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We- together to save yourself society e-ISSN 0976-7614

Volume 2, Issue 4, October 2011 Research Article

Standardization and Evaluation of Herbal Drug Formulations

Prashant Yadav*, Kanhiya Mahour and Ashok Kumar

*Animal Health Division, Central Institute for Research on Goats, Makhdoom, Farah, Mathura (U.P.), India.

Abstract: Herbal medicine has been utilized in many diseases since ancient times by saints and munis. Herbal
medicine has many active constituents for many diseases but the proper knowledge must be necessary for the
preparation of herbal formulation otherwise active constituents will be damaged. Considering this fact present article
emphasis the herbal formulation preparation. Knowing these facts we can use herbal formulation for most
complicated diseases and save humans from the effect of synthetic drug side effect.

Keywords: Herbal Formulation, Active Constituents, Test, Crude, Ethanol.

1. Herbal Drug Standardization and Evaluation respectively by adding excipients or by mixing herbal
drugs or herbal drug preparations (4). “Evaluation” of a
In recent years, there has been great demand for drug means confirmation of its identity and
plant derived products in developed countries. These determination of its quality and purity and detection of
products are increasingly being sought out as medicinal its nature of adulteration (5).
products, nutraceuticals and cosmetics (1). There are Standardization of herbal drugs is not an easy task
around 6000 herbal manufacturers in India. More than as numerous factors influence the bioefficacy and
4000 units are produced Ayurveda medicines. Due to reproducible therapeutic effect. In order to obtain
lack of infrastructures, skilled manpower reliable quality oriented herbal products, care should be taken
methods, and stringent regulatory laws, most of these right from the proper identification of plants, season
manufacturers produce their product on the very and area of collection and their extraction and
tentative basis (2). purification process and rationalizing the combination
In order to have a good coordination between the in the case of polyherbal drugs (3).
quality of raw materials, in process materials and the The Standardization of crude drug materials
final products, it has become essential to develop includes the following steps-
reliable, specific and sensitive quality control methods
using a combination of classical and modern Authentication: - Each and every step has to be
instrumental method of analysis. Standardization is an authenticated.
essential measurement for ensuring the quality control a) Stage of collection.
of the herbal drugs (3). “Standardization” expression is b) Parts of the collected plant.
used to describe all measures, which are taken during c) Regional status.
the manufacturing process and quality control leading d) Botanical identity like phytomorphology,
to a reproducible quality. It also encompasses the entire microscopical and histological analysis
field of study from birth of a plant to its clinical (characteristic of cell walls, cell contents,
application. It also means adjusting the herbal drug starch grains, calcium oxalate crystals,
preparation to a defined content of a constituent or a trichomes, fibers, vessels etc.) (6).
group of substances with known therapeutic activity

*Corresponding author:
E-mail: prashantcirg@gmail.com; Phone no.:+91-9058791388.
Standardization and Evaluation of Herbal Drug Formulations Yadav et al

Various histological parameter studies are:- matter, microscopical, histological, histochemical

