Inter. J. of Phytotherapy / Vol 2 / Issue 2 / 2012 / 74-88.

e - ISSN - 2249-7722
Print ISSN - 2249-7730

International Journal of Phytotherapy

www.phytotherapyjournal.com

A REVIEW ON STANDARDISATION OF HERBAL FORMULATION

Arun Rasheed*, Sravya Reddy B, Roja C
*Department of Pharmacognosy and Phytochemistry, Sree Vidyanikethan College of Pharmacy,
A. Rangampet, Tirupati, Andhra Pradesh, India.

ABSTRACT
Herbal medicines are not a simple task since many factors influence the biological efficacy and
reproducible therapeutic effect. Standardized herbal products of consistent quality and containing well-defined
constituents are required for reliable clinical trials and to provide consistent beneficial therapeutic effects.
Pharmacological properties of an herbal formulation depend on phytochemial constituents present therein.
Development of authentic analytical methods which can reliably profile the phytochemical composition, including
quantitative analyses of market/bioactive compounds and other major constituents, is a major challenge to scientists.
An overview covering various techniques employed in extraction and characterization of herbal medicines as well as
herbal nanomedicines standardization is reported. In addition, phytosomes increased bioavailability, bhasma as
ametal nanobarrier drug delivery system, potential of metabolomics in the development of improved
phytotherapeutic agents, DNA based molecular markers in adulterants, and SCAR markers for authentification and
discrimination if herbs from their adulterants are reported. Nanotechnology based herbal drugs possess improved
solubility and enhanced bioavailability.

Key words: WHO, Herbal formulation, Standardization, Quality control, Nanoherbal drugs, Phytosomes, DNA
markers.

INTRODUCTION develop pharmacopeial standards for ayurvedic

Standardization of herbal formulations is preparations. The subject of herbal drug standardization
essential in order to assess of quality drugs, based on the is massively wide and deep. There is so much to know
concentration of their active principles, physical, and so many seemingly contradictory theories on the
chemical, phyto-chemical, standardization, and In-vitro, subject of herbal medicines and their relationship with
In-vivo parameters. The quality assessment of herbal human physiology and mental function. India needs to
formulations is of paramount importance in order to explore the medicinally important plants. This can be
justify their acceptability in modern system of medicine achieved only if the herbal products are evaluated and
[1]. One of the major problems faced by the herbal analyzed using sophisticated modern techniques of
industry is the unavailability of rigid quality control standardization.
profiles for herbal materials and their formulations. In World Health Organization (WHO) encourages,
India, the department of Ayush, Government of India, recommends and promotes traditional/herbal remedies in
launched a central scheme to develop a standard natural health care programmes because these drugs are
operating procedures for the manufacturing process to easily available at low cost, safe and people have faith in

Corresponding Author:- Arun Rasheed Email: arunrasheed@rediffmail.com

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them. The WHO assembly in number of resolutions has 3) Safety assessment; documentation of safety based on
emphasized the need to ensure quality control of experience or toxicological studies.
medicinal plant products by using modern techniques and 4) Assessment of efficacy by ethnomedical informations
applying suitable standards [2]. and biological activity evaluations.
The bioactive extract should be standardized on the basis
Standardization of raw materials includes the of active principles or major compounds along with the
following steps:- chromatographic fingerprints (TLC, HPTLC, HPLC, and
Authentication- Each and every step has to be GC).
authenticated, area of the collection, parts of the plant
collection, the regional situation, as phytomorphology 1. Quality Control of Herbal Drugs
botanical identity, microscopic and histological Quality control for efficacy and safety of herbal products
analysis(characteristic features of cell walls, cell contents, is of paramount importance. Quality can be defined as the
starch grains, calcium oxalate crystals, hairs, fibers, status of a drug that is determined by identity, purity,
vessels etc.) content, and other chemical, physical, or biological
Several studies of the histological parameters are properties, or by the manufacturing processes. Quality
list of palisade ratio, vein islet number, vein termination, control is a term that refers to processes involved in
stomatal number, stomatal index, trichomes, stomata, maintaining the quality and validity of a manufactured
quantitative microscopy, taxonomic identity, foreign product.
matter. Loss on drying, swelling index, foaming index, The term “herbal drugs” denotes plants or plant
ash values and extractive values, Chromatographic and parts that have been converted into phytopharmaceuticals
spectroscopic evaluation, Determination of heavy metals, by means of simple processes involving harvesting,
pesticide residues, Microbial contamination, Radioactive drying, and storage [3]. Hence they are capable of
contamination. variation. This variability is also caused by differences in
The parameter stability of herbal formulations growth, geographical location, and time of harvesting. A
that includes pharmacognostic parameters, physico- practical addition to the definition is also to include other
chemical parameters, phyto-chemical parameters, crude products derived from plants, which no longer show
microbiological assay, chromatographic analysis. any organic structure, such as essential oils, fatty oils,
resins, and gums. Derived or isolated compounds (e.g.
Pharmacognostic evaluation strychnine from strychnous nux-vomica) or mixtures of
It includes color, odor, taste, texture, size, shape, compounds (e.g. abrin from Abrus precatorius).
microscopical characters, and histological parameters. In general, quality control is based on three important

 Identity- it should have one herb

Physico-chemical parameters It includes foreign pharmacopeial definitions

 Purity – it should not have any contaminant other

matter, total ash, acid-insoluble ash, swelling and foaming
index, assay, successive extractive values, moisture

 Content or assay-the active constituents should be

content, viscosity, PH, than herb
Disintegration time, friability, hardness, flow capacity,
flocculation, sedimentation, alcohol content. within the defined limits.
Chemical parameters It includes limit tests, chemical It is obvious that the content is the most difficult one to
tests etc. assess, since in most herbal drugs the active constituents
Chromatographic and spectroscopic analysis It includes are unknown. Sometimes markers can be used which are,
TLC, HPLC, HPTLC, GC, UV, IR, FT-IR, AAS, LC-MS, by definition, chemically defined constituents that are of
GC-MS, fluorimetry etc. interest for control purposes, independent of whether they
Microbiological parameters It includes the full content of have any therapeutic activity or not [4].
viable, total mould count, total coliforms count. Limiters Identity can be achieved by macro and
can be used as a quantitative tool or semi-quantitative to microscopical examinations. Voucher specimens are
determine and control the amount of impurities, such as reliable reference sources. Outbreaks of diseases among
reagents used in the extraction of various herbs, plants may result in changes to the physical appearance of
impurities ships directly from the manufacturing and the plant and lead to incorrect identification [5,6]. At
solvents etc. times an incorrect botanical quality with respect to the
labeling can be a problem.
WHO GUIDELINES FOR QUALITY Purity is closely linked with safe use of drugs
STANDARDIZED HERBAL FORMULATIONS and deals with factors such as ash values, contaminants
1) Quality control of crude drugs material, plant (e.g. foreign matter in the form of other herbs), and heavy
preparations and finished products. metals. However, due to the application of improved
2) Stability assessment and shelf life. analytical methods, modern purity evaluation also
includes microbial contamination, aflatoxins,

