{"id":50994,"date":"2023-09-30T11:42:38","date_gmt":"2023-09-30T11:42:38","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=50994"},"modified":"2023-10-12T04:53:32","modified_gmt":"2023-10-12T04:53:32","slug":"in-vitro-antioxidant-activity-pharmacognostical-evaluation-hptlc-and-ftir-fingerprinting-of-phyllanthus-acidus-l-stem-bark-extract-for-better-application-in-phytotherapy","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol16no3\/in-vitro-antioxidant-activity-pharmacognostical-evaluation-hptlc-and-ftir-fingerprinting-of-phyllanthus-acidus-l-stem-bark-extract-for-better-application-in-phytotherapy\/","title":{"rendered":"In Vitro Antioxidant Activity, Pharmacognostical Evaluation, HPTLC and FTIR Fingerprinting of Phyllanthus Acidus L. Stem Bark Extract for Better Application in Phytotherapy."},"content":{"rendered":"\n

Introduction<\/strong><\/p>\n\n\n\n

Post covid\npandemic phase has caused a paradigm shift into how herbal medicines are\nperceived all around world. With more and more people turning to alternate\nsystem of medicine, there is a dire need to provide referential information\nregarding the safe and authentic use of these herbal drugs. Due to lower cost,\nappreciable patience endurance, accessibility and availability of herbal\nproducts from local healers or from the market and the perception that herbal\nproducts do not contain detrimental chemicals and have  no side effects has led to a surge in the\ndemand of plant based pharmaceuticals. The use of plants for medicinal\ntreatment is also based on long established historical usage of that plant\nwhich is mostly indigenous and has deep roots in cultural heritage of a\nparticular community. This has led to an invigorating interest in plant based\nproducts and lead molecules as source of unconventional medicament for treating\nvarious ailments. Ground breaking work in the fields like ethnopharmacology,\nmolecular biology, medicinal chemistry and studies related to clinical trials\nof these herbal medicines have substantiated the clinical usage of these plant\nbased products.1<\/sup> Plant secondary metabolites like phenols and\nflavonoids have been reported to possess effective antioxidants , antidiabetic,\ncardioprotective, anti-inflammatory, immune-boosting activity  among several other pharmacological activities  and hence have emerge as an interesting\ncandidates for pharmaceutical and medicinal research.2,3,4<\/sup> \nHigh performance thin-layer chromatography (HPTLC) has evolved as an effective\ntool for chromatographic profiling of herbal drugs to determine the various\nphytoconstituents present in the plant extract. Because of its lucidity, and\nnegligible sample concoction, and the ease to analyse more than one sample at\nthe same time has also increased the popularity of this technique. Used along\nwith mass spectroscopy it is used to determine the phytoconstituents present on\nthe plate.5<\/sup> Fourier-transform infrared spectroscop (FTIR) is a sustainable technology which requires no solvent\nand negligible preparation of sample and provides spectrum with high resolution\nover a short period of time. Hence an extensive method used to elucidate\nphytoconstituents or determine their structure.6<\/sup> The\nneed to provide appropriate documentation, quality control measures and\nstandardisation of the crude, as well as finished herbal products has gained\nimportance.<\/p>\n\n\n\n

Phyllanyhus acidus\n<\/em>also\nknown as \u2018arinellikka\u2019, \u2018nellikkapuli\u2019\nin Malayalam \u2018hariful\u2019 in Bangla is also known by many other names like Star\ngooseberry, Otaheite gooseberry, and Malay gooseberry.7<\/sup> Although the\nplant initially originated in Madagasgar, is now spread across south-east Asia\nincluding Malaysia, Philippines and Indian Subcontinent.8<\/sup>An\nornamental plant with edible yellowish green berry fruits Phyllanthus.acidus<\/em>\nhas a long standing gastronomic use due to sour and tart taste of its fruit and\nis part of many culinary preparations like syrups, chutneys jellies and\nbeverages.9<\/sup> Different parts of these plants have well established ethno-pharmacological  and folklore uses. According to Ayurveda\npractices the plant is said to increase \u2018Vata\u2019.10 <\/sup>The roots of the\nplant are used as inhalations for headaches and congestion relief11<\/sup>,\nand an infusion is said to reduce asthma attacks.12 <\/sup>It is also used\nto manage  psoriasis, treat various skin diseases,\nantipyretic and relieve chronic constipation.13-14 <\/sup>The leaves are\nuse as vegetable in some parts of India, Malaysia and Thailand.15<\/sup> It\nis a common remedy for treating headaches, blood pressure and fever.16-18\n<\/sup>The bark is useful to treat catarrh and leaves are utilized to treat\nutricaria.19<\/sup> The fruit is acidic in nature and a rich source of\nvitamin C, it used to treat hepatic disorders by some tribal healers, and as a\nblood purifier, tonic for liver, remedy for hepatopathy and as an laxative.12,15,19,20,<\/sup>\nIt is also used to relieve cough, diabetes, asthma and also used as a\ncathartic.21-22<\/sup><\/p>\n\n\n\n

