Phytochemical, Microscopic and Standardization of Bergenia Ciliata for Authentication

2019J. Pl. Res. Vol. 17, No. 1, pp 118-124, 2019Journal of Plant Resources Vol.17, No. 1

Phytochemical, Microscopic and Standardization of Bergenia ciliata for Authentication

Laxman Bhandari1*, Bal Bahadur Bista2 and Chetana Khanal1 1Natural Products Research Laboratory, Department of Plant Resources, Thapathali, Nepal 2Department of Plant Resources, Thapathali, Nepal *E-mail: [email protected]

Abstract

The present research was conducted to investigate the Bergenia ciliata (Haw.) Sternb. (Family- Saxifragaceae) for pharmacognostic study including macroscopical and microscopical observations, UV-fluorescence characters, preliminary phytochemical screening and thin layer chromatography of methanolic extracts. The macroscopy revealed that rhizome was barreled shaped, fibrous fracture surface, brown in color, solid rough texture, pleasant in odor and astringent in taste. While microscopy of rhizome showed typical dicot histological differentiation, no endodermis and pericycle seen. Vascular bundles are arranged in ring. Tanniferous cells are seen abundantly in a cortical region and pith. Rhizome powder has rosette crystals, starch grains, and vessels with scalariform thickenings. The physico-chemical parameters of rhizome powder showed loss on drying 7.04, total ash value 10.23, acid insoluble ash 0.11, water soluble ash 0.92, pH at 10% water solution is basic, water soluble extractive value 74.12, alcohol soluble extractive value 53.23 and extractive value of methanolic extracts 4.234 g. Phytochemicals reveled the presences of flavonoids, terpenoids, tannins, phenols and fatty acids and Rf value 0.74 in Co- TLC with standard bergenin in methanolic extracts. Fluorescence study indicates different colors like dark brown, red, brown black, creamy black, dark black, dark brownish black etc. that revealed the presence of different fluorescent chemical compounds. The rhizome of this plant has a high reputation in indigenous systems of medicine for various medicinal properties so the current research was done in Nepal and might be beneficial for public on accurate identification, in authentication intentional and unintentional adulteration in raw materials of the sample, quality assurance and will give a basis for its phyto-pharmacognostic characterization of the plants.

Keywords: Co-TLC, Microscopic evaluation, Phytochemical screening, UV-Fluorescence

Introduction 30 cm or more, turning bright red in autumn fringed with long bristle like hairs, leaf stalk short. Flowers Bergenia ciliata in sanskrit: Pashanbheda (Medicinal are white, pinkish or purple in spreading or dense Plants of Nepal, 2007) is a plant species in the clusters borne on a stout leafless stem. Fruits capsule, genus Bergenia is a small perennial rhizomatous 2 mm long (Ghimire, 2014; Pokhrel, 2014).The root creeping herb of family saxifragaceae found is bitter and acrid (Ahmad, 2018). The flowering throughout the temperate himalayans at an altitude season of this plant is March to April. (Khanal, 2018; of 900-3000m (Khan, 2017; Khanal, 2018). It was Ahmad, 2018).The root is used as a tonic in the long since known for its use in Ayurveda and has treatment of fevers, diarrhea and pulmonary been used as medicines for the treatment of different affections and juice is used to treat coughs and colds, human ailments (Khan, 2016). Bergenin, catechin, asthma and urinary problems (Rafi, 2017). It is also gallic acid, tannic acid, gallicin, catechin-7-O- considered helpful in relieving backache (Rajkumar,  glucoside, limonene, afzelechin, arbutin, and - 2010). Besides its uses toxicity of this plant is sitosterol can be found in B.ciliata as a major reported (Ahmad, 2018). chemical constituents (Biradar, 2016; DPR, 2016). It is found on rocks ledges with stout rootstock The objectives of present study were microscopic (Ghimire, 2014). Leaves are simple, large with ovate study of plant materials and standardization of or rounded blade, 5 - 15 cm long but enlarging up to methanolic extract of these plants which is helpful

118 2019J. Pl. Res. Vol. 17, No. 1, pp 118-124, 2019Journal of Plant Resources Vol.17, No. 1 2019 Journal of Plant Resources Vol.17, No. 1

