phytochemical standardization and...

International Journal of Interdisciplinary Research Centre (IJIRC) ISSN: 2455-2275(E)

Volume III, Issue 3 March 2017

All rights are reserved 32

PHYTOCHEMICAL STANDARDIZATION AND

PHARMACOLOGICAL SCREENING OF BERBERIS ARISTATA

* Arshad Ahmad and * * Dr. Anil Middha

* Research Scholar, OPJS University, INDIA

* * Department of Pharmacy OPJS University, INDIA

Abstract

Aim of research work is focused on the Phytochemical and Pharamcological profile of the

drugCertain parameters has to be adopted for the standardization of Berberis Aristata viz:

macroscopic, microscopic evaluation of fresh drug. Further for Phytochemical profile some

of the analytical parameter such as HPTLC has been performed. From literature review

certain pharmacological activities has been reported. Result of current research revel the

aprox value given in the standard books and research papers published.

Introduction

Over the centuries, medicinal plants have been utilized in various cultures of the world as a

natural healing tool. Plants as a source of medication in the form of traditional and folklore

based on the rich experiences of innumerable healers over centuries, inherited from ancestors,

healer-to-healer transfer or developed through personal experiences over time. Modernity or

cultural revolutions has not altered the in-depth wisdom of this natural medical paradigm.

Consequently, no modern system of medicine can ordinarily lay claim to it. The traditional

system of treatment, differing in concept and protocol, exemplify welldeveloped systems

such as Allopathic, Homeopathic, Ayurvedic, Chinese system of treatment (Gurib-Fakim,

2006; Schippmann et al., 2002).

Extensive investigations have revealed that medicinal plants in different shapes, either in

crude form or pure molecules isolated from them, represent the most ancient mode of




medication. Archaeological studies have been provided reasonable evidences that the healing

properties of plants were known to peoples in prehistoric time (Halberstein, 2005).

The Traditional Chinese Medicine (TCM) represents one of the oldest systems of treatment.

TCM is unique in theories, treatment and therapies. This effective system of medicine has

tremendous importance in the history of medicine and now got global recognition due to

evidence basis approach (Patwardhan et al., 2005).

The interest in natural products as a source of new biologically active compounds decreased

in the last few decades as synthetic chemistry programmes expanded, natural products

continued to form a significant proportion of drugs in current use and of those under

investigation. Plant secondary metabolites are not only useful as potential drugs in their

natural unmodified form, but are also suitable as synthetic intermediate substances for the

production of useful drugs, e.g., the readily available plant steroids, diosgenin from

Dioscorea species may be synthetically converted to steroids with anabolic, anti-

inflammatory and oral contraceptive activity (Briggs, C.H. 1990).

PLANTS PROFIL

Plant Profile of Berberis Aristata

Table 1:Nomenclature of Berberis aristata (Sharma K et al, 2011)

Botanical name Berberis aristata

Synonyms Indian barberry

Family Berberidaceae




Table 2: Vernacular names Berberis aristata (Sharma K et al, 2011)

English Indian barberry

Hindi Daruhaldi

Tamil Gangeti

Malayalam Maramannal

Telugu Manupasupu

Kannada Daruhaladi

Bengali Daruharidra

Gujarati Daruharidra

Sanskrit Katamakateri

Marathi Daruhalad

Oriya Daruharidra

Urdu Darhald

Punjabi Sumalu

Table 3: Taxonomical Classification of Plant Berberis Aristata (Sharma K et al, 2011)

Kingdom Plantae

Division Magnoliophyta

Class Magnoliopsida

Order Ranunculales

Family Berberidaceae

Genus Berberis

Species Aristata




Figure 1: Picture of Whole Plant of Berberis Aristata With Flowers

Description of Berberis Aristata

Distribution: Berberis Indian barberry aristata is a spinous shrub native to mountainous

parts of North India and Nepal. These shrubs are distributed through out the Himalayas from

Bhutan to Kunawar (altitude 6- 10,000 ft), Nilgiri hills (altitude 6-7,000 ft) and Sri Lanka

(altitude 6-7,000 ft) (Shahid, M. et al., 2009). It grows at the height of 2000-3000 m

especially in Kumaon and Chammba region of Himachal Pradesh (Sharma, k. et al., 20

Morphology: A large deciduous shrub usually 1.8 to 3.6 m high but attaining 4.5 m with

stem 20 cm diam. Twigs whitish or pale yellowish brown. Bark pale brown, closely and

rather deeply furrowed, rough, Blaze 5 to 7.5 mm bright yellow with coarse reticulate fiber.