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radioactivity, and pesticide residues. Analytical methods botanist and should be stored for at least a 10-year period.
such as photometric analysis, Thin layer chromatography A lot number should be assigned and this should appear
(TLC), High performance liquid chromatography on the product label.
(HPLC), High performance thin layer chromatography
(HPTLC), and Gas chromatography (GC) can be Plant preparations
employed in order to establish the constant composition The manufacturing procedure should be
of herbal preparations. described in detail. If other substances are added during
Content or assay is the most difficult area of manufacture in order to adjust the plant preparation to a
quality control to perform, since in most herbal drugs the certain level of active or characteristics constituents or for
active constituents are unknown. Sometimes markers can any other purpose, the added substances should be
be used. In all other cases, where no active constituents or mentioned in the manufacturing procedures. A method for
marker can be defined for the herbal drug, the percentage identification and, where possible, assay of the plant
extractable matter with a solvent may be used as a form of preparation should be added. If identification of an active
assay, an approach often seen in pharmacopeia [7,8]. principle is not possible, it should be sufficient to identify
A special form of assay is the determination of a characteristic substance or mixture of substances to
essential oils by steam distillation. When active ensure consistent quality of the preparation.
constituents (e.g. sennosides in senna) or markers (e.g.
alkydamides in Echinacea) are known, a vast array of Finished product
modern chemical analytical methods such as The manufacturing procedure and formula,
ultraviolet/visible spectroscopy(UV/VIS), TLC, HPLC, including the amount of excipients, should be described in
HPTLC, GC, mass spectrometry, or a combination of GC detail. A finished product specification should be defined
and MS(GC/MS), can be employed [9]. to ensure consistent quality of the product. The finished
product should comply with general requirements for
2. Stability Assessment and Shelf Life particular dosage forms.
The past decade has seen a significant increase in the use
of herbal medicines. As a result of WHO‟s promotion of Stability
traditional medicine, countries have been seeking the The physical and chemical stability of the
assistance of the organization in identifying safe and product in the container in which it is to be marketed
effective herbal medicines for use in national health care should be tested under defined storage conditions and the
systems. shelf-life should be established.
Prolonged and apparently uneventful use of a
substance usually offers testimony of its safety. In a few Safety assessment:
instances, however, investigation of the potential toxicity Herbal medicines are generally regarded as safe
of naturally occurring substances widely used as based on their long-standing use in various cultures.
ingredients in these preparations has revealed previously However, there are case reports of serious adverse events
unsuspected potential for systematic toxicity, after administration of herbal products. In a lot of cases,
carcinogenicity and teratogenicity. Regulatory authorities the toxicity has been traced to contaminants and
need to be quickly and reliably informed of these adulteration. However, some of the plants used in herbal
findings. They should also have the authority to respond medicines can also be highly toxic. As a whole, herbal
promptly to such alerts, either by withdrawing or varying medicines can have a risk of adverse effects and drug-
the licences of registered products containing suspect drug and drug-food interactions if not properly assessed.
substances, or by rescheduling the substances to limit Assessment of the safety of herbal products,
their use to medical prescription [10]. therefore, is the first priority in herbal research.
These are various approaches to the evaluation of
Assesement of quality safety of herbal medicines. The toxic effects of herbal
All procedures should be in accordance with preparation may be attributed mainly to the following:
good manufacturing practices. Inherent toxicity of plant constituents and ingredients and
Manufacturing malpractice and contamination.
Crude plant material Evaluation of the toxic effects of plant
The botanical definition, including genus, constituents of herbal formulation requires detailed phyto-
species and authority, description, part of the plant, active chemical and pharmacological studies. It is, however, safe
and characteristics constituents should be specified and, if to assume that, based on human experiences in various
possible content limits should be defined. Foreign matter, cultures, the use of toxic plant ingredients has already
impurities and microbial content should be defined or been largely eliminated and recent reports of toxicity
limited. Voucher specimens, representing each lot of plant could largely ne due to misidentification and overdosing
material processed, should be authenticated by a qualified of certain constituents [11]. Substitution and

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misidentification of herbal substances, documented or Standardization of herbal formulation requires

regulatory approaches, development of monitoring and implementation of Good Manufacturing Practices (GMP)
surveillance systems, assessment of toxicity, risk [13,14,15]. In addition, study of various parameters such
assessment approach. as pharmacodynamics, pharmacokinetics, dosage,
The evaluation of new herbal products consists stability, shelf-life, toxicity evaluation, chemical profiling
of six steps, which define the following: Characteristics of of the herbal formulations is considered essential [16].
new substances, history and pattern of use, any adverse Other factors such as pesticide residue, aflatoxine content,
reaction, biological action, toxicity and carcinogenicity, heavy metals contamination, Good Agricultural Practices
and clinical trial data. (GAP) in herbal drug standardization are equally
The presence of impurities is either an intended important [17]. A schematic representation of herbal drug
addition, or accidental contamination via processing. standardization is shown in figure 1.
The substitution of plants arises because of similar plants/ The standardization of various marketed herbal
wrong identification, or the use of cheaper alternatives. and poly herbal formulation [kasisa bhasma, a traditional
formulation, containing the mixture of Emblica
Assessment of toxicity officianalis and lime juice used in the treatment of
Toxicity investigation will also be required anaemia, hepatotoxicity [18], Ashwagandhalehyam, a
because the analysis alone is unlikely to reveal the traditional formulation, used in the treatment of
contributions to toxicity itself. In assessing toxicity of an inflammation, and epileptic seizures have been reported
herbal medicine, the dose chosen is very important [12]. [19]. Ashwagandharistam, a traditional formulation,
Toxicity assessment involves one or more of the havind good anti-epileptic activity have been reported
following techniques- In vivo techniques, in vitro [20]. [Madhumehari churna (Baidynath) containing the
techniques, cell line techniques, micro- array and other mixture of eight herbal antidiabetic drugs [21]. Panacasa
modern technique Standardization techniques to churna known to be effective in gastrointestinal disorder
adequately model toxicity. [22], Dashamularishta, a traditional formulation, used in
the normalization of physiological processes after child
Assessment of efficacy birth [23], Gokshuradi churna, Megni, Jawarish-e
Herbal medicines are inherently different from Darchini [24,25] have been reported. But still there are
conventional pharmacological treatments, but presently many poly herbal formulations which require
there is no way to assess their efficacy other than by standardization as there are frequently used based only on
currently used conventional clinical trial methodologies, their ethanobotanical use [26]. Standarization minimizes
in which efficacy is conventionally assessed by clinical, batch to batch variation; assure safety, efficacy, quality
laboratory, or diagnostic outcomes: Clinical outcomes and acceptability of the poly herbal formulations [27].
include parameters such as improved morbidity, reduced Methiorep premix (a combination of herbs viz. Cicer
pain or discomfort, improved appetite and weight gain, arientinum, Phaseolus mungo, Mucuna pruriens, Triticum
reduction of blood pressure, reduction of tumor size or sativum, Allium cepa &richer source of protein with
extent, and improved quality of life. Laboratory /other highly bioavailable methionine) has been recommended
diagnostic outcomes include parameters such as reduction as a safe product to replace synthetic methionine in
of blood glucose, improvement of hemoglobin status, poultry ration and for supplementation in basal diet for
reduction of opacity as measured by radiological or regular usage [28]. TLC and HPTLC fingerprint were
imaging techniques, and improvement in used for deciding the identity, purity and strength of the
electrocardiogram (ECG) findings. poly herbal formulation and also for fixing standards for
Implementation of a standardized approach for this Ayurvedic formulation [29].
the herbal practitioners and collection of the prospective
data necessarily creates an interventional design which, if MODERN TECHNIQUES OF EXTRACTION
planned properly, may closely resemble single-blind METHODS
randomized trials. Even if it differs from double-blind Supercritical fluid extraction (SFE)
randomized trials in the degree of rigor, the design may Supercritical fluid extraction is the most
be the optimum, both biologically and economically, for preferable process for the extraction of the active
rapid evaluation of herbal products. Standardization, constituents from the medicinal and aromatic plants [30].
however, may sometimes be incompatible with the SFE has emerged as a highly promising technology for
existing legislative framework and caution is needed the production of herbal medicines and nutraceuticals
regarding the ethical implications of such studies. with high potency of active ingredients [31]. SFE
techniques have been found useful in isolating the desired
CONVENTIONAL METHODS FOR phytoconstituents from plant extracts [32].
STANDARDISATION OF HERBAL
FORMULATION