Phytochemical, Microscopic and Standardization of to identification of the plants parts and powder with freshly prepared acids, alkaline solution, Bergenia ciliata for Authentication material in public analysis. reagents and solvents and subjected to day light, short UV light 254nm and long UV light 366 nm for Laxman Bhandari1*, Bal Bahadur Bista2 and Chetana Khanal1 Materials and Methods visualization of color. 1Natural Products Research Laboratory, Department of Plant Resources, Thapathali, Nepal 2Department of Plant Resources, Thapathali, Nepal Plant material Soxhlet extraction *E-mail: [email protected] The plant material was collected in an appropriate 20 g each dried powder plant was extracted with Abstract stage of its growth from Pokhara, Kaski district near 250 ml methanol on Soxhlet Apparatus until the to Ghandruk Western part of Nepal in the month of solution of the sample in apparatus is colorless. The The present research was conducted to investigate the Bergenia ciliata (Haw.) Sternb. (Family- April. Plant material was authenticated through solution were evaporated in Rotatory Evaporator Saxifragaceae) for pharmacognostic study including macroscopical and microscopical under reduced pressure to get the viscous extract observations, UV-fluorescence characters, preliminary phytochemical screening and thin layer detail taxonomical study with compare to authentic chromatography of methanolic extracts. The macroscopy revealed that rhizome was barreled literatures and air- dried for study. Dried plant sample which was collected and dried for phytochemical shaped, fibrous fracture surface, brown in color, solid rough texture, pleasant in odor and was washed, cleaned, powdered in blender and screening and thin layer chromatography. astringent in taste. While microscopy of rhizome showed typical dicot histological differentiation, stored in air tight container for study of powder no endodermis and pericycle seen. Vascular bundles are arranged in ring. Tanniferous cells are microscopy, fluorescence, quality testing, extraction, Standardization for Quality Testing seen abundantly in a cortical region and pith. Rhizome powder has rosette crystals, starch grains, and vessels with scalariform thickenings. The physico-chemical parameters of rhizome powder thin layer chromatography profile and qualitative Total Ash: 2 g of sample was incarnated without showed loss on drying 7.04, total ash value 10.23, acid insoluble ash 0.11, water soluble ash 0.92, phytochemical screening. flaming at muffle furnace at 600ºc till residue is free pH at 10% water solution is basic, water soluble extractive value 74.12, alcohol soluble extractive from carbon (2 hrs.). The residue was cooled in value 53.23 and extractive value of methanolic extracts 4.234 g. Phytochemicals reveled the Macroscopic and organoleptic observations desiccators and amount of total ash is determined presences of flavonoids, terpenoids, tannins, phenols and fatty acids and Rf value 0.74 in Co- Z  X TLC with standard bergenin in methanolic extracts. Fluorescence study indicates different colors Macroscopic evaluations were carried out through by weighing. Percentage of total ash = x100 like dark brown, red, brown black, creamy black, dark black, dark brownish black etc. that revealed appearance, texture and fracture surface in both fresh Y  X the presence of different fluorescent chemical compounds. The rhizome of this plant has a high and dry states. Similarly organoleptic characters were where x = wt. of empty crucible, Y= wt. of crucible reputation in indigenous systems of medicine for various medicinal properties so the current identified through color, odor and taste of the sample with sample, Z= wt. of crucible with ash. research was done in Nepal and might be beneficial for public on accurate identification, in powder. authentication intentional and unintentional adulteration in raw materials of the sample, quality Acid Insoluble Ash: 25 mL of hydrochloric acid assurance and will give a basis for its phyto-pharmacognostic characterization of the plants. (about 70 g/L) was added to the crucible containing Histological evaluation the total ash, covered with watch glass and boiled Keywords: Co-TLC, Microscopic evaluation, Phytochemical screening, UV-Fluorescence Anatomy of fresh plant rhizome was studied through gently for 5 minutes. The watch glass was rinsed microscope by making permanent slide of transverse with 5 mL of hot water and added to crucible. The Introduction 30 cm or more, turning bright red in autumn fringed section of rhizome according standard method. The insoluble matter was collected on an ash less filter with long bristle like hairs, leaf stalk short. Flowers sections were observed through compound paper (Whatsmann 41) and washed with hot water Bergenia ciliata in sanskrit: Pashanbheda (Medicinal are white, pinkish or purple in spreading or dense microscope fitted with camera and photographs were until the filtrate was neutral. The filter-paper Plants of Nepal, 2007) is a plant species in the clusters borne on a stout leafless stem. Fruits capsule, taken. containing the insoluble matter was transferred to genus Bergenia is a small perennial rhizomatous 2 mm long (Ghimire, 2014; Pokhrel, 2014).The root the original crucible, ignited by gradually increasing creeping herb of family saxifragaceae found is bitter and acrid (Ahmad, 2018). The flowering Powder Microscopy the heat to 550ºC for 3 hours in the muffle furnace throughout the temperate himalayans at an altitude season of this plant is March to April. (Khanal, 2018; The powder was subjected in chemicals for analysis. to the constant weight. The residue was allowed to of 900-3000m (Khan, 2017; Khanal, 2018). It was Ahmad, 2018).The root is used as a tonic in the At first sufficient amount of powder was taken in cool in suitable desiccators for 30 minutes, and then long since known for its use in Ayurveda and has treatment of fevers, diarrhea and pulmonary chloral-hydrate solution on a slide, covered with weigh without delay. been used as medicines for the treatment of different affections and juice is used to treat coughs and colds, cover slip and was left for 18 hours and finally human ailments (Khan, 2016). Bergenin, catechin, Water Soluble Ash: 25 mL of water was added to asthma and urinary problems (Rafi, 2017). It is also observe under microscope. Starch test was also done gallic acid, tannic acid, gallicin, catechin-7-O- the crucible containing the total ash, covered with considered helpful in relieving backache (Rajkumar, with iodine solution.  the watch glass and boiled for 5 minutes. Insoluble glucoside, limonene, afzelechin, arbutin, and - 2010). Besides its uses toxicity of this plant is matter was collected on an ash less filter paper. sitosterol can be found in B.ciliata as a major reported (Ahmad, 2018). Fluorescence analysis in dried powder chemical constituents (Biradar, 2016; DPR, 2016). Washed with hot water and ignited in crucible for It is found on rocks ledges with stout rootstock The objectives of present study were microscopic The fluorescence analysis of dried powder of 15 minutes at temperature exceeding 450ºC in a (Ghimire, 2014). Leaves are simple, large with ovate study of plant materials and standardization of rhizome was carried out by treating with various muffle furnace. Allowed the residue to cool in or rounded blade, 5 - 15 cm long but enlarging up to methanolic extract of these plants which is helpful chemicals. 1 g of the sample powder was treated suitable desiccators for 30 min. and weighed without