Leaves 3.8 to 10 by 1.5 to 3.3 cm, obovate or elliptic, entire or spinout-toothed, base

gradually narrowed, with prominent reticulate nerve, glossy dark green above, glossy pale

green but not glaucous beneath. Petiole distinct up to 4 mm, Inflorescence a simple drooping

raceme, 2.5 to 7.5 cm long, dense-flowered. Pedicles stout 4 to 6 mm long. Fruit 7 to 10 mm




long, ovoid, blue-black with a hick pale bloom: style distinct (Kirtikar, K.R. et al.,

2005).Traditional use: The plant has been traditionally useful in all types of inflammations,

ENT infections, wound healing, dysentery, indigestion, uterine and vaginal disorders. It is

well known for its anti-inflammatory and immune-potentiating property. One of its active

constituent Berbamine effectively inhibits chemically-induced hepatocarcinogenesis. The

other uses of Berberis aristata DC. are as diaphoretic, laxative and useful in rheumatism. The

dried extract of the roots are applied externally to the eyelids to cure ophthalmia and other

eye diseases. It is also reported as tonic and useful in curing ulcers and fevers (Mazumder,

P.M. et al., 2010).

MATERIALS AND METHOD

Collection and Identification of Plant Material

Both Berberis Aristata and Hamelia Patens were collecte from the localsurrounding area of

Meerut India, in the month of September-October 2014. The plants were identified by Prof.

Dr. Anju Pal, Horticulture Department, Panthnagar University, Panthnagar, Uttarnchal,

India.

Processing of Plant Material

The plant materials were properly dried in shade for 5-6 days then dried in hot air oven at

400C after drying, the plant materials were milled to powder and passed through the sieve

(mesh size 40), this material were used for the identification of plant metabolite.




Macroscopic & Microscopic Identification

Thin section were made with the help of blade, stained and mounted following the usual

plant micro-techniques. For the study of isolated cells and tissues, small pieces of leaves,

roots, stem, were taken. Washed and mounted in glycerine. The anatomical sketches

were made with digital camera unit (Trease and Evans, 1992).

Fluorescence Analysis

To check the fluorescent property of plants were powdered leaf material obtained which is

used for to analysis under ultra and organic reagents like alcohol, 50% nitric acid and

water.16 Slides of powdered leaf material were also prepared and studied. Microphotography

on different magnifications was carried out with motic microscopic. The florescence

sulphuric acid, 10% sodium hydroxide, 50% behaviour as noted.

Quantitative Leaf Microscopy

Quantitative leaf microscopy to determine palisade ratio, stomata number, stomata index,

vein - islet number and vein let termination number were carried out on epidermal strips

Solubility Behaviour of Leaf Extracts of Plants

Solubility of plant leaf extracts has been observed with different solvents.VIZ non polar,

polar, polar acidic and polar basic.

Physico-Chemical Parameters For The Standardisation of Crude Drug

Determination of foreign matter: 50 g of drug sample examined was weighed and spread

out a thin layer. The foreign matter was detected by inspection with the unaided eye.




Separated and weighed it and calculated the percent present. Drug undertaken for further

study were free from moulds, insects, animal faecal matter and other contamination such

as soil, stones and extraneous material (A.O.A.C, 2000).

Determination of moisture content [Hot Air Oven Method]: To determine the amount of

moisture (water drying off from the drug) for substance appearing to contain water as the

only volatile constituent, the procedure given below, was used. 2.78g of drug (without

preliminary drying) after accurately weighing was placed in a tare evaporating dish. After

placing the above said amount of the drugs in the tared evaporating dish, dried at

105oC for 5 hrs, and weighed, percentage was calculated with reference to initial weight

(BP, 1980).