Compositional analyses of fruits of Phyllanthus acidus <\/em>shows that the plant is rich in mineral source of both macro and micro elements like calcium, potassium, phosphorous, zinc iron etc., it is also a rich source of ascorbic acid, carotenoids, fructose and glucose and therefore is advocated as a potential dietary supplement.23-26<\/sup> Phytochemical analysis has shown that various phytoconstituents have been isolated and characterised from various parts of this plant like leaf, fruits, roots and bark. Most of the constituents isolated are terpenoids, flavonoids, volatile oil component and phenolic compounds.27<\/sup>A plethora of pharmacological activities of bioactive components as well as extract of different parts of plants like antidiabetic cytotoxic, hepatoprotective antioxidant, anti-inflammatory etc., have been reported in both in-vivo and in-vitro experimental studies.28<\/sup><\/p>\n\n\n\n

Material and Methods<\/strong><\/p>\n\n\n\n

Material<\/strong><\/p>\n\n\n\n

The authenticated\nsample of dried bark of P acidus <\/em>was collected from the hills of Kerala.\nThe collected sample was identified with the help of a botanist at the National\nBotanical Research Institute, Lucknow, India.Chemicals that were used\nin the work were of analytical grade and procured\nfrom SD Fine Chemicals Ltd., Mumbai, India. Lead, arsenic, cadmium, and mercury\nwere obtained from Sigma Aldrich, Bangalore, India.<\/strong><\/p>\n\n\n\n

Microscopical studies<\/strong><\/p>\n\n\n\n

Morphological and organoleptic\nevaluation of the powdered stem bark of P.acidus<\/em> was carries out using\nmagnifying lens and naked eyes.29<\/sup> Fine\npowder was taken to observe microscopical features of the bark of P.acidus.<\/em>\n<\/strong><\/p>\n\n\n\n

Physiochemical study<\/strong><\/p>\n\n\n\n

 <\/strong>Evaluation of physiochemical\nparameters was evaluated based on WHO guidelines.30 <\/sup>Parameters such\nas loss on drying, extractive value, ash value, and moisture content by Karl\nFischer titration was determined. <\/p>\n\n\n\n

Preliminary phytochemical screening<\/strong><\/p>\n\n\n\n

Various\nchromophoric reagents were used to determine the existence of various classes\nof phytochemicals such carbohydrates alkaloids, phenolic and flavonoidal\ncompounds, saponnins, lipids, tannins and steroids.  The petroleum ether, chloroform and methanolic\nextracts were treated with chemical reagents in test tubes for the screening of\nvarious phytoconstituents.31-33<\/sup><\/p>\n\n\n\n

Preparation of extracts<\/strong><\/p>\n\n\n\n

The bark of P.acidus\n<\/em>was shade dried at 370<\/sup>C and powdered.  Powdered crude drug (10gm) each was soaked in\n100ml of methanol, water, and chloroform and petroleum ether respectively for\n24 hours. The extracts were filtered followed by evaporation to dryness under\nvacuum.<\/p>\n\n\n\n

DPPH radical scavenging assay<\/strong><\/p>\n\n\n\n

Estimation of scavenging of unbound DPPH radicals by the methanolic and aqueous extract of P.acidus <\/em>forms the basis for establishing its antioxidant potential. The in-vitro <\/em>antioxidant assay as stipulated by Nithianantham et al <\/em>with few alterations has been used.<\/em>34<\/sup> Different concentrations of methanolic and aqueous extract were added to 0.4mM DPPH solution (0.1ml) respectively. The mixture after vigorous shaken was placed in an unlit area for 15 min at 37o<\/sup>C and UV-Vis microplate reader was used determine the absorbance at 517 nm. Ascorbic acid was taken as the positive control. The degree of decolourisation of purple coloured DPPH into yellowish solution indicates the scavenging activity of the extracts.35 <\/sup>From the linear regression line of concentration versus inhibition (%) graph concentration that indicated 50% free radical scavenging (IC50<\/sub> value) was extrapolate.36<\/sup> Radical scavenging is expressed as percentage of inhibition which is deliberated by employing the formula:<\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

Where, A0<\/sub>\nis absorbance of control (DPPHsolution minus the extract) and A1<\/sub> is\nabsorbance of extract along with DPPH solution. The IC50<\/sub> values are\nused to express scavenging potential of the plant extracts. All the data were\nrepresented as mean values \u00b1 Standard deviation (n=3).<\/p>\n\n\n\n

