Attenuation of dermal wounds through topical application of ointment containing phenol enriched fraction of Caesalpinia mimosoides Lam.

Caesalpinia mimosoides Lam. is one of the important medicinal plants used by the traditional healers of Uttara Kannada district, Karnataka (India) for treating wounds. In our previous study ethanol extract of the plant was evaluated for its wound healing activity. In continuation, the present study was aimed to evaluate the phenol enriched fraction (PEF) of ethanol extract for wound healing activity along with its antioxidant, anti-inflammatory and antimicrobial properties. The potent wound healing activity of PEF was evidenced by observation of increased rate of cell migration in L929, 3T3L1 and L6 cells (92.59 ± 1.53%, 98.42 ± 0.82% and 96.63 ± 0.61% respectively) at 7.81 µg/ml doses in assays carried out in vitro. Significantly enhanced rate of wound contraction (97.92 ± 0.41%), tensile strength (973.67 ± 4.43 g/mm2), hydroxyproline (31.31 ± 0.64 mg/g) and hexosamine (8.30 ± 0.47 mg/g) contents were observed on 15th post wounding day in 5% PEF treated animals. The enzymatic and non-enzymatic cellular antioxidants (superoxide dismutase, catalase and reduced glutathione) were upregulated (15.89 ± 0.17 U/mg, 48.30 ± 4.60 U/mg and 4.04 ± 0.12 µg/g respectively) with the administration of 5% PEF. The significant antimicrobial, antioxidant and anti-inflammatory activities support the positive correlation of PEF with its enhanced wound healing activity. PEF contains expressive amounts of total phenolic and total flavonoid contents (578.28 ± 2.30 mg GAE/g and 270.76 ± 2.52 mg QE/g). Of the various chemicals displayed in RP-UFLC-DAD analysis of PEF, gallic acid (68.08 µg/mg) and ethyl gallate (255.91 µg/mg) were predominant. The results indicate that PEF has great potential for the topical management of open wounds.

Comparative Quantification of the Phenolic Compounds, Piperine Content, and Total Polyphenols along with the Antioxidant Activities in the Piper trichostachyon and P. nigrum.

India is the largest producer in the world of black pepper and it is the center of origin for Piper. The present study gives a comparative account of the chemical composition of the Piper nigrum and its wild putative parent the P. trichostachyon. Microextractions were performed and the quantification of six phenolic compounds (namely epicatechin, gallic acid, catechol, chlorogenic acid, caffeic acid, and catechin), piperine from leaves, petioles, and the fruits of both the species, were accomplished using the RP-UFLC system. The polyphenols (phenolic, flavonoid) and their antioxidant activities were also estimated. Among the six phenolic compounds studied, only three were detected and quantified. The polyphenol content correlating to the antioxidant activities was higher in the P. trichostachyon, whereas the piperine content was 108 times greater in the P. nigrum fruits. The Piper trichostachyon comparatively showed a higher content of polyphenols. The microextractions reduced the solvent consumption, the quantity of the plant material, and the amount of time used for the extraction. The first report on the TPC, TF, and the antioxidant activity of the P. trichostachyon has been described, and it also forms a scientific basis for its use in traditional medicine. The petioles of both species are good sources of phenolic compounds. A quantitative chemical analysis is a useful index in the identification and comparison of the species.

Evaluation of wound healing property of Caesalpinia mimosoides Lam.

ETHNOPHARMACOLOGICAL RELEVANCE: Caesalpinia mimosoides Lam. is one of the important traditional folk medicinal plants in the treatment of skin diseases and wounds used by healers of Uttara Kannada district of Karnataka state (India). However scientific validation of documented traditional knowledge related to medicinal plants is an important path in current scenario to fulfill the increasing demand of herbal medicine. AIM OF THE STUDY: The study was carried out to evaluate the claimed uses of Caesalpinia mimosoides using antimicrobial, wound healing and antioxidant activities followed by detection of possible active bio-constituents. MATERIALS AND METHODS: Extracts prepared by hot percolation method were subjected to preliminary phytochemical analysis followed by antimicrobial activity using MIC assay. In vivo wound healing activity was evaluated by circular excision and linear incision wound models. The extract with significant antimicrobial and wound healing activity was investigated for antioxidant capacity using DPPH, nitric oxide, antilipid peroxidation and total antioxidant activity methods. Total phenolic and flavonoid contents were also determined by Folin-Ciocalteu, Swain and Hillis methods. Possible bio-active constituents were identified by GC-MS technique. RP-UFLC-DAD analysis was carried out to quantify ethyl gallate and gallic acid in the plant extract. RESULTS: Preliminary phytochemical analysis showed positive results for ethanol and aqueous extracts for all the chemical constituents. The ethanol extract proved potent antimicrobial activity against both bacterial and fungal skin pathogens compared to other extracts. The efficacy of topical application of potent ethanol extract and traditionally used aqueous extracts was evidenced by the complete re-epithelization of the epidermal layer with increased percentage of wound contraction in a shorter period. However, aqueous extract failed to perform a consistent effect in the histopathological assessment. Ethanol extract showed effective scavenging activity against DPPH and nitric oxide free radicals with an expressive amount of phenolic and moderate concentration of flavonoid contents. Ethyl gallate and gallic acid were found to be the probable bio-active compounds evidenced by GCMS and RP-UFLC-DAD analysis. CONCLUSION: The study revealed the significant antimicrobial, wound healing and antioxidant activities of tender parts of C. mimosoides and proved the traditional folklore knowledge.

