Antiplasmodial diterpenoid alkaloid from Aconitum heterophyllum: Isolation, characterization, and UHPLC-DAD based quantification

Aconitum heterophyllum is a traditionally important medicinal plant having numerous therapeutic actions as documented in Ayurveda. This plant has been used alone as well as in combination with other plants for the preparation of different anti-malarial formulations. However, there is no report on the assessment of its anti-plasmodial activity, and the compound responsible for this activity. The main aim of this study was to conduct phytochemical investigation of A. heterophyllum roots for the preparation of extract, fractions and isolation of pure molecules to identify active fractions/molecules responsible for the anti-plasmodial activity, and development of UHPLC-DAD based analytical method which can be used for the quantification of marker compounds in the extracts and fractions. Hydro-alcoholic extract (1:1 v/v) and fractions (n-hexane, chloroform, ethyl acetate, n-butanol and water) were prepared from the dried powdered roots of A. heterophyllum. Fractions were further subjected to silica gel-based column chromatography to isolate pure specialized secondary metabolites from this plant. All extracts, fractions and pure molecules were evaluated against the chloroquine resistant Pf INDO and chloroquine sensitive Pf3D7 strains in culture for calculating their IC50 values. UHPLC-DAD based analytical method was also developed for the first time for the quantification and quality assessment of this commercially important Himalayan medicinal plant. Phytochemical investigation of A. heterophyllum root led to the isolation of six specialized metabolites named as 2-O-cinnamoyl hetisine (1), atisinium (2), 4-oxabicyclo [3.2.2] nona-1(7),5,8-triene (3), atisinium cinnamate (4), aconitic acid (5), and atisinium formate (6). Compound 1 is a new hetisine type diterpenoid alkaloid, compounds 4 and 6 are new counter ionic forms observed with atisinium ion, and compound 3 is being reported for the first time from this genus. Chloroform fraction was found to be the most active with IC50 (µg/mL) 1.01 (Pf INDO) and 1.32 (Pf3D7). The isolated molecule 2-O-cinnamoyl hetisine (1), a new diterpenoid alkaloid isolated from chloroform fraction, showed promising antiplasmodial activities with IC50 (µM) 1.92 (Pf INDO) and 10.8 (Pf 3D7). Activity of chloroform fraction was further validated by the developed UHPLC-DAD based method as the quantity of 2-O-cinnamoyl hetisine (1) was higher in the chloroform fraction (≅200 µg/mL) than in all other fractions (< 7µg/mL). Atisinium (2) and 2-O-cinnamoyl hetisine (1) were found to be the main marker compounds of this plant based on quantity and antiplasmodial activity, respectively. This study provides the scientific rational for the traditional use of this plant in treating malaria. Further, this study revealed that anti-malarial potential of this plant might be due to the presence of diterpenoid alkaloids.

Aconitum heterophyllum-An Overview

Aconitum heterophyllum also known as aruna, family Ranunculaceae is used in ayurvedic formulations for the treatment of diarrhoea, hepatic issues etc. The phytochemicals in this plant consists of alkaloids, proteins, carbohydrates, saponins, amino acids, quinones, flavonoids, glycosides, terpenoids, etc. In the present paper, not only are the various therapeutic properties of this plant has been discussed but the profiling of its toxicological studies done on rats have also been elaborated upon. Unlike other species of Aconitum, Aconitum heterophyllum has shown beneficial activity on a rat model of toxicity. Firstly, the ethanolic extract of the plant was evaluated for its oral toxicity but it came out be safe. The ethanolic extract of the plant reversed the activity of castor oil. Moreover, this plant has shown good anti-bacterial activity. After summing up of all the pharmaceutical features of this plant, it could turn out to be valuable source of knowledge for the academia, practioners of traditional medicines & for the pharma industry.