118 119 2019 Journal of Plant Resources Vol.17, No. 1 delay. The weight of the residue was subtracted in in triplicate mode. The standard operating procedures mg from the weight of total ash. Mathematically, % taken for analysis (Harborne, 1969;Sofowora, 1993). 550ºC for 3 hours in theWt. muffle of water furnace insoluble toash the (g) constant weight. The residue was allowed to cool in of water insoluble ash = suitable desiccators for 30 minutes,Wt. of sample and taken then (g) weigh withoutThin layerdelay. chromatography (TLC Profiling) X 100 and % of water soluble ash = Test method operated was Co-TLC with standard Water Soluble Ash: 25 mL of water was added to the crucible containing the total ash, covered with the Wt. of total ash (g) water insoluble ash (g) Bergenin and sample extract with standard extract watch glass and boiled for 5 minutes. Insoluble matter was collected on an ash less filter paper. Washed X 100. in Solvent System ethyl acetate: formic acid: acetic with hot waterWt. and of sample ignited taken in (g) crucible for 15 minutes at temperature exceeding 450ºC in a muffle acid: water in the ratio (100:11:11:27) and Scan at furnace. Allowed the residue to cool in suitable desiccators for 30 min. and weighed without delay. The 254 nm observe under UV and record the Rf value. Ethanolweight of soluble the residue extractive was subtracted and water in mg soluble from the weight of total ash. Mathematically, % of water extractive: 40 g of powdered material was subjected The TLC plates were TLC 1 and TLC 2 for standard insoluble ash = X 100 and % of water soluble ash = for percolation 200 mL of water and alcohol. After and extract precoated silica gel 60 F254of 0.2mm extraction for 72 hours the solvents extracts were thickness for repeatability. The Standard Solution concentrated and dried in vacuum desiccators.X 100. Then was made by dissolving standard Bergenin, standard the extractive values were calculated as % w/w of extract and sample extract in 1 ml methanol. The solvent soluble extractive with reference to the air procedure was followed from Quality Standards of Indian Medicinal Plants, Indian Council of Medical driedEthanol drug. soluble extractive and water soluble extractive: 40 g of powdered material was subjected for Research, New Delhi, Vol.1, 34-37, 2003. Losspercolation on drying 200 mL (LOD): of water For and the alcohol. 5g of drug After extraction for 72 hours the solvents extracts were concentrated and dried in vacuum desiccators. Then the extractive values were calculated as % w/w of powdered was taken and kept in an oven at 105ºC Results and Discussion tillsolvent a constant soluble weightextractive was with obtained. reference LOD to thein airthe dried drug. sample was calculated as reference to the air dried Loss on drying (LOD): For the 5 g of drug powderedMacroscopic was taken Featuresand kept in an oven at 105º c till a material. constant weight was obtained. LOD in the sample wasOrganoleptic calculated as reference (sensory) to theevaluations air dried material. are only pH range: The pH in 10% (10 g in 100 mL) of water parameters that required no involvement of solublePH range: portions The pH of inwhole 10% powder(10 g in of100 plants mL) wasof waterscientific soluble instrumentsportions of neitherwhole powderany expenses. of plants It gives was determined using standard pH meter. a valuable, simplest, quickest and easiest information regarding purity and quality for recognition of Preliminary Phytochemical Screening adulterants in crude drugs. The extracts were used for the preliminary The extracts were used for the preliminary phytochemical analysis. All the tests were performed in phytochemicaltriplicate mode. analysis. The standard All the operating tests were procedures performed taken for analysis (Harborne, 1969;Sofowora, 1993).

Table 1:1: TestTest Procedure Procedure of of Phytochemical Phytochemical Screening Screening Test Procedure/ Methods of phytochemical analysis Volatile Oils Methanolic solution of extracts was put on filter paper by means of capillary tube. Visualize. Alkaloids Test solution was tested with 2-3 drops of potassium mercuric iodide Flavonoids Test solution was tested with Mg metal and 5-6 drops of conc. HCl Steroids 1 mL of extracts were dissolved in 10 mL of chloroform and equal volume of conc. H 2SO4 was added by sides of the test tube Terpenoids Crude alcoholic extracts was dissolved in 2 mL chloroform and 3 mL conc. H2SO4 and heated for 2 minutes. Tannins/ Phenols To 0.5 mL of alcoholic extract add 1 mL water and 2-3 drops of 0.1% FeCl3 . Reducing Sugar 0.5 mL extract solution was added with 1 mL water acidified with dil. HCl, neutralized with alkali and heated with 0.5 mL Fehling solution A + B gently. Glycosides The extract was mixed with 2 mL chloroform, H2SO4 was added carefully and shaken gently. Saponins Extracts were diluted with distilled water to 20 mL and this was shaken in a graduated cylinder for 15 minutes. Protein Crude extract boiled with 2 mL of 025% w/v solution of Ninhydrin. Carbohydrate Filtrates were treated with 2 drops of alcoholic alpha napthol solution in a test tube. Shaken and add conc. sulphuric acid from side of the test tube.

120 Thin layer chromatography (TLC Profiling)

Test method operated was Co-TLC with standard Bergenin and sample extract with standard extract in Solvent System ethyl acetate: formic acid: acetic acid: water in the ratio (100:11:11:27) and Scan at 254 nm observe under UV and record the Rf value. The TLC plates were TLC 1 and TLC 2 for standard and extract precoated silica gel 60 F254of 0.2mm thickness for repeatability. The Standard Solution was made by dissolving standard Bergenin, standard extract and sample extract in 1 ml methanol. The procedure was followed from Quality Standards of Indian Medicinal Plants, Indian Council of Medical Research, New Delhi, Vol.1, 34-37, 2003.