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Figure 1. A schematic representation of herbal drug standardization

Colour odour Qualitative

taste Quantitative
texture&fractue SEMstudies
Powder studies

Moisture content
oistt Macroscopic Microscopic
Extractive, ash
KK Hh
value

Physical Botanical

Standardization of herbal drugs

Biological Invitro studies Chemical

Microbial contamination Chromatographic techniques

Pharmacological evaluation Heavy metal

Toxicological studies Pesticide residue

Mycotoxin

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Figure 2. Different types of extraction techniques

SFE
SPE

EXTRACTION

SBE
MAE
CCE

SFE- Super critical fluid extraction

SPE- solid phase extraction
MAE- microwave assisted extraction
CCE- counter current extraction
SBE- spouted bed extraction

Figure 3. A schematic representation of different techniques of chromatography

CAPILLARY TLC COLUMN

SUPER CRITICAL HPLC

LC-MS HPTLC
Techniques of
Chromatography

GC-MS
LC-NMR paper gel electrophoresis

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Figure 4. A Schematic representation of RAPD

Figure 5. A schematic representation of RFLP

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The use of supercritical fluids for the extraction comparision with more traditional sample preparation
of a range of materials including plant products of techniques [35]. The solid phase extraction was
medicinal, flavouring and cosmetic interest has during the introduced for determining thirteen organochlorine
last decade, become of increasing economic and research pesticide residues including alpha-benzene hexachloride
interest. In 1822, Cagniard de la Tour reported that above (BHC), beta BHC, gamma BHC, delta BHC,p,p‟-
a certain temperature and pressure, single substances do dichloro-diphenyldichloroethylene (pp‟DDE),
not condense or evaporate but exist as a fluid. Under these p,p‟dichloro-di-phenydichloroehane (pp‟DDD), o,p‟-
conditions the gas and liquid phase both possess the same dichloro-diphenytrichloroetane (op‟-DDT), pp‟-DDT
density and no division exist between the two phases. heptachlor (HEPT), aldrin, heptachlor epoxide (HCE),
This is the critical state. In phytochemistry these dieldrin and andrin in scutellaria baicalensis, Salvia
properties can be exploited to maximize the extraction of miltiorrhiza, Belamcanda chinensis, Paeoniae lactiflore,
plant constituents. Angelica dahurica, Arisaema erubescens, Fructus arctii,
Super critical fluid extraction (SFE) involves use Anemarrhena asphodelodes and Platycodon
of gases, usually CO2, and compressing them into a dense grandiflorum. The organochlorine pesticides were
liquid. This liquid is then pumped through a cylinder extracted from herbs with mixed solvents of acetone and
containing the material to be extracted. From there, the n-hexane by ultrasonic and cleaned up by Florosil solid
extract laden liquid is pumped into a separation chamber phase extraction column [36]. Solid phase extraction was
where the extract is separated from the gas and the gas is used to prepare the test solution for the analysis of
recovered for re-use. There are many other gases and aristolochic acid I and II in herbal medicines [37].
liquids that are highly efficient as extraction solvents
when put under pressure. Spouted bed extraction
Examples ivolving the extraction of phytochemicals with In certain instances, as in the production of
supercritical carbon dioxide follow: annatto powder from the seeds of Bixa orellana, the
(1) Alkaloids : decaffenation of green coffee physical removal of the pigment layer of the seed-coat
Isolation of vindoline from Cathranthus roseus. can yield a less impaired product than that produced by
(2) Pigments : extraction of annatto sees solvent extraction. Such methods can involve the use of
(3) Diterpene: extraction of Taxus brevifolia and T ball mill or a spouted bed unit. A development of the
.cuspidata latter, the conical spouted bed extractor, has been
(4) Acylphloroglucinols: oxygenated Hyperforin investigated for annatto production. Basically it consists
derivative of Hypericum [33]. of a cylinder tapered at both ends and containing the seeds
at the lower end through which a jet of hot air if forced.
Coupled SFE-SFC Seeds and pigment –loaded fine particles are propelled
System in which a sample is extracted with a into the space above from whence the seeds fall back to
supercritical fluid which then places the extracted be recirculated and the annatto powder moves to a
material in the inlet part of a supercritical fluid cyclone from which it is collected [38].
chromatographic system. The extract is than
chromatographic directly using supercritical fluid. Counter-current extraction
This is a liquid- liquid extraction process and the
Coupled SFE-GC and SFE-LC principle involved is similar to partition chromatography.
System in which a sample is extracted using a Briefly, a lower, stationary phase is contained in a series
supercritical fluid which is then depressurized to deposit of tubes and an upper, moving immiscible liquid is
the extracted material in the inlet part or a column of gas transferred from tube to tube along the series, the
or liquid chromatographic system respectively. SFE is immiscible liquids being shaken and allowed to separate
characterized by robustness of sample preparation, between each transference. The mixture to be fractionated
reliability, less time consuming, high yield and also has is placed in the first tube containing the immiscible
potential for coupling with number of chromatographic liquids and the apparatus is agitated and the layers are
methods [34]. allowed to separate. The components of the mixture will
be distributed between the two layers according to their
Solid phase extraction (SPE) partition coefficients. The upper phase is moved along to
SPE technique is applied for isolation of analyte the second tube containing lower phase and more moving
from a liquid matrix and purified herbal extracts. This phase is brought into contact with the lower phase of tube
technique has many advantages such as: high recovery of 1. Shaking and transference again takes place and
the analyte, concentrate of analyte, highly purified continues along a sufficient number of tubes to give a
extracts, ability to simultaneously extract analytes of high fractionation of the mixture.
polarity range, ease of automation, compatability with The distribution of each substance over a given
instrumental analysis and reduction in organic solvent in number of tubes can be ascertained by the terms of the