Pharmacognostic Screening of Piper trichostachyon Fruits and its Comparative Analysis with Piper nigrum Using Chromatographic Techniques.

BACKGROUND: Piper trichostachyon is a wild, endemic Piper species from Western Ghats of India. The folklore healers of Belagavi region use this plant, similar to Piper nigrum. AIMS: The present study investigates the comparison between P. nigrum and P. trichostachyon using pharmacognostic parameters. MATERIALS AND METHODS: Pharmacognostic evaluation was carried out in terms of morphological, microscopic characters, and phytochemical analysis using standard methods. Comparative physicochemical analysis between P. trichostachyon and P. nigrum was also carried out through estimation of micro-macro nutrients, high-performance thin layer chromatography (HPTLC) investigation and using piperine as a marker compound for reversed phase-ultra flow liquid chromatographic (RP-UFLC) technique. RESULTS: P. trichostachyon grows in the forests, and the fruits are morphologically similar to P. nigrum fruits, so the name in Kannada "Kaadu Kalu menasu" (wild/forest black pepper). The microscopy revealed the presence of stone cells, starch grains, oil cells and globules, beaker cells, and yellowish brown pigment layer, parenchymatous cells. The presence of alkaloids, oil, and tannins were observed in P. trichostachyon fruits. The HPTLC studies visibly indicated differences among two species with 12 peaks and varied banding pattern. RP-UFLC results showed less amount of piperine in P. trichostachyon (0.05 ± 0.002 mg/g) than in P. nigrum (16.14 ± 0.807 mg/g). CONCLUSION: The study reports on pharmacognostic parameters of P. trichostachyon for the 1(st) time and will be useful for the identification and authentication. The comparative HPTLC and RP-UFLC studies resolve the differentiation impasse among two species. However, further biological efficacy studies are required to establish its use in traditional medicine. SUMMARY: Piper trichostachyon grows in the forests, and the fruits are morphologically similar to Piper nigrum fruitsThe microscopy of P. trichostachyon revealed the presence of stone cells, starch grains, oil cells and globules, beaker cells and yellowish brown pigment layer, parenchymatous cellsThe high-performance thin layer chromatography studies visibly indicated differences among two species with varied banding patternReversed phase-ultra flow liquid chromatographic results showed less amount of piperine in P. trichostachyon than in P. nigrum. Abbreviation used: HPTLC: High Performance Thin Layer Chromatography, RP-UFLC: Reversed phase-ultra flow liquid chromatographic analysis, DST: Length of line, Maj: Length of large half axis for ellipse RDS - radius for circle, Rf: Retention Factor, TS: Transverse Section, TLC: Thin Layer Chromatography.

Determination of betulinic acid, oleanolic acid and ursolic acid from Achyranthes aspera L. using RP-UFLC-DAD analysis and evaluation of various parameters for their optimum yield.