Determination of total phenolics compounds <\/strong><\/p>\n\n\n\n

Folin\u2013Ciocalteu (FC) method has been employed to establish the total phenolic value of various concentrations of plant methanolic extracts using gallic acid as standard as stipulated by Singleton et al<\/em>.37<\/sup> A calibration curve was obtained by using different concentrations from 10 to 1000\u00b5g\/mL of gallic acid in methanol. Different concentrations of extract (10-1000\u00b5g\/mL) were prepared and thirty microlitre of standard and extract respectively was put in 150\u00b5L of FC reagent (10%) and after 10min, 120 \u00b5L of Na2<\/sub>CO3<\/sub> (7%) was added. After 2 hours absorbance were observed at 760nm compared to blank. The phenols present in P.acidus <\/em>extract are oxidised by FC reagent which changes the colour of the extract to bluish green that in turn is determined by UV-Vis spectrometer. The sample was prepared thrice for every examination. The mean value of absorbance was employed to mark calibration curve to obtain total phenols in extract. The total phenolic concentration is expressed as mg gallic acid equivalents (GAE) per gram of dry samples (mg\/g). The formula used to calculate total phenolic content was:<\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

Whereby, Cp<\/sub>\n<\/em>= total phenolic concentration mg GAE\/g dried sample, cg= gallic\nacid concentration evaluated from calibration curve in mg\/mL, V<\/em>= volume (mL)\nof extract, and m<\/em>= mass of dried extract in grams.<\/p>\n\n\n\n

Estimation of Total Flavonoid Content<\/strong><\/p>\n\n\n\n

 <\/strong>Aluminium chloride colorimetric assay is\nemployed to evaluate overall flavonoid concentration as stipulated by Chang et\nal<\/em>.38 <\/sup>Standard quercetin for calibration curve was prepared by\npreparing stock solution of quercetin (4mg\/ml) and diluting serially to make\nconcentrations from 0.1-1.0mg\/mL. Similarly the plant extract was also serially\ndiluted to make concentration ranging from 0.1- 1mg\/mL. Quercetin (1mL) from\neach concentration was diluted with 4mL distilled water and then 0.3mL of NaNO2<\/sub>\n(5%) and 0.3 mL of AlCl3<\/sub> (10%) was mixed after 5 minutes. Volume was\nmade up to 10mL by adding distilled water. Same procedure was followed with\ndifferent concentrations of extract. \nAbsorbance was measure at 510 nm using spectrophotometer. Triplicate\nreadings of extract were used to calculate average absorbance. Quercetin\nEquivalent (mg QE\/g) was determined by employing linear equation based on\nstandard calibration graph was employed to express total flavonoid concentration.<\/p>\n\n\n\n

 Heavy metal analysis<\/strong><\/p>\n\n\n\n

 <\/strong>Atomic absorption spectrophotometer was utilised\nto carry out the Heavy metal analysis of bark extracts of P.acidus.<\/em>\nStandards of Mercury (Hg), Lead (Pb), Arsenic (As) and Cadmium (Cd) were\nprepared and calibration plot was developed. The drug samples were analysed\nusing calibration plot.39<\/sup> <\/p>\n\n\n\n

HPTLC Screening<\/strong><\/p>\n\n\n\n

 <\/strong>HPTLC fingerprinting profile of P.acidus<\/em>\n(bark) methanolic extract was determined to establish presence of various\nphytoconstituents. 15mg bark extract was dissolved in 5mL of methanol and\nfiltered using nylon membrane filter. Various combinations of solvents of\nvarying polarity were combines to obtain a solvent system that gave excellent\nresolution and acute peaks for scanning. The solvent system that gave\nsatisfactory resolution of phytoconstituents present in methanolic extract of P.acidus<\/em>\nwas chloroform: methanol (19:1v\/v). HPTLC was performed on pre-coated aluminium\nsheet TLC Silica gel 60 F254<\/sub> (20 \u00d720 cm, Merck, Germany) using a\nCamag syringe(100\u00b5l). Samples (5\u00b5L) were spotted as 6mm bands, 10mm from bottom\nand 15 mm from left edge. The plates were developed in Twin Trough chamber were\ninitially saturated with mobile phase (chloroform: methanol: 19:1)  and then the plated were developed with\ndistance of solvent migration of 80 mm. The developed plates were placed in hot\nair to dry.<\/p>\n\n\n\n

Phytoconstituents like, terpenes, flavonoids, phenols terpenoids,\nand steroids, were ascertained succeeding chemical derivatization by\nanisaldehyde\/sulfuric acid reagent (p-anisaldehyde(0.5mL) in methanol (85mL),  acetic acid (10mL) and concentrated sulfuric\nacid(5mL).40<\/sup>\nChromatograms were recorded at 366 nm and 540 nm using CAMAG LINOMAT 5 spectro-\ndensitometer. The retention factor (Rf<\/sub> value) and peak area of each\nband were recorded. The plates were photo documented.41<\/sup><\/p>\n\n\n\n

 FTIR fingerprinting<\/strong><\/p>\n\n\n\n

 FTIR (PerkinElmer Version 10.03.06) was used\nto analyse the functional moieties existing in the etanolic extract in the\nrange of 400-4000 cm-1<\/sup>.<\/p>\n\n\n\n