a) Leaf constant: - Palisade ratio, Vein islet number, evaluation, quantitative measurements etc.
Vein termination, Stomatal number and Stomatal 2. Refers to the physicochemical character of the
index. drug. Physical and chemical identity,
b) Trichomes. chromatographic fingerprints, ash values,
c) Stomata. extractive values, moisture content, volatile oil and
d) Quantitative microscopy. alkaloidal assays, quantitative estimation protocols
e) Taxonomical identity. etc.
f) Foreign matter. 3. A reference to the pharmacological parameters,
g) Organoleptic evaluation. biological activity profiles, bitterness values,
h) Ash values and extractive values. hemolytic index, astringency, swelling factor,
i) Moisture content determination. foaming index etc.
j) Chromatographic and spectroscopic evaluation. 4. Toxicity details- pesticide residues, heavy metals,
k) Heavy metal determination. microbial contamination like total viable count,
l) Pesticide residue. pathogens like E. coli, Salmonella, P. aeruginosa,
m) Microbial contamination. S. aureus, Enterobacteria etc.
n) Radioactive contamination. 5. Microbial contamination.
6. Radioactive contamination.
The herbal formulation, in general, can be
standardized schematically as to formulate the 2.2 Modern herbal Ayurvedic monographs
medicament using raw materials collected from
different localities and a comparative chemical efficacy In the modern herbal Ayurvedic monographs the
of different batches of formulation are to be observed. standardization parameters are discussed in a
The preparations with better clinical efficacy are to be comprehensive way. According to the modern
selected. After all the routine physical, chemical and Ayurvedic monograph, the quality control protocols
pharmacological parameters are to be checked for all include the following:
the batches to select the final finished product and to The synonyms, publication related to the plant,
validate the whole manufacturing process (6). constituents present, analytical methods.
The stability parameters for the herbal formulations Descriptive evaluation: Description of the drug,
which include physical, chemical and microbiological phytomorphological, microscopical, organoleptic
parameters are as follows: evaluation and foreign matter, etc.
Physical parameters include color, odor, appearance, 3. Who Guidelines Monograph Title
clarity, viscosity, moisture content, pH, disintegration
time, friability, hardness, flowability, flocculation, Botanical: - Sensory evaluation, Foreign matter,
sedimentation, settling rate and ash values. Microscopy measurement.
Chemical parameters include limit tests, chemical Physicochemical TLC: - Ash, Extractable matter,
tests, chemical assays etc. Water content and volatile matter, Volatile oils.
Chromatographic analysis of herbals can be done Pharmacological: - Bitterness value, Haemolytic
using TLC, HPLC, HPTLC, GC, UV, GC-MS and activity, Astringency, Sterling index, Foaming index.
fluorimetry etc. Toxicological: - Pesticide residue, Arsenic, Metals.
Microbiological parameters include total viable Microbial contamination: - Total viable count,
content, total mold count, total enterobacterial and their Pathogens, Aflatoxins, Radioactive contamination.
count. Limiters can be utilized as a quantitative or semi-
quantitative tool to ascertain and control a number of 4. Standardization of Herbal Drug/Products
impurities like the reagents used during abstraction of
various herbs, impurities coming directly from the Commercial production of herbal medicines and
manufacturing vessels and from the solvents etc. their trade are the fastest growing sector of industry
today, due to increasing demand of medicinal plants;
2. Guidelines for Herbal Drug Standardization the supply line is adversely affected leading to the
adulteration and substitution for genuine drugs.
2.1 WHO Guidelines 1. Fluorescence quenching: When a plant extract is
spotted on a fluorescent silica gel layer and
The subject of herbal drug standardization is exposed to UV light, it appears as a spot on a
massively wide and deep. The guidelines set by WHO fluorescent background, thus causing quenching
can be summarized as follows:- and is directly proportional to the concentration of
1. Reference to the identity of the drug. Botanical the extract. The silica gel GF plate was used as an
evaluation- sensory characters, foreign organic adsorbent for fluorescence quenching. Solvents