Achyranthes aspera L. is a well known herb commonly used in traditional system of Indian medicine to treat various disorders, such as cough, dysentery, gonorrhea, piles, kidney stone, pneumonia, renal dropsy, skin eruptions, snake bite, etc. Here, we used RP-UFLC-DAD method for determining triterpenoids betulinic acid (BA), oleanolic acid (OA) and ursolic acid (UA) from A. aspera. Optimum yield of these compounds were studied and evaluated using parameters viz., method of extraction, time of extraction, age of plant and plant parts (leaves, stem and roots). Linear relationships in RP-UFLC-DAD analysis were obtained in the range 0.05-100 µg/mL with 0.035, 0.042 and 0.033 µg/mL LOD for BA, OA and UA, respectively. Of the variables tested, extraction method and parts used significantly affected content yield. Continuous shaking extraction (CSE) at ambient temperature gave better extraction efficiency than exposure to ultra sonic extraction (USE) or microwave assisted extraction (MAE) methods. The highest content of BA, OA and UA were determined individually in leaf, stem and root extracts with CSE. Collective yield of these triterpenoids were higher in leaf part exposed to 15 min USE method. To best of our knowledge, the study newly reports UA from A. aspera and the same was confirmed using ATR-FT-IR studies. This study explains the distribution pattern of these major triterpenoids and optimum extraction parameters in detail.

Tribes in Karnataka: Status of health research.

The south Indian State of Karnataka, once part of several kingdoms and princely states of repute in the Deccan peninsula, is rich in its historic, cultural and anthropological heritage. The State is the home to 42,48,987 tribal people, of whom 50,870 belong to the primitive group. Although these people represent only 6.95 per cent of the population of the State, there are as many as 50 different tribes notified by the Government of India, living in Karnataka, of which 14 tribes including two primitive ones, are primarily natives of this State. Extreme poverty and neglect over generations have left them in poor state of health and nutrition. Unfortunately, despite efforts from the Government and non-Governmental organizations alike, literature that is available to assess the state of health of these tribes of the region remains scanty. It is however, interesting to note that most of these tribes who had been original natives of the forests of the Western Ghats have been privy to an enormous amount of knowledge about various medicinal plants and their use in traditional/folklore medicine and these practices have been the subject matter of various scientific studies. This article is an attempt to list and map the various tribes of the State of Karnataka and review the studies carried out on the health of these ethnic groups, and the information obtained about the traditional health practices from these people.

Preliminary pharmacognostic screening of Achyranthes coynei stem.

Achyranthes coynei is a rare, endemic perennial shrub reported from Karnataka and Maharashtra states of India. The plant is used to treat various disorders by folk healers and was proven to have antimicrobial and antioxidant properties. The present study was undertaken to evaluate microscopic and macroscopic characters of A. coynei stem, along with its physicochemical parameters. ProgRes(®) CapturePro and Microsoft Excel were used for statistical analysis. Perennial, shrubby nature and woody stem were the distinguishing morphological characters observed. Transverse section (TS) illustrated quadrangular outline of the stem and showed the presence of two types of trichomes on the thick-walled epidermis. TS also showed number of rosette calcium oxalates crystals; prismatic and microsphenoid crystals; conjoint, collateral open secondary vascular bundles; and two amphixylic medullary bundles in the pith. Ash and extractive values, micro and macro elements and nutritive factors were estimated in the present study. The presence of alkaloids, saponins and triterpenoids were observed in preliminary phytochemical screening. High-performance thin layer chromatographic analysis yielded different bands and also indicated the presence of oleanolic acid. The studied parameters for A. coynei stem will be useful for identification and authentication of the plant material.

Total polyphenolic contents and in vitro antioxidant properties of eight Sida species from Western Ghats, India.

BACKGROUND: Sida L., is a medicinally important genus, the species of which are widely used in traditional systems of medicine in India. Pharmacologically, roots are known for anti-tumor, anti-HIV, hepatoprotective, and many other properties. Phenolic antioxidants help in reducing oxidative stress occurring during treatment of such diseases. OBJECTIVE: The study aimed to evaluate and compare polyphenol contents and antioxidant properties of eight selected species of Sida from Western Ghats, India. MATERIALS AND METHODS: Methanolic root extracts (10% w/v) of Sida species, viz., S. acuta, S. cordata, S. cordifolia, S. indica, S. mysorensis, S. retusa, S. rhombifolia, and S. spinosa were analyzed. RESULTS: Sida cordifolia possessed highest total phenolic content (TPC: 1.92 ± 0.10 mg Caffeic Acid Equivalent/g and 2.13 ± 0.11 mg Tannic Acid Equivalant/g), total flavonoid content (TF: 2.60 ± 0.13 mg Quercetin Equivalent/g) and also possessed highest antioxidant activities in 2,2-diphenylpicrylhydrazyl (DPPH) radical scavenging (51.31 ± 2.57% Radical Scavenging Activity, (RSA); Trolox Equivalent Antioxidant Capacity: 566.25 ± 28.31µM; Ascorbic acid Equivalent Antioxidant Capacity: 477.80 ± 23.89 µM) and Ferric Reducing Antioxidant Power assays (TEAC: 590.67 ± 29.53 µM; AEAC: 600.67 ± 30.03 µM). Unlike DPPH and Ferric Reducing Antioxidant Power (FRAP) activity, 2, 2'-Azinobis (3-ethyl Benzo Thiazoline-6-Sulfonic acid) ABTS(+) antioxidant activity was highest in S. indica (TEAC: 878.44 ± 43.92 µM; AEAC 968.44 ± 48.42 µM). It was significant to note that values of AEAC (µM) for all the antioxidant activities analyzed were higher than that of TEAC. CONCLUSION: The high contents of phenolic compounds in the root extracts of selected Sida species have direct correlation with their antioxidant properties. Conclusively, roots of S. cordifolia can be considered as the potential source of polyphenols and antioxidants.