 Statistical analysis<\/strong><\/p>\n\n\n\n

Inhibitory\nconcentration (IC50) i.e. <\/em>the concentration of the extract which\neffectively inhibited 50 % of free radicals, was estimated by regression\nanalysis between % inhibition and different concentrations of extract. All\nassays were performed in triplicates and presented as \u00b1SEM or \u00b1SD, n=3.<\/p>\n\n\n\n

Result\n<\/strong><\/p>\n\n\n\n

Organoleptic and Microscopical studies<\/strong><\/p>\n\n\n\n

 <\/strong>The powdered stem bark of P.acidus\n<\/em>is pale brown in colour. The stem bark powder had a stimulant odour and an\nastringent taste. The various diagnostic features observed during powder\nmicroscopy were elongated cork cells, cortex with sclereids, parenchyma cells,\nstone cell brown tannin masses, lignified fibres and non lignified fibres, and\nprismatic calcium oxylate crystals as shown in Figure 1.<\/p>\n\n\n\n\n\n
\"\"<\/td>\nFigure 1: <\/strong>Powder microscopy of P.acidus <\/em>(A)Elongated cork cells;(B) Cortex with Sclereids;(C) Parenchymatous cells ;(D) Brown Tannin Masses;(E) Xylem Fibres;<\/strong>

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

 Physiochemical parameters<\/strong><\/p>\n\n\n\n

 The various physiochemical parameters that\nwere evaluated to establish the purity and quality of the plant drug were, ash\nvalue, loss on drying, extractive value and moisture content. The parameters\nevaluate are represented in Table 1 along with standard deviation. The results\nobtained were within limits, corresponding with pharmacopeal standards.42,\n43<\/sup><\/p>\n\n\n\n

Table 1: Summary of phytochemical analysis of P.acidus<\/em>.<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Parameters<\/strong><\/p>\n<\/td>\n

\n

%w\/w(Mean \u00b1SD)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

LOD<\/strong><\/p>\n<\/td>\n

\n

25.23\u00b10.33<\/p>\n<\/td>\n<\/tr>\n

\n

Ash Values<\/strong><\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n<\/tr>\n

\n

Total Ash<\/p>\n<\/td>\n

\n

7.54\u00b10.45<\/p>\n<\/td>\n<\/tr>\n

\n

Water soluble Ash<\/p>\n<\/td>\n

\n

6.41\u00b10.36<\/p>\n<\/td>\n<\/tr>\n

\n

Acid insoluble Ash<\/p>\n<\/td>\n

\n

0.59\u00b10.074<\/p>\n<\/td>\n<\/tr>\n

\n

Extractive values<\/strong><\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n<\/tr>\n

\n

Petroleum ether<\/p>\n<\/td>\n

\n

3.4\u00b10.2<\/p>\n<\/td>\n<\/tr>\n

\n

Chloroform<\/p>\n<\/td>\n

\n

12.81\u00b10.19<\/p>\n<\/td>\n<\/tr>\n

\n

Methanol<\/p>\n<\/td>\n

\n

27.27\u00b10.35<\/p>\n<\/td>\n<\/tr>\n

\n

Water: alcohol (50:50)<\/p>\n<\/td>\n

\n

31.37\u00b11.15<\/p>\n<\/td>\n<\/tr>\n

\n

Moisture content<\/strong><\/p>\n<\/td>\n

\n

22.36\u00b10.27<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

 Preliminary phytochemical screening<\/strong><\/p>\n\n\n\n

Exploratory\nphytochemical analysis of petroleum ether, chloroform and methanolic extracts\nrespectively showed the existence of carbohydrates, glycosides, phenolic\ncompounds, flavonoids, tannins, lipids, saponins and steroids. Alkaloids and\nprotein were absent in all the extract, while lipids was positive only in\npetroleum ether extract. The conclusions are given in Table 2.<\/p>\n\n\n\n

Table 2: Qualitative Phytochemical analysis of P.acidus <\/em>stem bark extracts <\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Phytochemicals<\/strong><\/p>\n<\/td>\n

\n

Pet. ether <\/strong><\/p>\n

extract<\/strong><\/p>\n<\/td>\n

\n

Chloroform <\/strong><\/p>\n

extract<\/strong><\/p>\n<\/td>\n

\n

Methanolic <\/strong><\/p>\n

extract<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Alkaloid<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

Flavanoids<\/p>\n<\/td>\n

\n

+<\/p>\n<\/td>\n

\n

++<\/p>\n<\/td>\n

\n

+++<\/p>\n<\/td>\n<\/tr>\n

\n

Phenolic substances<\/p>\n<\/td>\n

\n

+<\/p>\n<\/td>\n

\n

+++<\/p>\n<\/td>\n

\n

+++<\/p>\n<\/td>\n<\/tr>\n

\n

Proteins<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

Tannins<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

++<\/p>\n<\/td>\n

\n

+++<\/p>\n<\/td>\n<\/tr>\n

\n

Glycosides<\/p>\n<\/td>\n

\n

++<\/p>\n<\/td>\n

\n

+++<\/p>\n<\/td>\n

\n

+++<\/p>\n<\/td>\n<\/tr>\n

\n

Carbohydrates<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

++<\/p>\n<\/td>\n

\n

+++<\/p>\n<\/td>\n<\/tr>\n

\n

Saponins<\/p>\n<\/td>\n

\n

++<\/p>\n<\/td>\n

\n

+++<\/p>\n<\/td>\n

\n

+++<\/p>\n<\/td>\n<\/tr>\n

\n

Fixed oil & Fats<\/p>\n<\/td>\n

\n

+++<\/p>\n<\/td>\n

\n

++<\/p>\n<\/td>\n

\n

+<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Key:+++: strong; ++: medium; +: weak; – : absent<\/p>\n\n\n