Results and Discussion Macroscopic Features Organoleptic (sensory) evaluations are only parameters that required no involvement of scientific instruments neither any expenses. It gives a valuable, simplest, quickest and easiest 2019 Journal of Plant Resources Vol.17, No. 1 2019 Journal of Plant Resources Vol.17, No. 1 information regarding purity and quality for recognition of adulterants in crude drugs. delay. The weight of the residue was subtracted in in triplicate mode. The standard operating procedures TableTable 2 :2 Macroscopic: Macroscopic features features (organoleptic (organoleptic evaluation) evaluation) of of Bergenia Bergenia ciliata ciliata (Haw.) (Haw.) Sternb Sternb rhizome rhizome mg from the weight of total ash. Mathematically, % taken for analysis (Harborne, 1969;Sofowora, 1993). Rhizome S.N. Parameter studied 550ºC for 3 hours in theWt. muffle of water furnace insoluble toash the (g) constant weight. The residue was allowed to cool in Fresh Dry ofsuitable water desiccatorsinsoluble ash for = 30 minutes, and then weigh withoutThin layerdelay. chromatography (TLC Profiling) 1 Shape barrel shaped Irregular cylindrical Wt. of sample taken (g) 2 Colour Brown Dark brown X 100 and % of water soluble ash = Test method operated was Co-TLC with standard 3 Taste Astringent Indistinct Water Soluble Ash: 25 mL of water was added to the crucible containing the total ash, covered with the Wt. of total ash (g) water insoluble ash (g) Bergenin and sample extract with standard extract 4 Odor Pleasant Indistinct watch glass and boiled for 5 minutes. Insoluble matter was collected on an ash less filter paper. Washed X 100. in Solvent System ethyl acetate: formic acid: acetic 5 Fracture Flexible Hard with hot waterWt. and of sample ignited taken in (g) crucible for 15 minutes at temperature exceeding 450ºC in a muffle acid: water in the ratio (100:11:11:27) and Scan at 6 Fracture surface Fibrous Uneven furnace. Allowed the residue to cool in suitable desiccators for 30 min. and weighed without delay. The 7 Texture solid rough ridge and groves 254 nm observe under UV and record the Rf value. Ethanolweight of soluble the residue extractive was subtracted and water in mg soluble from the weight of total ash. Mathematically, % of water extractive: 40 g of powdered material was subjected The TLC plates were TLC 1 and TLC 2 for standard MicroscopicMicroscopic characters characters Rhizome powder is dark brown in color, it shows insoluble ash = Xand 100 extract and precoated % of silica water gel soluble60 F of ash0.2mm = for percolation 200 mL of water and alcohol. After 254 Transverse section of rhizome shows multilayer of corkmany cells large followed rosette bycrystals, a layer starch of cork grain cambium and vessels and thickness for repeatability. The Standard Solution Transverse section of rhizome shows multilayer of extraction for 72 hours the solvents extracts were secondary cortex. Cortex is composed of parenchymatouswith cells.scalariform Endodermis thickenings. and pericycles are not seen. was made by dissolving standard Bergenin, standard cork cells followed by a layer of cork cambium and concentrated and dried in vacuum desiccators.X 100. Then Vascular bundles are arranged in a ring. They are conjoint, collateral and open. Pith is composed of secondary cortex. Cortex is composed of Preliminary Phytochemical Screening the extractive values were calculated as % w/w of extract and sample extract in 1 ml methanol. The rounded or oval parenchymatous cells. Taniferous cells are seen abundantly both in the cortical and the procedure was followed from Quality Standards of parenchymatous cells. Endodermis and pericycles solvent soluble extractive with reference to the air pith region. Phytochemical screening of the methanolic extract Indian Medicinal Plants, Indian Council of Medical are not seen. Vascular bundles are arranged in a ring. driedEthanol drug. soluble extractive and water soluble extractive: 40 g of powdered material was subjected for of B. ciliata rhizomes revealed the presence of Research, New Delhi, Vol.1, 34-37, 2003. TheyRhizome are conjoint,powder is collateraldark brown and in color,open. itPith shows is many large rosette crystals, starch grain and vessels percolation 200 mL of water and alcohol. After extraction for 72 hours the solvents extracts were various bioactive components of which phenolic, Loss on drying (LOD): For the 5g of drug composedwith scalariform of rounded thickenings. or oval parenchymatous cells. concentrated and dried in vacuum desiccators. Then the extractive values were calculated as % w/w of powdered was taken and kept in an oven at 105ºC Taniferous cells are seen abundantly both in the flavonoids, steroids, tannins, fatty acids, and steroids solvent soluble extractive with reference to the air driedResults drug. and Discussion till a constant weight was obtained. LOD in the cortical and the pith region. are the most prominenta components and the result sample was calculated as reference to the air dried of phytochemical test is presented in Table 3. Loss on drying (LOD): For the 5 g of drug powderedMacroscopic was taken Featuresand kept in an oven at 105º c till a material. constant weight was obtained. LOD in the sample wasOrganoleptic calculated as reference (sensory) to theevaluations air dried material. are only pH range: The pH in 10% (10 g in 100 mL) of water parameters that required no involvement of solublePH range: portions The pH of inwhole 10% powder(10 g in of100 plants mL) wasof waterscientific soluble instrumentsportions of neitherwhole powderany expenses. of plants It gives was determined using standard pH meter. a valuable, simplest, quickest and easiest information regarding purity and quality for recognition of Preliminary Phytochemical Screening adulterants in crude drugs. The extracts were used for the preliminary bc The extracts were used for the preliminary phytochemical analysis. All the tests were performed in phytochemical analysis. All the tests were performed triplicate mode. The standard operating procedures taken for analysis (Harborne, 1969;Sofowora, 1993). Plate 1: T.S. of rhizome of Bergenia ciliata. a. Section showing cork and cortex, b. Section showing pith region and c. Section showing vascular bundle. Table 1:1: TestTest Procedure Procedure of of Phytochemical Phytochemical Screening Screening a bc

Test Procedure/ Methods of phytochemical analysis Plate 1: T.S. of rhizome of Bergenia ciliata. a. Section showing cork and cortex, b. Section showing pith region and c. Section Volatile Oils Methanolic solution of extracts was put on filter paper by means of capillary tube. Visualize. showing vascular bundle. a Alkaloids Test solution was tested with 2-3 drops of potassium mercuric iodide Flavonoids Test solution was tested with Mg metal and 5-6 drops of conc. HCl Steroids 1 mL of extracts were dissolved in 10 mL of chloroform and equal volume of conc. H 2SO4 was added by sides of the test tube Terpenoids Crude alcoholic extracts was dissolved in 2 mL chloroform and 3 mL conc. H2SO4 and heated for 2 minutes. Tannins/ Phenols To 0.5 mL of alcoholic extract add 1 mL water and 2-3 drops of 0.1% FeCl3 . Reducing Sugar 0.5 mL extract solution was added with 1 mL water acidified with dil. HCl, neutralized with alkali and heated with 0.5 mL Fehling solution A + B gently. Glycosides The extract was mixed with 2 mL chloroform, H2SO4 was added carefully and shaken gently. Saponins Extracts were diluted with distilled water to 20 mL and this was shaken in a graduated cylinder for 15 minutes. a bcd Protein Crude extract boiled with 2 mL of 025% w/v solution of Ninhydrin. Carbohydrate Filtrates were treated with 2 drops of alcoholic alpha napthol solution in a test tube. Shaken and Plate 2: Powder characteristics of rhizome of Bergenia ciliata. a. Cork cells, b. Rosette crystals, c. Starch grains and d. Vessels. add conc. sulphuric acid from side of the test tube.