Determination of ASH

Determination of Total Ash: About 2.0 g of powder drug was incinerated in a redtop silica

dish at a temperature not exceeding 4500

C. until free carbon was left, cooled and final weight

was taken. The percentage of ash calculated with reference to the air-dried drug (PASF,

1987).

Determination of Acid Insoluble Ash: The ash obtained as above method was boiled for 5

minutes with 25 ml of dilute hydrochloric acid and collected the insoluble matter on the ash-

less filter paper, washed with hot water and ignited to constant weight. The percentage of

acid insoluble ash with reference to the air dried drug was calculated (PASF, 1987).

Determination of water soluble Ash: The ash was boiled for 5 minutes with 25 ml of

water, collected insoluble matter on the ash-less filter paper, washed with hot water,

and ignited for a temperature not exceeding 4500C. The weight of the insoluble matter was




subtracted from the weight of the drug ash. The difference in weight represents the water

soluble ash. Finally percentage of water-soluble ash with reference to the air dried drug was

calculated (PASF, 1987).

Determination of Extractable Matter

Method I. Hot Extraction: About 25.0 g accurately weighed air-dried drug coarse powder

was placed in thimble and refluxed with various organic solvents hexane, chloroform, ethyl

acetate and alcohol. After recovery solvents under vacuum and drying in desiccators, the

percentage extractable matter was calculated (WHO, 1980).

Method II. Cold Maceration: About 2.0 g of coarsely powdered air dried material, was

accurately weighed in a glass stopper conical flask and macerated with 100 ml of solvent for

6 hrs shaking frequently, then allowed to stand for 18 hrs, filtered rapidly taking care not to

lose solvent. The extracted matter was dried at 1050C for 6 hrs, cooled in desiccators for 30

minutes and then weighed. The percentage extractable matter was calculated (WHO, 1980).

Determination of Swelling Index: About 1.0 g fine powder accurately weighed was taken

into 25 ml of glass stoppered measuring cylinder. The internal diameter of the cylinder

was about 16 mm, the length of the graduate portion about 125 mm, marked in 0.2 ml in

division from 0 to 25 ml in upward direction. 25 ml, of water was taken and the mixture

thoroughly shaken every 10 minutes for 1 hrs. Kept for 3 hrs at room temperature and the

volume in ml occupied by the plant material, including any sticky mucilage was measured.

The mean value of the individual determination, related to 1.0g of plant material

was calculated (WHO, 1998).




Determination of Foaming Index: About 1.0 g a coarse powder of drug was placed into

a 500 ml conical flask containing 100 ml of boiling water. The moderate boiling was

maintained for 30 minutes. Cooled and filtered into a 100 ml volumetric flask and volume

was made up to the mark with distill water (WHO, 1998).

The decoction was poured into 10 stoppered test-tubes (height 16cm, diameter 16 mm) in

successive portion of 1ml, 2ml, 3ml, etc. Up to 10ml, and adjusted the volume of the liquid

in each tube with water to 10ml. The tubes were stopper and shaken them in length wise

motion for 15 seconds, two shake per second. After 15 minutes and height of the foam was

measured. The results are assessed as follows.

If the height of the foam in every tube is less than 1 cm, the foaming index is less

than 100 &

If the height of the foam 1 cm is measured in any tube, the volume of the plant

material decoction in this tube (a) is used to determine the index. If this tube is the

first or second tube in a series, prepare an intermediate dilution in a similar

manner to obtain a more precise result.

If the height of the foam is more than 1 cm in every tube, the foaming index is

over 1000. In this case repeat the determination using a new series of dilution of

the decoction in order to obtain a result.

Foaming index =1000\a

Where a = the volume in ml of the decoction used for preparing dilution in the tube

where foaming to a height of 1cm is observed.




Determination of Heavy Metals: About 0.504 g air dried material was accurately weighed

and placed in the test tubes for predigestion the test tube was contain 5ml nitric acid. It was

kept as such for a day.

Now digestion was perfomed by adding the nitric acid and perchloric acid in the ratio of

10:4. then 5ml sample for determination was made as 3.57 nitric acid 1.42 perchloric acid.