Aconitum heterophyllum Wall. ex Royle; an endemic, highly medicinal and critically endangered plant species of Northwestern Himalaya in peril

: Aconitum heterophyllum is a precious endemic medicinal plant of Northwestern Himalaya. It possesses a number of curative effects and is accounted for having diuretic, hepatoprotective, antipyretic, analgesic, antioxidant, alexipharmic, anodyne, anti-atrabilious, expectorant, immunostimulant, febrifuge, anthelminthic, anti- cancerous, anti-diarrhoeal, anti-emetic, anti-inflammatory, anti-flatulent, anti-periodic, anti-phlegmatic, anti-diabetic, antifungal, antimicrobial, antiviral and carminative properties. Further, it is an important ingredient of many Ayurvedic formulations. Its pharmacological potential may be attributed to of the presence of many biologically active phytochemicals like aconitine, mesaconitine, acetylaconitine, heterophylline A, and heterophylline B. Owing to its high therapeutic uses and market value, the plant is being exploited for its tubers from the wild. Further, there are some reproductive constraints due to which the population of the plant in the wild is waning at a rapid rate. Due to these factors, Aconitum heterophyllum has been categorized as critically endangered; demanding focused conservation strategies and cultivation efforts.

Inventorization of traditional ethnobotanical uses of wild plants of Dawarian and Ratti Gali areas of District Neelum, Azad Jammu and Kashmir Pakistan

Background An ethnobotanical expedition was conducted to document the traditional ethnobotanical (TEB) uses of wild flora of Dawarian and Ratti Gali villages of District Neelam, Azad Jammu and Kashmir (AJK) Pakistan. District Neelam has rich plant diversity and is hub of many endemic plant species while the study areas are not yet explored. The research area: Dawarian and Rati Gali (DRG) area is mountaineous terrain and villages are located on far and farther distances. DRG area has rich biocultural and plant diversity comprising of different ethnic groups of Kashmir state. The current research was aimed to explore and document traditional medicines (TEMs) and other domestic and commercial uses of wild plants. This study will assist to evaluate conservation and commercial worth of wild flora which can be potential candidate for drug discovery through ethnopharmacological analysis. Methods The current quantitative ethnobotanical research was carried out in 2018 by interviewing 150 indigenous informants (90 male and 60 female) of DRG area using questionnaire applying structured and semi structured interview methodology. Data analysis was analyzed by using quantitative ethnobotanical statistical tools such as fidelity level (FL), informant consensus factor (ICF), Spearman’s rank correlation (SRC) and data matrix ranking (DMR). Results The indigenous people of DRG area use wild plants in their daily life to cope life necessities i.e. food, vegetables, fodder, fuel, shelter, timber and herbal medicines. TEMs are primarily used to cure different infirmities like diabetics, asthma, dysentery, constipation, cold, fever, joint pain, wound healing, cancer, cardiovascular disorders, epilepsy, kidney infections and many types of skin diseases. Current study revealed the data of 103 wild plants species belonging to 46 plant families from selected areas of District Neelum, AJK. Results depicted that Asteraceae ranked 1st (12 plants spp). Among plant parts used leaf ranked 1st (18%), followed by seed (17%) and root (13%). While prevalent form recipe mode was decoction (20%), followed by powder (17%) and extract (14%) and fodder was highest (37%) EB use-form fodder, followed by food (32%) and fuel (17%). Quantitative ethnobotanical analysis (QEA) was carried to find the reliability and novelty of the study. Five plant species including Berberis lyceum (FL = 97.78%), Isodon rugosus (FL = 95.71%), Saussurea lappa (FL = 94.74%), Aconitum heterophyllum (FL = 92.71%) and Taxus baccata (91.58%) had shown high fidelity level which confirmed that these plants have high medicinal worth in study area. The highest value (0.94) of ICF was for diseases group “tuberculosis and leucorrhea”, followed by stomachache and flatulence (0.93), diabetics and blood pressure (0.92) and asthma and chest infections (0.88). For other uses fuel with ICF (0.83) ranked first and second was hedging and thatching (ICF = 0.82) where people use plants or their parts for construction. Spearman’s rank correlation (SRC) test indicated that number of TEB uses increases if number of species is increased. Jaccard index (JI) analysis depicted that 56.31% plants are being used as TEMs which are first time explored from the study area. While 26.21% plants are being used in different TEB uses which are different from past cited literature. These novel findings of research indicate that wild flora of the study area has great potential for novel drug discovery and provision of materialist services for the indigenous communities. Conclusion The present research revealed that TEMs uses of 58 plants are novel being first time reported from the study area (DRG) of District Neelam of AJK. The results showed that plants like Acer cappadocicum, Ajuga bracteosa and Swertia paniculata are used to cure diabetes, Viscum album, Viola canescens, Taxus baccata are used for cure of cancer, Isodon rugosus, Polygala chinensis are used in TEMs for treating cardiovascular disorders and Anaphalis triplinervis is used for epilepsy. Berberis lyceum, Ajuga bracteosa, Aconitum heterophyllum, Bistorta amplexicaule, Saussurea lapa and Jurinea dolomiaea are severely threatened and there is urgent need to do conservation measures for available of valuable MPs to the indigenous communities for life necessities and for future research. The current study will also be useful addition in ethnobotanical database, preservation of traditional culture and drug discovery and drug development through future ethnopharmacological research.