120 121 Thin layer chromatography (TLC Profiling)

Plate 2: Powder characteristics of rhizome of Bergenia ciliata. a. Cork cells, b. Rosette crystals, c. Starch grains and d. Vessels. Preliminary Phytochemical Screening Phytochemical screening of the methanolic extract of B. ciliata rhizomes revealed the presence of various bioactive components of which phenolic, flavonoids, steroids, tannins, fatty acids, and steroids Plate 2: Powder characteristics of rhizome of Bergenia ciliata. a. Cork cells, b. Rosette crystals, c. are the most prominent components and the result of phytochemical test is presented in Table 3. Starch grains and d. Vessels. Table 3: Preliminary phytochemical screening of rhizome of Bergenia ciliata Preliminary Phytochemical Screening S.N. Experiment Test Color Methanolic extracts 1. Volatile oils Spot /Residue test Transparent with no yellow color persist _ Phytochemical screening of the methanolic extract2. of B.Alkaloids ciliata rhizomes Mayers Reagent revealed Test the presence White yellowish of ppt. + 2019various bioactive components of whichJournal phenolic, of Plant flavonoids, 3.Resources Flavonoid steroids, tannins, Shinoda test fatty acids,Vol.17, and No. Pinksteroids 1 Scarlet +++ are the most prominent components and the result of phytochemical4. Steroids test is Steroid presented test in Table 3. Yellow green fluorescence ++ Table 3: Preliminary phytochemical screening of rhizome of Bergenia5. ciliataTerpenoids Terpenoids test A grayish colour +++ Table 3: Preliminary phytochemical screening of rhizome of Bergenia6. Tannins ciliata 0.1% FeCl Test Bluish black/greenish +++ S.N. Experiment Test 7. ReducingColor sugar Fehlings TestMethanolic extractsReddish brick ppt. + 1. Volatile oils Spot /Residue test Transparent8. withGlycosides no yellow color Salkowski’s persist test/ NaoH _ test Reddish brown ++ 2. Alkaloids Mayers Reagent Test White yellowish9. Phenols ppt. Phenolic test + Blue green +++ 3. Flavonoid Shinoda test Pink Scarlet10. Saponins Froth/ Foam Test +++ Foam _ 4. Steroids Steroid test Yellow green11. Proteinfluorescence Ninhydrin Test ++ Violet + 5. Terpenoids Terpenoids test A grayish12. colour Carbohydrate Molish Test +++ Violet ring at junction + 6. Tannins 0.1% FeCl3 Test Bluish b13.lack/greenish Fatty acids +++ +++ 7. Reducing sugar Fehlings Test ReddishIndications: brick ppt. Result + means presence in trace amount+ ++ means in moderate amount and +++ in high amount and – means 8. Glycosides Salkowski’s test/ NaoH test Reddishabsence brown of phytochemicals ++ 9. Phenols Phenolic test Blue green +++ 10. Saponins Froth/ Foam Test Foam Standardization quality testing parameters_ of Bergenia ciliata 11. Protein Ninhydrin Test Violet + 12. Carbohydrate Molish Test Violet ringPhysico-chemical at junction studies of the plant+ drugs are necessary for standardization, as it helps in 13. Fatty acids understanding the significance of physical+++ and chemical properties of the substance being analyzed in Indications: Result + means presence in trace amount ++ meansterms in moderate of their amount observed and +++ activities in high amount and especially and – means for the determination of their purity and quality. The absence of phytochemicals analysis include the determination of ash value, moisture content, pH valueat 10% solution, loss on StandardizationStandardization quality quality testing testing parameters parameters of of BergeniaTabledrying 4:ciliata Someand extractive Quality testing value. Parameters These wereof Bergenia carried out as per guidelines of WHO. ciliata (Haw). Sternb Bergenia ciliata Table 4: Some Quality testing Parameters of Bergenia ciliata (Haw). Sternb Physico-chemical studies of the plant drugs are necessary for standardization, as it helps in S.N. Experiment Result (%) Physico-chemicalunderstanding the studies significance of the ofplant physical drugs and are chemical properties of the substance being analyzed in necessary for standardization, as it helps in 1 Loss of weight on drying 7.04 terms of their observed activities and especially for 2.the determination Total Ash Value of their purity and quality.10.23 The understandinganalysis include the thesignificance determination of physical of ash value,and moisture3. Acid content, insoluble pHash valueat 10% solution,0.11 loss on chemicaldrying and properties extractive of the value. substance These being were analyzed carried out as4. per Waterguidelines soluble ofash WHO. 0.92 in terms of their observed activities and especially 5. pH at 10% water solution Basic forTable the 4: determination Some Quality testingof their Parameters purity and of quality. Bergenia The ciliata (Haw).6 Sternb Water soluble extractive 74.12 analysisS.N. include theExperiment determination of ashResult value, (%) 7. Alcohol soluble extractive 53.23 moisture1 Loss content, of weight pH on valueat drying 10% solution, loss7.04 on 8. Extractive value in methanol solvents 4.234 g drying2. and Total extractive Ash Value value. These were carried10.23 out 3. Acid insoluble ash 0.11 as per4. guidelines Water soluble of ashWHO. 0.92 FluorescenceUV and long Analysis wavelength UV light treated with 5. pH at 10% water solution Basic different chemical showed the presence of Fluorescence6 Water soluble Analysis extractive 74.12 Variationfluorescence color compound under day which light, is used short as wavelengthdiagnostic UV and long wavelength UV light treated with Variation7. Alcohol color soluble under extractive day light, short wavelength53.23 differenttool for testing chemical adulteration showed thein the presence sample of materials. fluorescence compound which is used as diagnostic tool for 8. Extractive value in methanol solvents 4.234 g testing adulteration in the sample materials. TableTable 5:5: FluorescenceFluorescence Analysis Analysis of of Powder Powder of of Bergenia Bergenia ciliata ciliata (Haw). (Haw). Sternb Sternb FluorescenceS.N. Powdered Analysis Rhizome (P.R.) Day light UV short (255nm) UV long (366nm) 1. P.R. + conc. HCl Brown Dark Brown Dark Black Variation2. P.R. + color conc. underH2SO4 day light, shortRed wavelength UV Red and long wavelength Brown UV light treated with different3. P.R. chemical + conc. HNO showed3 the presenceBrown of fluorescence compoundDark Brown which is usedB lackas diagnostic tool for 4. P.R. + Iodine soln Black Brown black creamy black testing5. P.R.adulteration + Acetic acid in the sample materials.pink Red Brown 6. P.R. + Picric acid Dark yellow Brown Black 7. P.R. + FeCl3 Dark brown Black Creamy black 8. P.R. + Ether Dark black Brown Dark brown 9. P.R. + Chloroform Brown Black Black 10. P.R. + NaOH 10% soln Brown Black Dark Black 11 P.R. + Lead acetate Black Brown Dark brown 12 P.R. + Ninhydrin solution Brownish blue Brown Black Brown 13. P.R. + Fehling solution Brownish red Black Dark Black 14. P.R. + Mayer's reagent Brownish white Brown black Dark Black 15. P.R. + CuSO4 5% solution Bluish Black Dark Brownish Black Black