Heavy metal evaluation<\/strong><\/p>\n\n\n\n

The\npresence of heavy metals like arsenic (As), cadmium (Cd), and lead (Pb), in P.acidus\n<\/em>was found to be within permissible limits. Level of Mercury (Hg) was found\nto be slightly     higher\nthan the permissible limit(i.e. over limit ratio of 2%) therefore carcinogenic\nand non carcinogenic risk assessment is requires before determining the dose of\nthe drug for human consumption.(Table 3).<\/p>\n\n\n\n

Table 3: Heavy metals analysis of P.acidus <\/em>stem bark<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n
\n

Metal<\/strong><\/p>\n<\/td>\n

\n

Concentration(ppm)<\/strong><\/p>\n<\/td>\n

\n

Limit(ppm)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Lead<\/p>\n<\/td>\n

\n

0.80\u00b10.04<\/p>\n<\/td>\n

\n

10<\/p>\n<\/td>\n<\/tr>\n

\n

Cadmium<\/p>\n<\/td>\n

\n

0.01\u00b1.001<\/p>\n<\/td>\n

\n

0.3<\/p>\n<\/td>\n<\/tr>\n

\n

Mercury<\/p>\n<\/td>\n

\n

0.52\u00b10.03<\/p>\n<\/td>\n

\n

0.5<\/p>\n<\/td>\n<\/tr>\n

\n

Arsenic<\/p>\n<\/td>\n

\n

1.50\u00b10.23<\/p>\n<\/td>\n

\n

3.0<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

n <\/em>= 3<\/p>\n\n\n

Antioxidant activity of extracts<\/strong><\/p>\n\n\n\n

Folin-Ciocalteu\nassay was employed to establish the Total Phenolic Content (TPC) of P.acidus<\/em>\nmethanolic extract. <\/em>Serial dilution of gallic acid was used to establish\na calibration graph. Calibration curve was used to establish regression\nequation (Y=0.012x; R2<\/sup>=0.894) which was used to calculate total\nphenolic concentration and indicated as mg gallic acid equivalents (GAE) per\ngram of dry extract (mg\/g). The total phenolic content was found to be\n189.74\u00b10.52mg GAE\/g. This is in conformity with the findings of Sulaiman and\nOoi44<\/sup>, where the phenolic content of the fruit juice extract was\nfound to be 204.75\u00b14.99mg GAE\/g. Comparably another research reported the total\nphenolic concentration of 41.801\u00b10.815mg GAE\/gm of the leaf extract which is\nmuch lower than the phenolic content of the stem bark.45 <\/sup>Phenolic\ncompounds are hydrogen donors and act as reducing agents. Therefore, the\nphenolic content is an indication of antioxidant property of the plant. The\npresent study showed a considerable existence of phenolic compounds in P.acidus<\/em>\nand may contribute to its antioxidant activity and also provides a validation\nto the ethno pharmacological use of this plant to treat various ailments.<\/p>\n\n\n\n

 Aluminium chloride colorimetric assay was used\nto establish the Total Flavonoid Content (TFC) present in the extracts. Serial\ndilution of quercetin was utilised as standard to establish the calibration\ncurve and regression equation(Y=0.031x; R2=0.865) was established to determine\nthe flavonoid concentration which was stated as mg quercetin equivalents(QE)\nper gram of dry extract weight(mg\/g).TFC was determined as to be 38.92\u00b10.47 mg\nQE\/g. This value is similar to the one obtained by Habib et al<\/em> which\nshowed flavonoid content of fruit as 30.05 mg QE\/g.46 <\/sup>The\nantioxidant potential of flavonoids is associated with the number hydroxyl\ngroups and their position in the phytoconstituent.47 <\/sup>It has been\nshown that flavonoid content may vary significantly due to genetic diversity,\nseasonal and biological differences.48<\/sup><\/p>\n\n\n\n