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binomial expansion. If buffer solutions are used as the The combination of HPLC and LC/MS is presently
stationary phase, the exploits differences in ionization powerful technique for the quality control of Chinese
constants of the various solutes. In some instances the medicine i.e. liquorice [47].
effectiveness of the separation can be improved by using Kankasava is a fermented polyherbal formulation
a series of buffer solutions of graded pH value as the prepared with Kanaka and other ingredients [48]. It is
lower phase. Solutions of acids and alkalis also find some used in chronic Bronchitis, asthmatic cough and
application as lower phase. breathlessness. Kankasava is analysed by RP HPLC. It is
A development of the extractor is the steady state a simple, precise, accurate RP- HPLC method was
distribution machine. This allows true counter-current developed for the quantitative estimation of atropine in
extraction and can be programmed so that both solvents kankasava polyherbal branded formulations. The
move in opposite directions. The mixture is to be separation was achieved with a column RP C-18
fractionated is fed continuously into the centre of the train (250mm×4.6mm×5 micron) using mobile phase mixture
of cells and, according to the solvents, the solutes may of methanol &10 mmol dihydrogen phosphate buffer in a
move in either direction [39]. ratio of 50:50 v/v at a flow rate of 1 ml/min, and analysis
Microwave –assisted extraction (MAE) was screened with UV detector at 254 nm. The retention
MAE technology includes the extraction of high- time for standard atropine sulphate was found to be
value compounds from natural sources including 4.0667 minutes. Linearity was found to be r 2 = 0.998.
phytonutrients, neutraceutical and functional food
ingridients and pharmaceutical actives from biomass [40]. High performance thin layer chromatography (HPTLC)
MAE find a utility in production of cost effective herbal TLC is the common fingerprint technique for
extracts and helpful in extraction of carotenoids from herbal analysis. The herbal compounds can easily
single cells, taxanes from taxus biomass, essential fatty identified by TLC [49]. In this technique, the
acids from microalgae and oilseeds, phytosterols from authentication of various species, evaluation of stability
medicinal plants, polyphenols from green tea, and and consistency of their preparations from different
essential oils from various sources. Compared to manufacturers [50]. HPTLC is the common fingerprint
conventional solvent extraction methods, advantages of mainly used to analyze the compounds which is having
this technology include: a) improved product,-purity of low or moderate polarities. HPTLC technique is widely
crude extracts, -stability of marker compounds and use of used in the pharmaceutical industry for process
minimal toxic solvents. b) reduced processing costs, development, identification and detection of adulterants,
increased recovery and purity of marker compounds, very substituent in the herbal products and also helps in the
fast extraction rates, reduced energy and solvent usage identification of pesticide content, mycotoxins and in
[41,42]. quality control of herb and health products [51]. HPTLC
technique was reported for simultaneous estimation of
Modern techniques in herbal drug identification and gallic acid, Rutin, Quercetin in terminalia chebula [52].
characterization The aqueous extract of Terminalia chebula, precoated
HPLC silica gel GF 254 as stationary phase and the mobile phase
The preparative and analytical HPLC are widely for tannins toluene: acetone: glacial acetic acid (3:1:2)
applicable in pharmaceutical industry for isolating and and mobile phase for rutin and quercetin, ethyl acetate:
purification of herbal compounds. They are of basically dichloromethane: formic acid: glacial acetic acid: water
two types in preparative HPLC: those are low pressure (10:2.5:1:1:0.1). Detection and quantification were
HPLC (typically under 5 bars) and high pressure HPLC reported densitometrically at λ = 254 for gallic acid and
(pressure greater than 20 bar) [43,44]. The most important 366nm for rutin and quercetin. The Rf values of gallic
parameters to be considered are of resolution sensitivity acid, rutin and quercetin are 0.30, 0.13, 0.93.
and fast analysis time in analytical HPLC however both The simultaneous estimation of withaferin A and
the degree of solute purity and the amount of compound beta- sinosterol- d- glucoside in four Ashwagandha
that can be produced per unit time that is recovery in formulations [53]. Syzygium jambolanum was
preparative HPLC [45]. quantitatively estimated in terms of stability, repeatability,
The main aim is to isolate the herbal compounds, accuracy and phytoconstistuents such as glycoside,
where as in analytical work the aim is to get the tannins, ellagic acid and gallic acid by HPTLC [54]. It
information about sample. The preparative HPLC is the was employed for detection, monitoring and
closest to analytical HPLC than the traditional PLC, quantification of bacoside A & B in Bacopa monnieria
because it is having higher column efficiencies and faster and its formulations. Simultaneous estimation of
solvent velocities permit more difficult separation to be Diosgenin and Levodopa was done by HPTLC method.
conducted more quickly [46]. This is most important in Quantification was carried out at 194 nm for Diosgenin
the pharmaceutical industries because newer formulations and 280 nm for levodopa using sbsorbance reflectance
have to be introduced in the market as early as possible. mode. The Rf value of levodopa and Diosgenin was found