122 Thin layer chromatography (TLC) Co-TLC of methanolic extract of B. ciliata rhizomes revealed the presence of Bergenin in standard and sample extract having Rf values of 0.74 when a solvent phase of Ethyl acetate: Formic Acid: Acetic acid: Water (100:11:11:27) was used. Rf values of sample and reference standard and extract were found to be identical.

Table 6: TLC Details of Methanolic Extract of Bergenia Ciliata Rhizome

Color of the Rf value of the standard Rf value of the Remarks band Bergenin sample extract Black 0.74 0.74 Rf values of sample and reference standard and extract were found to be identical. Sample of extract co-exist with Certified Reference Material (CRM).

Conclusions The present work will be helpful for the correct identification and authentication of crude drug available in Nepalese herbal market. Several other researchers also carried out similar research work on various other medicinal plant and documented similar observation which are in line with the present work and strongly agree with this work. Purity and identity standards in Nepal is yet unknown and highly focused in this matter of issue. Powder sample of B. ciliata is used by public in traditional medicine so its standardization and quality control is necessary. Some adulteration is mixed in the powder and extracts so its proper identification is recently important in Nepal for the benefits of customers and best medicine practice from the plant products in Nepal.

Acknowledgements The authors are to acknowledge Mr. Sanjeev Kumar Rai, Director General and Mrs. Jyoti Joshi Bhatta and Mr. Mohan Dev Joshi, Deputy Director General, Department of Plant Resources and Mr. Devi Prasad Bhandari, Chief, Natural Plant Research Laboratory for their kind support. We are also obliged to all the staff of NPRL and DPR for their co-operation and team work.