J. Adv. Lab. Res. Biol. 162

Standardization and Evaluation of Herbal Drug Formulations Yadav et al

took hexane toluene, ether, ethyl acetate, butanol, 6. Liquid chromatography UV-determination and
methanol and water (8). liquid chromatography-atmospheric pressure
2. Use of fingerprinting and marker compounds chemical ionization mass spectrometry
for identification and standardization of characterization of sitosterol and stigmasterol in
botanical drugs: Chemical and chromatographic soya bean oil: A narrow bore HPLC-UV method
techniques may be used to aid in identification of was developed for the analysis of two of the most
an herbal material or extract. Chromatographic abundant naturally occurring phytosterols in
technique such as HPLC, TLC, GC and capillary vegetable oils: sitosterol and stigmasterol. The
electrophoresis and spectroscopic methods such as method enabled detection of the compounds at a
IR, NMR and UV may also be used for concentration of 0.42µ/ml and quantization at a
fingerprinting. DNA fingerprinting has been concentration of 0.52 and 0.54µ/ml for sitosterol
widely used in many species, e.g. DNA and stigmasterol, respectively (14).
fingerprinting of Panax species and their 7. Simultaneous determination of cinnamaldehyde,
adulterants (9). Marker compounds may be used to eugenol and paeonol in traditional Chinese
help identify herbal materials, set specifications for medicinal preparations by capillary GC-FID: A
raw materials, standardize botanical preparations capillary GC method was established for
during all aspects of manufacturing processes and simultaneous determination of cinnamaldehyde
obtain stability profiles (10). (CNMD), eugenol (EL) and paeonol (PL) in two
3. Densitometric thin layer chromatographic traditional Chinese herbal medicinal preparations,
determination of aescin in an herbal medicinal Wei Tong Ding tablet (WTDT) and Guifu Dihuang
product containing Asculum and Vitis dry pill (GDHP). The assays were based on a
extract: A TLC method is developed to analyze programmed temperature GC in a 30m x 0.53mm
the total saponin content, also referred to as the capillary columns with nitrogen as the carrier and
aescin content, in an herbal medicinal product FID detector. Good linearity was obtained over
containing two dry extracts in capsules. After a ranges of 0.45-0.452mg/l CNMD, 0.31-0.625mg/l
purification step using C (18) solid phase EL and 0.30-610mg/l PL, respectively (15).
extraction, the samples are analyzed on a silica gel 8. HPTLC fingerprinting of marketed formulation
HPTLC plate with the upper layer of a mixture of containing Shankhpushpi: These are the
acetic acid/water/butanol (10/40/50v/v/v) as the important Ayurveda formulations used for perinatal
mobile phase. Spots are visualized by spraying care of mother and child health. Standardization of
with anisaldehyde reagent and heating the plate for churnas was carried out by organoleptic study,
5-10 min. (100-105oc) and measured at a phytochemical analysis; qualitative organic and
wavelength of 535nm (11). inorganic analysis, thin layer chromatography, UV-
4. Determination of stigmasterol, beta-sitosterol visible spectrophotometer and HPLC fingerprint
and stigmastanol in oral dosage forms using studies. Qualitative organic analysis of both the
HPLC with evaporative light scattering churnas revealed the presence of alkaloids,
detection: A validated and repeatable HPLC steroids, phenols, tannins, glycosides, resins,
method with online evaporative light scattering saponins and flavonoids (16).
was developed for the analysis of two sterols,
stigmasterol, beta-sitosterol and a stanol found to 5. Evaluation of Herbal Drug/Products
be common in many herbal formulations and health
care supplements. This method was used to assay 1. Biological parameter (bioassay): It is well
commercially available products formulated as oral established that the biological potency of the herbal
dosage forms purported to contain African potato constituents is due to not one but a mixture of
and associated sterols and stanol (12). bioactive plant constituents and the relative
5. Elemental analysis of herbal preparations for properties of a single bioactive compound can vary
traditional medicines by neutron activation from batch to batch while the biological activity
analysis with the kO standardization method: remains within the desirable limits (1). Some of the
Medicinal herb preparations prescribed for specific examples are:
treatment purposes were purchased from markets a. Evaluation of adaptogenic activity profiles of
and were analysed by instrumental neutron herbal preparation: Adaptogens help the body to
activation analysis with kO standardization. 500- come up with stress and enhance general health
700mg of each sample was palletized under a and performance. AVM is an herbal formulation.
pressure of six tones and irradiated together with Composition- Emblica officinalis, Withania
monitors for alpha and neutron flux ratio somnifera, Asparagus racemosus, Ocimum
determination for about 6h in a thermal flux of 2.29 sanctum, Tribulus terrestris and Piper longum.
x 10 (12) n/cm2/s (13). AVM shows significant antistress,
immunomodulatory and anabolic activities in

