics (50 mg kg(-1)) in arthritic rats, thereby indicating the extracts antioxidant property under arthritic conditions.

Free radical scavenging potential of Saussarea costus.

Saussurea costus (Falc.) Lipschitz from the family Asteraceae is an important medicinal drug, the roots of which are widely used in folk medicine. The antioxidant activity of the plant has been studied using its ability to scavenge DPPH, nitric oxide, superoxide radicals along with its ability to inhibit lipid peroxidation and GSH oxidation. The 1 mg mL(-1) extract had antioxidant activity with 85.2% reduction of DPPH and a 72.7% decrease in lipid peroxidation. It showed maximum inhibition of superoxide radical of 66.0%, and 58.4% inhibition of nitric oxide formation. The concentration of chlorogenic acid was 0.027% in the extract of S. costus. Thus, the therapeutic activity of the plant may be due to its antioxidant activity, probably as a result of the presence of chlorogenic acid.

Antioxidant potential of Anogeissus latifolia.

Anogeissus latifolia is widely used in the Indian indigenous system of medicine and is reported to contain leucocyanidins and tannoid principles like ellagic acid and its derivatives. In view of its wide use and its chemical composition, this study was aimed at examining the antioxidant activity of the extract of A. latifolia. The extract was studied for total antioxidant capacity, hydrogen-donating ability, nitric oxide, superoxide scavenging activity, hydrogen peroxide decomposition activity along with lipid peroxidation. Integral antioxidative capacity was determined by chemiluminescence assay. The extract was also studied for lipid peroxidation assay by thiobarbituric acid-reactive substances (TBARS) method using rat liver homogenate. The results indicate that A. latifolia extract has potent antioxidant activity. Also to ascertain the possible reason for the potent activity, percentage of gallic acid was estimated and was found to be 0.95%, which could be one of the reasons for potent antioxidant activity exhibited by the plant.

Healing potential of Anogeissus latifolia for dermal wounds in rats.

Wound healing potential of ethanolic extract of Anogeissus latifolia bark (ALE) for treatment of dermal wounds in rats was studied on excision and incision wound models. HPTLC of the total extract was recorded for the purpose of standardization. Various parameters of incision wound, viz. epithelization period, scar area, tensile strength and hydroxyproline measurements along with wound contraction, were used to evaluate the effect of A. latifolia on wound healing. The results obtained indicate that A. latifolia accelerates the wound healing process by decreasing the surface area of the wound and increasing the tensile strength. Nitrofurazone ointment was used as a positive control. Complete epithelization was observed within 15 days with ALE. Measurements of the healed area and the hydroxyproline level were in agreement. Antibacterial activity of ALE was studied against Gram-positive (Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae) compared to erythromycin and tetracycline. Moderate activity was observed against all organisms. The present study provides a scientific rationale for the traditional use of Anogeissus latifolia in the management of skin diseases such as sores, boils and itching.

Studies on the antioxidant activities of Desmodium gangeticum.

Desmodium gangeticum is herbal species which is widely used in the indigenous system of medicine and is reported to contain flavone and isoflavanoid glycosides. In view of its wide use and it's chemical composition, this study was aimed at examining the antioxidant activity of the extract of D. gangeticum. The extract was studied for diphenyl picryl hydrazyl (DPPH), nitric oxide, ferryl-bipyridyl and hypochlorous acid scavenging activity along with lipid peroxidation. Nitric oxide was generated using sodium nitroprusside and was studied using Griess reagent. In order to study the iron chelating capacity of the extract, the percentage ferryl-bipyridyl inhibition was studied. Hypochlorous acid scavenging activity was tested by measuring the inhibition of 5-thio-2-nitrobenzoic acid oxidation. The extract was also studied for lipid peroxidation assay by thiobarbituric acid-reactive substances (TBARS) method using rat brain homogenate. The results indicate that D. gangeticum extract has potent antioxidant activity.