References

Table 5: Fluorescence Analysis of Powder of Bergenia ciliata (Haw). Sternb S.N. Powdered Rhizome (P.R.) Day light UV short (255nm) UV long (366nm) 1. P.R. + conc. HCl Brown Dark Brown Dark Black 2. P.R. + conc. H SO Red Red Brown 3. P.R. + conc. HNO Brown Dark Brown Black 4. P.R. + Iodine sol Black Brown black creamy black 5. P.R. + Acetic acid pink Red Brown 6. P.R. + Picric acid Dark yellow Brown Black 7. P.R. + FeCl Dark brown Black Creamy black 8. P.R. + Ether Dark black Brown Dark brown 9. P.R. + Chloroform Brown Black Black 10. P.R. + NaOH 10% sol Brown Black Dark Black Plate 2: Powder characteristics of rhizome of Bergenia ciliata. a. Cork cells,11 b. P.R. Rosette + Lead crystals,acetate c. Black Brown Dark brown Starch grains and d. Vessels. 12 P.R. + Ninhydrin solution Brownish blue Brown Black Brown 13. P.R. + Fehling solution Brownish red Black Dark Black Preliminary Phytochemical Screening 14. P.R. + Mayer's reagent Brownish white Brown black Dark Black 15. P.R. + CuSO 5% solution Bluish Black Dark Brownish Black Black Phytochemical screening of the methanolic extract of B. ciliata rhizomes revealed the presence of various bioactive components of which phenolic, flavonoids, steroids, tannins, fatty acids, and steroids Plate 2: Powder characteristics of rhizome of Bergenia ciliata. a. Cork cells, b. Rosette crystals, c. are the most prominent components and the result of phytochemical testThin is presented layer chromatography in Table 3. (TLC) Starch grains and d. Vessels. Co-TLC of methanolic extract of B. ciliata rhizomes revealed the presence of Bergenin in standard and Table 3: Preliminary phytochemical screening of rhizome of Bergenia ciliata sample extract having Rf values of 0.74 when a solvent phase of Ethyl acetate: Formic Acid: Acetic acid: Preliminary Phytochemical Screening S.N. Experiment Test Color Water (100:11:11:27)Methanolic ewasxtracts used. Rf values of sample and reference standard and extract were found to be 1. Volatile oils Spot /Residue test Transparent with no yellow color persist _ Phytochemical screening of the methanolic extract of B. ciliata rhizomes revealed the presence of identical. 2. Alkaloids Mayers Reagent Test White yellowish ppt. + 2019various bioactive components of whichJournal phenolic, of Plant flavonoids, 3.Resources Flavonoid steroids, tannins, Shinoda test fatty acids,Vol.17, and No. Pinksteroids 1 Scarlet 2019 +++ Journal of Plant Resources Vol.17, No. 1 are the most prominent components and the result of phytochemical4. Steroids test is Steroid presented test in Table 3. Yellow green fluorescence ++ 5. Terpenoids Terpenoids test A grayish colour +++ Table 3: Preliminary phytochemical screening of rhizome of Bergenia ciliata Table 6: TLC Details of Methanolic Extract of Bergenia Ciliata Rhizome Table 3: Preliminary phytochemical screening of rhizome of Bergenia6. Tannins ciliata 0.1% FeCl Test Bluish black/greenish Table 6: TLC Details+++ of Methanolic Extract of Bergenia Ciliata Rhizome S.N. Experiment Test 7. ReducingColor sugar Fehlings TestMethanolic extractsReddish brick ppt. Color of the Rf+ value of the standard Rf value of the Remarks 1. Volatile oils Spot /Residue test Transparent8. withGlycosides no yellow color Salkowski’s persist test/ NaoH _ test Reddish brown band ++ Bergenin sample extract 2. Alkaloids Mayers Reagent Test White yellowish9. Phenols ppt. Phenolic test + Blue green Black +++ 0.74 0.74 Rf values of sample and reference standard and 3. Flavonoid Shinoda test Pink Scarlet10. Saponins Froth/ Foam Test +++ Foam _ extract were found to be identical. Sample of 4. Steroids Steroid test Yellow green11. Proteinfluorescence Ninhydrin Test ++ Violet + extract co-exist with Certified Reference 5. Terpenoids Terpenoids test A grayish12. colour Carbohydrate Molish Test +++ Violet ring at junction + Material (CRM). 6. Tannins 0.1% FeCl3 Test Bluish b13.lack/greenish Fatty acids +++ +++ 7. Reducing sugar Fehlings Test ReddishIndications: brick ppt. Result + means presence in trace amount+ ++ means in moderate amount andConclusions +++ in high amount and – means 8. Glycosides Salkowski’s test/ NaoH test Reddishabsence brown of phytochemicals ++ Thin layer chromatography (TLC) References The present work will be helpful for the correct identification and authentication of crude drug 9. Phenols Phenolic test Blue green +++ Co-TLC of methanolic extract of B. ciliata rhizomes 10. Saponins Froth/ Foam Test Foam Standardization quality testing parameters_ of Bergenia ciliata available in Nepalese herbal market. Several other researchersAhmad, M., also Butt, carried M.A., Zhanga,out similar G., Sultana,research S., work Triq, on 11. Protein Ninhydrin Test Violet + revealedvarious theother presence medicinal of Bergeninplant and indocumented standard and similar observationA. & Zafar, whichM. (2018) are in . Bergenialine with ciliata:the present A 12. Carbohydrate Molish Test Violet ringPhysico-chemical at junction studies of the plant+ drugs are necessary for sample standardization,work extractand strongly having as it agreeRf helps values with in of this 0.74 work. when Purity a andcomprehensive identity standards review in Nepal of its is traditional yet unknown uses, and 13. Fatty acids understanding the significance of physical+++ and chemical properties ofsolvent highlythe substance phase focused of being Ethyl in this analyzedacetate: matter Formic in of issue. Acid: Powder Acetic samplePhytochemistry, of B. ciliata is pharmacology used by public and in traditional safety, Indications: Result + means presence in trace amount ++ meansterms in moderate of their amount observed and +++ activities in high amount and especially and – means for the determinationacid: medicineof theirWater purity so(100:11:11:27) its and standardization quality. was The used. and Rf quality values controlof isBiomedicine necessary. and Some pharmacology, adulteration 97 is , mixed708-721. in the absence of phytochemicals analysis include the determination of ash value, moisture content, pHsamplepowder valueat and and 10% reference extracts solution, standard so loss its properon and extract identification were is recently important in Nepal for the benefits of Biradar, Y.S., & Mahadik, K.R. (2016). StandardizationStandardization quality quality testing testing parameters parameters of of BergeniaTabledrying 4:ciliata Someand extractive Quality testing value. Parameters These wereof Bergenia carried out as per guidelines offound customersWHO. to be identical.and best medicine practice from the plant products in Nepal. ciliata (Haw). Sternb Simulataneous Quantification of Bergenin, Bergenia ciliata Table 4: Some Quality testing Parameters of Bergenia ciliata (Haw). Sternb Physico-chemical studies of the plant drugs are necessary for standardization, as it helps in Acknowledgements Catechin and Gallic acid form Bergenia ciliate S.N. Experiment Result (%) Conclusions Physico-chemicalunderstanding the studies significance of the ofplant physical drugs and are chemical properties of the substance being analyzed in The authors are to acknowledge Mr. Sanjeev Kumar Rai,& BergeniaDirector General ligulata and by Mrs. using Jyoti Thin Joshi Layer Bhatta necessary for standardization, as it helps in 1 Loss of weight on drying 7.04 terms of their observed activities and especially for 2.the determination Total Ash Value of their purity and quality.10.23 The Theand present Mr. Mohan work will Dev be Joshi, helpful Deputy for the Director correct General,Chromatography, Department of PlantJ. Food Resources Comp Ana. and, 6 (2), Mr. 496- Devi understandinganalysis include the thesignificance determination of physical of ash value,and moisture3. Acid content, insoluble pHash valueat 10% solution,0.11 loss on identificationPrasad Bhandari, and Chief,authentication Natural Plant of crude Research drug Laboratory500. for their kind support. We are also obliged to chemicaldrying and properties extractive of the value. substance These being were analyzed carried out as4. per Waterguidelines soluble ofash WHO. 0.92 availableall the staff in Nepalese of NPRL herbal and DPR market. for their Several co-operation other and team work. in terms of their observed activities and especially 5. pH at 10% water solution Basic researchers also carried out similar research work DPR. (2007). Medicinal Plants of Nepal. Table 4: Some Quality testing Parameters of Bergenia ciliata (Haw). Sternb for the determination of their purity and quality. The 6 Water soluble extractive 74.12 onReferences various other medicinal plant and documented Kathmandu, Nepal. Bulletin No. 28, Kathmandu, analysisS.N. include theExperiment determination of ashResult value, (%) 7. Alcohol soluble extractive 53.23 Nepal: Government of Nepal, Department of 1 Loss of weight on drying 7.04 8. Extractive value in methanol solvents 4.234 g similar observation which are in line with the present moisture content, pH valueat 10% solution, loss on work and strongly agree with this work. Purity and Plant Resources. drying2. and Total extractive Ash Value value. These were carried10.23 out 3. Acid insoluble ash 0.11 identity standards in Nepal is yet unknown and DPR. (2016). Medicinal plants of Nepal. 2nd edition. as per guidelines of WHO. UV and long wavelength UV light treated with 4. Water soluble ash 0.92 Fluorescence Analysis highly focused in this matter of issue. Powder sample Kathmandu, Nepal: Government of Nepal, 5. pH at 10% water solution Basic different chemical showed the presence of of B. ciliata is used by public in traditional medicine Fluorescence6 Water soluble Analysis extractive 74.12 Variation color under day light, short wavelength UV and long wavelength UV light treated with Department of Plant Resources. fluorescence compound which is used as diagnostic so its standardization and quality control is necessary. 7. Alcohol soluble extractive 53.23 different chemical showed the presence of fluorescence compound which is used as diagnostic tool for Variation color under day light, short wavelength tool for testing adulteration in the sample materials. Some adulteration is mixed in the powder and Ghimire, B., Ghimire, B.K. & Heo, K. (2014). 8. Extractive value in methanol solvents 4.234 g testing adulteration in the sample materials. extracts so its proper identification is recently Anatomy of the Vegetative Parts of the Bergenin Table 5: Fluorescence Analysis of Powder of Bergenia ciliata (Haw). Sternb Table 5: Fluorescence Analysis of Powder of Bergenia ciliata (Haw). Sternb important in Nepal for the benefits of customers and ciliata (Haw). Sternb: A Potential medicinal Herb. FluorescenceS.N. Powdered Analysis Rhizome (P.R.) Day light UV short (255nm) UV long (366nm) best medicine practice from the plant products in Int. J. Bot., 8(3), 136-144. 1. P.R. + conc. HCl Brown Dark Brown Dark Black Nepal. Variation2. P.R. + color conc. underH2SO4 day light, shortRed wavelength UV Red and long wavelength Brown UV light treated with Harborne J. B. (1969). Phytochemical method: A different3. P.R. chemical + conc. HNO showed3 the presenceBrown of fluorescence compoundDark Brown which is usedB lackas diagnostic tool for guide to modern techniques of plant analysis. n 4. P.R. + Iodine sol Black Brown black creamy black Acknowledgements Phytochem, 8, 419-423. testing5. P.R.adulteration + Acetic acid in the sample materials.pink Red Brown 6. P.R. + Picric acid Dark yellow Brown Black The authors are to acknowledge Mr. Sanjeev Kumar ICMR (2003). Quality Standards of Indian 7. P.R. + FeCl3 Dark brown Black Creamy black Rai, Director General and Ms. Jyoti Joshi Bhatta 8. P.R. + Ether Dark black Brown Dark brown Medicinal Plants Vol.1. New Delhi, India: Indian 9. P.R. + Chloroform Brown Black Black and Mr. Mohan Dev Joshi, Deputy Director General, Council of Medicinal Research. 10. P.R. + NaOH 10% soln Brown Black Dark Black Department of Plant Resources and Mr. Devi Prasad 11 P.R. + Lead acetate Black Brown Dark brown Bhandari, Chief, Natural Plant Research Laboratory Khan, M.Y. & Kumar, V. (2016). 12 P.R. + Ninhydrin solution Brownish blue Brown Black Brown for their kind support. We are also obliged to all the Phytopharmacological and Chemical Profile of 13. P.R. + Fehling solution Brownish red Black Dark Black staff of NPRL and DPR for their co-operation and Bergenia Ciliata. International Journal of 14. P.R. + Mayer's reagent Brownish white Brown black Dark Black team work. Phytopharmacy, 6(5), 90-98. 15. P.R. + CuSO4 5% solution Bluish Black Dark Brownish Black Black

