STANDARDISATION AND EVALUATION OF
HERBAL DRUG FORMULATIONS
1Sunita Panchawat, 1Kamal Singh
Rathore, 2Dr.S.S.Sisodia, 3Dr. R.K.Nema
1BN PG Girls College of Pharmacy,
Udaipur-Raj.INDIA 313002
2BN PG College of Pharmacy, Udaipur
3Rishiraj College of Pharmacy, Udaipur
Email:kamalsrathore@yahoo.com
Mobile: +919887111211(Sunita P.);
+919828325713(Kamal)
Herbal Drug Standardization and
Evaluation:- In recent years, there has been great
demand for plant derived products in developed countries. These
products are increasingly being sought out as medicinal products,
nutraceuticals and cosmetics. (1) There are around 6000 herbal
manufacturers in India. More than 4000 units are producing Ayurveda
medicines. Due to lack of infrastructures, skilled manpower reliable
methods and stringent regulatory laws most of these manufacturers
produce their product on very tentative basis. (2)
In order to have a good coordination between the
quality of raw materials, in process materials and the final products,
it has become essential to develop reliable, specific and sensitive
quality control methods using a combination of classical and modern
instrumental method of analysis. Standardization is an essential
measurement for ensuring the quality control of the herbal drugs. (3)
“Standardization” expression is used to describe all measures, which
are taken during the manufacturing process and quality control leading
to a reproducible quality. It also encompasses the entire field of
study from birth of a plant to its clinical application. It also means
adjusting the herbal drug preparation to a defined content of a
constituent or a group of substances with known therapeutic activity
respectively by adding excipients or by mixing herbal drugs or herbal
drug preparations.(4) “Evaluation” of a drug means confirmation of its
identity and determination of its quality and purity and detection of
its nature of adulteration.(5)
Standardization of herbal drugs is not an easy
task as numerous factors influence the bio efficacy and reproducible
therapeutic effect. In order to obtain quality oriented herbal
products, care should be taken right from the proper identification of
plants, season and area of collection and their extraction and
purification process and rationalizing the combination in case of
polyherbal drugs.(3)
The Standardization of crude drug materials
includes the following steps:-
- Authentication: - Each and every step has to be
authenticated.
a) Stage of collection.
b) Parts of the plant collected.
c) Regional status.
d) Botanical identity like phytomorphology,
microscopical and histological analysis (characteristic of cell walls,
cell contents, starch grains, calcium oxalate crystals, trichomes,
fibers, vessels etc).(6) Various histological parameter studies are:-
- Leaf constant: - Palisade ratio, Vein islet
number, Vein termination, Stomatal number, and Stomatal index.
- Trichomes.
- Stomata.
- Quantitative microscopy.
- Taxonomical identity.
- Foreign matter.
- Organoleptic evaluation.
- Ash values and extractive values.
- Moisture content determination.
- Chrometographic and spectroscopic evaluation.
- Heavy metal determination.
- Pesticide residue.
- Microbial contamination.
- Radioactive contamination.
The herbal formulation in general can be
standardize schematically as to formulate the medicament using raw
materials collected from different localities and a comparative
chemical efficacy of different batches of formulation are to be
observed. The preparations with better clinical efficacy are to be
selected. After all the routine physical, chemical and pharmacological
parameters are to be checked for all the batches to select the final
finished product and to validate the whole manufacturing process. (6)
The stability parameters for the herbal
formulations which include physical, chemical and microbiological
parameters are as follow:
Physical parameters
include color, odor, appearance, clarity, viscosity, moisture content,
pH, disintegration time, friability, hardness, flow ability,
flocculation, sedimentation, settling rate and ash values.
Chemical parameters
include limit tests, chemical tests, chemical assays etc.
Chromatographic analysis of
herbals can be done using TLC, HPLC, HPTLC, GC, UV, GC-MS, fluorimetry
etc.
Microbiological parameters
include total viable content, total mold count, total enterobacterial
and their count. Limiters can be utilized as a quantitative or semi
quantitative tool to ascertain and control the amount of impurities
like the reagents used during abstraction of various herbs, impurities
coming directly from the manufacturing vessels and from the solvents
etc.