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to be 0.27+0.2 and 0.61+0.2. The content uniformity test quantitatively assess ME is the post –extraction
was carried out as per the USP specification. The fortification method.
proposed HPTLC method provides a faster and cost
effective quantitative control for the analysis of levodopa Supercritical fluid chromatography
and diosgenin [55]. The super critical fluid and microbore liquid
chromatography offer potential applications for the drug
Gas chromatography - mass spectroscopy (GC-MS) analysis. In this the mobile phase is a gas (CO2)
Gas chromatographic equipment can easily maintained at its supercritical state i.e., above its critical
interfaced with rapid scan mass spectrometer of various temperature and pressure. The SFC mobile phase has low
types. The flow rate of the capillary column is generally viscosity, approximating that of a gas, and high
low but enough that the column. Output can easily fed diffusivity, between those of capillary gas
directly into ionization chamber of MS. In this the chromatography and liquid chromatography.
simplest mass detector in GC is the Ion Trap Detector
[46]. The ions trap detector is remarkable compact and Capillary electrophoresis
less expensive than quadrapole instruments. The The methodology of CE was introduced to
identification and quantification of chemical constituents evaluate one drug in terms of specificity, sensitivity and
present in the polyherbal oil formulation was analyzed by precision, and the results were in agreement with those
GC-MS method [56]. An effective fast and accurate obtained by the HPLC method. Is morethan 100-fold less
capillary gas chromatography method was employed for [61]. Moreover the analysis time of CE method. The
determining the organochlorine pesticide residues. The hyphenated CE instruments, such as CE-diode array
SPE extract was separated by capillary column by using detection, CE-MS and CE-NMR, have been utilized,
electrochemical detector. The split ratio is of 1:2.2 using whereas, there are some limitations are being in CE
the carrier gas N2 with the flow rate of 1.4 ml/min. The hyphenations with respect to reproducibility were
Injector temperature is of 2200c and the detector reported [62].
temperature is of 3300c. Thus the good linearities were
obtained for organochlorine pesticides. It has been used Role of genetic markers in the standardization of herbal
for identification of large number of components present drugs
in natural and biological systems [57]. A DNA marker is a term used to refer a specific
DNA variation between individuals that has been found to
Liquid chromatography- Mass spectroscopy (LC-MS) be associated with a certain characteristic. These different
LC-MS is the one of the most prominent method DNA or genetic variants are known as alleles.DNA
of choice in many stages of drug development [58]. The marker testing or genotyping introduces which alleles an
chemical standardization of an aqueous extract of the animal is carrying for a DNA marker. DNA tests for
mixture of the herbs provided chemical compounds simple traits have been on the market for several years
serving as reference markers using LC-MS [59]. It is and include those for certain diseases, such as DUMPS
useful to analyze the aminoglycosides showed that these (Deficiency of Uridine Monophosphate Synthetase) and
drugs are highly soluble in water, showed low plasma BLAD (Bovine Leukocyte Adhesion Deficiency), coat
protein binding and more than 90 percent excreted colour, and horned status. It can be described as a
through the kidney[60]. The pharmacokinetic studies of variation, which may arise due to mutation in the genomic
Chinese medicinal herbs using LC-MS. Interference loci that can be observed.
peaks in biological samples are easily observed when Some of the commonly used of genetic markers
using HPLC coupled to ultraviolent, fluorescence and are: RAPD (Random amplification of polymorphic
electrochemical detectors. With the introduction of highly DNA), RFLP (Restriction fragment length
sensitive and selective LC-MS-based bioanalytical polymorphism), AFLP (Amplified fragment length
methods, sample preparation can usually be simplified to polymorphism), Micro satellite polymorphism SNP,
speed up the throughput of data. ME must be investigated (Single nucleotide polymorphism), SFP (Single feature
to ensure that accuracy, precision, selectivity and polymorphism), STR (Short tandem repeat).
robustness in the HPLC-MS- based procedures, so that
the sensitivity will not be compromised. On approach to Random Amplification of Polymorphic DNA
the identification of ME is the method of post- column RAPD is a type of PCR reaction, but the
infusion. In brief, the analyte of interest is infused segments of DNA that are amplified are random. The
constantly into the ion source of amass spectroscopy to scientist performing RAPD creates several arbitrary, short
form a steady signal. The blank matrix after sample primers (8-12 nucleotides), then proceeds with the PCR
preparation is then injected into the HPLC/MS system, using a large template of genomic DNA, hoping that
which it helps to determine the region in chromatogram fragments will amplify. By resolving the resulting
by the components of the matrix. Another approach to patterns, a semi-unique profile can be gleaned from a

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RAPD reaction. RAPD markers are decamer (10 helpful to identity cells, individuals or species as they can
nucleotide length) DNA fragments from PCR be used to produce normal, functioning proteins to replace
amplification of random segments of genomic DNA with defective ones. Moreover, these markers help in treatment
single primer of arbitrary nucleotide sequence and which of various diseases and help in distinguishing the genuine
are able to differentiate between genetically distinct herb from adulterated drug [65].
individuals, although not necessarily in a reproducible
way. It is used to analyse the genetic diversity of an ISSR (Inter-Simple Sequence Repeat)
individual by using random primers. Unlike traditional ISSR, a PCR- based application is unique and
PCR analysis, RAPD does not require any specific inexpensive popular technique of DNA finger printing
knowledge of the DNA sequence of the target organism which include the characterization of genetic
the identical 10- mer primers will or will not amplify a fingerprinting, gene tapping, detection of clonal variation,
segment of DNA, depending on positions that are phylogenetic analysis, detection of genomic instability,
complementary to the primers sequence. For example no and assessment of hybridization. Cannabis sativaand
fragment is produced if primers annealed too far apart of Arabidopsis thaliana L. Heyne have been differentiated
the primers are not facing each other. Therefore, if a from their adulterated species by using ISSR markers
mutation has occurred in the template DNA at the site that [65]. Molecular characterization by sequence-
was previously complementary to the primer, a PCR characterized amplified region (SCAR) markers allows
product will not be produced, resulting in a different effective and reliable authentification and discrimination
pattern of amplified DNA segments on the gel. A of herbs from their adulterants. In addition, morphological
schematic representation of RAPD as shown in the figure similar plant species can be differentiated using SCAR
4. markers [66].