122 123 Thin layer chromatography (TLC) Co-TLC of methanolic extract of B. ciliata rhizomes revealed the presence of Bergenin in standard and sample extract having Rf values of 0.74 when a solvent phase of Ethyl acetate: Formic Acid: Acetic acid: Water (100:11:11:27) was used. Rf values of sample and reference standard and extract were found to be identical.

Table 6: TLC Details of Methanolic Extract of Bergenia Ciliata Rhizome

References

2019 Journal of Plant Resources Vol.17, No. 1

Khan, S.A., Dastagir, G., Ullah,B. Ullah, S., Ali, U. Pharmacological effect of Bergenia ciliate. & Ahmad, I. (2017). Pharmacognostic evaluation Journal of Applied Pharmaceutical Research, of Bergenin ciliata (Haw). Sternb. Pure Appl. 2(1), 1-6. Biol. 6(2), 762-775. Rafi, S., Kamili, A.N., Ganai, B.A., Mir, M.Y. & Khanal, C., Swar, S. & Tandukar, U. (2018). Hand Parray, J.A. (2017). Phytochemical Analysis of Book of Pharmacognosy (Medicinal plants in Bergenia ciliata (Haw). Sternb. Journal of Nepal), Kathmandu, Nepal : Natural Product Research and Development, 17, 31-34. Research Laboratory, Department of Plant Resources, 21-23. Rajkumar, V., Guha, G., Kumar, R.A. & Lazar, M. (2010). Evaluation of antioxidant activities of Pokhrel, P., Parajuli, R.R., Tiwari, A.K.& Banerjee, Bergenia ciliata rhizome. Rec. Nat. Prod. , 4(1), J. (2014). A Short Glimpse on promising 38.

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