Methanolic\nand aqueous extract of P.acidus<\/em> bark was screened for radical scavenging\nactivity against DPPH radicals. The conversion of DPPH to DPPH2<\/sub> on\nreceiving a hydrogen ion from the plant extract was responsible for changing of\ncolour from purple to yellow.49<\/sup> This change brought about change in\nabsorbance which was measured spectrophotometrically to determine the\nconcentration of antioxidants. The DPPH radical scavenging assay of methanolic\nand aqueous extract showed IC50 <\/sub>values of 26.92 and 26.52\nrespectively. As shown in Table 4 ascorbic acid a well known antioxidant was\nused as standard with IC50<\/sub> value of 31.82 to measure the in-vitro<\/em>\nantioxidant activity of the extracts. Lower IC50<\/sub> value is indicative\nof higher free radical scavenging and therefore high antioxidant potential. The\nmethanolic extract showed marginally higher anti oxidant activity than aqueous\nextract and both were close when compared with the standard ascorbic acid as\nshown in Figure 2. Study of literature has showed that antioxidant activity of\ndifferent parts of P. acidus<\/em> has been studied previously. Habib et al<\/em>\nreported antioxidant capacity of chloroform extract of fruit of P.acidus <\/em>withIC50<\/sub> value of 2745.86 \u03bcg mL\u22121<\/sup>and that of standard ascorbic acid as 13.37\u03bcg\/mL.46<\/sup> However the\nIC50 <\/sub>value reported is more than 100 and therefore cannot be\nconsidered relevant.50 <\/sup>High phenolic content and presence of known\nantioxidants like gallic acid, ellagic acid rutin, quercetin and luteolin by\nHPLC analysis supports the present data.51<\/sup> The study also\ndemonstrated that high phenolic and flavonoid content in extracts\nscientifically validates the ethnological use of P.acidus <\/em>as natural\nantioxidant.<\/p>\n\n\n\n

Table 4: Mean absorbance and IC50<\/sub> determination of extracts and ascorbic acid <\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

% Inhibition (scavenging capacity)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Concentration \u00b5g\/ml<\/strong><\/p>\n<\/td>\n

\n

Ascorbic acid<\/strong><\/p>\n<\/td>\n

\n

Ethanolic extract<\/strong><\/p>\n<\/td>\n

\n

Aqueous extract<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

0<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

25<\/p>\n<\/td>\n

\n

49<\/p>\n<\/td>\n

\n

42<\/p>\n<\/td>\n

\n

45<\/p>\n<\/td>\n<\/tr>\n

\n

50<\/p>\n<\/td>\n

\n

67<\/p>\n<\/td>\n

\n

56<\/p>\n<\/td>\n

\n

58<\/p>\n<\/td>\n<\/tr>\n

\n

75<\/p>\n<\/td>\n

\n

80<\/p>\n<\/td>\n

\n

62<\/p>\n<\/td>\n

\n

67<\/p>\n<\/td>\n<\/tr>\n

\n

100<\/p>\n<\/td>\n

\n

89<\/p>\n<\/td>\n

\n

68<\/p>\n<\/td>\n

\n

71<\/p>\n<\/td>\n<\/tr>\n

\n

IC50<\/sub>(\u00b5g\/ml)<\/p>\n<\/td>\n

\n

31.82<\/p>\n<\/td>\n

\n

26.92<\/p>\n<\/td>\n

\n

26.52<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

\u00a0<\/p>\n\n\n\n
\"\"<\/td>\n\n

Figure 2<\/strong>:\u00a0A.DPPH\u00a0scavenging assay of P.acidus <\/em>extracts. B.IC50<\/sub> values for different extracts in DPPH radical scavenging assay<\/strong><\/p>\n

\u00a0<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

HPTLC Fingerprinting<\/strong><\/p>\n\n\n\n

In\norder to get a general idea of the various phytoconstituents found in the\nmethanolic extract of P.acidus <\/em>HPTLC screening was instrumental. Mobile\nphase comprising of chloroform: methanol (96:4) v\/v gave satisfactory\nseparation of compounds. After spotting of samples the chromatograms were\nscanned at 250nm, 360nm and 540nm (after derivatization with anisaldehyde in\nsulphuric acid). The chromatogram at 360nm and 540nm showed 9 and 6 peaks\nrespectively (Figure 3).\nTable 5 summarises Rf<\/sub> value of the various\nseparated constituents along with peak areas. Literature survey of P.acidus<\/em>\nrevealed that this study provides the first data pertaining to HPTLC\nfingerprinting of this plant.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 3: A. <\/strong>HPTLC chromatogram image of methanolic extract of P.acidus <\/em>at 360nm and 540nm respectively. B and C. HPTLC chromatograms of P.acidus<\/em> methanolc extract at 360nm and 540 nm showing various peaks (bands) of phytoconstituents.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Table 5: HPTLC data of different extracts of P.acidus<\/em><\/strong><\/p>\n\n\n\n\n\n\n\n
\n