J. Adv. Lab. Res. Biol. 163

Standardization and Evaluation of Herbal Drug Formulations Yadav et al

different animal models thereby proving a the color intensity at 532nm. Gallic acid was used
promising adaptogen (17). as a positive control (21).
b. Evaluation of antioxidant activity of herbal 2. Evaluation of marketed polyherbal antidiabetic
products: A new test method for measuring the formulations using biomarker charantin:
antioxidant power of herbal products, based on Charantin is one of the phytoconstituents present in
solid phase spectrophotometry using tetrabenzo-b, Momordica charantia. It is well known to possess
f, j, n, l, 5, 9, 13- tetraazacy-clohexadecin- Cu (II) antihyperglycemia, anticholesterol,
complex immobilized on silica gel is proposed. immunosuppressive, antiulcerogenic,
The method represents an alternative to the mostly antispermatogenic and androgenic activities.
used scavenging capacity assays. The method was HPTLC method is fast, precise, sensitive and
approved in the analysis of the most popular herbal reproducible with good recoveries for
beverages and drugs Echinacea determined standardization of polyherbal formulations. The
spectrophotometrically (18). recovery values of charantin were found to be about
c. Evaluation of microbial contamination 98.89% (2).
reduction on plants through a technological 3. In vivo and in vitro evaluation of hair growth
process of decoction and spray dry: The potential of Shoe flower: The leaves and flowers of
technological process of raw material has many Hibiscus rosa-sinensis are used as promoters of hair
stages, generally, adverse to microbial growth, but growth and as an aid in healing of ulcers. Petroleum
its complete elimination depends on the initial and ether extract of leaves and flowers of the plant was
work condition utilized. The aim of this work was evaluated for the potential growth in vivo and in
to verify the microbial contamination, such as vitro methods. In vivo, 1% extract of leaves and
extracting solution (SE) and spray dried extract flowers in liquid was applied topically over the
(PSA) with the purpose of evaluating the decrease shaved skin of albino rats and monitored and
of contamination after the decoction and the spray assessed for 30 days. The length of hair and
dry. The microbiological analysis of the products different cyclic phases of hair follicles, like anagen
was performed by total plate count and MPN and telogen phases were determined at different
coliform (19). time periods. In vitro, the hair follicles from albino
d. Evaluation of nitric oxide scavenging activity of rat neonates were isolated and cultured in DMEM
selected medicinal plants used in inflammatory supplemented with 0.01mg/ml petroleum ether
diseases: Four traditional medicinal plants, namely extract of leaves and flowers. It is concluded that the
Ventilago madraspatana Gaertn., Rubia cordifolia leaf extract, when compared to flower extract,
Linn., Lantana camara Linn. and Morinda exhibits more potency on hair growth (22).
citrifolia Linn. were selected for a study on the 4. Clinical evaluation to assess the safety and
inhibition of nitric oxide (NO), a key mediator in efficacy of code herbal medicine “Dysmo-off”
the phenomenon of inflammation, signifying the versus allopathic medicine “Diclofenac sodium”
presence of effective anti-inflammatory for the treatment of primary Dysmenorrhoea:
constituents therein. Plant samples were extracted The clinical study on primary Dysmenorrhoea to
with different solvents for evaluation of their comparatively examine the coded herbal drug
inhibitory activity on NO produced in vitro from formulation “Dysmo-off” with authentic allopathic
sodium nitroprusside, and in LPS- activated murine medicine “Diclofenac sodium”. A random
peritoneal macrophages, ex-vivo (20). controlled clinical trial was conducted. These
e. The lipid peroxidation inhibitory activity: The evaluations were based on a verbal rating scale so as
reaction mixture contained mice liver homogenate to ascertain the rate of analgesic effects on
(0.2ml, 10% w/v) in 0.15 KCl, KCl (0.1ml, dysmenorrhoeic pain. The patients were randomly
150µm), Tris buffer (0.4ml, Ph 7.5) and various allocated with the ratio of 1:2 for controlling
concentrations of test extracts. In vitro lipid treatment with (NSAIDS) (n=40) received
peroxidation was initiated by addition of Diclofenac sodium tablets twice daily for 4 days
FeSO4.7H2O (0.1ml, 10µm). The reaction mixture (50mg one day prior to and three days after the
was incubated at 37oC for 1 h. After the incubation menstruation), and test treatment with Dysmo-off
period, the reaction was terminated by addition of (n=80) received powdered Dysmo-off twice daily
thiobarbituric acid (TBA-2ml, 0.8%) and by for 4 days (5g one day prior to and three days after
heating the contents for 15 min. for development of the menstruation). Treatment lasted for 4
colored complex. The tubes were then centrifuged consecutive menstrual cycles. Haemoglobin, ESR
at 4000 rpm for 10 min. and cooled. The % and ultrasound were measured at baseline during the
inhibition of lipid peroxidation was determined by study. All subjects were clinically studied (23).
comparing the results of test compound with those 5. Thermographic evaluation: In the present study,
of controls not treated with extracts by monitoring the authors used Thermography to evaluate the
effects of herbal formulations based on “Sho”