RFLP (Restriction fragment length polymorphism) Phytosomes/ pharmacosomes: A novel drug delivery
This polymorphism consists of the presence or system for herbal drugs
absence of a restriction site for a bacterial restriction Pharmacosomes commonly known as phytosome
enzyme. This is an enzyme which breaks strands of DNA are drug- phospholipid complexes having active
wherever they contain they contain a certain sequence of ingredients of the herb and can be formulated in the form
half-a- dozen or so nucleotides. The locus of interest of solution, suspension, emulsion, syrup, lotion, gel,
could be probed using a radiolabelled piece of DNA with cream, aqueous microdispersion, pill, capsule, powder,
the same sequence as a part of the test locus. This would granules and chewable tablet [66,67]. Plants namely
selectively hybridise to the restriction fragment derived Silybium marianum, Ginkgo biloba and Ginseng showed
from the test locus. The whole process consisted of: better efficacy than conventional herbal formulations
Extracting DNA from white blood cells, digesting the [68]. In addition, the clinical trials of phytosomes have
DNA with a restriction enzyme into restriction fragments, shown increased bioavailabilty in comparision to
using gel electrophoresis to separate the fragments by conventional herbal formulations generally containing
size, denaturing the DNA so that the two strands of each polyphenols and flavonoids in humans [69]. Several
fragment separate, blotting the single –stranded DNA phytosomal herbal drug delivery systems have been
onto a filter to immobilize it, washing off excess probe. reported [70]. Phytosomal herbal drug delivery systems
are mainly used; to deliver systemic antioxidant, useful in
DNA fingerprinting technique treatment of the disease like blood pressure, liver disease,
DNA analysis has been proved as an important cancer, skin disease and helps in protecting the brain
tool in herbal drug standardization. This technique is lining [71].
useful for the identification of phytochemically
indistinguishable genuine drug from substituted or Standardization of herbal nanomedicines
adulterated drug. It has been reported that DNA Herbal nanotechnology helps incorporation of
fingerprint genome remain the same irrespective of the the active phytoconstituents to obtain desired therapeutic
plant part used while the phytochemical content will vary effect. The increased solubility, stability, bioavailabity,
with the plant part used, physiology and environment pharmacological activity of many popular herbal extracts
[63]. The other useful application of DNA fingerprinting including Milk thistle, Ginkgo biloba, grape seed, green
is the availability of intact genomic DNA specificity in tea, hawthorn, ginseng using nano dosage forms such as
commercial herbal drugs which helps in distinguishing polymeric nanoparticles nanospheres and nanocapsules,
adulterants even in processed samples [64]. Proper liposomes, proliposomes, solid lipid nanoparticles, and
integration of molecular techniques and analytical tools nanoemulsion has been reported [72]. Other advantage of
generated a comprehensive system of botanical herbal nanomedicine include protection from toxicity,
characterization that can be applied in the industry level improving tissue macrophages distribution, sustained
to ensure quality control of botanicals. DNA markers are delivery, protection from physical and chemical

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degradation [73]. Nanotechnology patents issue in due to the aggregation of the nanocrystals of the metallic
Chinese herbal medicine has been reported and oxides. It has been used in traditional medicines for
proliferation of nano – based Chinese herbal medicine treatment of anemia, hepatotoxicity [18].
patents in China was due to the illustrations of biomedical
technology progress extensively [74]. CONCLUSION
India can emerge as the major country and play
Bhasma as a nanoherbal medicine technology the lead role in production of standardized, therapeutically
Bhasma are the ayurvedic metallic preparations effective ayurvedic formulation. India needs to explore
in which metal act as a nanocarrier for drug delivery and the medicinally important plants. This can be achieved
are widely recommended for treatment of a variety of only if the herbal products are evaluated and analyzed
chronic ailments and are taken along with milk, butter, using sophisticated modern techniques of standardization
honey, or ghee to eliminate the harmful effects of metals such as UV-visible, TLC, HPLC, HPTLC, GC-MS,
and enhancing their biocompatibility in the body [75]. spectrofluorimetric and other methods. These guidelines
Neutron activation analysis of twenty metallic based for the assessment of herbal medicines are intended to
bhasmas such as calcium, iron, zinc, mercury, silver, facilitate the work of regulatory authorities, scientific
potassium, arsenic, Copper, tin, gemstones, confirmed the bodies and industry in the development, assessment and
purity of these bhasma as the other elements such as Na, registration of such products. The assessment should
K, Mg, V, Mn, Fe, Cu, Zn were found in microg/g reflect the scientific knowledge gathered in that field.
amounts and Au and Co in ultratrace (ng/g) amounts [76]. Such assessment could be the basis for future
Various techniques like atomic force microscope, classification of herbal medicines in different parts of the
scanning electron microscope, transmission electron world. Other types of traditional medicines in addition to
microscope and energy dispersive spectroscopy have been herbal products may be assessed in a similar way.
employed for the estimation and characterization of The advancement of analytical techniques will
bhasma. The Kasisa bhasma analyses qualitatively serve as a rapid and specific tool in the herbal research,
through X-ray diffraction, Fourier transform infrared thereby, allowing the manufacturers to set quality
spectroscopy correspond to inorganic metal (Fe2O3) standards and specifications so as to seek marketing
hydrated metal salt or oxide (FeO or Fe3O4). The FTIR approval from regulatory authorities for therapeutic
spectra showed no peak for any organic molecule or bond efficacy, safety and shelf- life of herbal drugs. The
corresponding it, there by confirming the absence of effective regulation and control of herbal medicines
organic matter and external organic contamination. moving in international commerce also requires close
The AAS study was conducted to determine the liaison between national institutions that are able to keep
concentration of elements present in the formulation. The under regular review all aspects of production and use of
results showed that the element iron was seen in the major herbal medicines, as well as to conduct or sponsor
concentration of 85.91%. AFM analysis confirmed the evaluative studies of their efficacy, toxicity, safety,
formulation has spherical morphology with an average acceptability, cost and relative value compared with other
particle size of 100 nm. The spherical morphology was drugs used in modern medicine.

REFERENCES
1. Satheesh Madhavi NN, Kumud Upadhya, Asha bishti. Phytochemical screening and standardization of poly herbal
formulation for Dyslipidemia. Indian journal of physiology and pharmacology, 3(3), 2011.
2. Sunita Panchawat, Kamal Singh Rathore, Sssisodia Nema RK. Standardization and evaluation of herbal drug
formulations. 2010.
3. EMEA. Quality of herbal medicinal products. Guidelines. European Agency for the Evaluation o Medicinal products
(EMEA), London, 1998.
4. WHO. Quality Control Methods for Medicinal Plant Materials. World Health Organisation, Geneva, 1992.
5. WHO. The International Pharmacopeia, Vol.3: Quality Specifications for Pharmaceutical Substances, Excipients, and
Dosage forms, 3rd edn. World Health organization Geneva, 1988.
6. De Smet. PAGM. Drug Information Journal, 33, 1999, 717-724.
7. WHO. Guidelines for the appropriate use of Herbal Medicines. WHO Regional publications, Western pacific series No
3, WHO Regional office for the Western Pacific, Manila, 1998.
8. WHO. Guidelines for the Assessment of Herbal Medicines. WHO Technical Report Series, No863. World Health
Organization, Geneva, 1996.
9. Watson DG. Pharmaceutical Analysis. Churchill Livingstone, Edinburgh, 1999.
10. WHO. Quality Assurance of Pharmaceuticals –A Compendium of Guidelines and Related Materials. 1, 1997.
11. ICDRA. 6th International Conference on Drug Regulatory Authorities, World Health Organization, 1991.

~ 85 ~
 Inter. J. of Phytotherapy / Vol 2 / Issue 2 / 2012 / 74-88.