Wavelength<\/strong><\/p>\n<\/td>\n

\n

Solvent System<\/strong><\/p>\n<\/td>\n

\n

No. of Peaks<\/strong><\/p>\n<\/td>\n

\n

Rf<\/sub> values<\/strong><\/p>\n<\/td>\n

\n

Peak Area (%)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

366 nm<\/p>\n<\/td>\n

\n

Chloroform: Methanol (96:4 v\/v)<\/p>\n<\/td>\n

\n

    9<\/p>\n<\/td>\n

\n

0.06,0.16,0.22,0.28, 0.35,0.42,0.49,0.66, 0.79<\/p>\n<\/td>\n

\n

4.81,3.44,37.85,9.55,<\/p>\n

17.68,8.74,3.07,<\/p>\n

9.83,5.04   <\/p>\n<\/td>\n<\/tr>\n

\n

540nm (derivatized in anisaldehyde sulphuric acid reagent )<\/p>\n<\/td>\n

\n

Chloroform: Methanol (96:4 v\/v)<\/p>\n<\/td>\n

\n

    6<\/p>\n<\/td>\n

\n

0.29,0.35,0.38,0.47, 0.82,0.93<\/p>\n<\/td>\n

\n

6.11,4.02,9.98,<\/p>\n

7.28,10.74,61.86<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Fourier Transform InfraRed (FTIR) Fingerprinting<\/strong><\/p>\n\n\n\n

FTIR being one of the most extensively employed methods for the determination of the various functional groups present in the plant extract. This spectroscopic method was used to elucidate the functional groups present in the methanolic extract of P.acidus. <\/em>The FTIR spectra showing characteristic peaks are shown in Figure 4. The broad peak at 3372cm-1<\/sup> is an indicative of the presence of phenolic compounds. The absorption bands at 2924cm-1<\/sup> and 2857cm-1<\/sup>are attributed to C-H symmetrical and asymmetrical stretching of methylene functional group. Sharp peak at 1722cm-1<\/sup> is indicative of stretching vibrations of carbonyl group (C=O). The bands at 1519-1453 cm-1<\/sup>  may be assigned to C=C aromatic stretching both symmetrical and asymmetrical related to unsaturated linkages and aromatic groups.52<\/sup>  The bands at 1264-1225 cm-1<\/sup> may be allocated to C-O-C group. The band at 1380 cm-1<\/sup> is indicative of bending vibration of O-H group. The bands at 800- 627 cm1<\/sup> is due to the substitutions at Ar-H group.53 <\/sup>Thus FTIR was used to determine the various functional groups present in the plant extract. Comparing the present spectra to the  spectra of some well known antioxidants like gallic acid, rutin, quercetin and tannic acid we see similarity in the region of 3400-2500 cm-1<\/sup> representing O-H and C-H stretching vibrations of phenolic groups present in these compounds.54<\/sup> which is similar to the FTIR spectra of P.acidus <\/em>, which also shows similar broad band at 3372cm-1<\/sup>. Since OH group plays a major role in the antioxidant activity of these standard compounds, it can be stipulated that the extract of P.acidus <\/em>contains similar phytoconstituents like phenols and flavonoids thus making it an effective anti-oxidant which has been further confirmed by DPPH assay in the above studies.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 4: a. <\/strong>FTIR spectra of stem bark of P.acidus<\/em> ethanolic extract.b.<\/strong> FTIR spectra of pure standard compound of 1) tannic acid 2) quercetin 3) rutin 4) gallic acid from literature data54<\/sup><\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Discussion<\/strong><\/p>\n\n\n\n

An\nincrease in use of herbal drugs as alternative or complimentary treatment of\nvarious diseases requires well established quality control parameters for their\nsafe and efficacious use. Bioassays play a pivotal role in establishing these\nquality control measures for the standardisation of herbal drugs. As per WHO\nGuideline the primary step towards establishing the authenticity and purity of\nherbal drugs is macroscopic and microscopic examination before taking any\nfurther tests or assays. Morphological study is one of the elementary and\neconomic means of evaluation of crude drug and involves organoleptic and\nsensory observations along with microscopical observations. It is a vital tool\nin authentication of herbal drugs.48<\/sup>The present study provides an\nanatomical and morphological description of bark of P.acidus<\/em> as\nreference for quality control.<\/p>\n\n\n\n