J. Adv. Lab. Res. Biol. 164

Standardization and Evaluation of Herbal Drug Formulations Yadav et al

scientifically. In the cases that were suitable for subject of herbal medicines and its relationship with
Keishi-bukuryo-gan, the so-called Keishi-bukuryo- human physiology and mental function.
gan Sho, a significant skin temperature rise was For the purpose of research work on
observed in the upper half of the body after the standardization of herbal formulations, a profound
intake of Keishi-bukuryo-gan. In a case that was knowledge of the important herbs found in India and
suitable for Hochuekkito, a marked elevation of skin widely used in Ayurvedic formulation is of utmost
temperature spread through the upper trunk. It importance (6).
suggested that thermography is useful for an Even when the chemical composition of a plant
objective evaluation of Sho in Kampo medicines, extract is known, the pharmacologically active moiety
and for identification of the action site of the herbal may not be. Environment, climate and growth
formulation (24). conditions influence the composition, as does the
6. Biochemical evaluation: Most of the herbal drugs specific part of the plant and its maturity. Monographs
are a mixture of a number of ingredients. Their detailing standardization of active ingredients would
cumulative effect increases the efficacy of the drug improve the marketplace. Even if an herbal product is
in curing the diseases. Muthu Marunthu is an herbal standardized to, for example, 4% of a constituent, the
formulation comprising of eight various plant remaining 96% of ingredients is not standardized and
ingredients and has been claimed to possess may affect the product’s solubility, bioavailability,
anticancer effect. It was observed that the growth stability, efficacy and toxicity. Just as controlled trials
rate in rats was normal and there was no change in are necessary to establish safety and efficacy,
blood parameters such as glucose, urea, proteins, manufacturing standards are required to ensure product
cholesterol and also in the activities of quality (28).
pathophysiological enzymes such as lactate Nowadays newer and advanced methods are
dehydrogenase (LDH), glutamate oxaloacetate available for the standardization of herbal drugs like
transaminase (GOT), glutamate pyruvate fluorescence quenching, the combination of
transaminase (GPT), alkaline and acid phosphatase chromatographic and spectrophotometric methods,
after Muthu Marunthu administration. The tumor biological assays, use of biomarkers in fingerprinting
weight was found to be reduced in etc. Bioassay can play an important role in the
methylcholanthrene induced fibrosarcoma rats after standardization of herbal drugs and can also become an
Muthu Marunthu treatment (25). important quality control method as well as for proper
7. Evaluation of Kutaj: Ghanavati for alkaloidal stability testing of the product (4).
principles- Kutaj-Ghanavati is a reputed Ayurvedic India can emerge as the major country and play the
preparation used in dysentery and diarrhea. It lead role in the production of standardized,
contains a water extract of Kurchi bark and fine therapeutically effective Ayurvedic formulation. India
powder of aconite roots. It was evaluated needs to explore the medicinally important plants. This
quantitatively and qualitatively employing TLC and can be achieved only if the herbal products are
titrimetric method. In the TLC study no interference evaluated and analyzed using sophisticated modern
of Kurchi and Aconite alkaloids with one another in techniques of standardization such as UV- visible, TLC,
their respective solvent systems. The formulation HPLC, HPTLC, GC-MS, spectrofluorimetric and other
was found to contain all alkaloids of Kurchi and methods (6).
Aconite (26).
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