12. TDR. Operational Guidance: Information Needed to Support Clinical Trials of Herbal Products, UNICEF/ UNDP/
WORLD Bank/WHO special program for Research and Training in Tropical Diseases (TDR) 2005.
13. Neeraj Choudary and Bhupinder Singh Sekhon. An overview of advances in the standardization of herbal drugs. Journal
of Pharmacognosy, 2, 2011, 55-70.
14. Indian Herbal Pharmacopoeia, Indian Drug Manufacturers Association, Mumbai, 2002.
15. Quality Control Methods for Medicinal Plant Materials, WHO, Geneva, 1996.
16. Mosihuzzaman M, Choudary MI. Protocols on safety, efficacy, standardization, and documentation of herbal medicine.
Pure Applied Chemistry, 80(10), 2008, 2195-2230.
17. Bauer R. Quality criteria and standardization of phytopharmaceuticals: Can acceptable drug standard can be achieved.
Journal of Drug Information, 32, 1998, 101-110.
18. Arun Rasheed, Anvesh Mari. Formulation, Characterization and Comparative Evaluation of Kasisa Bhasma. A Herbo-
Mineral Indian Traditional Medicine. Journal of Complementary and Integrative Medicine.
19. Arun Rasheed, K.V. Satyanarayana. Chemical and pharmacological standardization of Ashwagandhadi lehyam: An
ayurvedic formulation. Journal of Complementary and Integrative Medicine.
20. Arun Rasheed, Roja C. Formulation, standardization and pharmacological evaluation of a poly herbal traditional remedy-
Ashwagandharishtam. Oriental Pharmacy and Experimental medicine. 2012, 51-58.
21. Chandel HS, Pathak AK, Tailang M. Standardization of some herbal antidiabetic drugs in polyherbal formulation.
Journal of Pharmacognosy research, 3(1), 2011, 49-56.
22. Meena AK, Rao MM, Panda P, Kiran, Yadav A, Singh U, et al. Standardization of ayurvedic polyherbal formulation,
Pancasama Churna. International Journal of Pharmacognosy and Phytochemistry Research, 2(1), 2010, 11-14.
23. Sanjay J, Sweta S, Rakesh B, Praveen K. Standardization of „Dashamularishta‟: A polyherbal formulation. Journal of
Pharmacognosy, 1(3), 2009, 54-57.
24. Kumar T, Chandrasekhar KS, Tripati DK, Nagori K, Pure S, and Agarwal S. Standardization of “ Gokshuradi Churna”
An ayurvedic polyherbal formulation. Journal of Chemical and Pharmaceutical Research, 3(3), 2011, 742-749.
25. Meena R, Meena AK, Khan SA, Mageswari S. Standardization of Unani polyherbal drug-Jawarish-e-Darchini. Journal
of Pharmacognosy research, 7(3), 2010, 11-12.
26. Rajini M, Kanaki NS. Phytochemical standardization of herbal drugs and polyherbal formulations. Bioactive Molecules
and Medicinal Plants, 2008, 349-369.
27. Ahmad I, Aqil F, Owasis M. Turning medicinal plants into drugs. Modern Phytomedicine, 384, 2006, 67-72.
28. Rajurker S, Rekhe DS, Maini S, Ravikanth K. Acute toxicity studies of polyherbal formulation. Veterinary World, 2(2),
2009, 58-59.
29. Pattanaya P, Jena RK, Panda SK. HPTLC fingerprinting in the standardization of sulaharan yoga: An ayurvedic tablet
formulation. International Journal of Pharmaceutical Sciences Review and Research, 3(2), 2010, 33-36.
30. Chandrakant K., Dere Pravin J., Honde Bharat S.,Kothule Sachin Kote Amol P. An overview of supercritical fluid
extraction for herbal drugs. Pharmacologyonline, 2, 2011, 575-596.
31. VyasN, Khan MY, Panchal S, Butani A, Kumar V. Supercritical fluid technology- an unlimited frontier in herbal
research. International Journal of Pharmaceutical Sciences and Nanotechnology,1(4), 2009, 303-307.
32. Bertucco A, Franceschin G. Supercritical fluid extraction of medicinal and aromatic plants: Fundamentals and
applications in: Extraction technologies for medicinal and aromatic plants. International Centre for Science and High
Technology Trieste, 2008.
33. Trease and Evans. Text book of pharmacognosy. 15, 138.
34. Chaudary RD. Regulatory requirements. Herbal Drug Industry- A practical approach to industrial pharmacognosy.
Eastern publishers, New Delhi. 1, 1996, 537-546.
35. Wir-Ferenc A, Biziuk M. solid phase extraction technique_ Trends, oppurtunities and applications. Journal of
Environmental Studies, 15(5), 2006, 677-690.
36. Yan Z, Feng D, Li S, Zhao Y, Yang H. Determination of organochlorine pesticide residues in nine herbs by solidphase
extraction and capillary gas chromatography. National centre for Biotechnology information, 23(3), 2005, 308-311.
37. Lee MC, Tsao CH, lou SC, Chuang WC, Sheu SJ. Analysis of aristolochic acids in herbal medicines by LC/UV and
LC/MS. Journal of Seperation Science, 26, 2003, 818-822.
38. Trease and Evans. Text book of Pharmacognosy. 15,140.
39. Wang Y, Xi GS, Zheng YC, Miao FS. Microwave-assisted extraction of flavonoids from Chinese herb Radix puerariea.
U Med Plant Res, 4(4), 2010, 304-308.
40. Be‟atrice K, Philippe C. Recent extraction techniques for natural products: microwave-assisted extraction and
pressurized solvent extraction. Phytochemical Analysis, 13, 2002, 105-113.
41. Vivekananda M, Yogesh M, Hemalatha S. Microwave assisted extraction- an innovative and promising extraction tool
for medicinal plant research. Journal of Pharmacognosy, 1(1), 2007, 78-84.

~ 86 ~
 Inter. J. of Phytotherapy / Vol 2 / Issue 2 / 2012 / 74-88.