Now it kept for heating at 170-1800C for about 4 hrs. Now sample was cool and filtered

and volume was made up to 50 ml in volumetric flask with distilled water. With the

help of instrument named as I.C.P.[O.E.S.] Model optima 5300V. heavy metals was

determined.

Determination of heavy metals:

Reading __

Blank = y mg / L

Yx50/0.5 = Yx100 mg/kg (ppm). Where 0.5 is wt of drug taken.

Chromatographic Studies

Characterization of Non-Volatile and Polar Components of: Berberis AristataHPTLC

Fingerprinting of Berberis Aristata

Sample Prepration: 100 mg dried and finally milled tissues viz. aerial, flower, root, parts of

Berberis Aristata and Hamelia Patens were extracted with various organic solvents viz.

hexane, chloroform, ethyl acetate, methanol. (35ml, 10hrs for extraction everytime)

separately and exhaustively extracted residues were collected and dissolved in respective




solvents and centrifuged at 10,000rpm for 10 mins. The supernatant were filtered with

0.45m filter and used for TLC analysis.

Pre-coated TLC silica gel 60F254, (10 10 cm, 20 10cm) were used for optimization of

analytical protocol. Various system, TLC Scanner winctas-3, Reprostar-3, twin trough

chamber, immersion device-3, TLC plate heater, and Spin win were used for digital image

scanning for HPTLC fingerprint development.

Chromatographic Conditions: Different composition of organic solvent of varying polarity

were tried to achieve the separation on TLC plates. The suitable mobile solvent

composition for various tissues of Berberis Aristata is summarized in Table 4.




Pharmacological Screening

Antifungal Assay Test fungi such as Trichphyton longifusus, Candia albicans,

Aspergillus flavus, Microsproum canis, Fusarium solani and Candia glabrata were

employed for preliminary screening. Extracts were dissolved in sterile DMSO to serve

as stock solution. Sabouraud dextrose agar was prepared by mixing Sabouraud 4 %

glucose agar and agar in distilled water. Known amount of media was dispensed into

screw capped test tubes. Test tubes containing media were autoclaved 121 C° for 15

minutes. Tubes were allowed to cool to 50 C° and the desired concentration of extract

was added into non-solidified media. The tubes were allowed to solidify at room

temperature. Each tube was inoculated with a 4 mm diameter piece of inoculums removed

from a seven- day-old culture of fungi. All culture-containing tubes were inoculated at

Table 4: Summary of Solvent Composition Used For HPTLC Fingerprint of Various

Tissues of Berberis Aristata

Sr. No. Plant Part Mobile Phase Peak visualization procedure

1. Aerial part CHCl3:MeOH

(88:12 v/v)

254nm, 366nm

Vannilin-sulphuric acid reagent 620nm

2. Flower CHCl3:MeOH

(88:12 v/v)

254nm, 366nm


3. Root CHCl3:MeOH

(88:12 v/v)

254nm, 366nm





optimum temperature of 28-30 C° for growth for 7-10 days. Culture was examined at

least twice a weekly during the incubation. The concentration of the sample was 400ug /ml

of DMSO and incubation period was 7 days at 28 C° (Atta-ur-Rehman et al., 2001).

RESULTS AND DISCUSSION

Microscopic Characters of Stem of Berberis Aristata

The slides of T.S of different parts of plant were prepared and subjected to microscopical

examination. The histology of different parts of plant was examined and the

observations were recorded. The leaves showed groups of fibres with calcium oxalate

crystals and also exhibit outer and inner epidermis with round to oval cells, covered with

striated cuticle. A multilayered strip of collenchymas (3-4 layered) appear above the lower

epidermis and below the upper epidermis, midrib compose of xylem and phloem

arranged in an arc. Calcium oxalate crystals were numerous and mainly of cluster crystal

type. It contains numerous covering trichomes scattered in the powder. Some xylem

vessels (pitted vessels) were also visible which were lignified. Cells of palisade and

spongy parenchyma were also visible.