GUIDELINES FOR HERBAL DRUG
STANDARDISATION
WHO Guidelines:-
The subject of herbal drug standardization is
massively wide and deep. The guidelines set by WHO can be summarized as
follows:-
- Reference to the identity of the drug.
Botanical evaluation- sensory characters, foreign organic matter,
microscopical, histological, histochemical evaluation, quantitative
measurements etc.
- Reference to the physicochemical character of
the drug. Physical and chemical identity, chromatographic fingerprints,
ash values, extractive values, moisture content, volatile oil and
alkaloidal assays, quantitative estimation protocols etc.
- Reference to the pharmacological parameters,
biological activity profiles, bitterness values, hemolytic index,
astringency, swelling factor, foaming index etc.
- Toxicity details- pesticide residues, heavy
metals, microbial contamination like total viable count, pathogens likeE.coli, Salmonalla, P.aeroginosa, S. aureus,
Enterobacteria etc.
- Microbial contamination.
- Radioactive contamination.
Modern herbal ayurvedic monographs
In the modern herbal ayurvedic monographs the
standardization parameters are discussed in a comprehensive way.
According to the modern ayurvedic monograph the quality control
protocols include the following:
The synonyms, publication related to the plant,
constituents present, analytical methods.
Descriptive evaluation: Description of the drug,
phytomorphological, microscopical, organoleptic evaluation, foreign
matter etc.
WHO GUIDELINES MONOGRAPH TITLE (7)
Botanical: -
Sensory evaluation, Foreign matter, Microscopy measurement.
Physicochemical TLC: -Ash, Extractable matter, Water content and
volatile matter, Volatile oils.
Pharmacological: -
Bitterness value, Haemolytic activity, Astringency, Sterling index,
Foaming index.
Toxicological: -
Pesticide residue, Arsenic, Metals.
Microbial contamination:
- Total viable count, Pathogens, Aflatoxins, Radioactive contamination.
STANDARDIZATION OF HERBAL
DRUG/PRODUCTS
Commercial production of herbal medicines and
their trade are fast growing sector of industry today, due t6o
increasing demand of medicinal plants; the supply line is adversely
affected leading to the adulteration and substitution for genuine drugs.
1. Fluorescence
quenching:- When a plant extract is spotted on a
fluorescent silica gel layer and exposed to UV light, it appears as
spot on a fluorescent background, thus causing quenching and is
directly proportional to concentration of the extract. Silica gel GF
plate was used as an adsorbent for fluorescence quenching. Solvents
taken- hexane toluene, ether, ethyl acetate, butanol, methanol and
water.(8)
2. Use
of fingerprinting and marker compounds for identification and
standardization of botanical drugs:- Chemical and
chromatographic techniques may be used to aid in identification of an
herbal material or extract. Chromatographic technique such as HPLC,
TLC, GC and capillary electrophoresis and spectroscopic methods such as
IR, NMR, and UV-may also be used for fingerprinting. DNA fingerprinting
has been widely used in many species, e.g. DNA fingerprinting of Panaxspecies and their adulterants.(9) Marker
compounds may be used to help identify herbal materials, set
specifications for raw materials, standardize botanical preparations
during all aspects of manufacturing processes and obtain stability
profiles.(10)
3. Densitometric
thin layer chromatographic determination of aescin in an herbal
medicinal product containing Asculum and Vitis dry extract:-
A TLC method is developed to analyze the total saponin content, also
referred to as the aescin content, in an herbal medicinal product
containing two dry extract in capsules. After a purification step using
C(18) solid phase extraction, the samples are analyzed on a silica gel