Evaluating Nothapodytes nimmoniana population from three localities of Western Ghats using camptothecin as phytochemical marker and selection of elites using a new-content range chart method.

BACKGROUND: Nothapodytes nimmoniana (Grah.) Mabb. is a high valued medicinal plant endemic to Western Ghats of India, distributed in fragmented populations. The plant is valued for potent anticancer drug camptothecin (CPT). OBJECTIVE: The study compares and expounds variation in CPT content from leaves and stems of N. nimmoniana obtained from three populations of Western Ghats, India. The study also describes a method for categorizing these populations using content range chart (CRC) method for percent yield of CPT. MATERIALS AND METHODS: A total of 60 samples were investigated including ten each of leaves and stems from three localities. Micro-extraction method was implemented to extract CPT. reversed phase ultra-performance liquid chromatography photo diode array technique was used to quantify CPT. RESULTS: Leaf samples of an individual collected from Joida, yielded lowest CPT content (0.002 ± 0.000 g/100 g), whereas a stem sample from Amgaon, yielded highest CPT content (0.123 ± 0.006 g/100 g). The findings suggest great variation in individuals producing and accumulating CPT. Using this data along with earlier published work, five categories of CPT yielding plants were made viz. I: Very low: <0.020, II: Low: 0.021-0.039, III: Moderate: 0.040-0.059, IV: High: 0.060-0.079 and V: Very high: >0.080. Based on CPT content in leaves, majority of individuals were under very low category (I(st)) and on the other hand stem samples were in 'II' category. Besides, very few individuals were observed in category 'V'. CONCLUSION: The study expounds use of CRC method for identifying elite population and suggests the need for its conservation.

Accumulation and trends in distribution of three triterpenoids in various parts of Achyranthes coynei determined using RP-UFLC analysis.

BACKGROUND: Achyranthes coynei Sant. (Family: Amaranthaceae) is a rare endemic medicinal plant used by local traditional practitioners to treat various diseases. The plant has been reported for promising antibacterial and antioxidant activities. However, the plant is not explored for its phytocompounds, especially triterpenoids. OBJECTIVE: TO STUDY THE ACCUMULATION AND TRENDS IN DISTRIBUTION OF TRITERPENOIDS: betulinic, oleanolic and ursolic acids (BA, OA and UA) in leaf, stem, root and inflorescence of A. coynei. MATERIALS AND METHODS: Extraction was achieved using refluxing and reversed phase-ultra flow liquid chromatographic (RP-UFLC) technique was employed for determination. Separation of triterpenoids was achieved on a Hibar 250-4.6 mm, 5 µ, Lichrospher 100, C18e column using methanol and water (90:10) as mobile phase (pH adjusted to 5.0 using GAA) in an isocratic mode. RESULTS: Oleanolic acid was higher in leaf (0.172 ± 0.009%) followed by stem (0.035 ± 0.002%) and root (0.028 ± 0.001%). Ursolic acid was accounted to be highest in the inflorescence (0.099 ± 0.005%). The contents of BA and UA were lower than OA in leaf and stem while it was remained undetected in roots of A. coynei. CONCLUSION: THE TRITERPENOIDS: BA, OA and UA were detected, quantified and reported for the first time from A. coynei. In the present study leaves were found to be the major source of BA and OA, whereas inflorescence was for UA.

Non-codified traditional medicine practices from Belgaum Region in Southern India: present scenario.