Presence\nof moisture or volatile components in crude drug was analysed using Moisture\ncontent and loss on drying. High moisture content is an indicator of poor\nquality and efficacy of the drug as moisture may hydrolyse the active\ncomponents.55<\/sup> Presence of any possible foreign contamination like\nsand, water soluble salts etc in the crude drug is evaluated by ash value or\nash content. The residue left behind after ignition of crude drug and usually\nconsists of inorganic salts present in the drug or adulterants. Total ash value\nconsists of two types of ash \u2018physiological ash\u2019 comprising of plant tissues\nand \u2018non-physiological ash\u2019 which contains any adulterants present in the crude\ndrug. Acid insoluble ash is an indication of the amount of silica present in\nthe form of soil and earth materials, and is a component of total ash value.\nComponents of total ash which are water soluble are determined by water soluble\nash, also a part of total ash value.42,43 <\/sup>Total ash, water-soluble\nash, and acid-insoluble ash of P.acidus <\/em> were found to be 7.54 \u00b1 0.45, 6.41 \u00b1 0.36, and\n0.59\u00b1 0.74 respectively; the value of total ash indicates that the inorganic\ncontents of the crude drug are below the limits. Acid-insoluble ash\nvalue of P.acidus <\/em>(0.59 \u00b1 0.74) shows that a very small amount of the\ninorganic component is insoluble in acid. It indicates that adulteration by\nsubstances, such as silica etc., is very less, and a low acid-insoluble ash\nvalue may also affect the amount of the component absorbed in the\ngastrointestinal canal when taken orally.56<\/sup> Crude drugs are\nsometimes contaminated with metals like Cd, As, Hg and Pb which are toxic to\nhumans even in very minute quantities.57,58<\/sup> Gestational exposure to\ncadmium may cause ovo-toxicity, hepato-toxicity and renal disorders and\ntherefore they should be within acceptable limits in crude drug, in order for\nit to be safe and efficacious for consumption.59<\/sup> The extractive\nvalue is mostly used for the determination of exhausted drug. It also gives a\ngeneral idea about the chemical nature of different phytoconstituents found in\neach extract. For example petroleum ether extract contains non polar compounds\nlike fatty acids etc, while methanolic extract contains polar constituents like\nglycosides alkaloid, steroids, phenols etc. In recent times plants used in\nethno medicine and folk medicine have been backed by scientific investigations\nand data. The therapeutic properties exhibited by the herbal drugs are mostly\ndue to the phytoconstituents present in the plants. Therefore, a screening of\nphytochemicals present in the plant is of paramount importance and gives an\ninsight into the various chemical moieties present in the extract. In order to\ncreate a profile of phytoconstituents present in the P.acidus <\/em>a\npreliminary phytochemical screening was performed on methanolic extract. The\npreliminary screening also suggested that the polarity of the solvent also perform\na paramount part in the extraction of these phytoconstituents. The result shown\nin Table 2 showed that maximum constituents were extracted when ethanol was use\nas a solvent because of its higher polarity which enables the polar molecules\npresent in the plant to dissolve in it. However, the chloroform and petroleum\nether extract showed lesser amount of phytoconstituents as compared to the\netanolic extract. Therefore, polar solvents were found to be more effective for\nextraction as compared to semi-polar and non polar solvents.60<\/sup> This may be instrumental in further identification\nand quantitative estimation of pharmacologically active phytoconstituents.\nHPTLC is an instrumental quantitative tool to determine the phytochemical of\nherbal drugs.61<\/sup> HPTLC helped in creating a standard phytochemical\nfingerprint of P.acidus<\/em> for future reference for quality control and\nstandardisation.<\/p>\n\n\n\n

FTIR\nwas used to determine the various functional moieties that exist in the\netanolic extract. The presence of O-H functional group in plant extract has\nbeen associated to a plethora of pharmacological activities like antioxidant,\nanti-inflammatory, antidiabetic activity etc. the presence of OH- group also\ndemonstrates the phenols and flavonoid content of the plant, also associated\nwith above pharmacological effects.62<\/sup><\/sup><\/p>\n\n\n\n

Plants consisting of phytochemical like phenolic and\nflavonoids have been established to diminish the threat of neoplastic disorder,\nhepatic disorders, diabetes, inflammation, bacterial and viral diseases. This attribute\nof natural compounds comprising of phenols and flavonoids might be ascribed to\nthe free radical scavenging, diminishing oxidative stress and antioxidant potential\nof various polyphenolic bioactive components.63 <\/sup>Therefore analysis\nof the extract for total phenol and flavonoid content gives an insight into the\nmechanism of potential antioxidant and other pharmacological activities\nassociated with this plant. The DPPH assay which showed reasonable free radical\nscavenging may also be attributed to these polyhydroxy compounds present in P.acidus<\/em>\nextracts. The data pertaining to antioxidant activity may be instrumental in\ndetermining the compounds responsible for this action as well as advocating the\nuse of this plant to combat oxidative stress associated with chronic and acute\ndisorders like cancer, diabetes, neurodegenerative, liver disorders etc\nnormally associated with free radicals or reactive oxygen species (ROS).<\/p>\n\n\n\n

Conclusion<\/strong><\/p>\n\n\n\n

The plant-based pharmacologically active compounds are a valuable\nalternative resource for the treatment of various ailments. The\npharmacognostical tools used in this study have positive implications in\nproviding valuable information regarding the standardization parameters and\npharmacological use of P.acidus. <\/em>The study reveals that P.acidus <\/em>can\nbe used as a potential antioxidant due to its higher phenolic and flavonoid\ncontent. To the best of our knowledge this is the first study that has reported\nthe pharmacognostical parameters for quality control of P. acidus<\/em>,\nincluding HPTLC and FTIR fingerprinting. We recommend further screening and isolation of\nthe phytochemical constituents responsible for the high antioxidant activity\nof P.acidus <\/em> , especially in in vivo <\/em> free radical\nscavenging studies to establish a relationship between the antioxidant activity\nand isolated phenolic and flavonoid compounds and authenticate their possible\nuse as natural antioxidants<\/p>\n\n\n\n

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