Pharmacognostical and Phytochemical Evaluation of Aconitum heterophyllum Root

The present work aims to study the pharmacognostical and phytochemical characteristics of the roots of Aconitum heterophyllum. The study deals with the macroscopy, microscopy, powder analysis, physicochemical standardization, and preliminary phytochemical screening of Aconitum heterophyllum roots. Anatomical studies of the tuberous roots of Aconitum heterophyllum showed distinct endodermis, a wide zone of secondary phloem, and a ring of 4 to 9 vascular strands. The powder analysis revealed the presence of suberized cells of metaderm, reticulate xylem vessels, and starch grains. Physicochemical standardization such as moisture content, ash values, and extractive values of Aconitum heterophylluym was determined. For the identification of various chemical constituents, preliminary phytochemical screening was also studied.

PRELIMINARY COMPARATIVE PHARMACOGNOSTIC STUDY OF GENUINE AND ADULTERANT RAW MATERIAL IN ATIVISHA (ACONITUMHETEROPHYLLUM WALL. EX ROYLE)

Ativisha consists of the dried tuberous root of the aconitum heterophyllum wall. ex royle of ranunculaceae family. a perennial herb native and endemic to western himalayas. it is popular drug that is extensively used in ayurvedic pediatric medicine, specially used in respiratory disorders, fever, diarrhoea and vomiting in children. the fact that the suppliers of crude herbal materials are mostly traders having limited knowledge of their true identity. due to unavailability and high cost, they use their substitutes and adulterants in order to get more and more benefits. children's being more vulnerable, special care has to be taken in selecting the drugs. this paper includes preliminary pharmacognostical but very important aspects to ensure the standard and quality assurance of the raw drug of ativisha. preliminary comparative pharmacognostic study of genuine and adulterant raw material in ativisha. here the sample genuine ativisha with both small and big sized starch grains and taste is bitter followed by salivation in the mouth it may official ativisha. where the other sample with big starch grains with sweet taste may be an adulterated market sample sold under the name of ativisha.

Formulation, Evaluation and Comparison of the Poly Herbal Anti-Diabetic Tablet with the Commercial Tablets

Objective: To formulate a poly herbal anti-diabetic tablet and to evaluate and compare its physicochemical properties with the marketed herbal tablets. Materials and Methods: The poly herbal anti-diabetic tablet was formulated by adding the powder of extract of Enicostemma littorale in powder of roots of Aconitum heterophyllum rhizomes of Picrorhiza kurroa and fruits of Piper longum in different proportions to an aqueous 5 % Starch solution and Several tests such as visual inspection, ash values, moisture content, Water soluble extractive value and Alcohol soluble extractive value, disintegration time, Uniformity of weight of tablets, Determination of hardness of tablets, Determination of friability of tablets etc and Preliminary phytochemical screening and qualitative chemical examination were performed and compare Laboratory formulated tablets with commercial tablets. Results: The study showed that Laboratory formulated poly herbal anti-diabetic tablet has good flow property and compressibility. The moisture content of laboratory formulations was found to be 4.8%. Water extractive value was found to be 28.14% W/V result shows that laboratory formulation consisted of higher amount of water soluble substances like carbohydrates. Alcohol extractive value was found to be 20.08% W/V denotes the amount of alcohol soluble constituents present in the formulation. Disintegration of tablet was found to be 3 mins results shows laboratory formulation was within the limit as it was prepared with starch paste (5%w/v) as a binding and disintegrating agent. Disintegration of tablet is not more than 30 minutes. The laboratory formulation was found to have 3 (Kg/Cm2) hardness, 0.38 % Friability and weight variation within pharmaceutical limits. However, further research and development is required to improve its quality and safety. Conclusion: The aim of this study was to formulate a completely poly herbal antidiabetic tablet. Our Laboratory formulated tablets showed comparable good results as compare to that of marketed tablets but further research and development is required to improve its overall quality.