BACKGROUND: Traditional medicine in India can be classified into codified (Ayurveda, Unani, Siddha, Homeopathy) and non-codified (folk medicine) systems. Both the systems contributing equally to the primary healthcare in India. The present study is aimed to understand the current scenario of medicinal practices of non-codified system of traditional medicine in Belgaum region, India. METHODS: The study has been conducted as a basic survey of identified non-codified traditional practitioners by convenience sampling with semi structured, open ended interviews and discussions. The learning process, disease diagnosis, treatment, remuneration, sharing of knowledge and socio-demographic data was collected, analysed and discussed. RESULTS: One hundred and forty traditional practitioners were identified and interviewed for the present study. These practitioners are locally known as "Vaidya". The study revealed that the non-codified healthcare tradition is practiced mainly by elderly persons in the age group of 61 years and above (40%). 73% of the practitioners learnt the tradition from their forefathers, and 19% of practitioners developed their own practices through experimentation, reading and learning. 20% of the practitioners follow distinctive "Nadi Pariksha" (pulse examination) for disease diagnosis, while others follow bodily symptoms and complaints. 29% of the traditional practitioners do not charge anything, while 59% practitioners receive money as remuneration.Plant and animal materials are used as sources of medicines, with a variety of preparation methods. The preference ranking test revealed higher education and migration from villages are the main reasons for decreasing interest amongst the younger generation, while deforestation emerged as the main cause of medicinal plants depletion. CONCLUSION: Patrilineal transfer of the knowledge to younger generation was observed in Belgaum region. The observed resemblance in disease diagnosis, plant collection and processing between non-codified traditional system of medicine and Ayurveda require further methodical studies to establish the relationship between the two on a more objective basis. However, the practice appears to be at crossroads with threat of extinction, because of non-inheritance of the knowledge and non-availability of medicinal plants. Hence conservation strategies for both knowledge and resources at societal, scientific and legislative levels are urgently required to preserve the traditional wisdom.

Compound Specific Extraction of Camptothecin from Nothapodytes nimmoniana and Piperine from Piper nigrum Using Accelerated Solvent Extractor.

Effects of varying temperatures with constant pressure of solvent on extraction efficiency of two chemically different alkaloids were studied. Camptothecin (CPT) from stem of Nothapodytes nimmoniana (Grah.) Mabb. and piperine from the fruits of Piper nigrum L. were extracted using Accelerated Solvent Extractor (ASE). Three cycles of extraction for a particular sample cell at a given temperature assured complete extraction. CPT and piperine were determined and quantified by using a simple and efficient UFLC-PDA (245 and 343 nm) method. Temperature increased efficiency of extraction to yield higher amount of CPT, whereas temperature had diminutive effect on yield of piperine. Maximum yield for CPT was achieved at 80°C and for piperine at 40°C. Thus, the study determines compound specific extraction of CPT from N. nimmoniana and piperine from P. nigrum using ASE method. The present study indicates the use of this method for simple, fast, and accurate extraction of the compound of interest.

Ethnomedicinal plants used to treat bone fracture from North-Central Western Ghats of India.

ETHNOPHARMACOLOGICAL RELEVANCE: North Central Western Ghats in India comprises rich bio-cultural diversity and is also home to varied ethnomedicinal practices. The study was taken up for documentation and analysis of traditional knowledge regarding the practice and use of plants in the treatment of bone fracture. This is an effort to preserve the vanishing wealth of traditional knowledge. MATERIALS AND METHODS: Key informants identified in a preliminary survey and collection of information was through semi structured, open ended interviews. The details on age, place of practice, experience of key informants and learning of practice, disease they treat and mode of diagnosis, storage and usage of plants were collected. The identity of plants and their information was confirmed through repeated guided transect walks in different seasons with the informants and focus group discussions. Identified plants were deposited at the herbaria of Regional Medical Research Centre, Belgaum. RESULTS: Forty four key informants providing treatment for bone fracture in this region were identified. Thirty eight plant species belonging to 24 families have been documented in the present study. Highest number of species representation is found in families Euphorbiaceae and Fabaceae. The habit of the species showed that 45% of the herbal drugs were obtained from trees, followed by herbs, shrubs and climbers and majority of plants used were collected from wild (76%). The stem or stem bark (33%) was most commonly used plant part to prepare medicine. Twenty six formulations of 30 plant species were directly used in treating bone fracture, where Cissus quadrangularis has the highest use-value (0.14). Eleven plants were found to be administered for bone strengthening, pain relieving, inflammation reduction and speedy recovery and Gmelina arborea has the highest use value (0.27). CONCLUSIONS: The results indicated the importance of traditional herbal practices in community for their health needs. Both conservational strategies and further validation studies are the need of the hour for better utilization and sustenance of the documented knowledge.