HPTLC plate with the upper layer of a mixture of acetic
acid/water/butanol(10/40/50v/v/v) as the mobile phase. Spots are
visualized by spraying with anisaldehyde reagent and heating the plate
for 5-10 min.(100-105oc) and measured at a wavelength of 535 nm.(11)
4. Determination
of stigmasterol, beta-sitosterol and
stigmastanol in oral dosage forms using HPLC with evaporative light
scattering detection: - A validated and repeatable HPLC
method with online evaporative light scattering was developed for the
analysis of two sterols, stegmasterol, beta-sitosterol and a stanol
found to be common in many herbal formulations and health care
supplements. This method was used to assay commercially available
products formulated as oral dosage forms purported to contain African
potato and associated sterols and stanol. (12)
5. Elemental
analysis of herbal preparations for traditional medicines by neutron
activation analysis with the kO standardization method: -
Medicinal herb preparations prescribed for specific treatment purposes
were purchased from markets and were analysed by instrumental neutron
activation analysis with kO standardization. 500-700 mg of each sample
was palletized under a pressure of six tones and irradiated together
with monitors for alpha and neutron flux ratio determination for about
6h in a thermal flux of 2.29 x 10(12) n/cm2/s.(13)
6. Liquid
chromatography UV-determination and liquid chromatography-atmospheric
pressure chemical ionization mass spectrometric characterization of
sitosterol and stigmasterol in soyabean oil:- A narrow
bore HPLC-UV method was developed for the analysis of two of the more
abundant naturally occurring phytosterols in vegetable oils: sitosterol
and stigmasterol. The method enabled detection of the compounds at a
concentration of 0.42
µ/ml and quantization at concentration of 0.52 and 0.54 µ/ml for
sitosterol and stigmasterol, respectively.(14)
7. Simultaneous
determination of cinnamaldehyde, eugenol and paeonol in traditional
Chinese medicinal preparations by capillary GC-FID: - A
capillary GC method was established for simultaneous determination of
cinnamaldehyde(CNMD), eugenol(EL) and paeonol(PL) in two traditional
Chinese herbal medicinal preparations, Weitongding tablet (WTDT) and
Guifu Dihuang pill (GDHP). The assays were based on a programmed
temperature GC in a 30 m x 0.53 mm capillary column with nitrogen as
carrier and FID detector. Good linearity were obtained over ranges of
0.45-0.452 mg/l CNMD, 0.31-0.625 mg/l EL, and 0.30-610 mg/l PL,
respectively. (15)
8. HPTLC
fingerprinting of marketed formulation containing Shankhpushpi:
- These are the important Ayurveda formulations used for perinatal care
of mother and child health. Standardization of churnas was carried out
by organoleptic study, phytochemical analysis; qualitative organic and
inorganic analysis, thin layer chromatography, UV- visible
spectrophotometer and HPLC fingerprint studies. Qualitative organic
analysis of both the churnas revealed the presence of alkaloids,
steroids, phenols, tannins, glycosides, resins, saponins and
flavonoids.(16)
EVALUATION OF HERBAL DRUG/PRODUCTS
1. Biological
parameter (bioassay):- It is well established that the
biological potency of the herbal constituents is due to not one but a
mixture of bioactive plant constituents and the relative properties of
a single bioactive compound can vary from batch to batch while the
biological activity remains within the desirable limits. (1) Some of
the examples are:_
a. Evaluation
of adaptogenic activity profile of herbal preparation: - Adaptogens
help the body to come up with stress and enhance general health and
performance. AVM is an herbal formulation. Composition- Emblica
officinalis, Withania somnifera, Asparagus racemosus, Ocimum sanctum,
Tribulus terrestris and Piper longum.