42. Chimezie A, Ibukun A, Teddy E, Francis O. HPLC analysis of nicotinamide, pyridoxine, riboflavin and thiamin in some
selected food products in Nigeria. African Journal of Pharmacy and Pharmacology, 2(2), 2008, 29-36.
43. Saravanan J, Shajan A, Joshi NH, Varatharajan R, Valliapan K. Asimple and validated RP-HPLC method for the
estimation of methlycobalamin in bulk and capsule dosage form. International journal of Chemistry and Pharmaceutical
Sciences, 1(2), 2010, 323-324.
44. Rao Udaykumar B, Anna NP. Stability-indicating HPLC method for the determination of efavirenz in bulk drug and in
pharmaceutical dosage form. African Journal of Pharmacy and Pharmacology, 3(12), 2009, 643-650.
45. Rathod Shobhen, Patel N.M, Patel P.M. A Review on modification of analytical techniques in herbal research.
International Journal of Research in Ayurveda and Pharmacy, 2(5), 2011, 1483-1485.
46. Zhang Q, Ye M. Chemical analysis of the Chinese herbal medicine Gan-Cao (liquorice). Journal of Chromatography A,
1216(11), 2009, 1954-1969.
47. Manisha K, Gharate, Veena S. Kasture. Development and Validation of RP- HPLC method for determination of marker
in polyherbal marketed Kankasava formulations. Scholars research library, 3(5), 2011,28-33.
48. Vogel H, Gonzalez M, Faini F, Razmilic I, Rodriguez J and Martin JS. Journal of Ethnopharmacology, 97, 2005, 97-
100.
49. Xie P.S., ChenS.B, Liang Y.Z, Wang X.H., Tian R.T. &Roy Upton. Chromatographic fingerprint analysis – a rational
approach for quality assessment of traditional Chinese herbal medicine. Journal of Chromatography A, 1112, 2006, 171-
180.
50. Soni K, Naved T. HPTLC – Its applications in herbal drug industry. The Pharma Review, 2010, 112-117.
51. Ashok kumar, Lakshman .K, Jayaveera K.N., Mani Tripathi S.N., Satish K.V. Estimation of gallic acid, Rutin, and
Quercetin in Terminalia chebula by HPTLC. Jordan Journal of Pharmaceutical Sciences, 3(1), 2010.
52. Jirge SS, Tatke PA, Gabhe S. Development and validation of a novel HPTLC method for simultaneous estimation of
beta-sitosterol- d- glucoside and withaferin A. Internayional journal of Pharmacy and Pharmaceutical Sciences, (l2),
2011, 227-230.
53. Shanbhag DA, Khandagale NA. Application of HPTLC in the standardization of a homoeopathic mother tincture of
Syzygium jambolanum. Journal of Chemistry and Pharmaceutical Research, 3(1), 2011, 395-401.
54. Kshirsagar VB, Deokate UA, Bharkad VB, Khadabadi SS, HPTLC method development and validation for the
simultaneous estimation of Diosgenin and Levodopa in marketed formulation. Asian Journal of Research Chemistry,
1(1), 2008.
55. Kasthuri KT, Jayasree N, Anoop A, Shanthi P. Development of GC-MS for a polyherbal formulation- MEGNI.
International Journal of Pharmaceutical sciences, 2 (2), 2010, 81-83.
56. Binit DK, Sunil K, Nayak C, Mehta BK. Gas chromatography mass spectroscopy (GC-MS) analysis of the hexane and
benzene extracts of the Piper beetle from India. Journal of Medicinal Plants Research, 4(21), 2010, 2252-2255.
57. Mike Lee S, Edward Kerns H. LC-MS applications development. Milestone Development services, Pennington, New
Jersey, 1999.
58. Ip SP, Zhao M, Xian Y, Chen M, Zong Y, Tjong YW, ET AL. Quality assurance for Chinese herbal formulae:
Standardization of IBS-20, a 20- herb preparation. Journal of Chinese Medicine, 5, 2010, 8-9.
59. Yu-Tse Wu, Ming –Tsang Wu, Chia- Chun Lin, Chao- Feng Chien, and Tung-Hu Tsai. Pharmacokinetic studies of
Chinese Medicnal Herbs Using an Automated Blood sampling system and Liquid Chromatography-Mass spectroscopy.
Journal of Traditional Chinese medicine, 2(1), 2011, 33-40.
60. Sombra LL, Gomez MR, Olsina R, Martinez LD, Silva MF. Comparative study between capillary electrophoresis and
high performance liquid chromatography in guarana based phytopharmaceuticals. Journal of Pharmaceutical and
Biomedical Analysis, 36, 2005, 989-994.
61. Tistaert C, Dejaegher B, Heyden YV. Chromatographic separation techniques and data handling methods for herbal
fingerprints: A review. Analytica Chimica Acta, 690, 2011, 148-161.
62. Shikha S, Mishra N. Genetic markers - a cutting -edge technology in herbal drug research. Journal of Chemical and
Pharmaceutical Research, 1, 2009, 1-18.
63. Lazarowych NJ, Pekos P. The use of fingerprint and marker compounds for identification and standardization of
botanical drugs. Journal of Drug Information, 32, 1998, 497-512.
64. Jayvant K, Vaibhav S, Abhay H. Application of ISSR marker in pharmacognosy. Current Sciences, 3, 2009, 216-228.
65. Usha K, Salim K, Mirza KJ, Mauji R, Abdin MZ. SCAR markers: A potential tool for authentification of herbal drugs.
Fitoterapia, 81, 2010, 969-976.
66. Shalini S,Roy RK. Phytosomes : an emerging technology. International journal of Pharmaceutcal Research and
Development, 2, 2010, 14-15.
67. Shivanand P, Kinjal P. Phytosomes: Technical revolution in phytomedicine. International journal of Pharmaceutical
Technology and Research, 2(1), 2010, 627-631.

~ 87 ~
 Inter. J. of Phytotherapy / Vol 2 / Issue 2 / 2012 / 74-88.

68. Battachatya S, Ghosh A. Phytosomes: the emerging technology for enhancement of bioavailability of botanicals and
nutraceuticals. International journal of Aesthetic & Antiaging Medicine, 2(1), 2009, 87-91.
69. Darshan D, Satyaendra S, Shweta K, Gangwal A, Dubey PK. Phytosome: Anovel dosage structure. International journal
of Chemical Sciences, 1(1), 2007, 132-34.
70. Jagruti P, Rakesh P, Kapil K, Nirav P. An overview of phytosomes as an advanced herbal drug delivery system. Asian
Journal of Pharmaceutical Science, 4(6), 2009, 363-371.
71. Kidd PM. Bioavailability and activity of phytosome complexes from botanical poly phenols: the silymarin, curcumin,
greentea, and grape seed extracts. Alternative Medicine Review,14(3), 2009, 226-46.
72. Huang S, Chang WH. Advantages of nanotechnology- based Chinese herb drugs on biological activites. Current Drug
Metabolism, 10(8), 2009, 905-13.
73. Musthaba SM, Sayeed A, Alka A, Javed A. Nano approaches to enhance pharmacokinetic and pharmacodynamic
activity of plant origin drugs. Current Nanoscience, 5(3), 2009, 344-352.
74. Dong TP, Sung CH. Exuberence or bubble study of nano- based herbal medicines patients in the PR China. Journal of
Intellectual Property rights, 16, 2011, 225-234.
75. Kumar A, Nasair AG, Reddy AV, Garg AN. Bhasmas: unique Ayurvedic metallic- herbal preparations, chemical
characterization. Journal of Biological Trace Element Research, 109, 2006, 231-254.
76. Saper RB, Kales SN, Paquin J, Burns MJ, Eisenberg DM, Davis RB, et al. Heavy metal content of ayurvedic herbal
medicine products. Journal of Advanced Medical Research, 292, 2004, 2868-2873.

~ 88 ~