A B




C

Figure 2: (A) T.S of Stem, (B) T.S of Cork Cells, (C) TS of Fibers

Quantitative Leaf Microscopy

Table 5: Quantitative Leaf Microscopy of Berberis Aristata

Parameter Range

Palisade Ratio 06-10

Stomatal Number Upper surface 0

Stomatal Number Lower surface 12-15

Stomatal Index Upper surface 06

Stomatal Index Lower surface 10.44-12.32




Vein islet number 12-18

Veinlet Termination Number 6-10

Table 6: Physico-Chemical Parameters of Berberis Aristata

S.No. Parameters Observations

1.

PHYSICO-CHEMICAL

Foreign Organic matter No adulterants

Ash Values (% w/w)

(a) Total Ash Value 3.23 0.01

(b) Acid Insoluble Ash 2.33 0.01

(c) Water Soluble Ash 1.23 0.01

2.

Extractive Values (% w/w)

Cold percolation method

Petroleum ether(40-60) 2.03 0.13

Methanol (95%) 1.51 0.42

Methanol (50%) 1.20 0.11

Water 1.01 1.21

Soxhlet successive extraction

method

Hexane 2.11

Chlorofrom 3.30

Ethyl acetate 0.74

Methanol (95%) 1.14

Moisture content 7.03 0.02




Pharmacological

Swelling Index 0.26 0.14

Foaming index 126.45

Heavy metals & Minerals ( ppm)

Cadmium 2.6

Cooper 10.2

Iron 2.10

Chromium 12.4

Manganese 1.8

Nickel 9.7

Lead 5.6

Zinc 24.6

Calcium 14.02

Potassium 4.3

Sodium 5.6

Table 7 : Phytochemical test of Berberis Aristata Leaves Extracts

Active constituents CCR CPE CAC CME

Alkaloids ++ ++ +++ ++

Flavonoids +++ +++ ++ ++

Saponins +++ ++ +++ ++

Tannins +++ ++ +++ ++

Steroids +++ ++ +++ +++

Cardiac Glycosides + ++ +++ +++

Proteins ++ ++ ++ ++




HPTLC Fingerprint Analysis of Various Tissues of Berberis Aristata

The standardized herbal product ensures the consistency in the overall quality which

addresses the identity, safety, efficacy and stability. The undefined active compounds and the

abundance of phytomolecules in the herbs make the standardization of herbal products

challenging. It is impossible to determine potency measuring one or two marker compounds,

since the active principles have neither been conclusively identified nor can they be

accurately measured. The possibility in variation of extractability of pharmacological active

constituent cannot be ruled out during process and product development stages, thus can

affect the desired bioactivity. In order to overcome this issue, HPTLC fingerprinting is

practical solution to characterize the complex herbal extract. We have developed the HPTLC

fingerprints of various tissues of Berberis aristata and active extracts: hexane, chloroform,

ethyl acetate, methanol, of Aerial, flower, root parts by means of obtaining the Rf, colour of

the band and digital scanning.

Resins _ _ _ _ _ _ _

Starch _ _ _ _

Triterpenoids _ _ _ _ _

Steroids _ _ _ _




Figure 3: HPTLC Chromatograms of Extracts of Aerial, Flower and Root Part of

Berberis Aristata at 254nm Densitogram & TLC Image.




Figure 4: HPTLC Chromatograms of Extracts of Aerial, Flower And Root Part

of Berberis Aristata At 366nm Densitogram & TLC Image

Figure 5: HPTLC Chromatograms of extracts of aerial, flower and root part of

Berberis aristata at 620nm after derivatization with vanillin-sulphuric acid

Densitogram & TLC image.

Pharmacological Screening

Antifungal Assay of Berberis Aristata

Berberis Aristata (aerial parts) different extract exhibited significant activity against

Fusarium solani with 90 % inhibition & 60 % against Candida glabrata 80 % and 60%

inhibition against Aspergillus flavus and Microsporum canis and 50% & 70% Trichphyton

longifusus & Candida albicans respectively. While all other extracts under investigation

remain inactive as shown in table

Table 8: Results of antifungal activity of different extracts of Berberis Aristata




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