AVM shows significant antistress, immunomodulatory and anabolic
activities in different animal models there by proving a promising
adaptogen. (17)
b. Evaluation
of antioxidant activity of herbal products: - A new
test method for measuring the antioxidant power of herbal products,
based on solid phase spectrophptometry using tetrabenzo-b, f, j, n, l,
5, 9, 13- tetraazacy- clohexadecin- Cu (II) complex immobilized on
silica gel is proposed. The method represents an alternative to the
mostly used scavenging capacity assays. The method was approved in the
analysis of the most popular herbal beverages and drugs Echinacea
determined spectrophotometrically.(18)
c. Evaluation
of microbial contamination reduction on plants through technological
process of decoction and spray dry: - The technological
process of raw material has many stages, generally, adverse to
microbial growth, but its complete elimination depends on the initial
and work condition utilized. The aim of this work was to verify the
microbial contamination, such as extractive solution (SE) and spray
dried extract (PSA) with the purpose of evaluating the decrease of
contamination after the decoction and the spray dry. The
microbiological analysis of the products was performed by total plate
count and MPN coliform. (19)
d. Evaluation
of nitric oxide scavenging activity of selected medicinal plants used
in inflammatory diseases: - Four traditional medicinal
plants, namely Ventilago madraspatana Gaertn., Rubia cordifolia Linn.,
Lanatana camara Linn. And Morinda citrifolia Linn. Were selected for a
study on the inhibition of nitric oxide (NO), a key mediator in the
phenomenon of inflammation, signifying the presence of effective
anti-inflammatory constituents therein. Plant samples were extracted
with different solvents for evaluation of their inhibitory activity on
NO produced in vitro from sodium nitroprusside, and in LPS- activated
murine peritoneal macrophages, ex-vivo.(20)
e. The
lipid peroxidation inhibitory activity:- The reaction
mixture contained mice liver homogenate (0.2 ml, 10% w/v) in 0.15 KCl,
KCl (0.1 ml, 150 µm), Tris buffer (0.4 ml, Ph 7.5) and various
concentration of test extracts. In vitro lipid peroxidation was
initiated by addition of Feso4.7H2O (0.1 ml, 10 µm). The reaction
mixture was incubated at 37o for 1 h. After the incubation period,
reaction was terminated by addition of thiobarbituric acid (TBA-2 ml,
0.8%) and by heating the contents for 15 min. for development of
colored complex. The tubes were then centrifuged at 4000 rpm for 10
min. and cooled. The % inhibition of lipid peroxidation was determined
by comparing the results of test compound with those of control not
treated with extracts by monitoring the color intensity at 532 nm.
Gallic acid was used as a positive control. (21)
2. Evaluation
of marketed polyherbal antidiabetic formulatios using biomarker
charantin: - Charantin is one of the phytoconstituents
present in Momordica charantia. It is well known
to possess antihyperglycaemia, anticholesterol, immunosuppressive,
antiulcerogenic, antispermatogenic and androgenic activities. HPTLC
method is fast, precise, sensitive and reproducible with good
recoveries for standardization of polyherbal formulations. The recovery
values of charantin were found to be about 98.89%. (2)
3. In
vivo and in vitro evaluation of hair
growth potential of Shoe flower: - The leaves and
flowers of Hibiscus rosa-sinensis are used as
promoters of hair growth and as an aid in healing of ulcers. Petroleum
ether extract of leaves and flowers of the plant was evaluated for the
potential growth in vivo and in vitro
methods. In vivo, 1% extract of leaves and
flowers in liquid was applied topically over the shaved skin of albino
rats and monitored and assessed for 30 days. The length of hair and
different cyclic phases of hair follicles, like anagen and telogen
phases were determined at different time periods. In vitro,
the hair follicles from albino rat neonates were isolated and cultured
in DMEM supplemented with 0.01 mg/ml petroleum ether extract of leaves
and flowers. It is concluded that the leaf extract, when compared to
flower extract, exhibits more potency on hair growth. (22)
4. Clinical
evaluation to assess the safety and efficacy of coded herbal medicine
“Dysmo-off” versus allopathic medicine “Diclofenac sodium” for the
treatment of primary dysmenorrhoea: - The clinical study
on primary dysmenorrhoea to comparatively examine the coded herbal drug
formulation “Dysmo-off” with authentic allopathic medicine “Diclofenac
sodium”. A random controlled clinical trial was conducted. These
evaluations were based on verbal rating scale so as to ascertain the
rate of analgesic effects on dysmenorrhoeic pain. The patients were
randomly allocated with the ratio of 1:2 for controlled treatment with
(NSAIDS) (n=40) received Diclofenac sodium tablets twice daily for 4
days (50 mg one day prior to and three days after the menstruation),
and test treatment with Dysmo-off (n=80) received powdered Dysmo-off
twice daily for 4 days (5 g one day prior to and three days after the
menstruation). Treatment lasted for 4 consecutive menstrual cycles.
Haemoglobin, ESR and ultrasound were measured at baseline during study.
All subjects were clinically studied.(23)
5. Thermographic
evaluation: - In the present study, the authors used
thermography to evaluate the effects of herbal formulations based on
“Sho” scientifically. In the cases that were suitable for
Keishibukuryogan, the so called Keishibukuryogan Sho, a significant
skin temperature rise was observed in the upper half of the body after
the intake of Keishibukuryogan. In a case that was suitable for
Hochuekkito, a marked elevation of skin temperature spread through the
upper trunk. It suggested that thermography is useful for an objective
evaluation of Sho in Kampo medicines, and for identification of the
action site of the herbal formulation.(24)
6. Biochemical
evaluation: - Most of the herbal drugs are a mixture of
a number of ingredients. Their cumulative effect increases the efficacy
of the drug in curing the diseases. Muthu Marunthu is an herbal
formulation comprising of eight various plant ingredients, and has been
claimed to possess anticancer effect. It was observed that the growth
rate in rats was normal and there was no change in blood parameters
such as glucose, urea, proteins, cholesterol and also in the activities
of pathophysiological enzymes such as lactate dehydrogenase (LDH),
gluconate oxaloacetate transaminase (GOT), glutamate pyruvate
transaminase (GPT), alkaline and acid phosphatase after Muthu Marunthu
administration. The tumor weight was found to be reduced in
methylcholanthrene induced fibrosarcoma rats after Muthu Marunthu
treatment. (25)
7. Evaluation
of Kutaj-Ghanavati for alkaloidal principles:- Kutaj-Ghanavati
is a reputed Ayurvedic preparation used in dysentery and diarrhea. It
contains water extract of Kurchi bark and fine powder of aconite roots.
It was evaluated quantitatively and qualitatively employing TLC and
titrimetric method. In TLC study no interference of Kurchi and Aconite
alkaloids with one another in their respective solvent systems. The
formulation was found to contain all alkaloids of Kurchi and Aconite.
(26)
8. Organoleptic
evaluation: - Organoleptic evaluation of food products
plays an important role in judging the censoring acceptability or
rejection of food items in the market. Effect of various treatments
(blanching, pricking, and lye treatment), sugar concentration (50%,
60%, 70%) and storage on the color scores; flavor scores; texture
scores of intermediate moisture apricots. The overall acceptability of
the products was significantly higher in 70% sugar syrup but these
scores decreased as the storage period advanced. (27)
CONCLUSION: - The
subject of herbal drug standardization is massively wide and deep.
There is so much to know and so much seemingly contradictory theories
on the subject of herbal medicines and its relationship with human
physiology and mental function.
For the purpose of
research work on standardization of herbal formulations, a profound
knowledge of the important herbs found in India and widely used in
Ayurvedic formulation is of utmost importance.(6)
Even when the chemical composition of a plant
extract is known, the pharmacologically active moiety may not be.
Environment, climate, and growth conditions influence composition, as
does the specific part of the plant and its maturity. Monographs
detailing standardization of active ingredients would improve the
marketplace. Even if an herbal product is standardized to, for example,
4% of a constituent, the remaining 96% of ingredients is not
standardized and may affect the product’s solubility, bioavailability,
stability, efficacy and toxicity. Just as controlled trials are
necessary to establish safety and efficacy, manufacturing standards are
required to ensure product quality.(28)
Now a days newer and advanced methods are
available for the standardization of herbal drugs like fluorescence
quenching, combination of chromatographic and spectrophotometric
methods, biological assays, use of biomarkers in fingerprinting etc.
Bioassay can play an important role in the standardization of herbal
drugs and can also become an important quality control method as well
as for proper stability testing of the product.(4)
India can emerge as the major country and play
the lead role in the production of standardized, therapeutically
effective ayurvedic formulation. India needs to explore the medicinally
important plants. This can be achieved only if the herbal products are
evaluated and analyzed using sophisticated modern techniques of
standardization such as UV- visible, TLC, HPLC, HPTLC, GC-MS,
spectrofluorimetric and other methods.(6)
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Article Source: http://www.articlesbase.com/alternative-
medicine-articles/standardisation-and-evaluation-of-
herbal-drug-formulations-1317004.html About the Author
M.Pharm.(Pharmacognosy)
Lecturer at BN Girls college of Pharmacy, Udaipur-INDIA |
